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Relative Contribution of Food Folate and Folic Acid to Intake and Status of Young Men and Women

Permanent Link: http://ufdc.ufl.edu/UFE0021828/00001

Material Information

Title: Relative Contribution of Food Folate and Folic Acid to Intake and Status of Young Men and Women
Physical Description: 1 online resource (172 p.)
Language: english
Creator: Grabianowski, Melanie L
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: acid, cereal, dietary, enriched, equivalents, folate, folic, fortification, intake, status
Food Science and Human Nutrition -- Dissertations, Academic -- UF
Genre: Food Science and Human Nutrition thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Folate, a water-soluble vitamin essential for one-carbon metabolism, is available in the diet as naturally occurring food folate or synthetic folic acid. Since 1998, the United States (US) Food and Drug Administration has mandated folic acid fortification of all enriched cereal-grain products with the intent of increasing folic acid intake of women of reproductive potential to help reduce neural tube defect (NTD) risk. With the introduction of folic acid fortification, researchers have been interested in determining the impact of fortification on folate status and intake of the population. The aim of this study was to assess folate/folic acid intake and folate status of non-supplement consuming healthy young men and women, and to examine the relative contribution of different food categories to folate/folic acid intake. Furthermore, the relationship between folate status and methylenetetrahydrofolate reductase (MTHFR) 677 C > T genotype was examined because of previous reports suggesting a genotype effect on folate status. Folate status was determined for men (n = 140) and women (n = 162) (ages 18 to 49 years). Mean serum and red blood cell folate (RBC) concentrations for males (39.9 nmol/L; 810 nmol/L, respectively) did not differ (P > 0.2) from that of females (41.7 nmol/L; 767 nmol/L). The mean plasma homocysteine concentration was significantly higher for males (8.0 micromole/L) than females (6.6 micromole/L) (P < 0.0001). Comparisons of folate status by MTHFR 677 C > T genotype for all subjects revealed that individuals with the CC genotype had higher RBC folate and lower plasma homocysteine concentrations than individuals with the CT or TT genotypes. To estimate usual folate/folic acid intake, subjects completed a modified Dietary History Questionnaire. Mean total intake expressed as dietary folate equivalents (DFE) for males and females (652 mcg DFE/d; 512 mcg DFE/d, respectively) exceeded the Recommended Dietary Allowance of 400 mcg DFE/d. Average intake of folic acid for females was 128 mcg/d, with only 3% of females consuming at least 400 mcg/d of folic acid, the amount recommended by the Institute of Medicine to reduce the risk of an NTD-affected pregnancy. The largest contributors to folic acid intake for males and females included enriched cereal-grain products (41.1%; 41.9%, respectively), fortified ready-to-eat (RTE) cereals and bars (29.3%; 36.0%), and combination foods that included 'enriched' ingredients (17.5%; 14.8%). The food categories that provided the most naturally occurring food folate in the diets of males and females, respectively, were vegetables (31.6%; 38.4%) and legumes and nuts (16.2%; 14.4%). The present study is one of the first to use the US Department of Agriculture National Nutrient Database for Standard Reference (Release 17) to assess dietary folate intake expressed as food folate, folic acid, total folate, and DFEs. Dietary folic acid from enriched cereal-grain products and fortified RTE cereals positively affected total folate intake and status of males and females; however, folic acid intake for females was less than the level recommended for NTD risk reduction (i.e., 400 mcg/d). Daily consumption of fortified RTE cereals may be an effective intervention strategy for increasing folic acid intake in women of reproductive potential.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Melanie L Grabianowski.
Thesis: Thesis (M.S.)--University of Florida, 2007.
Local: Adviser: Kauwell, Gail P.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021828:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021828/00001

Material Information

Title: Relative Contribution of Food Folate and Folic Acid to Intake and Status of Young Men and Women
Physical Description: 1 online resource (172 p.)
Language: english
Creator: Grabianowski, Melanie L
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: acid, cereal, dietary, enriched, equivalents, folate, folic, fortification, intake, status
Food Science and Human Nutrition -- Dissertations, Academic -- UF
Genre: Food Science and Human Nutrition thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Folate, a water-soluble vitamin essential for one-carbon metabolism, is available in the diet as naturally occurring food folate or synthetic folic acid. Since 1998, the United States (US) Food and Drug Administration has mandated folic acid fortification of all enriched cereal-grain products with the intent of increasing folic acid intake of women of reproductive potential to help reduce neural tube defect (NTD) risk. With the introduction of folic acid fortification, researchers have been interested in determining the impact of fortification on folate status and intake of the population. The aim of this study was to assess folate/folic acid intake and folate status of non-supplement consuming healthy young men and women, and to examine the relative contribution of different food categories to folate/folic acid intake. Furthermore, the relationship between folate status and methylenetetrahydrofolate reductase (MTHFR) 677 C > T genotype was examined because of previous reports suggesting a genotype effect on folate status. Folate status was determined for men (n = 140) and women (n = 162) (ages 18 to 49 years). Mean serum and red blood cell folate (RBC) concentrations for males (39.9 nmol/L; 810 nmol/L, respectively) did not differ (P > 0.2) from that of females (41.7 nmol/L; 767 nmol/L). The mean plasma homocysteine concentration was significantly higher for males (8.0 micromole/L) than females (6.6 micromole/L) (P < 0.0001). Comparisons of folate status by MTHFR 677 C > T genotype for all subjects revealed that individuals with the CC genotype had higher RBC folate and lower plasma homocysteine concentrations than individuals with the CT or TT genotypes. To estimate usual folate/folic acid intake, subjects completed a modified Dietary History Questionnaire. Mean total intake expressed as dietary folate equivalents (DFE) for males and females (652 mcg DFE/d; 512 mcg DFE/d, respectively) exceeded the Recommended Dietary Allowance of 400 mcg DFE/d. Average intake of folic acid for females was 128 mcg/d, with only 3% of females consuming at least 400 mcg/d of folic acid, the amount recommended by the Institute of Medicine to reduce the risk of an NTD-affected pregnancy. The largest contributors to folic acid intake for males and females included enriched cereal-grain products (41.1%; 41.9%, respectively), fortified ready-to-eat (RTE) cereals and bars (29.3%; 36.0%), and combination foods that included 'enriched' ingredients (17.5%; 14.8%). The food categories that provided the most naturally occurring food folate in the diets of males and females, respectively, were vegetables (31.6%; 38.4%) and legumes and nuts (16.2%; 14.4%). The present study is one of the first to use the US Department of Agriculture National Nutrient Database for Standard Reference (Release 17) to assess dietary folate intake expressed as food folate, folic acid, total folate, and DFEs. Dietary folic acid from enriched cereal-grain products and fortified RTE cereals positively affected total folate intake and status of males and females; however, folic acid intake for females was less than the level recommended for NTD risk reduction (i.e., 400 mcg/d). Daily consumption of fortified RTE cereals may be an effective intervention strategy for increasing folic acid intake in women of reproductive potential.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Melanie L Grabianowski.
Thesis: Thesis (M.S.)--University of Florida, 2007.
Local: Adviser: Kauwell, Gail P.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021828:00001


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RELATIVE CONTRIBUTION OF FOOD FOLATE AND FOLIC ACID
TO INTAKE AND STATUS OF YOUNG MEN AND WOMEN





















By

MELANIE LYN GRABIANOWSKI


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
FOOD SCIENCE AND HUMAN NUTRITION

UNIVERSITY OF FLORIDA

2007































2007 Melanie Lyn Grabianowski









ACKNOWLEDGEMENTS

I would like to sincerely thank my supervisory committee members Lynn B. Bailey, PhD,

Gail P.A. Kauwell, PhD, RD, LDN, and Linda Young, PhD for the unique contributions that

each of them made to my research project. In particular, I would like to thank my committee co-

chairs, Drs. Lynn Bailey and Gail Kauwell, for guiding and encouraging me through this

challenging experience. They are both remarkable mentors and role models and have been an

inspiration from the very beginning. Without their expertise and support, I could not have

accomplished this endeavor. I have learned more in the past few years than I ever could have

anticipated, and I know that the knowledge and wisdom I have gained will take me great places

in the future.

I also would like to thank Dave Maneval for his assistance in the laboratory, James Colee

for his assistance with the statistical analyses of my data, and Karla Shelnutt, PhD, RD, Kristina

von-Castel-Roberts, PhD, and Amanda Wittmann, MS, RD, LDN for being great listeners. Each

of them played an important role in guiding me through this long journey.

My family and friends have been an amazing support system to me and have been a

constant source of encouragement and sage advice for which I am very grateful. Finally, I would

like to thank Dominick Savoca. He reminded me everyday how proud he was of my hard work

and accomplishments. His love and encouragement gave me the strength to complete this

program.









TABLE OF CONTENTS

Page

A CK N O W LED G M EN TS ........................................................ ....................................... 3

LIST O F TA BLE S ......................................... ...... .............. ...............................

LIST O F FIG U RE S ................................................................. 8

L IST O F A B B R E V IA TIO N S ....................................................................... ... ....................... 9

A B S T R A C T ......................................................................................................... 12

CHAPTER

1 INTRODUCTION ............... ..................................................... ..... 14

O v erall G o als ......................................................16
Specific Objectives .............. ................. ........... ..................... ......... 16

2 BACKGROUND AND LITERATURE REVIEW ....................................................17

F o la te ...................................................................................................................................... 1 7
C h em istry ................................................................................ 17
D dietary Sources ......................................................................................................17
B io av a ilab ility ........................................................................................................... 1 9
Absorption, Transport and Storage ............................................................ .....21
M etabolism and Excretion ............................................................................... 22
D dietary R reference Intakes ................................................................................. 23
Biochemical Functions.................... ......... ....................... 24
Key-Folate Dependent Reactions in One-Carbon Metabolism ...................................24
Methylenetetrahydrofolate Reductase Polymorphism .................................................26
Folate Status Assessment.............................. .................. 27
Serum and Red Blood Cell Folate Concentrations ........................................ ...27
Plasm a H om ocysteine Concentration ....................................................... 28
Folate D efficiency ..... ..................... ..... ........ ...................... 29
E tio lo g y ................................ .......................................................2 9
M egaloblastic A nem ia ............................................................................... 29
N eural T ube D effects .................................................. ................ ............................. 3 1
V ascular D disease and Stroke ..................................................................................34
C an cer ............................. ............................... ................................. ..........3 9
Folic A cid Fortification .......................... ................ ... .... .. ....... .................... 43
Effect of Fortification on Folate Status of Women of Reproductive Potential ................45
Effect of Fortification on Neural Tube Defects.............. ............... ...............47
Folic Acid Recommendations and Awareness ........................................... ...............49
A analytical M methodology ............................................................................................. 50
B lo o d F late A n aly sis .................................................................................... 5 1


4









Plasm a H om ocysteine A nalysis............................................. ............................. 53
M THFR Genotype Determination ...................................................................... 54
D dietary Intake A ssessm ent ....................................................................... ........................ 55
U SD A N national N utrient D atabase.......................................... ........................... 55
Food Frequency Questionnaire......... .. ... ......... .. ............. ............ ............... 55
Assessment of Folate/Folic Acid Intake in Women of Reproductive Potential.....................58

3 STUDY DESIGN AND METHODS .......................................................................63

Study D esign and M ethods Overview ......................................................... ............... 63
H um an Subject P procedures ......... ................. ......................................... ...........................64
D ietary H history Q questionnaire ....................................................................... ..................65
Processing the Dietary History Questionnaire ............................................ ............... 65
A analysis of N utrient Intakes......... ................. ..................................... ................... 66
Dietary History Questionnaire Instruction Pretest ...................................... ............... 66
Blood Sam ple Collection and Processing ........................................ ......................... 67
Analytical Methods for Assessing Folate Status.....................................68
Identification of Food Groups from the Dietary History Questionnaire ......................68
Serum and RB C Folate Concentrations.................................. ..................................... 69
Homocysteine Concentrations .................................. ......... ... ................. 69
MTHFR Genotype Determination...................... ...... ............................ 69
S statistic al M eth o d s ........................................................................................................... 6 9

4 R E S U L T S ............................................................................................7 2

Characteristics of Study Population ......................................................... .............. 72
D ietary Folate Intake and G ender ................................................ .............................. 73
D ietary Folate Intake and G enotype................................................................. ............... 73
Dietary Folate Equivalents, Folic Acid and Recommended Intakes................................ 74
Contribution of Folate from Food Groups ........................................ ........................ 74
Folate Intake from Fortified Ready-to-Eat Cereals .......... .................. .... ............76
Folate and Homocysteine Concentrations and Gender .................................. ...............77
Folate and Homocysteine Concentrations and Genotype ............................................... 78
Serum Folate and H om ocysteine..................................................... ........................... 79
Relationship Between Folate Intake and Status .......................................... ............... 79
Folate Status and Fortified Ready-to-Eat Cereal Consumption...................................80

5 DISCUSSION AND CONCLUSIONS ............................................................................95

Strengths and L im station s............................................................................ ................... 104
S u m m ary ..........................................................................10 5
Practical Application .......... ............. .. ............... ........ ............ 106

APPENDIX

A SUBJECT DATA COLLECTION FORM.................................... ................... 108

B DIETARY HISTORY QUESTIONNAIRE .................... ................ ............... 113









C DIETARY HISTORY QUESTIONNAIRE INSTRUCTIONS MATERIALS ...................153

L IST O F R E F E R E N C E S ..................................................................................... ..................159

B IO G R A PH IC A L SK E T C H ......................................................................... ... ..................... 172









LIST OF TABLES


Table Page

2-1 Definitions of Dietary Reference Intake recommendations ..............................................61

2-2 Folate intake recommendations for men and non-pregnant women >19 years ...............61

3-1 Description of foods categorized within each food group.........................................71

4-1 Demographic characteristics of males and females................................... ... ..................81

4-2 M ean intake ([g/d) by folate category and gender................................. ............... 82

4-3 Mean intake ([g/1,000 kcals/d) by folate category and gender .....................................83

4-4 Mean intake ([g/d) by folate category and genotype.....................................................84

4-5 Mean intake ([g/1,000 kcals/d) by folate category and genotype............................. 84

4-6 M ean total folate intake ([g/d) by food group ...................................... ............... 86

4-7 M ean DFE intake ([g DFE/d) by food group ....................................... ............... 86

4-8 M ean folic acid intake ( g/d) by food group................................... ....... ............... 87

4-9 M ean food folate intake ([g/d) by food group...................................... ............... 87

4-10 Comparison of serum and RBC folate and homocysteine concentrations by gender .......90

4-11 Comparison of mean serum folate concentrations (nmol/L) by genotype ........................92

4-12 Comparison of mean RBC folate concentrations (nmol/L) by genotype..........................92

4-13 Comparison of mean homocysteine concentrations ([mol/L) by genotype....................92










LIST OF FIGURES


Figure Page

2-1 F olate/folic acid structures........................................................................ ...................60

2-2 One-carbon metabolism folate-dependent pathway............................................... 62

4-1 Mean intake ([g/d) by folate category and gender ........................................................82

4-2 Mean intake ([g/1,000 kcals/d) by folate category and gender.........................................83

4-3 Percentage of subjects who consumed specific ranges of folate intake ([tg DFE/d)......... 85

4-4 Percentage of subjects who consumed specific ranges of folic acid ([tg/d) ......................85

4-5 Percentage of total intake derived from each food group by folate category for males ....88

4-6 Percentage of total intake derived from each food group by folate category for females.88

4-7 Mean percent contribution of fortified RTE cereal to total mean intake for each folate
categ ory for m ales ....................................................... ................ 89

4-8 Mean percent contribution of fortified RTE cereal to total mean intake for each folate
category for fem ales ......................... ............ .......... ... .............. .. 89

4-9 Mean serum folate concentration (nmol/L) by gender ............................................... 91

4-10 M ean RBC folate concentration (nmol/L) by gender ........................................................91

4-11 Mean homocysteine concentration ([tmol/L) by gender..................................................91

4-12 Mean serum folate concentration (nmol/L) by genotype........................................93

4-13 Mean RBC folate concentration (nmol/L) by genotype ..........................................93

4-14 Mean homocysteine concentration ([tmol/L) by genotype..............................................94









LIST OF ABBREVIATIONS


125I iodine-125

1-C one-carbon

AI Adequate Intake

ASCII American Standard Code for Information Interchange

BMI body mass index

C cytosine

C degrees Celcius

CDC Centers for Disease Control and Prevention

CH2 methylene

CH3 methyl

CHD coronary heart disease

CRC colorectal cancer

CSFII Continuing Survey of Food Intakes by Individuals

CVD cardiovascular disease

d day

DASHTM dynamic allele-specific hybridization

DFE dietary folate equivalents

DHF dihydrofolic acid

DHQ Dietary History Questionnaire

DNA deoxyribonucleic acid

DRI Dietary Reference Intakes

dTMP thymidylate

dUMP deoxyuridylate









EAR

F

FA

FAD

FDA

FF

FFQ

FIN

GLM

kcals

IOM

L

LC-MS/MS

M

mg

ml

MTHFR

NCI

NHANES

nmol

NTD

PCR

RBC


Estimated Average Requirement

females

folic acid

flavin adenine dinucleotide

Food and Drug Administration

food folate

food frequency questionnaire

food identification number

generalized linear models

kilocalories

Institute of Medicine

liter

liquid chromatography-tandem mass spectrometry

males

milligram

milliliter

methylenetetrahydrofolate reductase

National Cancer Institute

National Health and Nutrition Examination Surveys

nanomole

neural tube defect

polymerase chain reaction

red blood cell









RDA Recommended Dietary Allowance

RFC reduced folate carrier

RNA ribonucleic acid

RTE ready-to-eat

SAH S-adenosylhomocysteine

SAM S-adenosylmethionine

SD standard deviation

SE standard error

SNP single nucleotide polymorphism

T thymine

TF total folate

THF tetrahydofolic acid

Mg microgram

UL Tolerable Upper Intake Level

[mol micromole

UNC University of North Carolina

US United States

USDA United States Department of Agriculture









Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Food Science and Human Nutrition

RELATIVE CONTRIBUTION OF FOOD FOLATE AND FOLIC ACID
TO INTAKE AND STATUS OF YOUNG MEN AND WOMEN

By

Melanie Lyn Grabianowski

December 2007

Chair: Gail P.A. Kauwell
Major: Food Science and Human Nutrition

Folate, a water-soluble vitamin essential for one-carbon metabolism, is available in the

diet as naturally occurring food folate or synthetic folic acid (1). Since 1998, the United States

(US) Food and Drug Administration has mandated folic acid fortification of all enriched cereal-

grain products with the intent of increasing folic acid intake of women of reproductive potential

to help reduce neural tube defect (NTD) risk (2). With the introduction of folic acid fortification,

researchers have been interested in determining the impact of fortification on folate status and

intake of the population (3-6). The aim of this study was to assess folate/folic acid intake and

folate status of non-supplement consuming healthy young men and women, and to examine the

relative contribution of different food categories to folate/folic acid intake. Furthermore, the

relationship between folate status and methylenetetrahydrofolate reductase (MTHFR) 677 C-T

genotype was examined because of previous reports suggesting a genotype effect on folate

status.

Folate status was determined for men (n = 140) and women (n = 162) (ages 18 to 49

years). Mean serum and red blood cell folate (RBC) concentrations for males (39.9 nmol/L; 810

nmol/L, respectively) did not differ (P>0.2) from that of females (41.7 nmol/L; 767 nmol/L).

The mean plasma homocysteine concentration was significantly higher for males (8.0 [mol) than









females (6.6 imol/L) (P<0.0001). Comparisons of folate status by MTHFR 677 C-T genotype

for all subjects revealed that individuals with the CC genotype had higher RBC folate and lower

plasma homocysteine concentrations than individuals with the CT or TT genotypes.

To estimate usual folate/folic acid intake, subjects completed a modified Dietary History

Questionnaire. Mean total intake expressed as dietary folate equivalents (DFE) for males and

females (652 pg DFE/d; 512 pg DFE/d, respectively) exceeded the Recommended Dietary

Allowance of 400 pg DFE/d. Average intake of folic acid for females was 128 ig/d, with only

3% of females consuming at least 400 .g/d of folic acid, the amount recommended by the

Institute of Medicine to reduce the risk of an NTD-affected pregnancy (7). The largest

contributors to folic acid intake for males and females included enriched cereal-grain products

(41.1%; 41.9%, respectively), fortified ready-to-eat (RTE) cereals and bars (29.3%; 36.0%), and

combination foods that included "enriched" ingredients (17.5%; 14.8%). The food categories

that provided the most naturally occurring food folate in the diets of males and females,

respectively, were vegetables (31.6%; 38.4%) and legumes and nuts (16.2%; 14.4%).

The present study is one of the first to use the US Department of Agriculture National

Nutrient Database for Standard Reference (Release 17) to assess dietary folate intake expressed

as food folate, folic acid, total folate, and DFEs (8). Dietary folic acid from enriched cereal-

grain products and fortified RTE cereals positively affected total folate intake and status of males

and females; however, folic acid intake for females was less than the level recommended for

NTD risk reduction (i.e., 400 ig/d). Daily consumption of fortified RTE cereals may be an

effective intervention strategy for increasing folic acid intake in women of reproductive

potential.









CHAPTER 1
INTRODUCTION

Folate is a water-soluble B-complex vitamin that enables enzymes to transfer one-carbon

groups to a variety of target molecules that are essential to the regulation of one-carbon (1-C)

reactions involved in pyrimidine metabolism and methylation. Folate is an important nutrient

that is found in the diet as naturally occurring food folate or synthetic folic acid. Foods naturally

rich in food folate include dark green leafy vegetables, legumes, and orange juice.

In 1998, the United States (US) Food and Drug Administration (FDA) mandated folic

acid fortification of all enriched cereal-grain products (e.g., bread, pasta, flour, rice, corn meal,

and cereals, including ready-to-eat (RTE) breakfast cereals) with the intent of reducing neural

tube defect (NTD) risk by increasing daily folic acid intake of women of reproductive potential

(2). To decrease the risk of having a NTD affected pregnancy, it is currently recommended that

women of reproductive age consume 400 tg/d of folic acid from supplements or fortified foods,

in addition to dietary food folate (9). For the general population, it is recommended that both

males and females 19 years and older consume the Recommended Dietary Allowance (RDA) of

400 tg DFE/d. The RDA was based on controlled studies on the adequacy of folate intake to

maintain normal blood concentrations of folate status indicators, including serum and RBC

folate, and plasma homocysteine concentrations (10). A continuous public health challenge is to

ensure that adults, especially women capable of becoming pregnant, consume the recommended

amount of folic acid intake on a daily basis.

Since the FDA mandated folic acid fortification, there has been public interest in

determining the folate status and intake of adult men and women. Dietary intake of folate has

been assessed in adult men and women through a variety of methods, including food frequency

questionnaires. Research has consistently shown that folic acid fortification has improved the









folate status of the population compared to pre-fortification and dramatically decreased the

incidence ofNTDs (11-16). However, more recent studies have suggested that despite folate

status improvement, women of reproductive age still fail to consume the recommended amount

of folic acid daily (13). Furthermore, public health educational programs have focused attention

on the recommendation that folic acid supplements be taken daily by women of reproductive

age; however, recent surveys indicated that only 33% of women reported taking a daily

supplement containing folic acid (17).

The data from this study provide valuable information regarding the relative and total

contributions of dietary sources of folic acid to daily folic acid intake, which may help determine

improved strategies for helping women meet the recommended daily level of intake for folic

acid. In addition, this study adds valuable information concerning the folate status of healthy

young men and women. To evaluate folate status, this study was also designed to evaluate

plasma homocysteine concentration, which is a known folate status indicator. Blood folate and

homocysteine concentrations are inversely correlated indicating homocysteine concentration is

elevated in response to reduced folate status (18).

With regard to other factors that may influence folate status, this study provides

validating evidence of a genetic single-nucleotide polymorphism that has been extensively

studied and demonstrated to influence the folate-dependent metabolic pathway of 1-C

metabolism. This polymorphism is a cytosine (C) to thymine (T) substitution at base pair 677 in

the gene that encodes for 5,10-methylenetetrahydrofolate reductase (MTHFR) (19). The

MTHFR 677 C--T variant can be characterized as homozygous (TT) or heterozygous (CT) and

is prevalent in approximately 12% and 50%, respectively, of the overall population (20). In

individuals who are homozygous (TT) for the MTHFR 677 C--T polymorphism, the impaired









enzyme activity is associated with reduced blood folate and elevated plasma homocysteine

concentrations, both of which can be significantly improved in response to improved folate status

(21).

Overall Goal

The goal of the present study was to assess folate status of non-supplement consuming

healthy young men and women and to estimate the relative contribution of folate/folic acid from

different food categories to overall intake.

Specific Objectives

1. To assess folate/folic acid intake and folate status of young male and female non-supplement
users.

2. To evaluate the adequacy of folate intake relative to the RDA for men and women, and to
evaluate the adequacy of folic acid intake for women relative to the recommendation for
NTD risk reduction.

3. To compare the actual and relative contribution of specific food categories to folate/folic acid
intake in young male and female non-supplement users.

4. To compare folate status of young adults by folate intake and by MTHFR 677C--T
genotype.









CHAPTER 2
BACKGROUND AND LITERATURE REVIEW

Folate

Chemistry

Folate is a water-soluble B-complex vitamin that is consumed in the diet as either

naturally occurring food folate or synthetic folic acid. Folic acid is the synthetic, monoglutamate

form of folate used in fortified foods and supplements (1, 22). The chemical structure of folic

acid, which has a molecular weight of 441.4, is formed through the combination of three

structural features that are required for its functional activity: a para-aminobenzoic acid molecule

is linked at one end to a pteridine ring by a methylene bridge (C-9-N-10), and at the other end

joined by peptide linkage to a glutamic acid molecule (1, 23) (Figure 2-1). Naturally occurring

food folates exist in various chemical forms, containing a side-chain composed of two to ten

additional glutamate residues joined to the first glutamate residue (1, 9) (Figure 2-1).

The structure of the folate molecule can change through the reduction of the pteridine

ring to dihydrofolic acid (DHF) and tetrahydofolic acid (THF), elongation of the chain of

glutamate residues, and 1-C substitutions at the N-5, N-10, or both positions of THF (23). The

polyglutamyl form of the THF molecule can be converted to various forms of folate through the

transfer of methyl (CH3), methylene (-CH2-), methenyl (-CH=), formyl (-CH=O), or

formimino (-CH=NH) groups (1) (Figure 2-1). These one carbon entities can be transferred in

diverse pathways including those required for the synthesis of deoxyribonucleic acid (DNA),

amino acid interconversions (e.g., homocysteine to methionine), and DNA methylation.

Dietary Sources

Eukaryotic cells are unable to synthesize the folate molecule in the body. Thus, folate

must be consumed through dietary sources, which include naturally occurring food folate and









foods fortified with synthetic folic acid. Endogenous food folate is found in a variety of foods

including orange juice, dark green leafy vegetables, asparagus, strawberries, peanuts, and

legumes such as kidney beans, black beans and lima beans (9). Meat is not considered a good

source of food folate, except for liver. Additionally, folate concentrations are higher in raw

foods than cooked foods, due to folate losses that occur through cooking processes, with losses

dependent on oxygen exposure, ascorbic acid content, and the quantity of water used. Folate in

plant foods can lose up to 40% of their folate content during cooking (24).

Synthetic folic acid is another major source of folate in the diet and is found in enriched

cereal-grain products and fortified RTE breakfast cereals. Since January 1, 1998, the FDA has

mandated folic acid fortification of all enriched cereal-grain products (e.g., bread, pasta, flour,

rice, corn meal, and cereals, including RTE breakfast cereals) and mixed food products

containing these grains (9). The target folic acid concentration of cereal-grain products is 140 .g

folic acid per 100 g cereal-grain, with a range in recommended amount to be added to various

foods (95 to 309 pg per 100 g of product) (2). The majority of RTE breakfast cereals provide

25% of the Daily Value of folic acid (-100 Gg/serving), with some brands providing up to 100%

(400 Gg/serving) (9, 25). Naturally occurring food sources of folate are considered "excellent"

sources of folate if they contain 100 to 200 pg DFE/serving, "good" sources if they contain 50

to 99 pg DFE/serving, "moderate" sources if they contain 25 to 49 pg DFE/serving, and "fair to

poor" sources if they contain <25 pg DFE/serving (9).

Total folate is the term that refers to the combination of endogenous food folate and/or

folic acid from fortification. Total folate intake per day is expressed as ig/d. Total folate does

not take into account the difference in bioavailability between naturally occurring food folate and









folic acid, whereas the expression of folate intake in terms of DFE accounts for the higher

bioavailability of folic acid compared to food folate.

Bioavailability

The bioavailability of folate refers to the degree to which folate is absorbed and utilized,

including differences in folate absorption, transport, metabolism, catabolism, enterohepatic

circulation and urinary excretion (25). Physiological conditions, variability of food constituents,

and pharmaceutical drugs can possibly affect the efficiency of intestinal absorption of food folate

through a variety of ways: entrapment of food folate within the food matrix; the presence of

enzyme inhibitors required for the deconjugation of the polyglutamate form of food folate to the

monoglutamate form required for absorption; the instability of THF in the acidic pH of the

stomach before absorption; and the presence of food constituents that may inhibit folate

deconjugation (1, 25).

A great deal of ambiguity exists among food folate research with regard to potential

factors affecting bioavailability, particularly the difference in bioavailability between the

monoglutamate and polyglutamate folate forms, and the degree of bioavailability of food folates.

Research findings reported by McKillop and colleagues suggest that the extent of conjugation is

not a factor affecting the efficiency of absorption of food folate in the intestines (25). Variation

in bioavailability reported in previous research could be due to differences in folate digestion,

absorption and metabolism, as well as inconsistencies between protocols and analytical methods

used to measure and report bioavailability. Alcohol and pharmaceutical drugs also can inhibit

the absorption and metabolism of folate (26).

Estimates of the bioavailability of food folate relative to folic acid have varied in the

measured percentage of absorption, ranging from 10 to 98% (25, 27-32); however, the best









estimate is provided by data from Sauberlich and colleagues indicating that food folate is

approximately 50% bioavailable relative to folic acid (9, 30). When folic acid is consumed as a

supplement in the fasting state or without food, it is 100% bioavailable (33). Folic acid

absorption is slightly reduced and estimated at 85% bioavailable when folic acid is consumed

with food, which is the case with enriched cereal-grain products or fortified foods (10, 34, 35).

The physiological requirement for folate represents the amount the body requires post-folate

absorption. Considering the differences in folate bioavailability, a larger quantity of food folate

compared to folic acid must be ingested to meet the physiological requirement (9). These data

coupled with the estimate that food folate is approximately 50% bioavailable were used by the

Dietary Reference Intake (DRI) Committee of the Institute of Medicine (IOM) to develop the

concept of DFE, a unit of measure that acknowledges differences in bioavailability and

absorption among the various chemical forms of the vitamin (9). The DFE values for folic acid

in enriched cereal-grain products and vitamin supplements are calculated using conversion

factors that account for differences in bioavailability relative to naturally occurring food folate

(9). Folic acid in supplemental form taken while fasting is estimated to be twice as bioavailable

relative to food folate (i.e., 100:50). Folic acid from supplements taken with food or folic acid

from enriched or fortified food sources is estimated to be 1.7 times more bioavailable than

naturally occurring food folate (i.e., 85:50) (9). The total folate content of any food product in

DFE units can be calculated by multiplying the micrograms of synthetic folic acid by 1.7 then

adding this value to the micrograms of food folate present (9). No calculation or adjustment is

needed when solely determining food folate intake.









Absorption, Transport and Storage

Folate must be in the monoglutamate form in order for absorption to occur. Synthetic

folic acid is already a monoglutamate, and thus readily absorbed. However, since food folates

are polyglutamates, they must first be hydrolyzed by the enzymatic action of folypoly-y-

glutamate carboxypeptidase II, also known as pteroylpolyglutamate hydrolase or folate

conjugase, before absorption can occur (36). Following complete deconjugation of the

polyglutamate molecule, folates are transported across the brush border membrane in the

jejunum via an acidic pH-dependent (pH of 6.5 to 7.0) carrier-mediated mechanism (37, 38).

Drugs or diseases that impair jejunal pH may impair folate absorption (1).

Prior to entry into the portal system, the majority of folate is reduced by dihydrofolate

reductase to DHF and THF (36). Once folate is absorbed, it travels to the liver via portal

circulation, primarily in the form of 5-methylTHF. To allow for folate storage and retention in

the liver, folates are reduced and conjugated. A small fraction of 5-methylTHF in the plasma can

be present as free folate or bound with high affinity to a folate binding protein; however, the

majority of plasma folate is bound with low affinity to albumin (23, 39, 40).

Two mechanisms exist for transport of folate into the cells: a folate transporter and a

folate receptor (41, 42). These transport mechanisms are required for absorption of folate in the

small intestine, reabsorption of endogenous folate by the kidneys, and uptake of folate through

the plasma membrane into a developing embryo (41). The reduced folate carrier (RFC) is the

folate transporter in the intestinal epithelial cells and is a classic facilitated transport protein that

carries reduced folate (5-methylTHF) across the membrane; it is encoded by the RFC gene

(RFC1), which is expressed in most tissues and is capable of mediating bi-directional flux of

folate. The second transport mechanism is a folate receptor-mediated process by which folate









binds with high-affinity to a folate-receptor at the membrane surface and is transported into the

cells via a unidirectional system. The efficiency of cellular uptake of the carrier-mediated

process is much greater than that of the receptor-mediated process (41, 42). Pharmacological

quantities of folic acid are absorbed via diffusion as absorption by the RFC is a saturable process

(38).

Once cellular uptake of 5-methylTHF is complete, it is demethylated by methionine

synthase and converted to the polyglutamyl form through the action of folylpolyglutamate

synthetase. This mechanism allows for intracellular folate retention because polyglutamates are

unable to cross the cell membrane (43). Previous measurements of folate concentrations in liver

tissue indicate that the liver specifically stores approximately 6 to 14 mg of folate. Based on the

assumption that the liver stores 50% of total body folate, it is estimated that the body stores 15 to

30 mg of folate (44).

Metabolism and Excretion

When folate polyglutamates are released from tissues into circulation, the polyglutamate

form of folate molecules is reconverted to the monoglutmyl form by y-glutamylhydrolase. The

mechanisms of polyglutamation and monoglutamation are necessary for the function of 1-C

metabolism. The majority of circulating folate is reabsorbed in the proximal tubules of the

kidney primarily via a folate receptor (45-47). Folate may also be packaged as part of bile (-100

pg/d), concentrated and stored in the gall bladder, and subsequently secreted into the intestinal

tract. The majority of folate in bile is recycled during enterohepatic circulation, although some

may be excreted in the feces along with unabsorbed dietary folate, folate from endogenous

secretions, and folate synthesized by bacteria in the gut (48). Research findings have determined

that folate losses via fecal and urinary excretion are comparable and represent approximately 0.5









to 1% of folate (48, 49). These loses must be replaced daily through dietary folate or

supplemental folic acid to maintain normal folate metabolism.

Dietary Reference Intakes

Dietary Reference Intakes represent the most current recommendations for each vitamin

and mineral and include for each nutrient the Estimated Average Requirements (EAR), RDA,

Adequate Intake (AI), and Tolerable Upper Limit (UL). See Table 1-1 for definitions of each

DRI (10).

The most recent DRI recommendations were published by the National Academy of

Sciences IOM in 1998 (10). The previous recommendations for nutrient intake were established

to prevent clinical deficiencies. In establishing the 1998 DRI recommendations, the IOM shifted

the focus of recommendations from nutrient quantities needed to prevent clinical deficiencies to

intakes that ensure optimum health (10). Dietary Reference Intake recommendations for folate

are presented in Table 1-2. The RDA for men and non-pregnant women 19 years and older is

400 tg DFE/d. The recommendations are increased to 600 tg DFE/d and 500 tg DFE/d during

pregnancy and lactation, respectively. Since folate is not associated with toxic side-effects, the

UL of 1,000 [tg/d of synthetic folic acid is based solely on the concern that folic acid

supplementation can mask the diagnosis of a vitamin B12 deficiency, which is prevalent in

approximately 10 to 15% of the elderly population over 60 years of age (10, 50).

The IOM also recommends that all women of childbearing age consume 400 tg/d of

synthetic folic acid from fortified foods and/or supplements in addition to consuming food folate

from a varied diet to reduce the risk of having an NTD-affected pregnancy (9). However, the

achievement of the latter is problematic as compliance with the supplementation

recommendation is often suboptimal. It is estimated that approximately 33% of women of









reproductive age take a daily folic acid-containing supplement (17). Evidence for an association

between folic acid intake and NTD risk reduction provided the basis for the mandated folic acid

fortification of cereal-grain products.

Biochemical Functions

Interconnected reactions requiring specific forms of folate to accept and transfer one-

carbon units, together referred to as 1-C metabolism, include amino acid metabolism,

homocysteine remethylation, purine and pyrimidine synthesis, and the generation of S-

adenosylmethionine (SAM) (1). One-carbon metabolism is a series of folate-dependent

pathways that requires the donation of one-carbon units from individual folate coenzymes

resulting in the regeneration of THF. An illustration of the 1-C metabolism pathway is presented

in Figure 2-2. In 1-C metabolism, THF is the primary acceptor molecule and is required for

continuation of the cycle allowing for nucleotide biosynthesis and methylation reactions (1).

Key-Folate Dependent Reactions in One-Carbon Metabolism

Nucleotide biosynthesis. Through a reversible reaction, THF is converted to 5,10-

methyleneTHF in association with the conversion of serine to glycine, both of which are non-

essential amino acids, by the vitamin B6-dependent enzyme serine hydroxymethyltransferase

(SHMT). DNA synthesis is dependent upon the availability of 5,10-methyleneTHF, which can

donate a one-carbon unit to deoxyuridylate (dUMP) through the action of thymidylate synthase

to irreversibly synthesize thymidylate (dTMP). This rate-limiting step is essential for DNA

synthesis to proceed. During the process of donating a one-carbon group, 5,10-methyleneTHF is

oxidized to form DHF and reduced back to THF by dihydrofolate reductase. Additional

nucleotide production requires 5,10-methyleneTHF for the formation of 10-formylTHF by 10-









formylTHF synthetase, which can be used to donate multiple one-carbon units for purine

synthesis and the regeneration of THF for recirculation (1, 51).

Methylation reactions. The folate-dependent reactions of 1-C metabolism hinge on the

production of 5,10-methyleneTHF, which is also required for several other functional outcomes.

In order for the remethylation of methionine from homocysteine to occur, 5,10-methyleneTHF is

first reduced by MTHFR to 5-methylTHF. The 5-methylTHF form of folate, in conjunction with

methionine synthetase and cobalamin (vitamin B12), irreversibly donates a one-carbon group to

cobalamin to become methyl cobalamin. The methyl group from methyl cobalamin is

transferred to homocysteine to form methionine, and 5-methylTHF is ultimately converted to

THF. Regeneration of methionine and THF is required for the 1-C metabolism cycle to

continue. During a cobalamin deficiency, a secondary folate deficiency often occurs. The

"methyl-trap hypothesis" suggests the latter emerges because there is not enough cobalamin to

accept and donate a one-carbon unit to homocysteine (52). Thus, folate is "trapped" within 5-

methylTHF, and THF is not regenerated to continue the cycle through the synthesis of dTMP.

Consequently, DNA replication and cellular mitosis are interrupted.

Methionine is essential for the formation of S-adenosylmethionine (SAM) by the enzyme

SAM synthase and activation by adenosine 5'-triphosphate (ATP). Methionine, an essential

amino acid, can be derived from dietary protein intake or by generation from homocysteine.

SAM is the primary methylating agent that donates a methyl group, originally accepted from 5-

methylTHF, that is used in more than 100 methyltransferase reactions, including the methylation

of DNA, ribonucleic acid (RNA), protein, phospholipids and neurotransmitters (53). Once SAM

donates its methyl group, it is converted to S-adenosylhomocysteine (SAH) by various cellular

methyltransferases. SAH is hydrolyzed by SAH hydrolase to homocysteine and adenosine









through a reversible reaction in which SAH is favored in the balance. SAM also regulates the

production of 5-methylTHF. When dietary methionine is adequate, SAM will inhibit MTHFR

from producing 5-methylTHF and thus methionine; whereas, when the availability of methionine

is low, the concentration of SAM will be decreased (54). This releases the inhibition of MTHFR

to allow for greater production of 5-methylTHF and regeneration of methionine (54).

SAM also regulates the pyridoxal phosphate-dependent reaction in which homocysteine

is catabolized to cystathionine in combination with serine and the enzyme cystathionine P-

synthase. The transsulfuration pathway catabolizes excess homocysteine not required for methyl

transfer reactions. This transsulfuration pathway ultimately leads to the synthesis of cysteine

(non-essential amino acid) and glutathione (antioxidant) (1).

Methylenetetrahydrofolate Reductase (MTHFR) Polymorphism

Methylenetetrahydrofolate reductase, the enzyme required to reduce 5,10-methyleneTHF

to 5-methylTHF, is the main circulating form of folate and the major methyl donor for the

remethylation of homocysteine to methionine. The most extensively studied polymorphisms

related to folate metabolism are MTHFR polymorphisms. Frosst and colleagues discovered that

individuals homozygous for the 677C--T polymorphism (TT genotype) have decreased MTHFR

enzyme activity resulting in reduced levels of 5-methylTHF available to donate a methyl group

for the remethylation of homocysteine to methionine (19). This substitution renders the enzyme

"thermolabile" and may cause elevated plasma levels of the amino acid homocysteine. The

homozygous TT genotype is termed thermolabile because the enzymatic activity is decreased

compared to the CC genotype (55). Kang and colleagues discovered the TT genotype had a

specific enzymatic activity of approximately 50% of the normal levels seen with the CC

genotype (55). Decreases in MTHFR function have been associated with mild









hyperhomocysteinemia and may therefore be a risk factor for the development of cardiovascular

disease (CVD) (56). Approximately 12% of the population is homozygous for the 677C--T

variant (i.e., TT genotype), whereas approximately 50% is heterozygous (i.e., CT genotype) (20).

Matthews and colleagues characterized the decrease in MTHFR enzyme activity through

an increased dissociation of flavin adenine dinucleotide (FAD) from thermolabile MTHFR (56).

Furthermore, these researchers found that folic acid supplementation provided protection by

increasing the affinity of the enzyme for FAD. These results agreed with the findings by

Guenther and colleagues (57) who also found that dissociation of FAD resulted in a loss in

MTHFR enzyme activity. These investigators monitored enzyme activity and found the TT

genotype loses activity at a rate ten times faster compared to the enzyme activity associated with

the CC genotype (57).

Folate Status Assessment

Serum and Red Blood Cell Folate Concentrations

Serum folate concentration best reflects current and recent intakes of folate and is an

early and sensitive measure of short-term folate status (58). Controlled, metabolic studies have

shown that serum folate concentrations decrease quickly within a period of one to three weeks

when folate intake is limited (21, 52, 59). These metabolic changes precede decreases in RBC

folate concentrations. The lower limit of normal used to define inadequate serum folate status

using the microbiological assay is <13.6 nmol/L (60). An alternative method for assessing folate

status is the radiobinding assay, which has been shown to yield lower blood folate values relative

to the microbiological assay (61). The lower limit of normal for the radiobinding assay is <7

nmol/L.









Red blood cell folate is considered a better indicator of long-term folate status than serum

folate concentration as this marker reflects tissue folate storage. A previous study using liver

tissue biopsies found that RBC folate concentrations correspond to liver folate concentrations

(62). Uptake of folate into erythrocytes only occurs in the bone marrow during the early stages

of erythropoeisis. Since folate cannot permeate the membrane of a mature RBC during its 120-

day lifespan, RBC folate reflects folate status over the preceding three months. The lower limit

of normal used to define inadequate RBC folate status using the radiobinding assay is <317

nmol/L (10, 52).

Plasma Homocysteine Concentration

In addition to measuring blood folate concentrations, other "functional" indicators should

be used to evaluate folate status as these indices may reflect abnormalities in metabolic function

that may or may not be reflected in altered blood folate concentrations. The most notable

"functional" indicator is total plasma homocysteine concentration (63). An elevated

homocysteine concentration reflects not only a reduction in blood folate concentration secondary

to inadequate folate intake, but also an insufficient concentration of 5-methylTHF required to

convert homocysteine to methionine in the 1-C metabolism pathway (10, 18). Plasma

homocysteine concentration has been shown to be directly and inversely correlated with folate

status; however, it is not specific to folate deficiency because other nutrient deficiencies (i.e.,

vitamins B12 and B6), genetic abnormalities, renal insufficiency, and dietary and lifestyle factors

may influence homocysteine concentration (64, 65). Although discrepancies exist when defining

acceptable homocysteine concentrations, individuals with a plasma homocysteine concentration

>12 mol/L are often considered to be at higher risk for adverse health effects, including

vascular disease (4).









To assess homocysteine status in the US, Ganji and colleagues (66) presented the

distribution of plasma homocysteine concentrations of adults in the US by using data from the

National Health and Nutrition Examination Surveys (NHANES) 1999-2000 and 2001-2002.

These researchers studied the homocysteine distribution by age, gender and race-ethnicity in

9,196 subjects. Results showed that plasma homocysteine concentrations were higher in men

than in women (aged 19-70 years) and in older persons compared to younger persons (66). In a

preceding study, these researchers also reported that mean circulating homocysteine

concentrations in the period since folic acid fortification were 7.6 itmol/L in 1999-2000 and 7.9

pmol/L in 2001-2002, as compared with 9.5 imol/L during the period prior to fortification (1994

to 1998) (67).

Folate Deficiency

Etiology

In addition to inadequate folate in the diet, a folate deficiency can develop through other

causes, such as drug-nutrient interactions, genetic variations, impaired intestinal folate

absorption or folate metabolism secondary to high alcohol consumption, and increased renal

folate excretion. High doses of nonsteroidal anti-inflammatory drugs (i.e., aspirin, ibuprofen,

and acetaminophen) have been shown to exert antifolate activity (68-71). However, impaired

folate status has not been reported with low doses of these drugs. Folate deficiency associated

with genetic variations, alcohol consumption and dietary deficiency are discussed in the

following sections.

Megaloblastic Anemia

The development of a folate deficiency is associated with a progression of events that can

eventually lead to megaloblastic anemia seen with chronic severe folate deficiency. The









sequence of events begins with a reduction in serum folate concentration, which can rapidly

decrease within one to three weeks. Following a long phase of folate deficiency, RBC folate

concentration will also decline (58).

The most common etiology of megaloblastic anemia is impaired DNA synthesis, linked

to abnormalities in the folate pathway (1-C metabolism). A decrease in the availability of folate

from circulation and tissues will lead to a decline in DNA synthesis, reducing cell division, and

ultimately resulting in the formation of large, immature RBCs characteristic of megaloblastic

anemia. Abnormalities in RBC formation will lead to decreases in hemoglobin, hematocrit, and

RBC number.

Clinical manifestations of megaloblastic anemia can develop after a period of

compromised folate status. Folate status can be compromised and a folate deficiency can

develop through various factors including: inadequate folate intake; malabsorption induced by

abnormalities of the small intestine, drugs or alcohol; altered metabolism; increased requirement

(i.e., pregnancy, diseases); increased losses (i.e., dialysis, some skin diseases); abnormalities of

folate metabolism; and inherited abnormalities of folate absorption and metabolism (72).

Pregnancy is a common cause of megaloblastic anemia in women. Folate requirements

increase five to ten fold for pregnant women relative to non-pregnant women. The higher folate

requirement during pregnancy is related to increases in cellular proliferation secondary to the

growth and development of the fetus, placenta, and maternal tissues (73, 74), rather than an

increase in folate catabolism as substantiated by Caudill and colleagues (75). This elevated

demand for folate must be met by adequate dietary intake.









Neural Tube Defects

Neural tube defects are congenital malformations that occur in the brain and spinal

column that are caused primarily when closure of the neural tube fails during embryonic

development (76). The development and closure of the neural tube of an embryo occurs within

28 days after conception, often before women are aware they are pregnant. The extent of an

NTD varies with different degrees of tissue protrusion and neural involvement. The two most

common forms of NTDs are anencephaly (failure of the brain to develop) and spina bifida

(exposure of the spinal cord due to defective closure of the neural tube). In some instances,

defects in the orofacial cleft can develop from an abnormal increase in cerebrospinal fluid

pressure on the closed neural tube during the first trimester of embryonic development (76). The

incidence of NTDs (anencephaly or spina bifida) is 0.75 cases per 10,000 births in the US, with

roughly 400,000 cases per year worldwide (77) .

It has long been suspected that a cause-and-effect relationship exists between diet and

NTDs. Hibbard was one of the first researchers to address the importance of folate during

pregnancy. He proposed that the incidence of megaloblastic anemia in mid to late pregnancy

may have resulted from a defect in folate metabolism (78). Shortly after, Hibbard hypothesized

that fetal spontaneous abortions and placental abruption were associated with folate deficiencies

during pregnancy (79). In 1976, Smithells and colleagues further suggested that folate

deficiency was linked to NTDs because women who gave birth to NTD-affected infants had low

blood folate concentrations (80). These investigators reported that supplementation with folic

acid consumed periconceptionally reduced the recurrence of NTD pregnancies in high-risk

mothers. These results spawned further research studies that have continued to explore the

benefits of folic acid on reducing the incidence of NTDs. The powerful driving force in folate









research was the report from the Medical Research Council (MRC) Vitamin Study Research

Group (81). In 1983, the MRC launched a large-scale, randomized, double-blind, prevention

trial evaluating the effect of multivitamin supplementation with and without folic acid on the

recurrence of NTD-affected pregnancies. The study was ended early because 72% of NTDs

were prevented with 4 mg/d of supplemental folic acid (81). Moreover, in 1992, Czeizel and

Dudas found that periconceptional folic acid supplementation (800 .g/d) decreased first time

occurrence of NTDs in Hungarian women compared to subjects receiving no folic acid (82).

Further research led to the conclusion that folic acid supplementation can prevent up to 70% of

NTDs (83).

A subsequent intervention study conducted by Berry et al. (84) supported the work of

earlier investigators who observed a relationship between folic acid and NTDs. Berry and his

team investigated the effect of periconceptional use of folic acid in Chinese women living in

northern regions of China where NTDs rates were high and southern regions of China where

NTDs rates were low (84). Among the fetuses or infants of women who did not take folic acid,

NTDs rates were 4.8 and 1.0 per 1,000 pregnancies in the northern and southern regions of

China, respectively; whereas, 400 .g/d of folic acid reduced the occurrence of NTDs in the

northern and southern regions of China to 1.0 and 0.6 per 1,000 pregnancies, respectively. These

NTD rate decreases are equivalent to a respective 79% and 40% reduction in the incidence of

NTDs in the northern and southern regions.

In a study of 56,000 Irish women, Daly and colleagues (85) examined the relationship

between blood folate concentration and number of NTDs. The investigators observed that as

RBC folate concentration increased, the risk for NTDs decreased. They also reported an eight-

fold difference in the incidence of NTDs between women with RBC concentrations <341 nmol/L









and women with concentrations >908 nmol/L (85). It was estimated that a RBC folate

concentration of 908 nmol/L could be achieved with 400 pg/d of folic acid.

In a case-control study by Moore and colleagues, investigators reported an NTD risk of

3.4 cases per 1,000 pregnancies for women who consumed <150 ig DFE/d. Risk for NTDs

significantly decreased by 77% to 0.8 cases per 1,000 pregnancies for women who consumed

>1,200 pg DFE/d. These researchers concluded that the prevalence of NTDs decreased by 0.78

cases per 1,000 pregnancies with folate intake increments of 500 .g DFE/d (86). These data

suggest that NTD risk declined significantly with modest increases of total folate intake during

early pregnancy. Overall, an overwhelming amount of scientific evidence supports an

association between adequate folate status/folic acid intake and NTD risk reduction (31).

MTHFR polymorphism and neural tube defects. The MTHFR 677C-T

polymorphism was identified as the first genetic risk factor for NTDs. The MTHFR 677C--T

mutation, as previously reviewed, causes reduced activity of the enzyme. The homozygous

mutation (TT genotype) is associated with an elevated plasma homocysteine concentration and

reduced RBC and plasma folate concentrations. It has also been suggested that individuals with

the TT genotype have higher folate requirements (87).

Proposed mechanisms for folate-responsive NTDs include decreased rates of DNA

synthesis and cell division due to impaired dTMP synthesis, and a reduction in DNA methylation

(83). Embryonic tissues grow very rapidly, with high requirements for DNA synthesis and

methyl groups from SAM (88). Individuals homozygous for the MTHFR genotype may need

more folate in their diets to provide adequate amounts of methyl groups and to provide substrate

for the DNA methylation and synthesis via 1-C metabolism. During early pregnancy, a mild

deficiency can impair DNA synthesis and methylation, the latter of which can affect gene









expression. Disordered gene expression may explain how a relatively mild deficiency can

initiate the development of severe neural tube deformities in an embryo (88).

van der Put and colleagues (89) examined the frequency of the MTHFR 677C--T

polymorphism in patients with spina bifida (n = 55) and their parents (n = 130) and controls who

did not have spina bifida or offspring with spina bifida (n = 207). Only 5% of the control

subjects were homozygous for the TT genotype, whereas 13% of patients with spina bifida had

the TT genotype, as well as 16% and 10% of their mothers and fathers, respectively. The

researchers found that all subjects and controls with the TT genotype had decreased MTHFR

activity, low plasma folate concentrations, and high plasma homocysteine and RBC folate

concentrations (89). These researchers later reported that the risk of having offspring born with

spina bifida was strongest when both the mother and her child carried the homozygous variant

genotype (TT) (90). van der Put and colleagues concluded that the 677C--T polymorphism

should be regarded as a genetic risk factor for spina bifida.

Vascular Disease and Stroke

Several epidemiological studies have confirmed an inverse association between folate

and homocysteine concentrations based on the metabolic role of folate as a coenzyme in the

regulation and methylation of homocysteine in 1-C metabolism. Furthermore, studies have

indicated the role of an elevated plasma homocysteine concentration, also known as

hyperhomocysteinemia, as a significant risk factor for atherosclerotic vascular and coronary

heart disease. Homocysteine may increase vascular and coronary heart disease risk and

atherogenesis through direct toxicity to endothelial cells via lipid peroxidation, increased

coagulation and platelet aggregation, decreased endothelial cell reactivity, and stimulation of

smooth muscle cell proliferation (91-94).









To establish if there is a dose-dependent effect of folic acid supplementation on plasma

homocysteine concentration, meta-analyses of randomized controlled trials have been conducted

(95). Specifically, the Homocysteine Lowering Trialists' Collaboration reported results from

randomized controlled trials in two different meta-analyses (1998 and 2005) to determine the

size of reduction in homocysteine concentration achieved by different daily supplemental doses

of folic acid, with or without vitamins B12 or B6 (96, 97). The first meta-analysis revealed that

the effect of folic acid on blood homocysteine concentration seemed to depend on the

pretreatment blood homocysteine and folate concentrations (97). Participants with lower blood

folate concentration or higher baseline plasma homocysteine concentration experienced the

greatest improvement from supplements containing folic acid. After adjusting for pretreatment

blood homocysteine and folate concentrations, results indicated that daily folic acid doses of <1

mg/d (mean = 0.5 mg/d), 1-3 mg/d, and >3 mg/d lowered homocysteine concentration by

approximately 25%.

The second meta-analysis provided new data regarding a folic acid dose-dependent

lowering effect on homocysteine concentrations (96). Baseline plasma folate and homocysteine

concentrations before treatment with folic acid were standardized at 12 nmol/L and 12 [imol/L,

respectively. Results suggested that doses >800 [g/d of folic acid are typically required to

achieve maximum reductions in plasma homocysteine concentrations produced by folic acid

supplementation. Folic acid doses of 200 and 400 [g/d were associated with homocysteine

reductions of 60% and 90%, respectively, from baseline levels. The results revealed that 400

[g/d of folic acid produced near-maximum lowering effects in plasma homocysteine

concentration, which may be significant to public health (96).









Considering the role of supplemental folic acid in lowering homocysteine, the main

unanswered question is whether folic acid supplementation will reduce the overall rate of

vascular disease. Cardiovascular disease is the leading cause of death in the US, accounting for

approximately 37% of all deaths (98). Between 1980 and 1994, Rimm and colleagues (99)

examined the intakes of folate and vitamin B6 of 80,082 women in the Nurses' Health Study

Cohort in relation to the incidence of nonfatal myocardial infarction (MI) and fatal coronary

heart disease (CHD). The results revealed an approximate 30% lower relative risk of CHD for

women with higher folate intake (-700 tg/d) compared to intakes at or below the RDA at that

time (-160 ig/d), after controlling for vascular disease risk factors (i.e., smoking, hypertension,

alcohol consumption, fiber, vitamin E, and saturated, polyunsaturated, and trans fat) (99).

Because this study was conducted before implementation of folic acid fortification, the largest

contributor to the overall intake of folate was multivitamins, followed by RTE cereals, orange

juice, lettuce, eggs, broccoli, and spinach (99). The researchers noted that their findings were

also consistent with previous data linking higher intakes of folate with lower homocysteine

concentrations. However, one limitation to this study was that it was an observational study, and

the individuals who fell into the highest range of folate intake might have had overall "healthier

diets" and were taking supplements that might have contained other nutrients that could have

affected CHD risk.

In addition, Refsum and colleagues (100) conducted a meta-analysis in 1998 including

data from 80 clinical and epidemiological studies (-10,000 patients). The results of this analysis

provided unequivocal evidence that hyperhomocysteinemia is a common, independent CVD risk

factor in the general population, and may also enhance the effect of other conventional risk

factors (100). Because supplementation with B vitamins, in particular folic acid, is an efficient,









safe, and inexpensive means to reduce an elevated homocysteine level, studies have tried to

establish whether such therapy will reduce CVD risk. A meta-analysis conducted by the

Homocysteine Studies Collaboration assessed the relationship of homocysteine concentrations

with primary prevention of vascular disease and determined that an estimated 25% lower usual

homocysteine concentration was associated with approximately 11% lower ischemic heart

disease risk and 19% lower stroke risk (101).

Moreover, a meta-analysis by Bazzano and colleagues (102) summarized the results of

twelve randomized, controlled clinical trials on dietary supplementation with folic acid on CVD

risk reduction. These studies included data from 16,958 participants with preexisting vascular

disease. Folic acid supplementation was not shown to reduce recurrence risk of CVD or all-

cause mortality among subjects with a prior history of vascular disease (102). It is possible that

folic acid supplementation may have a protective effect on risk reduction and prevention of a

primary rather than secondary occurrence of a vascular event. Several ongoing randomized,

controlled, clinical trials in the US, Canada, Europe and Australia with large sample sizes as part

of the B-Vitamin Treatment Trialists' Collaboration might provide a more definitive answer to

the role of folic acid on primary risk reduction (103). Although the results of these studies are

uncertain, subjects enrolled in trials conducted in the US and Canada are expected to have lower

baseline homocysteine concentrations because folic acid fortification is mandatory in these

regions and has previously been associated with significant reductions in plasma homocysteine

concentrations. These ongoing intervention trials in the US and Canada may not observe the

same decreases in homocysteine that might appear in populations not exposed to fortification

(11). Furthermore, it is difficult to detect a cause-and-effect relationship between folic acid

supplementation and vascular disease risk secondary to difficultly in controlling for other









nutrients in the diet (i.e., vitamins B12 and B6) that may significantly lower homocysteine, as

well as the fact that the underlying causes of vascular disease are multifactorial (99, 100, 102).

Research studies have also been conducted to investigate the efficacy of folate therapy on

stroke prevention. Wang and colleagues conducted a meta-analysis of eight randomized trials of

folic acid that reported stroke as an endpoint (104). The data indicated that folic acid

supplementation significantly reduced the risk of stroke by 18%, and an even greater beneficial

effect was seen in those trials in which homocysteine concentration was reduced by more than

20%, treatment duration was more than 36 months, no fortification program or partial

fortification of grains had been introduced, and there was no history of stroke. Investigators

concluded that the inverse relationship detected between the duration of folic acid

supplementation and the risk of stroke suggests that folic acid supplementation can effectively

reduce the risk of stroke in primary prevention (104).

MTHFR polymorphism and vascular disease. Studies have also indicated that

individuals with the homozygous variant (TT) of the MTHFR 677 C--T polymorphism have

elevated plasma homocysteine concentrations compared to individuals who express either the

heterozygous (CT) or normal (CC) genotypes when folate status is low. Jacques and colleagues

(105) assessed the potential interaction between the MTHFR 677 C--T polymorphism and folate

status in homocysteine metabolism. This study included 365 individuals from the National

Heart, Lung, and Blood Institute Family Heart Study. The investigators found that among

individuals with lower plasma folate concentrations (<15.4 nmol/L), those with the TT genotype

had a mean total fasting homocysteine concentration (12.1 [imol/L) that was 24% greater than

individuals with the CC genotype (9.8 [mol/L). A difference in homocysteine concentrations

between the TT and CC genotypes (7.9 and 7.8 [mol/L, respectively) was not seen for









individuals with a folate concentration >15.4 nmol/L (105). A meta-analysis by Brattstrom and

colleagues (106) combined the results of studies that have documented plasma homocysteine

concentrations in relation to the MTHFR genotypes (CC, CT and TT). Similar to previous

findings, the meta-analysis revealed that individuals with the TT genotype had higher mean

homocysteine concentration (25% or 2.6 imol/L increase) than subjects with the CC genotype

when both populations had suboptimal folate status. A difference in homocysteine

concentrations among the MTHFR genotypes was not detected when folate status was high.

Ultimately, these studies concluded that the MTHFR 677C--T mutation is a major cause of mild

hyperhomocysteinemia and that individuals are at greater risk for hyperhomocysteinemia when

they have the MTHFR 677 TT genotype coupled with low serum folate concentrations.

However, there appears to be little evidence that individuals with the TT genotype are at greater

risk for vascular disease.

Similarly, Kauwell and colleagues reported data suggesting that older women with the

TT genotype may be at greater risk for elevated plasma homocysteine in response to low dietary

intake of folate in comparison to older women with the CC or CT genotypes (59). Studies

investigating the effects of the MTHFR 677 C--T polymorphism on homocysteine metabolism

suggest that individuals with the TT genotype may have higher folate requirements to maintain

adequate folate status and prevent hyperhomocysteinemia.

Cancer

Compelling evidence from epidemiologic, animal and human studies suggests that folate

status is associated with risk reduction for developing several types of cancers, including breast,

cervical, pancreatic, brain, lung, and colorectal cancer (CRC) (107), although more recent

evidence is contradictory to the latter. Research also suggests that folate depletion enhances









carcinogenesis, whereas large doses of supplemental folic acid, above what is presently

recommended for the general population, appear to have a protective effect. Although the exact

mechanism is still uncertain, the beneficial effect of folate on carcinogenesis is speculated to

come from the metabolic role of folate in DNA methylation and stability (107). When there is a

deficiency of folate in circulation, dTMP synthesis is impaired and results in nucleotide errors

that incorporate uracil into DNA stands. This may lead to the instability of DNA strands that are

associated with increased cancer risk. Moreover, folate can affect cellular SAM levels, which

regulate DNA methylation and gene transcription by methylating specific cytosines in DNA

(108). As a consequence of folate deficiency, SAM is depleted, which in turn induces DNA

hypomethylation and potentially induces expression of cancer-prone genes (109). Research has

shown that folate administration can repair DNA synthesis by reversing excessive DNA uracil

misincorporation thereby reducing the number of chromosomal breaks, and supplying adequate

methyl groups for DNA methylation and reducing expression of cancer-related genes (110).

Significant evidence from population cohorts from the Nurses' Health Study (15,984

women) and Health Professional Follow-Up Study (9,490 men) supports the association between

folate and CRC risk (111). These epidemiological studies revealed a 30 to 40% reduction in the

risk for CRC and adenocarcinomas in participants with the highest folate intake (>700 ig/d), the

majority of who were supplement users, compared to those with the lowest intake of folate (166

and 241 pg/d for women and men, respectively) (111).

In the Nurses' Health Study, a reduction in the risk for CRC was not associated with

folate intake from food sources alone (111). However, when total folate intake exceeded 400

pg/d, a 30% lower risk was observed compared to the group of women who consumed <200

Lig/d. It was noted that of the group of women who consumed >400 pg/d of folate, 86%









consumed multivitamins. An apparent association between length of supplement use and risk for

cancer was also noted. Women who had been consuming supplements containing folic acid for

at least 15 years had a 75% decreased risk for CRC compared to non-supplement users; whereas

no significant reductions in risk were observed in women who had been taking multivitamins for

shorter lengths of time (111).

In contrast, more recent population-based observations from two data sets have suggested

that the incidence of CRC in North America increased as a result of folic acid fortification.

Mason and colleagues (112) noted a surge in absolute rates of CRC in 1996 to 1998 (US) and

1998 to 2000 (Canada), despite a downward trend in CRC incidence in both countries during the

decade prior to fortification. These investigators attributed this incidence to the role of folate in

nucleotide synthesis, including the vitamin's role as a potential growth factor for neoplastic cells.

Evidence indicating that high doses of folic acid can accelerate the growth of established

neoplasms has been documented in previous research (113, 114). Subsequent research also

noted that supplemental folic acid is only protective before neoplastic cells appear in the

intestine. Once these proliferative cells are established, microscopic and macroscopic tumors

develop faster as more folic acid is administered (115, 116). The observations by Mason and

colleagues that rates in CRC spiked as a result of fortification suggest that fortification

"unveiled" a significant number of suppressed neoplasms that would have otherwise never

transformed into cancers or would have gradually evolved into cancer over a longer period of

time (112).

In a double-blind, placebo-controlled, randomized clinical trial (the Aspirin/Folate Polyp

Prevention Study) conduced by Cole and colleagues (117), 1,021 participants with recently

removed colorectal adenomas were randomized to receive either 1 mg of folic acid or a placebo









over a period of three to five years. Analysis of the final follow-up colonoscopy did not reveal a

protective effect of folic acid supplementation; rather, incidence of at least 1 recurrent advanced

lesion was significantly higher for folic acid (11.6%) compared to the placebo (6.9%) (P = 0.05)

(117). Further research is needed to investigate the potential of folic acid supplementation to

accelerate the growth of existing neoplasms and increase risk of CRC.

In contract to the studies by Mason et al. (112) and Cole et al. (117), the American

Cancer Society, the Centers for Disease Control and Prevention (CDC), the National Cancer

Institute, and the North American Association of Central Cancer Registries found that for all

races/ethnicities combined in the US, favorable trends in lower incidence and mortality were

noted for lung and CRC in men and women and for breast cancer in women (118). Cancer

incidence data were available for up to 82% of the US population, and cancer deaths were

available for the entire US population. Overall cancer death rates decreased by 2.1% per year

from 2002 through 2004, nearly twice the annual decrease of 1.1% per year from 1993 through

2002. Among men and women, death rates declined for most cancers. Breast cancer incidence

rates in women decreased 3.5% per year from 2001 to 2004, the first decrease observed in 20

years. Colorectal cancer incidence and death rates and prostate cancer death rates declined, with

CRC death rates dropping more sharply from 2002 through 2004.

The role of folic acid supplementation in risk reduction for other cancers also is

supported by evidence of the role of adequate folate intake in the reduction of breast cancer in

relation to alcohol consumption. Folate status may be compromised in chronic alcohol abusers

secondary to inadequate folate intake. Ethanol may also aggravate folate deficiency by

impairing intestinal folate absorption, interfering with folate metabolism, or increasing renal

folate excretion (119). Excessive alcohol consumption is defined as >14 to 15 g/d, which is









equivalent to 5 to 6 ounces of wine and 13 to 14 ounces of beer. Data from the Nurses' Health

Study revealed that for women consuming >15 g/d of alcohol, risk for breast cancer was lowest

in those women consuming >600 pg/d of folate from both food sources and supplements

compared to women who consumed 150 to 200 pg/d of folate (61). Furthermore, a 26%

reduction in risk for breast cancer was observed in women consuming alcohol and taking

multivitamins compared to similar women who were not taking supplements. Additional data

from the Canadian National Breast Screening Study (56,837 women) showed a 43% reduction in

breast cancer risk associated with folate consumption (>300 pg/d) in women consuming >14 g/d

of alcohol compared to women with equivalent alcohol intake who consumed <225 pg/d of

folate (120). These results suggest that a diet high in folate may compensate for the negative

effects on folate metabolism caused by alcohol intake and may consequently reduce breast

cancer risks (11). Ongoing research is still being conducted with the hope of providing better

insight into the potential role of folate in cancer prevention.

Folic Acid Fortification

Following reports from clinical intervention studies identifying a direct association

between periconceptional folic acid intake and NTD risk reduction, the US Public Health Service

issued a recommendation in September 1992 that women of childbearing age (i.e., 15 to 44

years) capable of becoming pregnant should consume 400 pg of the folic acid daily to reduce the

number of cases of NTD. Since then, an ongoing national public health campaign has

encouraged women to consume dietary supplements containing folic acid. In addition, the FDA

mandated in 1996 that all "enriched" cereal-grain products be fortified with folic acid by 1998

(2). Although the effective date was January 1, 1998, the majority of food manufacturers had

implemented folic acid fortification by mid-1997. The predicted increase of daily folic acid









consumption attributable to fortification of enriched cereal-grain products was estimated to be

approximately 100 ig/d.

Originally, the FDA considered several options that included fortification of enriched

cereal-grain products, RTE breakfast cereals, dairy products, and fruit juices. However, analysis

of folic acid intake by consumers in some non-target groups (i.e., men and elderly) showed that

when fruit juices and dairy products were fortified with folic acid, in addition to cereal-grain

products, RTE breakfast cereals and dietary supplements, folic acid intake exceeded the UL of

1,000 .g/d even at the lowest level of fortification. As a result of FDA's analysis, the FDA

determined that fortification should be limited to cereal-grain products, RTE breakfast cereals

and dietary supplements (2).

The FDA reviewed information showing that cereal-grain products and RTE breakfast

cereals are consumed on a daily routine basis by 90% of the target population (i.e., women of

childbearing age). Furthermore, representatives of manufacturers in the cereal-grain industry

stated that these products could be easily fortified with folic acid and that a fortification mandate

would not be unfair to the industry. Thus, the FDA determined that mandatory folic acid

fortification of cereal-grain products with 140 pg per 100 g, along with voluntary fortification of

RTE breakfast cereals up to 400 pg per serving and dietary supplements up to 400 pg per unit or

per serving, would provide increased intakes of folic acid for women in reproductive years while

keeping intakes for the non-target population below the UL (2). In addition to the US, other

countries that have implemented a mandated folic acid fortification program include Canada

(121), Chile (122), and some Latin American countries (123).









Effect of Fortification on Folate Status of Women of Reproductive Potential

The initiation of fortification of the US food supply with folic acid has had a positive

effect on the folate status of the entire population and has decreased the incidence of NTD-

affected pregnancies in the US by approximately 26% (13). Dietrich and colleagues have

assessed the benefit of fortification by comparing serum and RBC folate concentrations for

women of reproductive age from two separate NHANES data sets. NHANES III, conducted

during 1988 to 1994, reflects the time prior to folic acid fortification, and NHANES 1999-2000,

reflects the time period post-fortification. Compared to pre-fortification concentrations, both

serum and RBC folate concentrations increased significantly post-fortification. For women

between the ages of 20 to 39 years and 40 to 59 years who did not use supplements, the mean

serum folate concentration increased more than two-fold from 10.3 to 26.0 nmol/L and 11.4 to

27.1 nmol/L, respectively. These increases represent a respective 153% and 137% increase in

serum folate concentrations (13).

The mean RBC folate concentration increased for women 20 to 39 years old and 40 to 59

years old from 341 to 556 nmol/L and 386 to 629 nmol/L, respectively, between NHANES III to

NHANES 1999-2000. A 63% increase in RBC folate concentration was observed for both age

groups of women. These increases appear to be associated with the consumption of folic acid in

enriched and fortified foods (i.e., enriched cereal-grain products and fortified RTE breakfast

cereals) because data for supplement users were evaluated separately (11, 17).

Jacques and colleagues (4) evaluated the effect of fortification on indices of folate status

in a cross-sectional study with participants in the Framingham Offspring Cohort (men and

women, ages 30 to 59). These researchers found that among nonusers of folic acid supplements,

mean plasma folate concentration among individuals examined after fortification increased 46%









(10.4 to 22.7 nmol/L; P <0.001) relative to individuals examined before fortification.

Furthermore, the prevalence of low folate concentrations (<7 nmol/L) significantly decreased

from 22.0 to 1.7% (P <0.001). Choumenkovitch and colleagues also evaluated folate status of

participants in the Framingham Offspring Cohort and observed a 38% increase in mean RBC

folate concentration of subjects comparing pre- to post-fortification (3).

In addition, Lawrence and colleagues reviewed pre-existing data on serum folate

concentrations in more than 98,000 blood samples submitted to Kaiser Permanente's Southern

California Endocrinology Laboratory between 1994 and 1998 to evaluate changes in serum folate

concentrations since implementation of folic acid fortification of cereal-grain products (5).

These researchers observed a median pre-fortification serum folate value of 28.6 nmol/L in 1994,

which gradually increased by 48% during the transition period between initiation of fortification

in 1996 and full implementation in 1998 to 42.4 nmol/L.

Recently the CDC assessed the trends in folate status of women and reported that blood

folate concentrations among non-pregnant US women of childbearing age declined from

NHANES 1999-2000 through NHANES 2003-2004 (124). Based on data from NHANES 1999-

2000, 2001-2002, and 2003-2004, the median serum folate concentrations among women 15 to

44 years old were 28.6 nmol/L, 25.8 nmol/L and 24.0 nmol/L, respectively. This reduction in

medium serum folate concentrations reported from 1999-2000 through 2003-2004 represented a

statistically significant reduction of 16% (P <0.001). Similarly, RBC folate concentration

decreased 8% (P = 0.03) from 578 nmol/L reported during NHANES 1999-2000 to 533 nmol/L

reported from NHANES 2003-2004 data. Although a decline has been noted, the majority of the

women participating in NHANES 2003-2004, particularly non-Hispanic Caucasian women,

achieved the 2010 national health objective of a median RBC folate concentration of 500 nmol/L









(124). Nevertheless, this recent decline in folate status emphasizes the need for enhanced folic

acid awareness for women of reproductive age.

Effect of Fortification on Neural Tube Defects

Folic acid fortification has a direct positive and significant effect on the reduction of

NTD birth prevalence. Honein and colleagues (14) estimated reductions in NTDs in the US by

evaluating data from birth certificates from 45 states and Washington, DC and reporting the

number of infant birth certificates reporting births affected by either spina bifida or anencephaly.

Data post-fortification revealed a decline in the prevalence of spina bifida and anencephaly by

23% and 11%, respectively. However, the data included only live births, since NTD-affected

infants who were either miscarried or stillborn were not reported (14). The CDC reported that

rates of NTDs have decreased 26% in the US since folic acid fortification was mandated. The

prevalence of anencephaly reported on birth certificates declined from 18.38 cases per 100,000

live births in 1991 to 9.40 in 2001. The prevalence of spina bifida reported on birth certificates

declined from 24.88 cases per 100,000 live births in 1991 to 20.09 in 2001 (15). Although the

level NTD risk reduction observed in the US was not as high as originally expected, research has

shown that the apparent positive reduction in NTD incidence has been associated with improved

folate status post-fortification and supports food fortification as an effective intervention strategy

for individuals (11).

In 2006, Botto and colleagues (12) assessed two crucial issues relative to the benefits and

impact of folic acid in the prevention of birth defects: whether folic acid supplementation alone,

without fortification, is effective in reducing the population-wide rates of NTDs, and whether the

current policies can reduce the occurrence of other birth defects. These investigators used data

from 15 birth registries from areas with either official supplementation recommendations of 400









tg/d of folic acid or a folic acid fortification program to assess the effectiveness of both on the

rates and trends of major birth defects, including NTDs. The results revealed significant changes

in the incidence of NTDs in areas with folic acid fortification, but not in areas with folic acid

supplementation recommendations and no fortification program. The investigators concluded

that recommending folic acid supplementation alone remains an ineffective approach in

translating the known protective effect of folic acid in reducing NTD rates. In contrast,

fortification appears to be effective in reducing NTDs (12).

When folic acid fortification was first implemented, the estimated increase of folic acid

intake in the US food supply that resulted from consumption of folic acid enriched and fortified

products was approximately 200 tg/d or two times the quantity originally predicted at 100 tg/d

(16). Actual measurements of total folate content in enriched cereal-grain products have shown

that many of these products contained total folate levels that were higher than the amounts

required by federal regulation (125-127). Although there is a lack of consistent research

evaluating the folic acid content of enriched cereal-grain products, recent research groups have

reported data suggesting a decline in the amount of folic acid added to foods since the initiation

of fortification. Johnston and Tamura (128) measured the folate content of white sandwich

breads containing enriched flour during 2001 to 2003 and found that the mean folate content in

enriched white bread significantly declined from 2001 to 2002 or 2003. In addition, P6o-Prieto

and colleagues (129) analyzed the folic acid content of numerous enriched foods and found no

evidence of folic acid overages in enriched products contrary to previous reports. In fact, many

of the fortified foods tested tended to contain less folic acid than required by law (129).









Folic Acid Recommendations and Awareness

Despite significant reductions in the incidence ofNTDs observed since folic acid

fortification, the majority of women capable of becoming pregnant have not achieved the level of

intake associated with NTD risk reduction (13). Therefore, increasing the number of women

who consume 400 pg of folic acid daily from dietary supplements or folic acid fortified foods

remains an important public health goal for NTD prevention.

Numerous public health policies have been implemented worldwide based on the strength

of the evidence relating periconceptional folic acid supplementation to NTD risk reduction. In

2005, the CDC and the March of Dimes sponsored the first National Summit on Preconception

Care and launched the Preconception Health and Health Care Initiative, the goal of which is to

improve both the health of mothers before pregnancy and maternal and infant health outcomes

(130). The recommendations from this summit were published on April 21, 2006 (131). The

recommendations noted that the time has come to ensure that efforts promoting adequate folic

acid intake and improved pregnancy outcomes, including NTD risk reduction, are not limited to

prenatal care but should be expanded to include preconception health and health care. The panel,

which included an array of health care providers, public health practitioners, and researchers,

developed strategies to implement the recommendations across three areas: clinical practice,

consumer roles, and public health practice (130). Current and future plans to promote folic acid

include developing and distributing clinical guidelines and tools, educating consumers,

integrating preconception care activities into clinical and public health programs, educating and

training clinical and public health care providers, developing a research agenda, identifying,

documenting, and promoting best practices, and supporting state and local initiatives (130).









However, despite these recommendations and other folic acid awareness campaigns,

there does not appear to be a significant increase in the number of women taking folic acid

supplements. A survey conducted by the March of Dimes recently provided evidence that only

33% of women of reproductive age (18 to 45 years) take a folic acid containing supplement

daily, compared to 40% in 2004 and 28% in 1995 (17). Furthermore, reports have indicated that

women not contemplating pregnancy, women between the ages of 18 to 24 years, non-white

women, those less educated, and those of lower socioeconomic status have both lower folic acid

knowledge and lower folic acid supplement use (132-134). The March of Dimes has also

reported that only 24% of women aged 18 to 24 years took a supplement with folic acid daily,

yet this population accounts for at least one third of all US births (135). In addition, although

84% of women reported awareness of folic acid, up from 78% in 2004, only 25% of women

reported knowing that folic acid prevents birth defects, and only 7% of women reported knowing

that folic acid should be taken preconception (17). Comparable data were reported in a survey

conducted in Puerto Rico, where nationwide campaigns had been promoting folic acid for four

years prior to the study. Despite the majority of pregnant women surveyed who understood the

importance of folic acid (88%), regardless of whether or not they planned their pregnancy, only

32% actually took a folic acid supplement prior to conception (136).

Analytical Methodology for Assessing Folate Status

There are several analytical methods used to assess blood folate status. Not all

researchers have used the same methodology for detecting folate concentrations, and even when

research groups have used the same method, there may be differences in protocols between labs.

This section reviews the two most commonly used methods.









Blood Folate Analysis

Serum and RBC folate concentrations are most often measured using the radiobinding or

the microbiological assays. The microbiological assay is considered the gold standard method

for determining folate concentrations in blood, urine, tissue, and food samples (137, 138). The

microbiological assay uses the test organism Lactobacillus rhamnosis as it metabolizes the

highest number of folate derivatives, including 5-methylTHF, the predominant folate form in

plasma and RBC (22). Ascorbic acid and a phosphate buffer (pH 6.1) are added to the test

sample and micro-organism to offer better stability and prevent the oxidative loss of labile

reduced folates. Each 96-well flat bottom microtiter plate contains serial dilutions of a control

sample, samples of folic acid standard, and subject samples. Samples are inoculated with L.

rhamnosis and incubated at approximately 370C to allow for growth of the organism. Growth of

the folate-dependent microorganism is assessed by comparing the degree of turbidity of the

sample compared to the turbidity of known concentrations of folic acid standard. Cell growth is

determined by absorbance at 650 nanometers using a computer-interfaced microtiter plate reader

and data reduction software (137). A standard curve is generated by plotting the log-linear

absorbance against the folic acid standard concentration to interpolate unknown folate

concentrations of samples.

The radiobinding assay also is used for blood folate analysis (139). The radiobinding

assay uses a competitive folate binding protein attached to microbeads and iodine-125 [1251]-

labeled folic acid, which are used to quantify serum or RBC folate concentrations (140). In the

assay, the folate binding protein has an equal affinity for the standard and the folate present in

the serum or plasma. The unlabeled folate competes with the labeled folate for the limited









number of available binding sites on the folate binding protein. Thus, the level of radioactivity

bound is inversely related to the concentration in the sample or standard.

The radiobinding assay is conducted by adding dithiothreitol solution to the folate tracer

(125I, borate buffer with human serum albumin, dextran, potassium cyanide, dye and

preservative). This mixture is added to serum or RBC samples and heated in a water bath at

100C for 15 minutes. Once cooled, the folate binder is added to the mixture, which is protected

from exposure to light, and incubated for one hour. During incubation, endogenous folate and

125I compete for binding sites to the folate binder. Samples are centrifuged and bound folates

and microbeads precipitated. The bound folate (labeled and unlabeled) accumulates in a pellet,

while unbound folate is in the supernatant, which is gently discarded. The radioactivity of the

pellet is measured using a scintillation gamma counter.

The microbiological assay has been the preferred method for measuring folate

concentrations. However, the method is tedious and time consuming, requires microbiological

expertise, and growth of the test organism may be inhibited by non-folate substances, such as

prescription medications (e.g., antibiotics or methotrexate) taken by subjects. The advantages of

using the radiobinding assay include the fact that it is relatively inexpensive, easy to perform,

and has a high specificity towards folate isomers. One limitation of the radiobinding assay is

that it gives lower folate values than the microbiological assay since this method underestimates

certain folate forms as evidenced by recovery analysis (61).

Another method used to assess folate concentrations in biological samples uses liquid

chromatography-tandem mass spectrometry (LC-MS/MS). Fazili and colleagues (61) compared

serum folate species analyzed using LC-MS/MS with total folate measured by the

microbiological and Bio-Rad radiobinding assays. Advantages of the LC-MS/MS method









compared to the microbiological assay are that it provides information on the different folate

species in addition to total folate and is less prone to interference such as antibiotics. Mean and

median total folate concentrations measured by LC-MS/MS and microbiological assays were

generally in agreement, but the radiobinding assay values were much lower (-29% relative to

LC-MS/MS values). Fazili and colleagues concluded that the radiobinding assay produces much

lower results, on average, probably due to underrecovery of 5-methylTHF, which is the main

circulating form of folate (61). The Bio-Rad QuantaPhase II radiobinding assay has been used to

measure blood folate in NHANES for 25 years. Due to the fact that the radiobinding assay is

being discontinued in 2007, NHANES is switching to the microbiological assay for all samples,

and will use the LC-MS/MS method for a subset of the population.

Plasma Homocysteine Analysis

Determination of plasma homocysteine concentration is generally measured as total

homocysteine concentration. The most commonly used approach for quantifying homocysteine

concentration is capillary gas chromatography-mass spectrometry with selected ion monitoring

(141). The term "total homocysteine" as applied to biological samples (i.e., serum or plasma)

refers to the sum of homocysteine "that is linked via disulfide bond formation in a variety of

compounds that include homocysteine, homocysteine-cysteine mixed disulfide, proteins via their

cysteine moieties, and peptides such as glutathione via their cysteine moieties (141)." The assay

is conducted by adding 2-mercaptoethanol to the sample in order to chemically reduce and

release endogenous homocysteine from proteins and other disulfides. The samples are analyzed

using a gas chromatograph-mass spectrometer equipped with a falling needle injector.

Quantitation is based on the ratio of the areas of the base peak ion for homocysteine to the areas

of the base peak ion for the derivative of the respective stable isotope internal standards (141).









MTHFR Genotype Determination

Since the discovery of single nucleotide polymorphisms (SNP), technological advances

have been made in methods for determining subject genotypes for these SNPs. A common

method for genotype determination involves polymerase chain reaction (PCR) to amplify the

desired region or target sequence of the DNA strand. Once the preferred region is magnified,

specific restriction enzymes are added depending on the SNP being studied. The SNP of greatest

interest with regard to folate metabolism is the MTHFR 677 C--T that results in the substitution

of the amino acid alanine with valine in the gene product (19).

In order to achieve unambiguous determination of all SNP variations, Howell and

colleagues (142) created a new SNP scoring method known as dynamic allele-specific

hybridization (DASHTM). Using this approach, the target sequence is amplified by PCR with one

biotinylated primer. The biotinylated product strand is bound to a streptavidin-coated microtiter

plate well, and the non-biotinylated, unbound strand is removed with alkali. An oliogonucleotide

probe, specific for the desired allele, is hybridized to the bound DNA at low temperature. This

forms a probe-target duplex that interacts with a double strand-specific intercalating dye. The

temperature is increased and the denaturation temperature and fluorescence emitted from the

dye, which is proportional to the amount of double-stranded DNA present, are recorded. For

analysis, the negative derivatives of the melting curves are plotted to show a lower temperature

peak for a homozygous allele mismatch, a higher temperature peak for a homozygous match, or

both peaks for a heterozygous sample (142).









Dietary Intake Assessment

USDA National Nutrient Database

The USDA National Nutrient Database for Standard Reference is the major source of

food composition data in the US and provides the foundation for most public and private sector

databases. The 2007 release (Release 20) contains nutrient data for 7,517 food items for up to

140 food components including vitamins, minerals, amino acids, and fatty acids (143). Major

applications of the USDA National Nutrient Database include the development of the Food and

Nutrient Database for Dietary Surveys by the Food Surveys Research Group, which is used to

process dietary data records from the survey "What We Eat in America", the dietary intake

component of NHANES. The version of the USDA National Nutrient Database used to estimate

nutrient intakes for subjects in the present study was Release 17 (2004), which contains nutrient

data for 6,839 food items for up to 128 food components (8). In contrast to older database

releases, a unique component of the Release 17 nutrient composition database is that it provides

data for four categories of dietary folate including folic acid (ig/d), natural food folate (ig/d),

total folate (ig/d), and DFE (.g DFE/d). Until recently, researchers reporting folate intake from

NHANES data used an older version of the nutrient database that only provided nutrient

composition data for total folate (ig/d).

Food Frequency Questionnaire

A food frequency questionnaire (FFQ) is often used as one method for determining usual

dietary intake. The FFQ is the most practical and economical method for collection of

comprehensive dietary data and is most commonly used in large epidemiological studies (144).

Compared with other dietary assessment methods, such as 24-hour dietary recalls or multiple-day

food records, the FFQ obtains less detailed information regarding food type or portion size.









Although alternative methods are more detailed, FFQ is self-administered and is designed to

capture usual dietary intake, unlike records or recalls. The FFQ often generalizes food intake

into food groupings, such as fortified cereal, whereas a 24-hour recall method or multiple-day

food record provides more detailed information such as brand names of foods consumed. The

FFQ has predetermined questions and portion sizes to which an individual responds. This type

of questionnaire can be electronically scanned and the information uploaded by software for

further analysis (144). The limitation with this dietary assessment method is that there is little

opportunity for individuals to list dietary intake data not included as part of the questionnaire.

Concern regarding measurement error has stimulated numerous validation studies comparing

nutrient intakes estimated from FFQs with those estimated by other methods.

Validation of this method, which is the process of determining if estimated nutrient intake

is true to actual intake, is essential to providing valid estimates of dietary intake for observational

epidemiological studies and clinical trials. The National Cancer Institute (NCI) Diet History

Questionnaire (DHQ) is an FFQ developed by staff at the Risk Factor Monitoring and Methods

Branch (144). The DHQ captures data on frequency of consumption and estimated portion size

from a list 124 individual food and beverage items over the past year. The 124 food items

currently included in the DHQ were selected based on the work of Subar and colleagues (145)

who originally categorized 5,261 individuals foods with 170 food groups. The DHQ has been

refined over the years based on results from intensive cognitive interviewing. Subar and

colleagues (146) conducted cognitive evaluation of various approaches to asking about usual

dietary intake and identified ways to improve the FFQ so completing the FFQ would be easier

while the accuracy of responses would be enhanced. Numerous cognitive issues in FFQs have

been addressed in the DHQ, including comprehension, order of food items, intake of seasonal









foods (e.g., fruits and vegetables), specificity of low-fat or fat-free food items, intake averages

from multiple food items, and format. Other improvements to the DHQ include modification of

portion size categories based on analysis of data and responses from 10,019 adults in the CSFII

(1994-1996) study (147). Rather than categorizing portion sizes as small, medium, or large,

portion size choices were changed to provide more detail about specific intake of a food item,

such as "less than 1 cup", "1 to 2 cups", and "more than 2 cups". These changes have ultimately

improved the validity and accuracy of nutrient intake estimated by the DHQ (146, 148).

In 2001, Subar and colleagues (144) evaluated the NCI DHQ against repeated 24-hour

recalls and compared to the Block and Willett FFQ, and ultimately validated the DHQ as a

suitable method for estimating nutrient intake. The Block FFQ is an eight-page questionnaire

that inquires about 106 food items, and categorizes portion size choices as small, medium, or

large (144). This questionnaire also has eight questions concerning use of added fats or low-fat

foods, 13 dietary supplement questions, five summary questions, and six questions on eating at

restaurants. The Willett FFQ asks about 126 foods over four pages, including ten questions each

on supplement fat intake. This questionnaire does not include separate portion size questions,

but rather categorizes responses into intake frequency of a reference portion size. This validation

study consisted of 1,301 men and women who completed four 24-hour recalls during one session

over the telephone. The subjects were then randomized to complete two FFQs, either the DHQ

and Block FFQ or the DHQ and Willett FFQ. Researchers found that the DHQ was more

accurate compared to the 24-hour recall method than the Block or Willett FFQ in determining

nutrient intake (144).









Assessment of Folate/Folic Acid Intake in Women of Reproductive Potential

Assessment of folate/folic acid intake among men and women typically has been

evaluated using data from NHANES or the Framingham Offspring Cohort Studies. However,

there are few studies that have evaluated folate/folic acid intake for women of reproductive

potential who do not consume supplements containing folic acid.

Choumenkovitch and colleagues (125) examined food and nutrient intake of 1,480

individuals who participated in the Framingham Offspring Cohort Studies to assess folic acid

intake from fortification in the US. Participants completed a 126-item semi-quantitative FFQ

developed by Willett et al. (149) that allowed for estimation of usual nutrient intakes during the

previous year. Among the 186 women (ages 30 to 80 years) who did not use supplements, intake

of folic acid after exposure to fortification increased by a mean of 192 .g/d (125). Furthermore,

9% of non-supplement consuming women post-fortification compared to 55% of women pre-

fortification consumed less than the EAR (<320 pg DFE/d) (125). A study by Dietrich and

colleagues (13) who also used data from NHANES 1999-2000 estimated mean total folate intake

for women between the ages of 20 to 39 years and 40 to 59 years to be 294 pg/d and 302 ig/d,

respectively.

A study by Yang and colleagues (6) used NHANES 2001-2002 data to compare

differences in folic acid intake in women of childbearing age (ages 15 to 49 years) in the US

after folic acid fortification. Information on folic acid intake from fortified foods and intake of

food folate was obtained from a single 24-hour food recall questionnaire. Nutrient values were

calculated using the USDA National Nutrient Database (Release 17). Yang and colleagues

reported that estimated mean consumption of folic acid from fortified foods in women of

childbearing age was 128 ig/d. Approximately 8% of non-supplement consuming women









reported consuming >400 pg/d of folic acid from fortified foods alone, and 26% of the women

surveyed reported taking >400 pg/d of folic acid from supplements in the previous month. The

mean food folate intake from dietary sources was 151 ig/d. Yang and colleagues concluded that

at the present level of folic acid fortification, most women need to take a supplement containing

folic acid to achieve the IOM recommendation for 400 .g/d of folic acid (6). However, a

limitation to this study is that it did not characterize sources of folate/folic acid. Further research

is needed to identify which components of the diet contribute to folic acid intake in women of

reproductive potential who do not consume a folic acid supplement or supplements containing

this nutrient.









OH H 0 H COOH
N N CH2--( I-C-N-C-CH2-CH2-COOH

H2 >. Folic (Pteroyl-L-Glutamic) Acid


P olyglutamyl Tetrahydrofolates

SH COOH 0 H COOH
OH R-N N.--t--CH2-CH2- --,CHH2--CH2-COOH

HKH2 H n H
NC j H

H2N-N N H Substituent(R) Position
H -CH3 (methyl) 5


-CHO (formyl)
CH=NH (formimino)
-CH--- (methylene)
-CH= (methenyl)


Figure 2-1.


5or10
5
5 and 10
5 and 10


Folate/folic acid structures adapted from Present Knowledge in Nutrition (1). Folic
acid consists of a para-aminobenzoic acid molecule linked on one side by a
methylene bridge to a pteridine ring, and joined by peptide linkage to a glutamic
acid molecule on the other side. Naturally occurring food folates exist in various
chemical forms, containing a side-chain composed of two to ten additional
glutamate residues (n) joined to the first glutamic acid. The pteridine ring of the
folate/folic acid structure can be reduced to form dihydrofolic acid and
tetrahydrofolic acid (THF). Folate coenzymes are formed by substitution of one-
carbon units at the N5, N10, or both positions (R) to the polyglutamyl form of THF.









Table 2-1. Definitions of Dietary Reference Intake (DRI) recommendations.


DRI recommendation
Estimated Average
Requirement (EAR)
Recommended Dietary
Allowance (RDA)


Adequate Intake (AI)



Tolerable Upper Intake
Level (UL)


Definition
A daily nutrient intake value that is estimated to meet the
requirement of half the healthy individuals in a group
The average daily dietary intake level that is sufficient to meet
the nutrient requirement of nearly all (97 to 98%) healthy
individuals in a particular life stage and gender group.
Individuals should aim for this intake level.
A recommended daily intake value based on observed or
experimentally determined approximations of nutrient intake
by a group (or groups) of healthy people that are assumed to
be adequate-used when an RDA cannot be determined.
The highest level of daily nutrient intake that is likely to pose no
risk of adverse health effects to almost all individuals in the
general population. As intake increases above the UL, the
risk for adverse health effects increases.


Adapted from the Institute of Medicine (10)

Table 2-2. Folate intake recommendations for men and non-pregnant women >19 years.
DRI recommendation Physiological folate requirement
Estimated Average Requirement (EAR) 320 gg DFE/d
Recommended Dietary Allowance (RDA) 400 gg DFE/d
Adequate Intake (AI) Not applicable
Tolerable Upper Intake Level (UL) 1,000 gg synthetic folic acid/d
Adapted from the Institute of Medicine (10)













DNA Synthesis


DNA Methylation + Othermehylation
reactions


dUMP


Cystarhionine
Purine Synthesis + THF I Transulfuration
Cysteine

Glutathione

Figure 2-2. One-carbon metabolism folate-dependent pathway adapted from Present
Knowledge in Nutrition (1).









CHAPTER 3
MATERIALS AND METHODS

Study Design and Methods Overview

Approximately 1,000 healthy male and female adult volunteers were recruited through

newspaper advertisements, flyers, and radio broadcasts. Prospective subjects were screened by

phone using a screening questionnaire (Appendix A) to determine their eligibility. Individuals

were included in the study if they met the following criteria: (a) age 18 to 49 years, (b) currently

not using prescription medications (birth control medication was permitted), (c) low alcohol

intake (< one drink per day), (d) non-smoker, (e) no history of chronic disease, (f) non-pregnant,

(g) non-lactating, (h) no use of supplements within the last 6 months, and (i) no major dietary

changes within the last 3 years. Individuals also were screened for consumption of highly

fortified RTE cereals containing 400 .g/d of folic acid (e.g. Total, and were not eligible to

participate if they indicated they consumed these highly fortified RTE cereals. Eligible male and

female volunteers (n = 388) were chosen to participate in the study.

Subjects were scheduled to have a fasting blood sample drawn by a certified phlebotomist

and to receive comprehensive detailed verbal and written instructions lasting 15 to 20 minutes

regarding the completion of the DHQ (Appendix B). Subjects took the DHQ and prepaid

mailing envelopes home with them and were asked to mail the completed DHQ to the primary

investigator within 2 weeks of receiving the in-person instructions.

Fasting blood samples were collected and processed for multiple analyses, including serum

and RBC folate concentrations, plasma homocysteine concentration, and MTHFR genotype.

Folate intake (total folate, DFE, folic acid, and food folate), in addition to other nutrients were

assessed using the DHQ. If subjects completed all aspects of the study they were compensated









$50 for their participation. The University of Florida Institutional Review Board approved this

protocol, and all subjects signed informed consent forms.

Human Subject Procedures

Eligible subjects enrolled in the study were scheduled for an appointment and were

instructed to report to the Food Science and Human Nutrition building on the University of

Florida campus following an overnight fast. Upon arrival, the purpose of the study was

discussed with each subject who was then asked to sign the approved informed consent form. To

ensure the privacy of personal health information, subjects who granted consent were assigned a

subject identification number. As a follow up to questions asked during the phone screening,

subjects were again asked about supplement use during the past 6 months. Subjects

acknowledging that they had taken supplements were asked to provide more detailed

information. This information was recorded on each subject's data form.

Blood samples were collected by a phlebotomist for analysis of biochemical indices.

Following the blood draw, subjects were given a snack and were instructed on how to complete

the DHQ. During the 20 minute DHQ instruction session, each small group of participants

received detailed verbal and written descriptions of how to complete the DHQ. Subjects were

also taught how to report accurate portion sizes by reviewing how to read food labels and how to

use measuring cups and spoons to visually interpret portion sizes correctly.

Additional handouts were reviewed and given to subjects to assist them in properly

completing the DHQ. These handouts included a detailed drawing illustrating the proper method

for marking or changing an answer (to ensure that the forms would scan correctly) and handouts

on "Caffeinated versus Non-caffeinated Beverages," "Fortified Cereals", and "Seasonal Fruits









and Vegetables" (Appendix C). Subjects were given the opportunity to ask questions regarding

the study and the DHQ.

Dietary History Questionnaire

This study was a collaborative effort between researchers at the University of Florida and

the University of North Carolina (UNC), Chapel Hill. The original DHQ from NCI was

modified with the addition of questions pertaining to beef consumption, beef-containing food

products, and vitamin B 12 fortified foods since the primary objective of this study was to assess

vitamin B 12 intake in the diet. The DHQ data also were used to evaluate the contributions of

folate and folic acid containing foods to overall folate intake. The total intake of each nutrient

was analyzed using the Diet*Calc Analysis program provided by NCI. This software program

can be downloaded free of charge from the NCI website (www.riskfactor.cancer.gov). The

nutrient database within the Diet*Calc software is from the USDA National Nutrient Database

Standard Reference, Release 17 (8). The Diet*Calc Analysis program nutrient database had

been updated in August 2004.

Processing the Dietary History Questionnaire

Subjects were instructed to mail the completed DHQ within 2 weeks of the initial

instruction. Subjects who failed to return the DHQ within this time frame were contacted by

phone or email. All returned DHQs were reviewed to ensure they were completed entirely and

that no questions were missing a response. Marked responses were not reviewed at this time to

prevent bias. Subjects who neglected to respond to one or more DHQ questions were contacted

to obtain the missing information. The completed paper versions of the DHQ were mailed to

Optimal Solutions Corporation, Lynbrook, New York, in groups of 100 where they were scanned

electronically. After the scanning process was finished, Optimal Solutions Corporation mailed









the DHQs and electronic data in the form of an American Standard Code for Information

Interchange (ASCII) text file to collaborators at UNC. Investigators at UNC uploaded the files

into the Diet*Calc Analysis program to analyze the nutrient intake of each subject based on their

DHQ responses.

Analysis of Nutrient Intakes

After the paper version of the DHQ was electronically scanned, each food item or question

included in the DHQ was linked to a predetermined Food Identification Number (FIN). Each

FIN had defined nutrients for gender and serving size. The FINs corresponding to the DHQ food

items and questions were linked to the Diet*Calc Nutrient and Food Group database. The

Diet*Calc Analysis program used the FIN associated with each response on the DHQ to evaluate

the nutrient intake for each participant based on gender and serving size. When Diet*Calc

identified a FIN from the ASCII text file, the software used the database to calculate an

individual's nutrient intake. The FINs and corresponding nutrient values estimated using each

individual's responses to questions on the DHQ were entered into a Microsoft Excel spreadsheet.

The Diet*Calc software produced data files that were available for analysis: the details.txt file

and the results.txt file. The details.txt file was an expanded version of the data calculated from

the ASCII text file and allowed investigators to compare FINs to individual nutrient intake and

daily frequency of intake. The results.txt file was a condensed version that provided combined

daily nutrient intake values for each subject regardless of the food source.

Dietary History Questionnaire Instruction Pretest

To ensure that the instructions and handouts on how to complete the DHQ would be clear

to the subjects, a pretest of the DHQ instructions was conducted with a group of 20 graduate

students in the Food Science and Human Nutrition Department and five vegetarian and vegan









consumers prior to starting the study. This pretest group was instructed using the same script to

be used during the study. The students were asked to follow the instructions and complete the

DHQ as if they were participants in the study. They were also given an additional survey that

asked questions regarding the length of time needed to complete the DHQ, if any regularly

consumed foods were missing from the DHQ, and if they had any other comments to improve

the instructions and/or the DHQ instrument. Feedback from this group was reviewed and

incorporated into the DHQ and DHQ instructions.

Blood Sample Collection and Processing

Fasting blood samples were collected for each participant by a phlebotomist (Vacutainer

Blood Collection Set; Becton Dickinson, Vacutainer Systems; Franklin Lakes, NJ). All blood

samples were processed within one hour of collection. A total of 70 ml of blood were obtained

from each subject.

Blood for serum samples was collected in 8.3 ml serum separator gel clot activator tubes

(Vacutainer, Becton Dickinson, Rutherford, NJ) and kept at room temperature for 30 to 60

minutes to allow time for clotting. Serum was obtained by centrifuging the tubes at 650 x g for

15 minutes at 210C (International Equipment Compant; Model HN-S II Centrifuge, Needham

Heights, MA). Supernatant sera were mixed with sodium ascorbate (1 mg/ml), aliquoted into

200 gl samples, and stored at -300C until analysis.

Whole blood was collected in 7 ml tubes containing K3 ethylenediaminetraacetic acid

(Vacutainer, Becton Dickinson, Rutherford, NJ). Blood for plasma homocysteine was kept on

ice prior to processing. A small aliquot of whole blood held at room temperature was diluted 20-

fold in 1 mg/ml ascorbic acid and aliquoted into 200 gl samples and frozen for measurement of

RBC folate concentration. The iced blood was centrifuged at 2000 x g at 40C for 30 minutes.









The plasma from these samples was frozen and used to measure the plasma homocysteine

concentration. Following removal of the plasma, the samples were used to extract DNA to be

used to determine genotype for the MTHFR 677 C--T polymorphism. Aliquots were frozen at

-30C for subsequent analysis of serum and RBC folate concentrations.

Analytical Methods

Identification of Food Groups from the Dietary History Questionnaire

All foods included in the DHQ were categorized into specific food groups: beef,

pork/other meat, poultry, dairy, eggs, seafood, cereal, soy, meal replacements, mixed dishes with

meat type unknown, non-dairy fats, beans, rice/pasta, soups/sauces, breads/crackers/cakes/pies,

nuts/seeds, vegetables, fruit, syrup/honey/gelatin/candy, and beverages. A new set of food

categories were derived including the following: (1) enriched cereal-grains, (2) fortified cereals

and bars, (3) vegetables, (4) fruit, (5) juice (only orange and grapefruit), (6) legumes and nuts,

(7) dairy, (8) eggs, (9) meat, (10) combination foods, (11) snacks, (12) miscellaneous, (13) other.

Foods that were fortified with folic acid but consumed minimally were grouped together as (12)

miscellaneous, and foods that were not fortified and contained minimal levels of food folate were

grouped together in the (13) other category. Table 3-1 describes the types of food included in

each food category. Although we attempted to screen out individuals who consumed highly

fortified RTE cereals, individuals that later reported consumption of these cereals were included

in the final analysis. Intake of highly fortified RTE cereals was included in the fortified cereals

and bars food category. The nutrient composition database used to calculate nutrient estimates

from the DHQ was the USDA National Nutrient Database Standard Reference Release 17

(Diet*Calc Nutrient and Food Group database). The nutrient database provided data for four









categories of dietary folate intake, including folic acid (ig/d), food folate (ig/d), total folate

(pg/d) and DFE (pg DFE/d), from foods in each of these new food categories.

Serum and RBC Folate Concentrations

The serum and RBC folate concentrations of all subjects were determined using the MP

Biomedicals, Inc. SimulTRAC-S Radioassay Kit (Orangebury, New York), a competitive

protein binding assay previously described in detail. Folate concentrations were inversely

related to the measured radioactivity. Serum and RBC folate concentrations >12 nmol/L and

>317 nmol/L, respectively, represented the lower limit of normal values for this study.

Homocysteine Concentrations

Samples to be analyzed for homocysteine were sent to Dr. Sally Stabler, University of

Colorado Health Sciences Center, Division of Hematology (Denver, CO). Total homocysteine

concentration was quantified using a gas chromatograph-mass spectrometer (141). A plasma

homocysteine concentration >12 imol/L was considered elevated when interpreting the results

for this study.

MTHFR Genotype Determination

Samples were sent to DynaMetrix Limited, University of Leicester (United Kingdom,

where primers and probes were designed for the MTHFR 677C--T polymorphism and analyses

were performed using the DASHTM method with DynaScore Software v. 0.7.7

(http://www. dynametrix-ltd. com).

Statistical Methods

The initial statistical analysis determined basic means and standard deviations for

demographic data, folate intake, and blood folate and homocysteine concentrations for the

overall population. Another initial statistical analysis was conducted to determine if there was a









relationship among any of the covariates of interest, which included gender, genotype, age, and

body mass index (BMI). For the continuous variables (i.e., BMI and age), a one-way analysis of

variance was used to determine whether the mean BMI and mean age differed between males

and females. A Chi-square test was used to determine whether the proportion of responses in

each of the categories differed among groups when analyzing categorical response variables. All

possible pairings were run, and only two pairs were found to be correlated. Significant

differences in BMI were detected between males and females with males having a higher mean

BMI compared to females. BMI also was found to be positively correlated with age. Since BMI

and gender and BMI and age were both highly correlated, BMI was not used in the analysis as

correlations could lead to confusing results. Therefore, only gender and age were used as

covariates in the analysis. Linear regression analysis was used to test for relationships between

folate/folic acid intake and folate and homocysteine concentrations. Generalized linear models

(GLM) were used to conduct the rest of the analyses to determine correlations for given response

variables.

The data used in the analyses were naturally bound at zero, because individuals cannot

have either negative intake or blood concentrations. This resulted in data that were non-normal

and positively skewed to the right. To correct for this, the data were transformed to the natural

log scale. The GLM were used on the transformed data. The estimated least squares means

obtained from the GLM were retransformed to original scale along with their appropriate

confidence bounds. The estimated least squares means also are the means that are corrected for

the covariates of interest (age and gender). Normalized data were used consistently in reporting

the results of the analyses.









Table 3-1. Description of foods categorized within each food group.
Food group Type of foods included
Enriched cereal- Breads (enriched breads, rolls, bagels, muffins, croissants, dumplings,
grains biscuits, dessert breads, pancakes, waffles, French toast)
Cakes, cookies, doughnuts, Danish, sweet rolls, pies, cobblers, crisps,
enriched with cereal-grain
Enriched rice
Enriched pasta, noodles, pasta with meatless red sauce
Fortified cereals Fortified ready-to-eat cereals, hot breakfast cereals, cereal bars, granola
and bars bars, power bars, slim fast bars
Vegetable Vegetables, vegetable juice, tomato juice, tomato sauce without meat,
vegetable medley
Fruit Fruit, fruit juice, fruit drinks (other than orange/grapefruit juice)


Juice (orange and
grapefruit)
Legumes and nuts


Dairy

Eggs


Meat


Combination





Snacks
Miscellaneous



Other


Orange and grapefruit juice

Legumes, soup with legumes, chili without meat, nuts/seeds, nut butters

Dairy products (cheese, yogurt, milk, milkshakes, ice cream, frozen
yogurt, cheesecake, sour cream, puddings/custards)
Whole eggs, egg salad, egg whites, egg substitute

Meat, fish, poultry, meat/fish sauces, liver (all meats except products
prepared with enriched cereal-grains [e.g., breaded, batter dipped,
and meatloaf])
Combination foods (e.g., Mexican food, pizza, macaroni and cheese,
chili with meat, lasagna with and without meat, pot pies, egg rolls,
pasta salad, pasta with meat sauce)
Breaded meats (meats and fish, breaded, batter-fried or meatloaf with
enriched flour)
Soups (creamed, noodles/rice, vegetables, no beans or meat)
Snacks (chips, pretzels, crackers, popcorn)
Other fortified foods eaten minimally (beer, fortified vegetarian meals
(e.g., Boca entree, burger, breakfast sausage, chicken, ground),
tofu/soy products, soymilk, liquid meal replacements, fortified egg
substitute)
Other foods with negligible folate (fats/oils, alcohol [except beer],
dressings, sugars, gelatins/jams/jelly, candy, soda, tea, coffee
without cream, rice milk)









CHAPTER 4
RESULTS

Characteristics of Study Population

Three hundred eighty-eight subjects were enrolled in this study and 302 subjects (140

males, 162 females) were eligible for inclusion in the final analyses. Subjects excluded after

enrollment included 62 subjects who reported supplement use within the past six months and six

who failed to return their DHQ. Data from another 18 subjects were excluded because estimates

of their total caloric intake derived from their DHQ data was less than 600 calories (n = 9

females) or greater than 5,000 calories (n = 9 males). These calorie levels are considered

improbable, so these subjects were excluded from the study. Of the 302 eligible subjects, the

mean daily caloric intake (mean SD) for males and females was 2,382 958 and 1,744 805,

respectively; males consumed significantly more calories per day than females (P <0.0001).

The demographic characteristics of males and females in this study population are

presented in Table 4-1. The mean age (years) and BMI (kg/m2) (mean SD) for all study

participants were 26.0 7.7 and 23.5 4.1, respectively. The education level of all subjects was

15.0 2.4 years, which is equivalent to status as a junior at the university level. Body mass

index was the only variable that was significantly different between genders with males having a

higher BMI (P <0.0001) than females.

All eligible participants were categorized by their genotype for the MTHFR 677C--T

single nucleotide polymorphism. The number of subjects within each genotype group was as

follows: CC genotype (n = 151), CT genotype (n = 118), and TT genotype (n = 31). Two

subjects had unknown genotypes. The subjects with known genotype data included 140 men (68

CC, 56 CT, 16 TT) and 160 women (83 CC, 62 CT, 15 TT).









Dietary Folate Intake and Gender

In relation to objective 1, four categories of folate/folic acid intake were analyzed from the

DHQ data. These categories included total folate, DFE, folic acid, and food folate. After

adjusting for differences in age, the mean intake for each folate category (.g/d and .ig/1,000

kcals/d) was evaluated for males and females. Mean daily folate/folic acid intake (.g/d) for

males and females among the four folate categories is presented in Table 4-2 and illustrated in

Figure 4-1. Unadjusted for calories, mean total folate, DFE, folic acid and food folate intake

were significantly (P <0.01) higher for males than females.

Mean folate/folic acid intake for males and females was further analyzed after adjusting for

calories (.g/1,000 kcals/d), the data for which are presented in Table 4-3 and illustrated in Figure

4-2. After adjusting for caloric intake, females had significantly higher mean intakes (.g/1,000

kcals/d) of total folate (P = 0.02), DFE (P = 0.05), and food folate (P <0.01) than males. There

was no statistically significant difference between males and females for mean folic acid intake

(pg/1,000 kcals/d) (P = 0.9).

Dietary Folate Intake and Genotype

Mean folate intake (pg/d) of subjects by MTHFR 677C-*T genotype was compared for

each folate category (Table 4-4) and adjusted for age and gender. As expected, no significant

differences were detected among genotype groups for intakes of total folate (P = 0.7), DFE (P =

0.9), folic acid (P = 0.9), or food folate (P = 0.8). Similarly, when intake was expressed as

.ig/1,000 kcals/d significant differences among genotype groups were not detected for total folate

(P = 0.6), DFE (P = 0.9), folic acid (P = 0.9), or food folate (P = 0.9) intake (Table 4-5).









Dietary Folate Equivalents, Folic Acid and Recommended Intakes

Relative to objective 2, total daily folate intake of males and females expressed as DFE

was compared to the Recommended Dietary Allowance (RDA) of 400 pg DFE/d. A

significantly higher proportion of males (81.4%) consumed the RDA for folate compared to

females (67.9%) (P <0.01) (Figure 4.3). In addition, female subjects' intake of diet-derived folic

acid, including folic acid from fortified ready-to-eat (RTE) cereals, was compared to the

recommended folic acid intake for women of reproductive potential (i.e., 400 ig/d). Only 3.1%

of females (n = 5) met this recommendation (Figure 4-4). Of the remaining female subjects,

24.7% (n = 40) consumed 200 to 399 pg/d folic acid, and 72.2% (n = 117) consumed <200 pg/d

folic acid. Consumption of folic acid did not exceed the UL (>1,000 pg/d) for any of the male or

female participants. The highest estimated intake of folic acid for any male or female subject

was 947 pg/d and 665 ig/d, respectively.

Contribution of Folate from Food Groups

In relation to objective 3, to analyze the contribution of various foods to dietary folate

intake, foods included in the DHQ were categorized into thirteen categories. A comprehensive

descriptive list of foods within each food category is presented in Table 3-1. Foods were

categorized based on type, folic acid enrichment/fortification status, and the amount of naturally

occurring food folate. Mean daily total folate intake (.g/d) contributed by various food sources

is presented in Table 4-6. Both genders consumed the greatest amount of total folate from

enriched grain products, and fortified cereals and bars, including fortified RTE breakfast cereals,

vegetables, and legumes and nuts. Mean daily DFE intake (pg DFE/d) contributed from various

food categories is presented in Table 4-7. The largest contributors of DFE for both genders

included fortified cereals and bars, enriched grain products, vegetables, and combination foods.









Mean daily folic acid intake (.g/d) contributed from various food categories is presented in

Table 4-8. Both males and females primarily consumed folic acid in their diet from enriched

grain products, fortified cereals and bars, and combination foods. Lastly, mean daily food folate

intake ([g/d) contributed from various food categories is presented in Table 4-9. Vegetables

were the largest contributor of food folate, two-fold higher than any other food source.

However, other good sources of food folate for both genders included legumes and nuts, and

fruits.

Within each folate category, no significant differences between males and females were

detected in the folate/folic acid contribution from various food categories except for the snack

category. The folate/folic acid contribution from snack foods was significantly higher (P <0.02)

for females than males; however, snack foods were not a large contributor to the overall intake of

folic acid or food folate.

The proportion of total folate, DFE, folic acid and food folate intake provided by different

food categories to total intake is illustrated for males and females in Figures 4-5 and 4-6,

respectively. Enriched grains (24.4%), vegetables (21.8%), and fortified RTE cereals and bars

(12.5%) provided the majority of total folate for males, whereas vegetables (27.6%) provided the

most total folate in females, followed by enriched grains (20.4%) and fortified RTE cereals and

bars (13.3%). Enriched grains contributed the most to DFE intake for both males (26.2%) and

females (22.0%), followed by vegetables (17.0%) and fortified RTE cereals and bars (16.4%) for

males, and similarly, vegetables (21.6%) and fortified RTE cereals and bars (19.0%) for females.

The food categories that supplied the most folic acid for males and females included enriched

grains (41.1%; 41.9%, respectively), fortified RTE cereals and bars (29.3%; 36.0%,

respectively), and combination foods (17.5%; 14.8%, respectively). The largest contributors of









food folate for males and females included vegetables (31.6%; 38.4%, respectively) and legumes

and nuts (16.2%; 14.4%, respectively).

With the exception of vegetables, the greatest contributors of folic acid intake (i.e.,

enriched cereal-grain products, fortified cereals and bars, and combination foods that included

"enriched" ingredients) were also the greatest contributors to DFE intake. Without folic acid

fortification of these products, DFE intake would have been solely provided by food folate intake

from dietary sources (e.g., vegetables, legumes and nuts, and fruit). Furthermore, DFE intake for

males and females would have been 293 pg DFE/d and 249 pg DFE/d, respectively, which

would have been insufficient to meet the RDA.

Folate Intake from Fortified Ready-to-Eat Cereals

Intake of DFE and folic acid consumed from fortified RTE cereals was compared to the

total daily DFE and folic acid intake to determine what proportion of DFE and folic acid in the

diet comes from fortified RTE cereals. The percentage of males and females who consumed

fortified RTE cereals was 91.4% and 96.9%, respectively. Of these subjects, 50% and 58% of

males and females, respectively, consumed highly fortified RTE cereals. For males, 41.6% of

total DFE intake and 23.0% of total folic acid intake came from consumption of fortified RTE

cereals (Figure 4-7); for females, 37.8% of total DFE intake and 20.9% of total folic acid intake

came from consumption of fortified RTE cereals (Figure 4-8). The mean DFE intake from

fortified RTE cereals alone for males and females was 103 pg DFE/d and 97 pg DFE/d,

respectively. The mean folic acid intake from fortified RTE cereals for males and females was

59 pg/d and 56 ig/d, respectively.









Folate and Homocysteine Concentrations and Gender

Relative to objective 1, mean serum folate, RBC folate, and homocysteine concentrations

were determined for males and females, adjusted for age, and are presented in Table 4-10. No

significant differences in serum folate (P = 0.3) or RBC folate (P = 0.2) concentrations were

detected between males and females. Mean serum folate and RBC folate concentrations for both

genders were above the limits considered normal for the radiobinding assay (>13.6 nmol/L; >317

nmol/L, respectively) (Figures 4-9 and 4-10). Although males had a significantly higher mean

homocysteine concentration compared to females (P <0.0001), mean homocysteine

concentrations for males and females were in an acceptable range (<12.0 .imol/L) (Figure 4-11).

Each subjects' serum folate, RBC folate, and homocysteine concentrations were compared

against the limits considered normal for each metabolite. Only one male subject fell below the

recommended limit for serum folate (>13.6 nmol/L) with a concentration of 11.7 nmol/L. The

participants whose values were below the minimum recommended concentration for RBC folate

(>317 nmol/L) included 1 male subject (289 nmol/L) and 3 female subjects (179, 259, and 278

nmol/L). Nine male subjects had elevated (>12.0 imol/L) homocysteine concentrations (12.4 to

45.7 pmol/L). Of these males, 4 subjects had homocysteine concentrations >14 imol/L (21.6,

27.0, 29.6, 45.7 imol/L). Three female subjects had high homocysteine concentrations (12.4,

12.9, 13.0 pmol/L). The one male subject with a low serum folate concentration (11.7 nmol/L)

had a high homocysteine concentration (29.6 imol/L) and normal RBC folate concentration (460

nmol/L). The male subject with the highest homocysteine concentration (45.7 imol/L) had a

borderline low serum folate concentration (14.9 nmol/L) and normal RBC folate concentration

(589 nmol/L).









Folate and Homocysteine Concentrations and Genotype

Relative to objective 4, the relationship between MTHFR 677 C--T genotype and serum

folate concentration and MTHFR 677 C--T genotype and RBC folate concentration also were

determined. Mean serum and RBC folate concentrations by genotype for all subjects and for

males and females are presented in Tables 4-11 and 4-12, respectively, and illustrated in Figures

4-12 and 4-13. Significant differences were not detected in serum folate concentrations among

the genotype groups for all subjects or for males or females. However, for all participants,

individuals with the CT or TT genotypes had significantly lower mean RBC folate

concentrations (P <0.01) relative to the mean RBC folate of individuals with the CC genotype

(Table 4-12). No significant difference was detected for mean RBC folate concentration

between individuals with the CT and TT genotypes (P = 0.5). When mean RBC folate

concentrations among genotype groups were compared within each gender, the trend for RBC

folate concentrations by genotype for males was similar to the trend for all subjects. However,

the same genotype effect on RBC folate was not detected for female subjects.

When mean homocysteine concentrations were compared among the MTHFR 677 C-T

genotype groups for all subjects, significant differences (P <0.05) were detected between the CC

versus CT genotypes and the CC versus TT genotypes. No significant differences were detected

between individuals with the CT and TT genotypes (P = 0.4) (Table 4-13). As presented in

Table 4-13, the genotype effect on homocysteine concentrations observed for all subjects was

different when data were separated by gender. Significant differences in mean homocysteine

concentrations were detected among genotype groups for males, but not for females. Male

subjects with the TT or CT genotypes had the highest mean homocysteine concentrations (9.2









.imol/L; 8.5 .mol/L, respectively), whereas, male subjects with the CC genotype had the lowest

mean homocysteine concentration (7.5 .imol/L). These data are also illustrated in Figure 4-14.

Serum Folate and Homocysteine

When the association between serum folate and homocysteine concentrations was

evaluated, a significant inverse association was found for all participants (P <0.001), as well as

within gender groups (i.e., males, P = 0.02; females, P = 0.02). These associations indicate that

as the concentration of serum folate increased, homocysteine concentration decreased.

This association was further tested within each gender and by genotype group. For males,

a significant association between serum folate and homocysteine concentration was not detected

for either the CC or TT genotype groups (P = 0.5; P = 0.3, respectively). There was a trend

toward a significant association for CT genotype group (P = 0.06). A significant association also

was not detected for females within the CC genotype group (P = 0.4) or the TT genotype group

(P = 0.1). However, a significant inverse association between serum folate and homocysteine

concentration was detected for females within the CT genotype group (P = 0.03).

Relationship Between Folate Intake and Status

Relative to objective 4, the association between serum or RBC folate concentrations and

folate/folic acid intake (.g DFE/d) was assessed using linear regression analysis to determine

whether the level of DFE intake impacted serum or RBC folate concentrations. As hypothesized,

a significant positive association between serum folate concentration and DFE intake for all

subjects was detected (P = 0.03). When this relationship was analyzed by gender, there was

insufficient power to detect whether significant differences existed for males or females (P = 0.2;

P = 0.4, respectively). No relationship was detected between RBC folate and DFE intake for all

subjects (P = 0.5) or by gender (P = 0.07).









The association between homocysteine concentration and folate/folic acid intake (.g

DFE/d) also was examined to determine if the level of folate/folic acid intake impacted

homocysteine concentration. A significant inverse association between homocysteine

concentration and DFE intake was found for all participants (P <0.0001), as well as for males (P

= 0.02). The behavior of this relationship was not constant between genders as an association

was not detected in females.

Folate Status and Fortified Ready-to-Eat Cereal Consumption

Although no significant difference was detected, serum folate concentration tended to be

different (P = 0.08) between consumers and non-consumers of fortified RTE cereals for all

subjects. Gender differences in serum folate and RBC folate concentrations between consumers

and non-consumers of fortified RTE cereals were not detected. Comparison of homocysteine

concentrations of consumers and non-consumers of fortified RTE cereals identified a significant

difference in mean homocysteine concentration between female consumers (6.5 .imol/L) and

non-consumers (8.4 imol/L; P <0.05). A significant difference in mean homocysteine

concentration between male consumers (8.0 imol/L) and non-consumers (8.4 imol/L) was not

detected (P >0.5).









Table 4-1. Demographic characteristics of males and females.a
Demographic variable Males Females P-value
(n = 140) (n = 162)
Age (years)bc 26.0 + 8.2 25.3 7.1 0.6
BMI (kg/m2)bc 24.5 + 4.3 22.6 + 3.6 <0.0001
Education level (years)bc 16.0 + 0.2 15.2 + 2.5 0.4
Ethnicity (%)d 0.4
White 58 63
Asian 17 12
African American 7 4
Indian 2 3
Hispanic 11 15
Other 4 1
Unknown 1 1
Marital Status (%)d 0.7
Single 79 77
Married 15 17
Divorced 4 3
Separated 1 2
Refused 2 1
Student Status (%)d 0.2
Full-Time 67 65
Part-Time 4 7
Not a student 9 15
Unknown 19 13
Employed (%)d 0.2
Yes 55 59
No 34 36
Unknown 11 5
aDue to rounding, percentages may not always sum to 100. bMean + standard deviation (SD).
cOne-way ANOVA was used for statistical comparisons between genders. dChi-square test was
used for statistical comparisons between genders and demographic variables.










Table 4-2. Mean intake (pg/d) by folate category and gender.a'b'c
Folate group Males Females P-value
(n = 140) (n = 162)
Total folate 462 (426, 501) 371 (344, 400) 0.0001
DFE 652(603,705) 512 (476, 551) <0.0001
Folic acid 175 (156, 195) 128 (115, 142) <0.0001
Food folate 293 (268, 321) 249 (229, 270) 0.01
aMean (5%, 95% CI). bOne-way ANOVA was used for statistical comparisons between genders
after adjusting for age. cP-values were based on normalized (log transformed) data. The results
have been back-transformed to original scale.


750-
I Males
3 Females
t *
w 500-



0 250-
U-
C)


TF DFE FA FF
Folate Category

Figure 4-1. Mean intake (pg/d) by folate category and gender. TF = total folate; DFE = dietary
folate equivalents; FA = folic acid; FF = food folate. *Significant difference (P
<0.01) in mean intake (.g/d) by folate category between males and females after
adjusting for age.










Table 4-3. Mean intake (.g/1,000 kcals/d) by folate category and gender.a'b'c
Folate group Males Females P-value
(n = 140) (n = 162)
Total folate 210 (197, 224) 233 (219, 247) 0.02
DFE 296(279,314) 321(304,340) 0.05
Folic acid 79 (72, 87) 80 (73, 88) 0.9
Food folate 133 (124, 143) 156 (146, 167) <0.01
aMean (5%, 95% CI). bOne-way ANOVA was used for statistical comparisons between genders
after adjusting for age. cP-values were based on normalized (log transformed) data. The results
have been back-transformed to original scale.


I Males
- Females


TF DFE FA FF
Folate Group


Mean intake (.g/1,000 kcals/d) by folate category and gender. TF = total folate;
DFE = dietary folate equivalents; FA = folic acid; FF = food folate. *Significant
difference (P <0.05) in mean intake (.g/1,000 kcals/d) by folate category between
males and females after adjusting for age.


350-
ro
o
. 300-

S250-

S200-

S150-
I-
S100
-I

o 50-
0-
0-


Figure 4-2.









Table 4-4. Mean intake (pg/d) by folate category and genotype.a'b'c
Folate group CC genotype CT genotype TT genotype P-value
(n = 151) (n = 118) (n= 31)
Total folate 423 (391, 456) 395 (361, 429) 410 (344, 477) 0.7
DFE 581(538, 625) 559 (512, 606) 583 (490, 675) 0.9
Folic acid 151 (135, 167) 141 (125, 158) 161 (126, 196) 0.9
Food folate 273 (250, 296) 262 (238, 287) 265 (218, 311) 0.8
aMean (5%, 95% CI). bOne-way ANOVA was used for statistical comparisons between genders
after adjusting for age. cP-values were based on normalized (log transformed) data. The results
have been back-transformed to original scale.

Table 4-5. Mean intake (.g/1,000 kcals/d) by folate category and genotype.a'b'c
Folate group CC genotype CT genotype TT genotype P-value
(n = 151) (n = 118) (n= 31)
Total folate 227 (213,241) 215 (200, 229) 224 (195,252) 0.6
DFE 312(294, 330) 304(285,323) 317(279, 356) 0.9
Folic acid 81 (74, 88) 77 (69, 84) 88 (71, 104) 0.9
Food folate 146 (136, 157) 143 (132, 154) 144 (123, 165) 0.9
aMean (5%, 95% CI). bOne-way ANOVA was used for statistical comparisons between genders
after adjusting for age. cP-values were based on normalized (log transformed) data. The results
have been back-transformed to original scale.













<200 pg/d
Males- 200-399 pg/d
400-999 pg/d
_> 11,000 pg/d

Females



0% 25% 50% 75% 100%
Percentage of males/females consuming
specific ranges of folate expressed as pg DFE/d

Figure 4-3. Percentage of subjects who consumed specific ranges of folate expressed as ig
DFE/d.


Males




Females


25% 50% 75%
Percentage of males/females consuming
specific ranges of folic acid (pg/d)


- <200 pg/d
I 200-399 pg/d
400-999 pg/d


100%


Figure 4-4. Percentage of subjects who consumed specific ranges of folic acid (ig/d).









Table 4-6. Mean total folate contribution (pg/d) by dietary source and gender.a
Dietary source Males Females
(n = 140) (n = 162)
Enriched cereal-grains 104 + 6 94 5
Fortified cereals and bars 66 9 63 8
Vegetable 117 9 127 9
Fruit 25 2 27 2
Juice (orange and grapefruit) 16 + 2 14 + 2
Legumes and nuts 58 6 51 5
Meat 4 0 3 0
Combination 18 2 19 2
Snacks 7 2b 13 2
Dairy 14 1 15 + 1
Eggs 5 1 5 1
Miscellaneous 24 4 25 + 4
Other 9 1 9 1
aMean SE. One-way ANOVA was used for statistical comparisons between genders after
adjusting for age and calories. bSignificantly lower than females (P = 0.01).

Table 4-7. Mean DFE contribution (pg DFE/d) by dietary source and gender.a
Dietary source Males Females
(n = 140) (n = 162)
Enriched cereal-grains 158 9 143 9
Fortified cereals and bars 128 + 17 129 15
Vegetable 119 + 10 130 + 9
Fruit 25 2 27 2
Juice (orange and grapefruit) 16 + 2 14 + 2
Legumes and nuts 59 6 52 5
Meat 5 1 4 1
Combination 66 4 59 4
Snacks 10 2b 18 2
Dairy 14 1 15 1
Eggs 5 1 5 1
Miscellaneous 35 5 36 5
Other 9 1 10+ 1
aMean SE. One-way ANOVA was used for statistical comparisons between genders after
adjusting for age and calories. bSignificantly lower than females (P = 0.01).









Table 4-8. Mean folic acid contribution (pg/d) by dietary source and gender.a
Dietary source Males Females
(n = 140) (n = 162)
Enriched cereal-grains 78 5 71 5
Fortified cereals and bars 73 10 73 9
Vegetable 0 + 0 0 + 0
Fruit 0 + 0 0 + 0
Juice (orange and grapefruit) 0 + 0 0 + 0
Legumes and nuts 0 + 0 0 + 0
Meat 0 0 0 0
Combination 26 2 24 2
Snacks 4 lb 7 1
Dairy 0 + 0 0 + 0
Eggs 0 + 0 0 + 0
Miscellaneous 7 2 6 2
Other 0 + 0 0 + 0
aMean SE. One-way ANOVA was used for statistical comparisons between genders after
adjusting for age and calories. bSignificantly lower than females (P = 0.02).

Table 4-9. Mean food folate contribution (pg/d) by dietary source and gender."
Dietary source Males Females
(n = 140) (n = 162)
Enriched cereal-grains 25 1 23 1
Fortified cereals and bars 4 1 4 0
Vegetable 117 9 127 9
Fruit 25 2 27 2
Juice (orange and grapefruit) 16 + 2 14 + 2
Legumes and nuts 58 6 51 5
Meat 4 0 3 0
Combination 8 1 8 1
Snacks 3 1b 6 1
Dairy 14 1 15 1
Eggs 5 1 5 1
Miscellaneous 23 3 27 3
Other 9 1 9 1
aMean SE. One-way ANOVA was used for statistical comparisons between genders after
adjusting for age and calories. bSignificantly lower than females (P = 0.02).














K'S fliiiii


K^^* S11IIIIIIIIr


K ~~III~


TF-


DFE-


FA-


FF-

0%


Food groups
I Enriched cereal grains
\ g Fortified cereals and bars
Vegetables
Em Fruit
Juice (orange and grapefruit)
1XXS Legumes and nuts
Dairy
XX Eggs
Meat
fI Combination
EEE1 Snacks
LQn Miscellaneous
Other


Percentage of total intake derived from each food group by folate category for
males. TF = total folate, DFE = dietary folate equivalents, FA = folic acid, FF =
food folate. The relative contribution of each food group to TF, DFE, FA and FF
intake was calculated by dividing the mean intake of each food group by total intake
for each folate category (e.g., mean DFE intake from enriched cereal grains divided
by total mean DFE intake).


Food groups
SEnriched cereal grains
[ g Fortified cereals and bars
Vegetables
Em Fruit
Juice (orange and grapefruit)
XS Legumes and nuts
Dairy
XXEggs
Meat
FII Combination
m 1Snacks
LQ Miscellaneous
Other


Percent of folate and folic acid intake by food group (females)

Percentage of total intake derived from each food group by folate category for
females. TF = total folate, DFE = dietary folate equivalents, FA = folic acid, FF =
food folate. The relative contribution of each food group to TF, DFE, FA and FF
intake was calculated by dividing the mean intake of each food group by total intake
for each folate category (e.g., mean DFE intake from enriched cereal grains divided
by total mean DFE intake).


100%


e 4-5.


Percent of folate and folic acid intake by food group (males)


Figur












TF-


DFE-


FA-


FF-

0%


SA11111111111


Figure 4-6.


~ fi~IN


I











I I Fortified RTE cereals
All other dietary sources


TF

DFE

FA-

FF-

01


Percent contribution of enriched RTE cereal
to the folate/folic acid intake (males)


Mean percent contribution of fortified RTE cereal to the total mean intake for each
folate category for males. TF = total folate, DFE = dietary folate equivalents, FA =
folic acid, FF = food folate.


SFortified RTE cereals
All other dietary sources


20% 40% 60% 80%
Percent contribution of enriched RTE cereal
to the folate/folic acid intake (females)


100%


Mean percent contribution of fortified RTE cereal to the total mean intake for each
folate category for females. TF = total folate, DFE = dietary folate equivalents, FA
= folic acid, FF = food folate.


100%


40%


60%


Figure 4-7.


Figure 4-8.


I


%a









Table 4-10. Comparison of serum and RBC folate, and homocysteine concentrations by
gender'ab,c
Males Females P-value
Serum folate (nmol/L) 39.9 (37.8, 42.0) 41.7 (39.6, 43.8) 0.3
RBC folate (nmol/L) 810 (766, 857) 767 (727, 809) 0.2
Homocysteine (pmol/L) 8.0 (7.7, 8.4) 6.6 (6.3, 6.9) <0.0001
aConcentrations expressed as mean (5%, 95% CI). bOne-way ANOVA was used for statistical
comparisons between genders after adjusting for age. cP-values were based on normalized (log
transformed) data. The results have been back-transformed to original scale.






















Males
Males


Normal
(>13.6 nmol/L)


Females


Mean serum folate concentration (nmol/L) by gender after adjusting for age.
Normal serum folate status was defined as a serum folate concentration >13.6
nmol/L.


Males
Males


Normal
(>317 nmol/L)


Females


Figure 4-10. Mean RBC folate concentration (nmol/L) by gender after adjusting for age.
Normal RBC folate status was defined as a RBC folate concentration >317 nmol/L.


Acceptable cutoff
12------------------- (<12 pmol/L)


Males
Males


Females


Figure 4-11. Mean homocysteine concentration (imol/L) by gender after adjusting for age.
Normal homocysteine concentration defined as <12 imol/L. *Significant
difference (P <0.0001) in mean homocysteine concentration (imol/L) between
males and females.


Figure 4-9.









Table 4-11. Comparison of mean serum folate concentrations (nmol/L) by genotype.a'b
Gender CC genotype CT genotype TT genotype
All subjects' 42.5 (40.3, 44.7) 39.3 (37.1, 41.6) 38.5 (34.3, 42.6)
Malesd 41.7 (38.6, 44.9) 38.6 (35.4, 41.8) 36.9 (31.3, 42.4)
Femalesd 43.3 (40.2, 46.3) 40.0 (36.8, 43.2) 40.2 (34.1, 46.3)
aMean (5%, 95% CI). bGLM procedure was used for statistical comparisons of mean serum
folate concentrations between genotypes for each gender after adjusting for age. P-values were
based on normalized (log transformed) data. The results have been back-transformed to original
scale. cUnable to detect significant differences in serum folate concentrations among genotype
groups (P >0.05). dNo significant differences detected in serum folate among genotype groups
for males or females (P >0.1).

Table 4-12. Comparison of mean RBC folate concentrations (nmol/L) by genotype. ab
Gender CC genotype CT genotype TT genotype
All subjected 848 (803, 895) 744 (700, 790) 713 (634, 801)
Males ef 878 (811, 944) 756 (693, 819) 733 (622, 845)
Femalesgh 821 (762, 880) 732 (673, 790) 691 (584, 798)
aMean (5%, 95% CI). bGLM procedure was used for statistical comparisons of mean RBC folate
concentrations between genotype groups after adjusting for age. P-values were based on
normalized (log transformed) data. The results have been back-transformed to original scale.
CCC genotype significantly higher than CT genotype (P = 0.002). dCC genotype significantly
higher than TT genotype (P <0.01). eCC genotype significantly higher than CT genotype (P
0.01). fCC genotype significantly higher than TT genotype (P = 0.05). gCC genotype
significantly higher than CT genotype (P = 0.04). hUnable to detect a significant difference for
CC versus TT genotype (P = 0.07).

Table 4-13. Comparison of mean homocysteine concentrations (pmol/L) by genotype.a,b
Gender CC genotype CT genotype TT genotype
All subjects' 6.9 (6.6, 7.2) 7.5 (7.1, 7.8) 7.8 (7.1, 8.6)
Malesde 7.5 (7.0, 7.9) 8.5 (7.9, 9.0) 9.2 (8.0, 10.3)
Femalesft 6.5 (6.1, 6.8) 6.6 (6.2, 7.1) 6.6 (5.8, 7.5)
aMean (5%, 95% CI). bGLM procedure was used for statistical comparisons of mean
homocysteine concentrations between genotypes for each gender after adjusting for age.
P-values were based on normalized (log transformed) data. The results have been back-
transformed to original scale. "CT and TT genotypes significantly higher than CC genotype (P =
0.03). dCT genotype significantly higher than CC genotype (P = 0.01). eTT genotype
significantly higher than CC genotype (P <0.01). fNo significant differences detected in
homocysteine concentrations among genotype groups for females (P >0.05).











I I All subjects
- Males
- Females


CC CT TT


MTHFR 677C-*T Genotype


Figure 4-12.


1000-


Mean serum folate concentration (nmol/L) by genotype after adjusting for age.


I IAll subjects
r ** **Males
-- -i- LT -- T Females


CC CT TT


MTHFR 677C->T Genotype

Figure 4-13. Mean RBC folate concentration (nmol/L) by genotype after adjusting for age.
*Significant difference (P <0.01) in mean RBC folate concentration compared to
all subjects with the CC genotype. **Significant difference (P <0.05) in mean
RBC folate concentration compared to males with the CC genotype.
***Significant difference (P = 0.04) in mean RBC folate concentration compared
to females with the CC genotype.












"' I I IAll subjects
10-
** Males
1T 9-

-a
h8- Females
o8- .
7-
6-
5-
4-
0
oE 3-
0 2
1-

0-
CC CT TT

MTHFR 677C-*T Genotype

Figure 4-14. Mean homocysteine concentration (pmol/L) by genotype after adjusting for age.
*Significant difference (P = 0.03) in mean homocysteine concentration compared
to all subjects with the CC genotype. **Significant difference (P <0.01) in mean
homocysteine concentration compared to males with the CC genotype.









CHAPTER 5
DISCUSSION AND CONCLUSIONS

The focus of this study was to assess folate intake and status of non-supplement

consuming healthy young men and women and to estimate the relative contribution of

folate/folic acid from different food categories to overall intake. Previous research studies have

addressed the impact of folic acid enrichment of cereal-grain products and folic acid fortification

of breakfast cereals on folate status of men and women (3-6, 13, 125, 150), but the present study

is the first to include an estimate of the relative contribution of folate/folic acid from different

food categories to overall intake. Information related to the contribution of different food

categories to folate/folic acid intake can be used to develop targeted intervention strategies

directed at improving folate/folic acid intake.

Recent updates of the USDA National Nutrient Database for Standard Reference

(Release 17), which reflects folic acid fortification of foods, separates dietary folate intake into

four categories: food folate, folic acid, total folate, and DFEs (8). Unlike most previous

investigations that only reported total folate intake (i.e., [g of food folate plus [g of folic acid

without adjustment for differences in bioavailability), our study provides separate data on folic

acid and food folate intake enabling a comparison with the recommended level of folic acid for

women of reproductive potential, as well as intake expressed as DFE, a calculated value essential

for comparison to the RDA for males and females.

The majority of reports in the literature have used data from NHANES to evaluate the

adequacy of folate intake in the US post-fortification (4, 13, 151). One of the limitations of that

database is that nutrient intake data are estimated for each individual from a single 24-hour

dietary recall. In the present study, the previously validated DHQ was used to assess dietary

intake based on recall of food frequency over a 12 month period (144). Another limitation









associated with using the NHANES database is the fact that intake analysis data are reported as

total folate intake (tg/d) and do not provide folate intake expressed as DFEs; consequently, the

researchers using that database were unable to compare intake relative to the RDA.

A study by Dietrich et al. (13), in which total folate intake (tg/d) of males and females

was compared before and after fortification, reported that the absolute increase in total folate

(tg/d) were larger among men than women. These investigators attributed this to the fact that

men, in general, have higher dietary intakes of folate than women. Although our analysis also

indicated that males consumed more folate per day than females, the average total folate intake

(tg/1,000 kcals/d) was significantly higher for females. Adjusting for calories allows for more

equal comparison between genders as the DHQ revealed that males consume more daily calories,

and for this reason, males are more likely to consume more folate/folic acid than females on any

given day.

Based on data from NHANES 1999-2000, Briefel et al. (152) reported that among

women of childbearing age (20 to 39 years), mean total folate intake was 327 ig/d. Dietrich et

al. (13) also evaluated folate intake of NHANES 1999-2000 participants and found that the

mean total folate intake of males and females combined (aged >20 years) was 351 ig/d. In

comparison, our data revealed a slightly higher average total folate intake of 371 tg/d for

women. The average total folate intake for men was 462 ig/d. However, since these data do not

account for the form of the vitamin consumed (i.e., food folate versus folic acid from enriched

and fortified products), it is not possible to compare these intake levels fairly, nor is there a way

to determine if they would meet the current RDA for folate. Our data, however, revealed that the

mean total intake expressed as DFEs for males and females (652 tg DFE/d; 512 tg DFE/d,

respectively) exceeded the RDA of 400 tg DFE/d, with the majority of males (81%) and females









(68%) meeting the RDA. Interestingly, after adjusting for caloric intake, females in our study

had a significantly higher average intake of food folate compared to males, but no difference was

detected for folic acid intake between genders.

The present study estimated the actual and relative contribution of different food

categories to overall folate/folic acid intake. This unique aspect of the present study identified

the major contributors of daily folate intake (i.e., .g DFE/d) to be enriched cereal-grains

products, fortified cereals and bars, and combination foods that include "enriched" ingredients

for both males and females. When comparing the actual contribution of food categories to total

folate intake, vegetables contributed the highest amount of total folate, followed by enriched

cereal-grains products for men and women alike. The differences noted in average intake

between total folate and DFE are due to the way DFEs are calculated, where more weight is

given to folic acid than endogenous food folate (pg DFE/d = 1.7 x pg/d folic acid + pg/d food

folate).

The IOM recommends that all women of reproductive potential consume 400 [g of folic

acid daily from vitamin supplements and/or fortified foods in addition to consuming food folate

from a varied diet to reduce the risk of having an NTD-affected pregnancy (7). Yang et al. (6)

examined folic acid intake in women of childbearing age in the US using NHANES 2001-2002

data and nutrient intakes estimated from the USDA National Nutrient Database Standard

Reference, Release 16. Mean consumption of folic acid from fortified foods was 128 .g/d in

non-supplement consuming, non-pregnant women, and only 8% of the women reported

consuming >400 pg/d of folic acid from fortified foods. Similar to the findings by Yang et al.,

the average intake of folic acid from fortified foods for women in the present study who were

non-supplement users was 128 pg/d or approximately one-third of the amount recommended for









NTD risk reduction. Furthermore, only 3% of female subjects consumed >400 pg/d of folic acid

from enriched and fortified foods. This falls well short of the Healthy People 2010 goal of 80%

of women of reproductive potential achieving a daily folic acid intake of 400 Gg. However, even

though folic acid intake was inadequate relative to the amount recommended for NTD risk

reduction, and only two-thirds of the women in our study exceeded the folate RDA, the

estimated levels of folate/folic acid intake reported in our study were associated with normal

blood folate status. Whether or not the level of intake that was associated with normal blood

folate status in our study is adequate to achieve the level of NTD risk reduction associated with

400 pg of folic acid is not known.

Since folic acid enrichment of cereal-grain products was designed to augment folic acid

intake from supplements, it is understandable that the majority of women would not be

consuming the recommended 400 pg/d of folic acid from dietary sources alone. What the data

from this investigation illustrate so clearly is that when women do not take supplements, which is

the case for the majority of women, dependence on dietary sources of folic acid to meet the

recommended intake is totally inadequate. The CDC, the March of Dimes, and the National

Council on Folic Acid have organized the National Folic Acid Campaign for the prevention of

NTDs, with the goal of teaching all women about the importance of folic acid and ensuring

women consume 400 [g of folic acid daily (17). The campaign targets all women of

reproductive age, the health care professionals who serve these women, and community

advocacy groups (153). However, despite all of these major folic acid awareness campaigns,

these data illustrate that more attention needs to be focused on increasing folic acid intake from

dietary sources for women of childbearing age who do not take supplements.









In 2005, the CDC and the March of Dimes launched the Preconception Health and Health

Care Initiative, the aims of which are to improve the health of mothers before pregnancy and

maternal and infant health outcomes (130). As a result of this summit and initiative, strategies

for improving preconception health incorporating folic acid awareness have been incorporated

into numerous programs throughout the US, including areas in clinical practice, consumer roles,

and public health (130, 154). However, many of these campaigns have proven to be ineffective

as recent data from the 2005 March of Dimes Gallup Survey reported by the CDC suggested

only 33% of the general population of women of reproductive age take a folic acid supplement

daily (17). Therefore, the sole source of folic acid in the diets of the majority of this target group

is enriched and fortified foods. Because only 3% of women in our study consumed 400 pg/d of

folic acid from fortified foods alone, it can be concluded that non-supplement users are at risk for

not meeting the recommended level of folic acid intake shown to result in NTD risk reduction.

Women of reproductive potential who do not take supplements should be encouraged to

consume adequate amounts of fortified or enriched cereal grain products daily to achieve the

recommended level of folic acid intake.

The present study also evaluated the impact of folate intake on folate status of male and

female non-supplement users. A significant positive association was detected between intake of

DFE and serum folate concentrations for all subjects, and a significant inverse association was

found between intake of DFE and homocysteine concentrations for all subjects and for male

subjects only. No relationship was detected between intake of DFE and RBC folate

concentrations for all subjects or by gender. The mean folate/folic acid intake observed in this

study was associated with adequate mean serum folate concentrations for both males (39.9

nmol/L) and females (41.7 nmol/L). These concentrations are higher than reported by Jacques et









al. (4), who reported that the mean plasma folate concentration for both males and females who

were non-supplement users in the Framingham Offspring Study (aged 32 to 80 years) was 23

nmol/L. Lawrence et al. (5) reported a median serum folate concentration of 42.4 nmol/L post-

fortification; however, no information was given on subjects' folic acid supplement use. Caudill

et al. (155) investigated 135 women (aged 18 to 45 years) who were non-supplement users post-

implementation of folic acid fortification. These women were reported to have a mean serum

folate concentration of 50 nmol/L. Possible explanations for differences in blood folate

concentrations among studies could be due to various ages of study participants, differences in

analytical methodology for measuring blood folate, and the fact that folic acid fortification had

been in effect longer when Caudill et al. completed their study and when blood samples used for

folate analysis were collected in the current study.

Based on data from the NHANES, Pfeiffer et al. (156) reported that the prevalence of low

serum folate concentrations (<7 nmol/L) for women of childbearing age (15 to 45 years) was

<1%. Serum folate samples collected as part of the NHANES study were analyzed using the

Bio-Rad QuantaPhase II radiobinding assay. This method yields lower serum folate values than

the microbiological method, the latter of which uses a cutoff value of 13.6 nmol/L (61).

Although the cutoff value for serum folate concentration of 13.6 nmol/L was used in the present

study, none of the women in this investigation fell below this level. If the Bio-Rad norm for

serum folate that was established with the radiobinding assay had been used in this study, then

none of the male or female subjects would have had a low serum folate concentration. Taken

together, the data examining folate status provide convincing evidence to indicate that a

significant proportion of the population, particularly women of reproductive age, have more than

adequate folate status.









With respect to RBC folate, mean RBC folate concentrations in the present study for

males (810 nmol/L) and females (767 nmol/L) were well above the lower limit of normal (i.e.,

317 nmol/L) based on the value established for use with the radiobinding assay RBC folate

concentration is a better indicator of long-term folate status than serum folate concentration.

Only 2% of females and <1% of males had RBC folate concentrations <317 nmol/L. Our data

are similar to those reported by Choumenkovitch et al. (3) indicating that 96.1% of women >45

years who were non-supplement users had adequate RBC folate concentrations (>362.6 nmol/L).

Despite the fact that the majority of our female subjects did not meet the recommended level of

intake for NTD risk reduction, 98% of them had adequate RBC folate concentrations. However,

whether or not their status is sufficient to reduce NTD risk is unknown. Research from an Irish

population indicated that RBC folate concentrations >908 nmol/L were associated with an eight-

fold difference in NTD incidence compared to concentrations <341 nmol/L (85). However,

these results may not be applicable to a US population so further research is warranted.

The benefits of adequate blood folate status may confer benefits related to chronic

disease risk reduction. Mild folate deficiency has been associated with an increased plasma

homocysteine concentration, an independent risk factor for vascular disease (100, 157, 158).

Homocysteine is also recognized to be a "functional indicator of folate status" (63). The data

from our study confirm that serum folate concentration is inversely related to plasma

homocysteine concentrations. Although no significant differences in mean serum or RBC folate

concentrations were detected between males and females, males had a significantly higher mean

homocysteine concentration (8.0 .imol/L) compared to females (6.6 .imol/L). These data agree

with findings reported by Ganji et al. (66) that plasma homocysteine concentrations are often

higher in males than females. Of the 6.5% of males (n = 9) with elevated homocysteine (>12









.imol/L), five subjects fell below the mean serum folate concentration for males, six were below

the mean RBC folate concentration for males, and four subjects consumed <400 ag DFE/d.

Although not evaluated as part of this study, a possible explanation for the elevated

homocysteine concentrations of these subjects could be related to vitamin B12 deficiency (159,

160). Because elevated plasma homocysteine concentrations are considered a risk factor for

cardiovascular disease, the association between homocysteine and folate broadens the health-

related consequences of low folate status and has implications for both men and women.

Therefore, marketing folic acid consumption should not be limited to prevention of NTDs as

folate status plays a role in various health-related problems.

Our study also investigated the relationship between folate status, homocysteine and the

MTHFR 677C--T polymorphism. Comparable to the findings ofBrattstrom et al. (106),

Kauwell et al.(59), and Shelnutt et al. (21), subjects with the TT genotype had higher

homocysteine concentrations and lower RBC folate concentrations than individuals with the CC

genotype. However, in contrast to these reports, serum folate concentrations in the present study

population did not differ among genotype groups. One explanation for this could be due to the

fact that the majority of our population consumed sufficient dietary folate to maintain normal

serum folate concentrations. Previous research by Shelnutt et al. (21) determined that a

predisposition for reduced blood folate and elevated plasma homocysteine concentration could

be significantly improved in response to improved folate status. Other research has also

confirmed the ability of increased food folate and folic acid intake to positively modify the

negative effects of the MTHFR polymorphism on folate status (161, 162). Ashfield-Watt and

colleagues (161), however, determined that individuals with the TT genotype require higher









intakes of folic acid from fortified foods or supplements than individuals with the CC or CT

genotype to achieve a similar lowering effect on homocysteine concentrations.

Another aim of our study was to characterize the relative contribution of specific food

categories to food folate/folic acid intake. The findings from this investigation indicate that the

intake of folic acid from fortified RTE cereals alone represented 20% of the total folic acid

intake for women of reproductive age who consumed fortified cereals. Seventy-eight percent of

total folic acid in their diets was provided by a combination of enriched cereal-grain products

and fortified cereals and bars, including RTE cereals. For females, RTE cereal consumption also

accounted for 13.3% of total folate intake and 19.0% of DFE intake. Our findings are similar to

those of Dietrich et al. (13) who reported that fortified RTE breakfast cereals provided 12.1% of

total folate intake. However, estimates of folic acid intake from our study population may not be

representative of women of reproductive potential because we used the data collected for a study

designed to assess vitamin B12 intake from dietary sources. The protocol for that study excluded

women who consumed highly fortified RTE cereals on a daily basis, but not those who

consumed these products less frequently. These results may not be observed in other parts of the

world where fortified RTE cereals are not a prevalent component of the diet.

Although actual intake of fortified cereals might be greater than reported in the present

study, our study identified fortified RTE cereal as an important source of folic acid in the diet.

Most RTE cereals contain 100 .g of folic acid per serving, and some highly fortified cereals

contain as much as 400 pg per serving. Examples of specific brands of highly fortified RTE

cereal include All-Bran, Multigrain Cheerios, Complete, KASHI Heart-to-Heart, Mueslix,

Product 19, Smart Start', Special K, and Total (all varieties). In this study, 58% of women

who consumed fortified cereals reported consuming highly fortified RTE cereals. A note of









caution is that cereals are often consumed in amounts twice the labeled serving size (163);

therefore, individuals who consume highly fortified breakfast cereals in large quantities have the

potential to exceed the UL (1,000 .g/d of folic acid). Nevertheless, no subjects in this study

were found to consume the UL for folic acid.

Since the majority of women of childbearing age are not taking folic acid supplements,

promoting folic acid consumption from fortified RTE cereals and enriched cereal-grain products

represents an important approach to the delivery of folic acid in the diet. Ensuring women

consume the recommended quantities of folic acid daily is fundamental as more than half of all

pregnancies are unplanned and because the development of NTDs occurs during the first 28 days

of pregnancy, often before many women realize they are pregnant.

Strengths and Limitations

Weaknesses of the current investigation include limitations associated with the use of a

FFQ to assess dietary folate intake, including the fact that there is no option for individuals to

add personal responses related to food intake on the questionnaire. More accurate approaches to

assess dietary intake include weighed food records or multiple-day food records. However, these

methods are time consuming and tedious and are generally discouraged for larger studies. The

DHQ used in this study has been validated as an appropriate method of assessing usual dietary

intake (144, 164). Nevertheless, this subgroup may not be representative of men and women in

the US population in contrast to NHANES, a population based study designed to be

representative of the US population. Furthermore, the criteria used for the study from which our

data were obtained attempted to exclude women who consumed highly fortified RTE cereals on

a daily basis. This may have resulted in an underestimation of folic acid intake from fortified

RTE cereals.









Another limitation of this study was the lack of statistical power due to variability and

limited observations when comparing folate status of subjects by MTHFR 677C--T genotype

within the same gender group. Another limitation of this study is that differences in measured

blood folate concentrations have been noted between the radiobinding assay, the method used in

this study, and the microbiological assay method used by many other investigators. Fazili et al.

(61) reported that the vitamin B12/folate radioassay kit may underestimate serum folate

concentrations compared to the LC-MS/MS and microbiological assay. An advantage of using

the radioassay in this study is the fact that this was the method used for analysis of blood folate

concentrations in the most recent NHANES, making comparisons between the data in this study

and data reported in the NHANES study possible.

Strengths of the present study included the ability to estimate DFE intake, thus enabling

us to compare intake to the RDA, and the ability to compare the relative contribution of specific

food categories to total folate/folic acid intake in non-supplement users. Moreover, the fact that

supplement users were excluded from the study made it possible to assess the impact of dietary

folate/folic acid intake on folate status. It would have been ideal to compare folate status

between consumers and non-consumers of RTE cereals; however, since 90% of our subjects

consumed these products, it was not feasible to make this comparison.

Summary

This study supports previous research that has shown a positive relationship between

folate/folic acid intake and folate status. The estimated average dietary folate/folic acid intake of

our study population exceeded the current RDA, and average blood folate concentrations were

well within the acceptable range. Although male subjects had a significantly higher mean

homocysteine concentration compared to females, the majority of all subjects had plasma









homocysteine concentrations within the acceptable limit. In relation to the MTHFR 677 C--T

polymorphism, the data from all subjects are consistent with previous studies in which the TT

genotype was associated with significantly lower RBC folate and higher homocysteine

concentrations compared to the CC genotype.

Based on the findings from this study, it appears that folic acid fortified foods are

important contributors to the diet in terms of meeting the folate RDA of non-supplement users.

The major food categories that contributed toward meeting the RDA for males and females were

enriched cereal-grain products, fortified cereals and bars and combination foods that include

"enriched" ingredients. This highlights the integral role of folic acid fortification in achieving an

adequate intake (based on the RDA) and status of this vitamin in non-supplement users;

however, despite the relative contribution of folic acid fortified foods toward meeting the RDA,

intake of these foods was not sufficient among our study population to meet the IOM

recommendation (400 pg/d of folic acid) for women of reproductive potential. The extremely

low proportion of females (3%) who met this recommendation is disappointing considering the

extensive efforts to promote awareness and behavior change among women of reproductive

potential.

Practical Application

Since public health educational programs and strategies designed to market daily folic

acid supplementation to women of reproductive age have not been effective, women might be

more responsive to food advertisements, including promotion of fortified cereals and snack bars

as good to excellent dietary sources of folic acid. Rather than solely promoting folic acid

supplementation, researchers, dietitians, health educators and food industry manufacturers should

forge efforts to create and market educational outreach programs promoting intake of fortified









RTE cereals, breakfast and snack bars, and enriched cereal-grain products as easy alternatives to

help women consume 100% of the RDA, meet the IOM recommendation for folic acid and

maintain adequate folate status. However, folic acid fortification will not ensure adequate folic

acid intake in target women unless women are educated to change the way they eat. Health

professionals and health educators could promote eating one serving of cereal that has been

fortified with 100% of the daily value of folic acid as a means of consuming the recommended

amount of folic acid daily.

Health professionals and researchers should make an effort to explore the reasons why

women of reproductive age are not consuming sufficient levels of folic acid to prevent NTDs.

These future findings may allow researchers to better understand barriers to folic acid

consumption in this population. Previous surveys have shown that the biggest barrier to

overcome with women is the lack of knowledge about folic acid usage. Many women who have

heard of the vitamin do not know that they need to take it before pregnancy to reduce NTD risk.

Therefore, educational awareness about the importance of consuming supplemental folic acid

and/or folic acid-fortified foods every day is another objective that should be taken into

consideration. Targeted messages aimed at younger women, regardless of pregnancy plans,

should include campaigns focused on promoting consumption of fortified RTE cereals to help

increase the number of women of childbearing age who consume adequate folic acid daily.









APPENDIX A
SUBJECT DATA COLLECTION FORM

Introduction

I am calling in regard to your interest in our nutrition study; do you have a few minutes right
now?

This is a UF Nutrition department study and involves coming in one morning for about 1 hour
for a fasting blood sample, we take about 1 12 ounces of blood, and you only need to fast 8 hours.
We will give you a breakfast snack right afterward, and then give a brief explanation of a food
frequency questionnaire you will be taking home. You will be asked to mail it back in the
provided envelope, and once we receive the questionnaire you would get paid the $50. I just
have to ask you some questions to see if you are eligible for our study and to get background
information, OK?

How old are you? 18-49
Do you smoke? no
Are you pregnant or breastfeeding? no
Do you take any prescription medications other than oral contraceptives? no

If not ii hin the age range or if they answer yes to any question:
I am very sorry, but you do not meet our exclusion criteria, but thank you for your interest.

Now I just have a few questions about your diet to see what specific category of our study
you would fit in to. Please answer as best you can, estimates are ok and consider all
instances of when you might eat the items I will ask about, even if only occasionally.

Do you take a multi-vitamin, complex, red star nutritional yeast, or any other supplement
or additive ever?

If they take a multivitamin, B complex, red star nutritional yeast, complete the session through
all diet info but do not record. Conclude by confirming their name and saying "This has been a
preliminary screening call, your information will be reviewed by the principal investigator based
on need, and our selection criteria at this time. If you are chosen you will be called again to
schedule an appointment over the next two weeks. Thank you very much for your interest and
your time.

Do you eat breakfast cereals? (If so) What Kind do you eat mostly?

If they eat a 100% fortified cereal or eats a 50% cereal daily complete the through all diet info
but do not record. Conclude by confirming their name and saying "This has been a preliminary
screening call, your information will be reviewed by the principal investigator based on need,
and our selection criteria at this time. If you are chosen you will be called again to schedule an
appointment over the next two weeks. Thank you very much for your interest and your time.









If the interviewee fulfills all selection criteria continue with the questionnaire, record info on
moderate/non-fortified cereal consumption below.
Do you eat breakfast cereals?
o Yes
o No

Name/Brand Quantity Frequency



Are you a vegan, vegetarian or meat eater?
Vegan this means you eat NO animal derived foods intentionally (if they eat small amount
like in cake then OK)
Vegetarian this means you eat NO beef, chicken, turkey, pork, or fish
How often do you eat...
Never Rarely Occasionally Frequently Always
(<1 x/mo) 1-4 x/mo) (2-4 x/wk) (5-7 x/wk)
Beef
Chicken
Turkey
Pork
Fish
Eggs
Cheese
Cow's Milk
Yogurt
Other Dairy

Do you follow a restricted diet such as:
o No red meat o Low salt
o Lactose-free o Low fat
o Kosher o Low cholesterol
o Weight loss o Low carbohydrate
o Weight gain o Hypoallergenic

(If so) How long have you consumed this type of diet?


Have you made any major dietary changes within the last 3 years?
o No
o Yes; How long ago did you make changes and what changes did you make?


NO YES
Do you consume alcoholic beverages?
> How often/quantity









Health Information


I am going to ask you a few questions about your health to determine if you are eligible for
our study. I will be recording this information, but it will be kept confidential and is this
ok with you?

Height: Weight:

Have you do you currently have any of the following? NO YES
Alcoholism
Anemia
Blood clots
Bronchitis
Cystic Fibrosis
Dermatitis
Diabetes
Eating disorders/Chronic nausea or vomiting
Food allergy
Gall bladder disease
GI problems/ Lactose intolerance
Gout
Migraines
Hemorrhoids
Hepatitis/Liver disease
Heart disease/High cholesterol/High blood pressure
HIV
Kidney disease
Neurological disorder
Obesity
Seizures/Stroke
Thyroid problem
Tumors/Cancer
Ulcers
Other
Have you been hospitalized within the last 5 years?
> Cause

Do you have a history of more than 1 miscarriage?
o Yes
o No

If you are selected to participate in this study are you willing to sign an informed consent
understanding we have access to medical information on you?
o Yes
o No









Demographic Information


What is your birth date? /
Month Day


Year


How would you describe your race or ethnic background?
o White
o Black or African American
o American Indian or Alaska Native
o Hispanic or Latino
o Asian
o Native Hawaiian or Other Pacific Islander
o Other

What is the highest level of school or training that you have completed? [Circle only one
response]


Grade school
High school
Technical school or college
Graduate or professional
Don't know

Marital status?
o Single/never married
o Married
o Separated
o Divorced
o Widowed


Are you a full-time or part-time student?
o Full time
o Part time
o Not a student


05 06 07 08


Are you employed?
o Yes
o No
o Student employee









Contact Information


Name

M / F Last First Middle
Address

Street Apt. #


City Zip code
Phone


Day Evening Cell
E-mail





Name of person and phone number to call in case of an emergency if you are invited to
participate in this study:


If we need to contact you, and can not reach you where/with who can a message be left?


How did you hear about our study?









APPENDIX B
DIETARY HISTORY QUESTIONNAIRE

NATIONAL INSTITUTES OF HEALTH


Diet History Questionnaire


Today's date:

MONTH DAY YEAR
010 Jan I I| R 2002
020 Feb 0 E o0 2003
030 Mar 0 2004
040 Apr 1l E] 2005
050 May R2 W2 W 2006
060 Jun [3 03
070 Jul E04
080 Aug 05
090 Sep D6
100 Oct 07
110 Nov 08
120 Dec 09


In what month were
you born?

010 Jan
020 Feb
030 Mar
040 Apr
050 May
060 Jun
070 Jul
08] Aug
09] Sep
100 Oct
110 Nov
120 Dec


In what year were
you born?


191

E01
W2
W3
04
05
W6
07
W8
W9


01
WIl
W2
W3
04
05
W6
07
W8
W9


Are you male or
female?
OMale
OFemale


BAR CODE LABEL OR SUBJECT ID
HERE

L I


GENERAL INSTRUCTIONS


* Answer each question as best you can. Estimate if you are not sure. A
guess is better than leaving a blank.
* Use only a black ball-point pen. Do not use a pencil or felt-tip pen. Do not
fold, staple, or tear the pages.
* Put an X in the box next to your answer.
* If you make any changes, cross out the incorrect answer and put an X in
the box next to the correct answer. Also draw a circle around the correct
answer.
* If you mark NEVER, NO, or DON'T KNOW for a question, please follow
any arrows or instructions that direct you to the next question.

BEFORE TURNING THE PAGE, PLEASE COMPLETE THE FOLLOWING QUESTIONS.










1. Over the past 12 months, how often did you drink
tomato juice or vegetable juice?

--I NEVER (GO TO QUESTION 2)


1 time per month or less
2-3 times per month
1-2 times per week
3-4 times per week
5-6 times per week


1 time per day
2-3 times per day
4-5 times per day
6 or more times per day


la. Each time you drank tomato juice or
vegetable juice, how much did you usually
drink?

D Less than % cup (6 ounces)
S%3to 1% cups (6 to 10 ounces)
F More than 1 cups (10 ounces)

. Over the past 12 months, how often did you drink
orange juice?

- R NEVER (GO TO QUESTION 3)


1 time per month or less
2-3 times per month
1-2 times per week
3-4 times per week
5-6 times per week


1 time per day
2-3 times per day
4-5 times per day
6 or more times per day


2a. Each time you drank orange juice, how
much did you usually drink?

F Less than % cup (6 ounces)
% 3/to 1% cups (6 to 10 ounces)
D More than 1 cups (10 ounces)

3. Over the past 12 months, how often did you drink
other 100% fruit juice or 100% fruit juice
mixtures (such as apple, grape, pineapple,
grapefruit or others)?

-- NEVER (GO TO QUESTION 4)


1 time per month or less
2-3 times per month
1-2 times per week
3-4 times per week
5-6 times per week


1 time per day
2-3 times per day
4-5 times per day
6 or more times per day


3a. Each time you drank other fruit juice or fruit
juice mixtures, how much did you usually
drink?

D Less than % cup (6 ounces)
3 %to 1 cupss (6 to 12 ounces)
D More than 11/ cups (12 ounces)


Over the past 12 months...

4. How often did you drink other fruit drinks (such
as cranberry cocktail, Hi-C, lemonade, or Kool-
Aid, diet or regular)?

r-E NEVER (GO TO QUESTION 5)


1 time per month or less
2-3 times per month
1-2 times per week
3-4 times per week
5-6 times per week


E 1 time per day
F 2-3 times per day
D 4-5 times per day
F 6 or more times per day


4a. Each time you drank fruit drinks, how much
did you usually drink?

E Less than 1 cup (8 ounces)
D 1 to 2 cups (8 to 16 ounces)
D More than 2 cups (16 ounces)

4b. How often were your fruit drinks diet or
sugar-free drinks?


F Almost never or never
D About of the time
D About / of the time
D About 3/4 of the time
F Almost always or always


5. How often did you drink milk, including lactose-
free milk (but NOT milk substitutes) as a
beverage (NOT in coffee, NOT in cereal)?
(Please include chocolate milk, flavored milk like
Ovaltine or Quick, and hot chocolate.)

r-D NEVER (GO TO QUESTION 6)


1 time per month or less
2-3 times per month
1-2 times per week
3-4 times per week
5-6 times per week


1 time per day
2-3 times per day
4-5 times per day
6 or more times per day


5a. Each time you drank milk as a beverage,
how much did you usually drink?

E Less than 1 cup (8 ounces)
R 1 to 1I cups (8 to 12 ounces)
F More than 1/ cups (12 ounces)



Please continue on next page.





5b. What kind of milk did you usually drink?











F Whole milk (including lactose-free variety)
D 2% fat milk (including lactose-free variety)
F 1 % fat milk (including lactose-free variety)
D Skim, nonfat, or /2% fat milk (including lactose-free
variety)

6. How often did you drink a milk substitute such
as soy or rice milk as a beverage (NOT in
coffee, NOT in cereal)? (Please include milk
substitute used to make other beverages such as
chocolate "milk", Ovaltine, Quick and hot
chocolate.)

-- FD NEVER (GO TO QUESTION 7)


1 time per month or less
2-3 times per month
1-2 times per week
3-4 times per week
5-6 times per week


1 time per day
2-3 times per day
4-5 times per day
6 or more times
per day


6a. Each time you drank a milk substitute as a
beverage, how much did you usually drink?

D Less than 1 cup (8 ounces)
D 1 to 112 cups (8 to 12 ounces)
F More than 11 cups (12 ounces)

6b. What kind of milk substitute did you usually
drink?

D Soy milk (8th Continent)
D Soy milk (Whitewave Silk)
D Soy milk (VitaSoy)
D Soy milk (other brand)
F Rice milk


Over the past 12 months...

7. How often did you drink meal replacement,
energy, or high-protein beverages such as
Instant Breakfast, Ensure, Slimfast, AdvantEdge,
Boost or others?

-0D NEVER (GO TO QUESTION 8)


1 time per month or less
2-3 times per month
1-2 times per week
3-4 times per week
5-6 times per week


1 time per day
2-3 times per day
4-5 times per day
6 or more times per day


7a. Each time you drank meal replacement,
energy, or high protein beverages, how
much did you usually drink?

D Less than 1 cup (8 ounces)
D 1 to 12 cups (8 to 12 ounces)
F More than 1 / cups (12 ounces)

8. Over the past 12 months, did you drink soft
drinks, soda, or pop?

-- NO (GO TO QUESTION 9)

D YES


8a. How often did you drink soft drinks, soda,
or pop IN THE SUMMER?

D NEVER


1 time per month or less
2-3 times per month
1-2 times per week
3-4 times per week
5-6 times per week


1 time per day
2-3 times per day
4-5 times per day
6 or more times
per day


8b. How often did you drink soft drinks, soda,
or pop DURING THE REST OF THE YEAR?

D NEVER


1 time per month or less
2-3 times per month
1-2 times per week
3-4 times per week
5-6 times per week


1 time per day
2-3 times per day
4-5 times per day
6 or more times
per day


8c. Each time you drank soft drinks, soda, or
pop, how much did you usually drink?

O Less than 12 ounces or less than 1 can or bottle
D 12 to 16 ounces or 1 can or bottle
] More than 16 ounces or more than 1 can or bottle

8d. How often were these soft drinks, soda, or
pop diet or sugar-free?

D Almost never or never
D About 14 of the time
D About 1 of the time
D About 34 of the time
D Almost always or always










8e. How often were these soft drinks, soda, or
pop caffeine-free? (See list of caffeine and
caffeine-free sodas provided by the
researcher.)

D Almost never or never
D About of the time
D About / of the time
D About 34 of the time
F Almost always or always

9. Over the past 12 months, did you drink beer?

D NO(GO TO QUESTION 10)

D YES


9a. How often did you drink beer IN THE
SUMMER?

D NEVER


D 1 time per month or less
D 2-3 times per month
D 1-2 times perweek
D 3-4 times per week
D 5-6 times per week


1 time per day
2-3 times per day
4-5 times per day
6 or more times
per day


9b. How often did you drink beer DURING THE
REST OF THE YEAR?

O NEVER


D 1 time per month or less
D 2-3 times per month
D 1-2 times perweek
D 3-4 times per week
D 5-6 times per week


1 time per day
2-3 times per day
4-5 times per day
6 or more times
per day


9c. Each time you drank beer, how much did you
usually drink?

D Less than a 12-ounce can or bottle
D 1 to 3 12-ounce cans or bottles
D More than 3 12-ounce cans or bottles


Over the past 12 months...

10. How often did you drink wine or wine coolers?


- D NEVER (GO TO QUESTION 11)


1 time per month or less
2-3 times per month
1-2 times per week
3-4 times per week
5-6 times per week


D 1 time per day
E 2-3 times per day
E 4-5 times per day
D 6 or more times
per day


10a. Each time you drank wine or wine coolers,
how much did you usually drink?

D Less than 5 ounces or less than 1 glass
E 5 to 12 ounces or 1 to 2 glasses
D More than 12 ounces or more than 2 glasses

I. How often did you drink liquor or mixed drinks?

- D NEVER (GO TO QUESTION 12)


1 time per month or less
2-3 times per month
1-2 times per week
3-4 times per week
5-6 times per week


D 1 time per day
E 2-3 times per day
D 4-5 times per day
F 6 or more times
per day


11a. Each time you drank liquor or mixed drinks,
how much did you usually drink?

F Less than 1 shot of liquor
D 1 to 3 shots of liquor
E More than 3 shots of liquor

12. Over the past 12 months, did you eat oatmeal,
grits, or other cooked cereal?

NO (GO TO QUESTION 13)

D YES


12a. How often did you eat oatmeal, grits, or
other cooked cereal IN THE WINTER?

D NEVER


E 1-6 times per winter
D 7-11 times per winter
E 1 time per month
E 2-3 times per month
D 1 time per week


D 2 times per week
D 3-4 times per week
D 5-6 times per week
D 1 time per day
D 2 or more times
per day










12b. How often did you eat oatmeal, grits, or
other cooked cereal DURING THE REST
OF THE YEAR?

D NEVER


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


2 times per week
3-4 times per week
5-6 times per week
1 time per day
2 or more times
per day


12c. Each time you ate oatmeal, grits, or other
cooked cereal, how much did you usually
eat?

D Less than % cup
D/4 to 1 cups
D More than 1 cups

13. How often did you eat cold cereal?

- D NEVER (GO TO QUESTION 14)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


2 times per week
3-4 times per week
5-6 times per week
1 time per day
2 or more times per day


13a. Each time you ate cold cereal, how much
did you usually eat?

D Less than 1 cup
D 1 to 212 cups
D More than 21/ cups

13b. How often was the cold cereal you ate a
fortified cereal? (See list of fortified
cereals provided by the researcher.)

F Almost never or never
D About % of the time
F About / of the time
F About 34 of the time
D Almost always or always


13c. How often was the cold cereal you ate any
other type of cold cereal (such as Corn
Flakes, Rice Krispies, Frosted Flakes, Fruit
Loops, Cap'n Crunch, or others)?

D Almost never or never
F About of the time
F About / of the time
D About 3/4 of the time
F Almost always or always

13d. Was milk or a milk substitute added to your
cold cereal?

-- NO (GO TO QUESTION 14)

r YES

13e. What kind of milk or milk substitute was
usually added?

D Whole milk (including lactose-free variety)
E 2% fat milk (including lactose-free variety)
D 1% fat milk (including lactose-free variety)
E Skim, nonfat, or 1/2% fat milk (including
lactose-free variety)
D Soy milk (8th Continent)
D Soy milk (Whitewave Silk)
D Soy milk (VitaSoy)
D Soy milk (other brand)
D Rice milk
D Other

13f. Each time milk or a milk substitute was
added to your cold cereal, how much was
usually added?

D Less than 12 cup
D 1 to 1 cup
D More than 1 cup

14. How often did you eat applesauce?

F- D NEVER (GO TO QUESTION 15)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


2 times per week
3-4 times per week
5-6 times per week
1 time per day
2 or more times per day


Please continue on next page.










14a. Each time you ate applesauce, how much
did you usually eat?

F Less than 12 cup
D 1 to 1 cup
D More than 1 cup

15. How often did you eat apples?

- D NEVER (GO TO QUESTION 16)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


F times per week
E 3-4 times per week
D 5-6 times per week
E 1 time per day
E 2 or more times per day


15a. Each time you ate apples, how many did you
usually eat?

F Less than 1 apple
D 1 apple
+ More than 1 apple

16. How often did you eat pears (fresh, canned, or
frozen)?

F- D NEVER (GO TO QUESTION 17)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


F times per week
D 3-4 times per week
E 5-6 times per week
D 1 time per day
E 2 or more times per day


16a. Each time you ate pears, how many did you
usually eat?

F Less than 1 pear
0 1 pear
D More than 1 pear

17. How often did you eat bananas?

I- D NEVER (GO TO QUESTION 18)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


2 times per week
3-4 times per week
5-6 times per week
1 time per day
2 or more times per day


Over the past 12 months...

17a. Each time you ate bananas, how many did
you usually eat?

D Less than 1 banana
D 1 banana
D More than 1 banana

18. How often did you eat dried fruit, such as prunes
or raisins (not including dried apricots)?

D NEVER (GO TO QUESTION 19)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


E 2 times per week
D 3-4 times per week
F 5-6 times per week
E 1 time per day
E 2 or more times per day


18a. Each time you ate dried fruit, how much did
you usually eat (not including dried apricots)?

F Less than 2 tablespoons
D 2 to 5 tablespoons
F More than 5 tablespoons

19. Over the past 12 months, did you eat peaches,
nectarines, or plums?

NO (GO TO QUESTION 20)

D YES


19a. How often did you eat fresh peaches,
nectarines, or plums WHEN IN SEASON?
(See list for description of "in season".)

D NEVER


1-6 times per season
7-11 times per season
1 time per month
2-3 times per month
1 time per week


2 times per week
3-4 times per week
5-6 times per week
1 time per day
2 or more times
per day


19b. How often did you eat peaches, nectarines,
or plums (fresh, canned, or frozen) DURING
THE REST OF THE YEAR?

D NEVER


D 1-6 times per year
D 7-11 times per year
D 1 time per month
D 2-3 times per month
D 1 time per week


2 times per week
3-4 times per week
5-6 times per week
1 time per day
2 or more times
per day










19c. Each time you ate peaches, nectarines, or
plums, how much did you usually eat?

D Less than 1 fruit or less than 12 cup
D 1 to 2 fruits or 1/ to % cup
D More than 2 fruits or more than % cup

20. How often did you eat grapes?

F- D NEVER (GO TO QUESTION 21)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


2 times per week
3-4 times per week
5-6 times per week
1 time per day
2 or more times per day


20a. Each time you ate grapes, how much did
you usually eat?

D Less than 12 cup or less than 10 grapes
i 1 to 1 cup or 10 to 30 grapes
D More than 1 cup or more than 30 grapes

21. Over the past 12 months, did you eat
cantaloupe?

NO (GO TO QUESTION 22)

D YES


21a. How often did you eat fresh cantaloupe
WHEN IN SEASON? (See list for description
of "in season".)

D NEVER


1-6 times per season
7-11 times per season
1 time per month
2-3 times per month
1 time per week


2 times per week
3-4 times per week
5-6 times per week
1 time per day
2 or more times
per day


21b. How often did you eat fresh or frozen
cantaloupe DURING THE REST OF
THE YEAR?

F NEVER


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


2 times per week
3-4 times per week
5-6 times per week
1 time per day
2 or more times
per day


Over the past 12 months...

21c. Each time you ate cantaloupe, how much
did you usually eat?

F Less than % melon or less than 12 cup
D melon or to 1 cup
F More than % melon or more than 1 cup

22. Over the past 12 months, did you eat melon,
other than cantaloupe (such as watermelon or
honeydew)?

D NO (GO TO QUESTION 23

D YES


22a. How often did you eat fresh melon, other
than cantaloupe (such as watermelon or
honeydew) WHEN IN SEASON? (See list for
description of "in season".)

D NEVER


1-6 times per season
7-11 times per season
1 time per month
2-3 times per month
1 time per week


2 times per week
3-4 times per week
5-6 times per week
1 time per day
2 or more times
per day


22b. How often did you eat fresh or frozen
melon, other than cantaloupe (such as
watermelon or honeydew) DURING THE
REST OF THE YEAR?

D NEVER


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


2 times per week
3-4 times per week
5-6 times per week
1 time per day
2 or more times
per day


22c. Each time you ate melon other than
cantaloupe, how much did you usually eat?

D Less than 12 cup or 1 small wedge
D 12 to 2 cups or 1 medium wedge
E More than 2 cups or 1 large wedge










23. Over the past 12 months, did you eat
strawberries?

-- NO (GO TO QUESTION 24)

D YES

23a. How often did you eat fresh strawberries
WHEN IN SEASON? (See list for description of
"in season".)

D NEVER


1-6 times per season
7-11 times per season
1 time per month
2-3 times per month
1 time per week


2 times per week
3-4 times per week
5-6 times per week
1 time per day
2 or more times
per day


23b. How often did you eat fresh or frozen
strawberries DURING THE REST OF THE
YEAR?

D NEVER


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


2 times per week
3-4 times per week
5-6 times per week
1 time per day
2 or more times
per day


23c. Each time you ate strawberries, how much
did you usually eat?

F Less than % cup or less than 3 berries
D 4 to cup or 3 to 8 berries
F More than % cup or more than 8 berries

24. Over the past 12 months, did you eat oranges,
tangerines, or tangelos?

-D NO (GO TO QUESTION 25)

T YES


24a. How often did you eat fresh oranges, tangerines,
or tangelos WHEN IN SEASON? (See list for
description of "in season".)

D NEVER


F 1-6 times per season
D 7-11 times per season
F 1 time per month
D 2-3 times per month
F 1 time per week


2 times per week
3-4 times per week
5-6 times per week
1 time per day
2 or more times
per day


Over the past 12 months...

24b. How often did you eat oranges, tangerines,
or tangelos (fresh or canned) DURING THE
REST OF THE YEAR?

D NEVER


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


2 times per week
3-4 times per week
5-6 times per week
1 time per day
2 or more times
per day


24c. Each time you ate oranges, tangerines, or
tangelos, how many did you usually eat?

D Less than 1 fruit
D 1 fruit
D More than 1 fruit

25. Over the past 12 months, did you eat grapefruit?

D NO (GO TO QUESTION 26)

D YES


25a. How often did you eat fresh grapefruit
WHEN IN SEASON? (See list for description
of "in season".)

D NEVER


1-6 times per season
7-11 times per season
1 time per month
2-3 times per month
1 time per week


2 times per week
3-4 times per week
5-6 times per week
1 time per day
2 or more times
per day


25b. How often did you eat grapefruit (fresh or
canned) DURING THE REST OF THE YEAR?

D NEVER


D 1-6 times per year
D 7-11 times per year
D 1 time per month
D 2-3 times per month
D 1 time per week


25c. Each time you ate grapefruit,
you usually eat?

E Less than 12 grapefruit
D 12 grapefruit
F More than 12 grapefruit


2 times per week
3-4 times per week
5-6 times per week
1 time per day
2 or more times
per day

how much did










26. How often did you eat other kinds of fruit such
as pineapple, mangoes, blueberries, or others?

I- NEVER (GO TO QUESTION 27)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


D 2 times per week
D 3-4 times per week
E 5-6 times per week
D 1 time per day
E 2 or more times per day


26a. Each time you ate other kinds of fruit, how
much did you usually eat?

D Less than % cup
D /4 to cup
D More than % cup

!7. How often did you eat COOKED greens (such as
spinach, turnip, collard, mustard, chard, or kale)?

- D NEVER (GO TO QUESTION 28)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


F 2 times per week
D 3-4 times per week
D 5-6 times per week
E 1 time per day
E 2 or more times per day


27a. Each time you ate COOKED greens, how
much did you usually eat?

O Less than 12 cup
D 1 to 1 cup
F More than 1 cup

28. How often did you eat RAW greens (such as
spinach, turnip, collard, mustard, chard, or kale)?
(We will ask about lettuce later.)

r- NEVER (GO TO QUESTION 29)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


D 2 times per week
] 3-4 times per week
D 5-6 times per week
D 1 time per day
D 2 or more times per day


28a. Each time you ate RAW greens, how much
did you usually eat?

F Less than 12 cup
D 1 to 1 cup
F More than 1 cup


Over the past 12 months...

29. How often did you eat coleslaw?

-- D NEVER (GO TO QUESTION 30)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


D 2 times per week
] 3-4 times per week
E 5-6 times per week
D 1 time per day
D 2 or more times per day


29a. Each time you ate coleslaw, how much did
you usually eat?

E Less than % cup
SY4 to cup
F More than % cup

0. How often did you eat sauerkraut or cabbage
(other than coleslaw)?

- D NEVER (GO TO QUESTION 31)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


D 2 times per week
E 3-4 times per week
D 5-6 times per week
F 1 time per day
D 2 or more times per day


30a. Each time you ate sauerkraut or cabbage,
how much did you usually eat?

E Less than % cup
D 0to 1 cup
D More than 1 cup

31. How often did you eat carrots (fresh, canned, or
frozen)?

-- D NEVER (GO TO QUESTION 32)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


E 2 times per week
D 3-4 times per week
E 5-6 times per week
D 1 time per day
D 2 or more times per day


31a. Each time you ate carrots, how much did
you usually eat?

D Less than % cup or less than 2 baby carrots
SY to 12 cup or 2 to 5 baby carrots
D More than 12 cup or more than 5 baby carrots










32. How often did you eat string beans or green
beans (fresh, canned, or frozen)?

-- D NEVER (GO TO QUESTION 33)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


D 2 times per week
F 3-4 times per week
D 5-6 times per week
F 1 time per day
F 2 or more times per day


32a. Each time you ate string beans or green
beans, how much did you usually eat?

D Less than 12 cup
D 1 to 1 cup
D More than 1 cup
F
23. How often did you eat peas (fresh, canned, or
frozen)?

- D NEVER (GO TO QUESTION 34)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


33a. Each time you ate peas,
usually eat?


2 times per week
3-4 times per week
5-6 times per week
1 time per day
2 or more times per day

how much did you


F Less than % cup
D]4 to cup
F More than % cup

W4. Over the past 12 months, did you eat corn?

- D NO (GO TO QUESTION 35)

D YES


34a. How often did you eat fresh corn WHEN IN
SEASON? (See list for description of "in
season".)

D NEVER


1-6 times per season
7-11 times per season
1 time per month
2-3 times per month
1 time per week


2 times per week
3-4 times per week
5-6 times per week
1 time per day
2 or more times
per day


Over the past 12 months...

34b. How often did you eat corn (fresh, canned, or
frozen) DURING THE REST OF THE YEAR?

D NEVER


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


34c. Each time you ate corn,
usually eat?


2 times per week
3-4 times per week
5-6 times per week
1 time per day
2 or more times
per day


how much did you


D Less than 1 ear or less than 12 cup
F 1 ear or 1 to 1 cup
D More than 1 ear or more than 1 cup


35. Over the past 12 months, how often did you eat
broccoli (fresh or frozen)?

-- D NEVER (GO TO QUESTION 36)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


D 2 times per week
F 3-4 times per week
D 5-6 times per week
D 1 time per day
D 2 or more times per day


35a. Each time you ate broccoli, how much did
you usually eat?

D Less than % cup
D 4to 1 cup
D More than 1 cup

.6. How often did you eat cauliflower or Brussels
sprouts (fresh or frozen)?

- D NEVER (GO TO QUESTION 37)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


F 2 times per week
D 3-4 times per week
F 5-6 times per week
D 1 time per day
E 2 or more times per day


36a. Each time you ate cauliflower or Brussels
sprouts, how much did you usually eat?

F Less than % cup
D4 to 1 cup
D More than 12 cup














2 times per week
3-4 times per week
5-6 times per week
1 time per day
2 or more times per day


37a. Each time you ate mixed vegetables, how
much did you usually eat?

F Less than 12 cup
D 1 to 1 cup
F More than 1 cup

38. How often did you eat onions?

- D NEVER (GO TO QUESTION 39)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


2 times per week
3-4 times per week
5-6 times per week
1 time per day
2 or more times per day


38a. Each time you ate onions, how much did
you usually eat?

D Less than 1 slice or less than 1 tablespoon
F 1 slice or 1 to 4 tablespoons
D More than 1 slice or more than 4 tablespoons

19. Now think about all the cooked vegetables you
ate in the past 12 months and how they were
prepared. How often were your vegetables
COOKED WITH some sort of fat, including oil
spray? (Please do not include potatoes.)

- D NEVER (GO TO QUESTION 40)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


2 times per week
3-4 times per week
5-6 times per week
1 time per day
2 or more times per day


39a. Which fats were usually added to your
vegetables DURING COOKING? (Please do
not include potatoes. Mark all that apply.)


Margarine
(including low-fat)
Butter (including
low-fat)
Lard, fatback, or
bacon fat
Olive oil
Corn oil


F Canola or rapeseed oil

E Oil spray, such as Pam
or others
D Other kinds of oils

D None of the above


40. Now, thinking again about all the cooked
vegetables you ate in the past 12 months, how
often was some sort of fat, sauce, or dressing
added AFTER COOKING OR AT THE TABLE?
(Please do not include potatoes.)

- D NEVER (GO TO QUESTION 41)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1-2 times per week


3-4 times per week
5-6 times per week
1 time per day
2 times per day
3 or more times per day


40a. Which fats, sauces, or dressings were
usually added AFTER COOKING OR AT
THE TABLE? (Please do not include
potatoes. Mark all that apply.)


E Margarine including
low-fat)
E Butter (including
low-fat)
O Lard, fatback, or
bacon fat
D Salad dressing


D Cheese sauce

D White sauce

D Other


40b. If margarine, butter, lard, fatback, or bacon
fat was added to your cooked vegetables
AFTER COOKING OR AT THE TABLE, how
much did you usually add?

F Did not usually add these
D Less than 1 teaspoon
D 1 to 3 teaspoons
F More than 3 teaspoons

40c. If salad dressing, cheese sauce, or white
sauce was added to your cooked vegetables
AFTER COOKING OR AT THE TABLE, how
much did you usually add?

D Did not usually add these
D Less than 1 tablespoon
F 1 to 3 tablespoons
D More than 3 tablespoons


- D NEVER (GO TO QUESTION 38)


37. How often did you eat mixed vegetables?


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


Over the past 12 months...










41. Over the past 12 months, how often did you eat
sweet peppers (green, red, or yellow)?

- D NEVER (GO TO QUESTION 42)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


D 2 times per week
D 3-4 times per week
E 5-6 times per week
D 1 time per day
D 2 or more times per day


41a. Each time you ate sweet peppers, how
much did you usually eat?

E Less than 1/8 pepper
D 1/8 to % pepper
D More than % pepper

42. Over the past 12 months, did you eat fresh
tomatoes (including those in salads)?

D NO (GO TO QUESTION 43)

D YES


42a. How often did you eat fresh tomatoes
(including those in salads) WHEN IN
SEASON? (See description of "in season".)

D NEVER


1-6 times per season
7-11 times per season
1 time per month
2-3 times per month
1 time per week


2 times per week
3-4 times per week
5-6 times per week
1 time per day
2 or more times
per day


42b. How often did you eat fresh tomatoes
(including those in salads) DURING THE
REST OF THE YEAR?

D NEVER


D 1-6 times per year
D 7-11 times per year
D 1 time per month
D 2-3 times per month
D 1 time per week


2 times per week
3-4 times per week
5-6 times per week
1 time per day
2 or more times
per day


42c. Each time you ate fresh tomatoes, how
much did you usually eat?

F Less than % tomato
SY4 to 2 tomato
D More than 12 tomato


Over the past 12 months...

43. How often did you eat lettuce salads (with or
without other vegetables)?

E- NEVER (GO TO QUESTION 44)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


D 2 times per week
F 3-4 times per week
D 5-6 times per week
D 1 time per day
D 2 or more times per day


43a. Each time you ate lettuce salads, how much
did you usually eat?

E Less than % cup
D to 11/4 cups
E More than 11/4 cups

44. How often did you eat salad dressing (including
low-fat) on salads?

- D NEVER (GO TO QUESTION 45)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


E 2 times per week
D 3-4 times per week
] 5-6 times per week
E 1 time per day
E 2 or more times per day


44a. Each time you ate salad dressing on salads,
how much did you usually eat?

D Less than 2 tablespoons
F 2 to 4 tablespoons
D More than 4 tablespoons

.5. How often did you eat sweet potatoes or yams?

- D NEVER (GO TO QUESTION 46)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


F 2 times per week
D 3-4 times per week
] 5-6 times per week
D 1 time per day
E 2 or more times per day


45a. Each time you ate sweet potatoes or yams,
how much did you usually eat?

D 1 small potato or less than % cup
l 1 medium potato or % to % cup
E 1 large potato or more than % cup










46. How often did you eat French fries, home fries,
hash browned potatoes, or tater tots?


- D NEVER (GO TO QUESTION 47)


D 1-6 times per year D 2 times per week
D 7-11 times per year D 3-4 times per week
F 1 time per month E 5-6 times per week
D 2-3 times per month D 1 time per day
D 1 time per week D 2 or more times per day

46a. Each time you ate French fries, home fries,
hash browned potatoes, or tater tots how
much did you usually eat?

F Less than 10 fries or less than 1/ cup
D 10 to 25 fries or 1 to 1 cup
F More than 25 fries or more than 1 cup

17. How often did you eat potato salad?

- D NEVER (GO TO QUESTION 48)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


D 2 times per week
] 3-4 times per week
D 5-6 times per week
D 1 time per day
D 2 or more times per day


47a. Each time you ate potato salad, how much
did you usually eat?

F Less than 12 cup
D 1 to 1 cup
F More than 1 cup
F
18. How often did you eat baked, boiled, or mashed
potatoes?

- D NEVER (GO TO QUESTION 49)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


D 2 times per week
F 3-4 times per week
D 5-6 times per week
E 1 time per day
D 2 or more times per day


48a. Each time you ate baked, boiled, or
mashed potatoes, how much did you
usually eat?

F 1 small potato or less than 12 cup
D 1 medium potato or 12 to 1 cup
F 1 large potato or more than 1 cup


Over the past 12 months...

48b. How often was sour cream (including low-
fat) added to your potatoes, EITHER IN
COOKING OR AT THE TABLE?

-- Almost never or never (GO TO QUESTION 48d)
D About of the time
E About / of the time
D About 3/4 of the time
F Almost always or always

48c. Each time sour cream was added to your
potatoes, how much was usually added?

D Less than 1 tablespoon
E 1 to 3 tablespoons
D More than 3 tablespoons

48d. How often was margarine (including low-fat)
added to your potatoes, EITHER IN
COOKING OR AT THE TABLE?

F Almost never or never
D About of the time
D About / of the time
D About 34 of the time
F Almost always or always

48e. How often was butter (including low-fat)
added to your potatoes, EITHER IN
COOKING OR AT THE TABLE?

F Almost never or never
D About of the time
F About /of the time
F About 3/4 of the time
D Almost always or always

48f. Each time margarine or butter was added to
your potatoes, how much was usually
added?

D Never added
D Less than 1 teaspoon
F 1 to 3 teaspoons
D More than 3 teaspoons

48g. How often was cheese or cheese sauce
added to your potatoes, EITHER IN
COOKING OR AT THE TABLE?

-- Almost never or never (GO TO QUESTION 49)
D About % of the time
D About 2 of the time
D About 34 of the time
F Almost always or always










48h. Each time cheese or cheese sauce was
added to your potatoes, how much was
usually added?

D Less than 1 tablespoon
D 1 to 3 tablespoons
D More than 3 tablespoons

49. How often did you eat salsa?

-- D NEVER (GO TO QUESTION 50)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


E 2 times per week
D 3-4 times per week
F 5-6 times per week
D 1 time per day
D 2 or more times per day


49a. Each time you ate salsa, how much did you
usually eat?

F Less than 1 tablespoon
D 1 to 5 tablespoons
+ More than 5 tablespoons

50. How often did you eat catsup?

I- D NEVER (GO TO QUESTION 51)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


D 2 times per week
D 3-4 times per week
F 5-6 times per week
D 1 time per day
D 2 or more times per day


50a. Each time you ate catsup, how much did you
usually eat?

D Less than 1 teaspoon
F 1 to 6 teaspoons
+V More than 6 teaspoons

51. How often did you eat stuffing, dressing, or
dumplings?

|- D NEVER (GO TO QUESTION 52)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


F 2 times per week
D 3-4 times per week
] 5-6 times per week
D 1 time per day
E 2 or more times per day


51a. Each time you ate stuffing, dressing, or
dumplings, how much did you usually eat?

D Less than 12 cup
D 1 to 1 cup
D More than 1 cup


Over the past 12 months...

52. How often did you eat chili made with beef?
(DO NOT include chili made with soy or
vegetable protein substitute. We will ask about
this later.)

-- D NEVER (GO TO QUESTION 52b)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


D 2 times per week
E 3-4 times per week
D 5-6 times per week
E 1 time per day
D 2 or more times per day


52a. Each time you ate chili made with beef,
how much did you usually eat?

E Less than 12 cup
D 1 to 13/4 cups
] More than 13/4 cups

52b. How often did you eat chili made with meat
other than beef? (DO NOT include chili
made with soy or vegetable protein
substitutes. We will ask about these later.)

r- NEVER (GO TO QUESTION 52d)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


E 2 times per week
D 3-4 times per week
E 5-6 times per week
D 1 time per day
F 2 or more times per day


52c. Each time you ate chili made with meat
other than beef, how much did you usually
eat?

E Less than 12 cup
D 1 to 13/4 cups
D More than 13/4 cups

52d. How often did you eat chili without meat?
(DO NOT include chili made with a soy or
vegetable protein meat substitute. We will
ask about meat substitutes later.)

- D NEVER (GO TO QUESTION 53)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


F 2 times per week
F 3-4 times per week
D 5-6 times per week
E 1 time per day
F 2 or more times per day


126 *











52e. Each time you ate chili made without meat,
how much did you usually eat?

D Less than 12 cup
D 2 to 13/4 cups
D More than 13/4 cups

53. How often did you eat Mexican foods (such as
tacos, tostados, burritos, tamales, fajitas,
enchiladas, quesadillas, and chimichangas)
made with beef? (DO NOT include Mexican
foods made with soy or vegetable protein
substitutes. We will ask about these later.)

r- NEVER (GO TO QUESTION 53b)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


2 times per week
3-4 times per week
5-6 times per week
1 time per day
2 or more times per day


53a. Each time you ate Mexican foods made
with beef, how much did you usually eat?

F Less than 1 taco, burrito, etc.
D 1 to 2 tacos, burritos, etc.
F More than 2 tacos, burritos, etc.

53b. How often did you eat Mexican foods (such
as tacos, tostados, burritos, tamales, fajitas,
enchiladas, quesadillas, and chimichangas)
made with meat other than beef? (DO NOT
include Mexican foods made with soy or
vegetable protein substitutes. We will ask
about these later.)

- D NEVER (GO TO QUESTION 53d)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


2 times per week
3-4 times per week
5-6 times per week
1 time per day
2 or more times per day


53c. Each time you ate Mexican foods made
with meat other than beef how much did
you usually eat?

D Less than 12 cup
D/2 to 13/4 cups
D More than 13/4 cups


53d. How often did you eat Mexican foods (such
as tacos, tostados, burritos, tamales, fajitas,
enchiladas, quesadillas, and chimichangas)
made without meat? (DO NOTinclude
Mexican foods made with a soy or vegetable
protein meat substitute.)

-- NEVER (GO TO QUESTION 54)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


2 times per week
3-4 times per week
5-6 times per week
1 time per day
2 or more times per day


53e. Each time you ate Mexican foods made
without meat, how much did you usually
eat?

D Less than 12 cup
D 2 to 13/4 cups
F More than 13/4 cups


54. How often did you eat cooked dried beans
(such as baked beans, pintos, kidney, blackeyed
peas, lima, lentils, soybeans, refried beans, or
chick peas/garbanzo beans, including hummus)?
(Please don't include bean soups or chili.)

r- NEVER (GO TO QUESTION 55)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


2 times per week
3-4 times per week
5-6 times per week
1 time per day
2 or more times per day


54a. Each time you ate beans, how much did you
usually eat?

D Less than 12 cup
D 2 to 1 cup
D More than 1 cup


54b. How often were the beans you ate refried
beans, beans prepared with any type of
fat, or with meat added?

] Almost never or never
E About % of the time
F About /of the time
D About 3/4 of the time
D Almost always or always










55. How often did you eat other kinds of
vegetables such as asparagus, mushrooms,
zucchini or others ?

-- D NEVER (GO TO QUESTION 56)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


2 times per week
3-4 times per week
5-6 times per week
1 time per day
2 or more times per day


55a. Each time you ate other kinds of
vegetables, how much did you usually eat?

F Less than % cup
D]4 to 1 cup
F More than 12 cup

3. How often did you eat rice or other cooked
grains (such as bulgur, cracked wheat, or
millet)?

- D NEVER (GO TO QUESTION 57)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


2 times per week
3-4 times per week
5-6 times per week
1 time per day
2 or more times per day


56a. Each time you ate rice or other cooked
grains, how much did you usually eat?

F Less than 12 cup
D 1 to 11/2 cups
O More than 11/2 cups

56b. How often was butter, margarine, or oil
added to your rice IN COOKING OR AT THE
TABLE?

F Almost never or never
D About % of the time
D About / of the time
D About 3/4 of the time
F Almost always or always


Over the past 12 months...

57. How often did you eat pancakes, waffles, or
French toast?

D NEVER (GO TO QUESTION 58)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


2 times per week
3-4 times per week
5-6 times per week
1 time per day
2 or more times per day


57a. Each time you ate pancakes, waffles, or
French toast, how much did you usually
eat?

O Less than 1 medium piece
D 1 to 3 medium pieces
E More than 3 medium pieces

57b. How often was margarine (including low-fat)
added to your pancakes, waffles, or French
toast AFTER COOKING OR AT THE
TABLE?

O Almost never or never
D About % of the time
D About / of the time
D About 3/4 of the time
E Almost always or always

57c. How often was butter (including low-fat)
added to your pancakes, waffles, or French
toast AFTER COOKING OR AT THE
TABLE?

D Almost never or never
E About % of the time
E About / of the time
D About /4 of the time
E Almost always or always

57d. Each time margarine or butter was added to
your pancakes, waffles, or French toast, how
much was usually added?

E Never added
D Less than 1 teaspoon
E 1 to 3 teaspoons
D More than 3 teaspoons

57e. How often was syrup added to your
pancakes, waffles, or French toast?

-- D Almost never or never (GO TO QUESTION 58)
D About % of the time
D About /of the time
D About 3/4 of the time
F Almost always or always

57f. Each time syrup was added to your
pancakes, waffles, or French toast, how
much was usually added?

E Less than 1 tablespoon
D 1 to 4 tablespoons
E More than 4 tablespoons










58. How often did you eat lasagna, stuffed shells,
stuffed manicotti, ravioli, or tortellini made
with beef? (DO NOT include spaghetti or other
pasta or products made with soy or vegetable
protein substitutes. We will ask about these
later.)

D NEVER (GO TO QUESTION 58b)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


2 times per week
3-4 times per week
5-6 times per week
1 time per day
2 or more times per day


58a. Each time you ate lasagna, stuffed shells,
stuffed manicotti, ravioli, or tortellini made
with beef, how much did you usually eat?

F Less than 1 cup
D 1 to 2 cups
D More than 2 cups

58b. How often did you eat lasagna, stuffed
shells, stuffed manicotti, ravioli, or
tortellini made with meat other than beef?
(DO NOT include spaghetti or other pasta or
products made with soy or vegetable protein
substitutes. We will ask about these later.)

- D NEVER (GO TO QUESTION 58d)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


2 times per week
3-4 times per week
5-6 times per week
1 time per day
2 or more times per day


58c. Each time you ate lasagna, stuffed shells,
stuffed manicotti, ravioli, or tortellini made
with meat other than beef, how much did
you usually eat?

F Less than 1 cup
D 1 to 2 cups
D More than 2 cups


58d. How often did you eat lasagna, stuffed
shells, stuffed manicotti, ravioli, or
tortellini made without meat? (DO NOT
include spaghetti or other pasta or products
made with a soy or vegetable protein
substitutes. We will ask about these later.)

- D NEVER (GO TO QUESTION 59)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


2 times per week
3-4 times per week
5-6 times per week
1 time per day
2 or more times per day


58e. Each time you ate lasagna, stuffed shells,
stuffed manicotti, ravioli, or tortellini made
without meat, how much did you usually
eat?

F Less than 1 cup
D 1 to 2 cups
F More than 2 cups

59. How often did you eat macaroni and cheese?

F- NEVER (GO TO QUESTION 60)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


2 times per week
3-4 times per week
5-6 times per week
1 time per day
2 or more times per day


59a. Each time you ate macaroni and cheese,
how much did you usually eat?

F Less than 1 cup
O 1 to 11/2 cups
] More than 11/2 cups

60. How often did you eat pasta salad or macaroni
salad?

-- NEVER (GO TO QUESTION 61)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


2 times per week
3-4 times per week
5-6 times per week
1 time per day
2 or more times per day


Over the past 12 months...

60a. Each time you ate pasta salad or macaroni
salad, how much did you usually eat?

F Less than 12 cup
D 1 to 1 cup
F More than 1 cup










61. Other than the pastas listed in Questions 58, 59,
and 60, how often did you eat pasta, spaghetti,
or other noodles?

S-D NEVER (GO TO QUESTION 62)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


D 2 times per week
] 3-4 times per week
F 5-6 times per week
D 1 time per day
D 2 or more times per day


61a. Each time you ate pasta, spaghetti, or
other noodles, how much did you usually
eat?

D Less than 1 cup
F 1 to 3 cups
D More than 3 cups


61b. How often did you eat your pasta, spaghetti, or
other noodles with tomato sauce or spaghetti
sauce made with beef? (DO NOT include
tomato or spaghetti sauce made with soy or
vegetable protein substitutes. We will ask about
these later.)

D Almost never or never
D About /4of the time
D About / of the time
D About 34 of the time
D Almost always or always

61c. How often did you eat your pasta, spaghetti, or
other noodles with tomato sauce or spaghetti
sauce made with meat other than beef? (DO
NOT include tomato or spaghetti sauce made
with soy or vegetable protein substitutes. We
will ask about these later.)

D Almost never or never
D About /4of the time
D About /of the time
D About 3/4 of the time
F Almost always or always

61d. How often did you eat your pasta, spaghetti,
or other noodles with tomato sauce or
spaghetti sauce made WITHOUT meat?
(DO NOT include tomato sauce or spaghetti
sauce made with a soy or vegetable protein
meat substitute. We will ask about these
later.)

D Almost never or never
D About /4 of the time
D About 1 of the time
D About 3/4 of the time
F Almost always or always


61e. How often did you eat your pasta, spaghetti,
or other noodles with margarine, butter, oil,
or cream sauce?
R Almost never or never
D About /4 of the time
D About /of the time
D About 3/4 of the time
F Almost always or always

62. How often did you eat bagels or English
muffins?

r--D NEVER (GO TO INTRODUCTION TO QUESTION 63)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


D 2 times per week
F 3-4 times per week
D 5-6 times per week
E 1 time per day
D 2 or more times per day


62a. Each time you ate bagels or English
muffins, how many did you usually eat?

F Less than 1 bagel or English muffin
F 1 bagel or English muffin
D More than 1 bagel or English muffin

62b. How often was margarine (including low-fat)
added to your bagels or English muffins?

E Almost never or never
D About /4 of the time
D About 1 of the time
D About 3/4 of the time
D Almost always or always

62c. How often was butter (including low-fat)
added to your bagels or English muffins?

] Almost never or never
D About 14 of the time
D About 12 of the time
D About 3/4 of the time
E Almost always or always


62d. Each time margarine or butter was added to
your bagels or English muffins, how much
was usually added?

F Never added
D Less than 1 teaspoon
E 1 to 2 teaspoons
D More than 2 teaspoons










62e. How often was cream cheese (including low-
fat) spread on your bagels or English
muffins?

-- Almost never or never (GO TO INTRODUCTION
TO QUESTION 63)
D About % of the time
D About / of the time
D About 3/4 of the time
D Almost always or always


Over the past 12 months...

62f. Each time cream cheese was added to your
bagels or English muffins, how much was
usually added?

F Less than 1 tablespoon
D 1 to 2 tablespoons
D More than 2 tablespoons


The next questions ask about your intake of
breads other than bagels or English muffins. First,
we will ask about bread you ate as part of
sandwiches only. Then we will ask about all other
bread you ate.

63. How often did you eat breads or rolls AS PART
OF SANDWICHES (including burger and hot dog
rolls)?

R- NEVER (GO TO QUESTION 64)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


2 times per week
3-4 times per week
5-6 times per week
1 time per day
2 or more times per day


63a. Each time you ate breads or rolls AS PART
OF SANDWICHES, how many did you
usually eat?

D 1 slice or 1/ roll
F 2 slices or 1 roll
D More than 2 slices or more than 1 roll

63b. How often were the breads or rolls that you
used for your sandwiches white bread
(including burger and hot dog rolls)?

D Almost never or never
D About of the time
D About / of the time
D About 3/4 of the time
D Almost always or always


63c. How often was mayonnaise or
mayonnaise-type dressing (including low-
fat) added to your sandwich bread or rolls?

Almost never or never (GO TO QUESTION 63e)
E About % of the time
D About / of the time
D About 3/4 of the time
E Almost always or always

63d. Each time mayonnaise or mayonnaise-type
dressing was added to your sandwich
breads or rolls, how much was usually
added?

D Less than 1 teaspoon
E 1 to 3 teaspoons
E More than 3 teaspoons

63e. How often was margarine (including low-fat)
added to your sandwich bread or rolls?

D Almost never or never
E About % of the time
D About / of the time
D About 3/4 of the time
E Almost always or always

63f. How often was butter (including low-fat)
added to your sandwich bread or rolls?

D Almost never or never
D About % of the time
D About /of the time
D About 3/4 of the time
D Almost always or always

63g. Each time margarine or butter was added to
your sandwich breads or rolls, how much
was usually added?

E Never added
D Less than 1 teaspoon
F 1 to 2 teaspoons
D More than 2 teaspoons


64. How often did you eat breads or dinner rolls,
NOT AS PART OF SANDWICHES?

--- NEVER (GO TO QUESTION 65)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


2 times per week
3-4 times per week
5-6 times per week
1 time per day
2 or more times per day










64a. Each time you ate breads or dinner rolls,
NOT AS PART OF SANDWICHES, how
much did you usually eat?

D 1 slice or 1 dinner roll
D 2 slices or 2 dinner rolls
F More than 2 slices or 2 dinner rolls



Over the past 12 months...


64b. How often were the breads
white bread?


or rolls you ate


F Almost never or never
F About of the time
F About / of the time
D About 3/4 of the time
F Almost always or always

64c. How often was margarine (including low-fat)
added to your breads or rolls?

D Almost never or never
D About of the time
D About / of the time
D About 3/4 of the time
D Almost always or always

64d. How often was butter (including low-fat)
added to your breads or rolls?

D Almost never or never
D About of the time
D About / of the time
D About 34 of the time
D Almost always or always

64e. Each time margarine or butter was added to
your breads or rolls, how much was usually
added?

F Never added
D Less than 1 teaspoon
F 1 to 2 teaspoons
D More than 2 teaspoons

64f. How often was cream cheese (including low-
fat) added to your breads or rolls?

-- Almost never or never (GO TO QUESTION 65)
F About % of the time
D About 2 of the time
D About 34 of the time
D Almost always or always


64g. Each time cream cheese was added to your
breads or rolls, how much was usually
added?

D Less than 1 tablespoon
D 1 to 2 tablespoons
E More than 2 tablespoons


65. How often did you eat jam, jelly, or honey on
bagels, muffins, bread, rolls, or crackers?

D NEVER (GO TO QUESTION 66)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


2 times per week
3-4 times per week
5-6 times per week
1 time per day
2 or more times per day


65a. Each time you ate jam, jelly, or honey, how
much did you usually eat?

E Less than 1 teaspoon
D 1 to 3 teaspoons
E More than 3 teaspoons

66. How often did you eat peanut butter or other
nut butter?

r- D NEVER (GO TO QUESTION 67)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


2 times per week
3-4 times per week
5-6 times per week
1 time per day
2 or more times per day


66a. Each time you ate peanut butter or other
nut butter, how much did you usually eat?

j Less than 1 tablespoon
E 1 to 2 tablespoons
E More than 2 tablespoons

67. How often did you eat roast beef or steak IN
SANDWICHES, SUBS or WRAPS?

1-- NEVER (GO TO QUESTION 68)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


2 times per week
3-4 times per week
5-6 times per week
1 time per day
2 or more times per day










67a. Each time you ate roast beef or steak IN
SANDWICHES, SUBS or WRAPS, how
much did you usually eat?

D Less than 1 slice or less than 2 ounces
D 1 to 2 slices or 2 to 4 ounces
D More than 2 slices or more than 4 ounces


Over the past 12 months...

68. How often did you eat turkey or chicken COLD
CUTS (such as loaf, luncheon meat, turkey ham,
turkey salami, or turkey pastrami), including
those used in sandwiches, subs or wraps? (DO
NOT include turkey or chicken cold cuts made
with soy or vegetable protein substitutes. We will
ask about these later.)

- D NEVER (GO TO QUESTION 69)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


E 2 times per week
D 3-4 times per week
F 5-6 times per week
D 1 time per day
D 2 or more times per day


68a. Each time you ate turkey or chicken COLD
CUTS, how much did you usually eat?

D Less than 1 slice
D 1 to 3 slices
D More than 3 slices


69. How often did you eat luncheon or deli-style
ham including luncheon or deli-style ham used in
sandwiches, subs or wraps? (DO NOTinclude
luncheon or deli-style ham made with soy or
vegetable protein substitutes. We will ask about
these later.)

-- D NEVER (GO TO QUESTION 70)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


D 2 times per week
F 3-4 times per week
D 5-6 times per week
F 1 time per day
F 2 or more times per day


69a. Each time you ate luncheon or deli-style
ham, how much did you usually eat?

F Less than 1 slice
D 1 to 3 slices
F More than 3 slices


69b. How often was the luncheon or deli-style
ham you ate light, low-fat, or fat-free?

D Almost never or never
O About % of the time
D About / of the time
O About 34 of the time
F Almost always or always


70. How often did you eat other cold cuts or
luncheon meats (such as beef bologna, corned
beef, pastrami, or others, including low-fat)?
Include other cold cuts and luncheon meats used
in sandwiches, subs or wraps. (DO NOTinclude
ham, turkey, salami, chicken cold cuts or cold
cuts made with soy or vegetable protein.)

r- NEVER (GO TO QUESTION 71)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


D 2 times per week
E 3-4 times per week
D 5-6 times per week
E 1 time per day
E 2 or more times per day


70a. Each time you ate other cold cuts or
luncheon meats, how much did you usually
eat?

E Less than 1 slice
D 1 to 3 slices
E More than 3 slices

70b. How often were the other cold cuts or
luncheon meats you ate light, low-fat, or fat-
free cold cuts or luncheon meats? (Please
do not include ham, turkey, or chicken cold
cuts.)

F Almost never or never
D About % of the time
D About /of the time
D About 3/4 of the time
F Almost always or always






Please continue on next page.










71. How often did you eat canned tuna (including in
salads, sandwiches, or casseroles)?

-0 NEVER (GO TO QUESTION 72)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


E 2 times per week
D 3-4 times per week
E 5-6 times per week
D 1 time per day
E 2 or more times per day


71a. Each time you ate canned tuna, how much
did you usually eat?

F Less than % cup or less than 2 ounces
D Y to 1/cup or 2 to 3 ounces
D More than 12 cup or more than 3 ounces

71b. How often was the canned tuna you ate
water-packed tuna?

F Almost never or never
F About of the time
F About / of the time
F About 3/4 of the time
F Almost always or always


Over the past 12 months...

71c. How often was the canned tuna you ate
prepared with mayonnaise or other
dressing (including low-fat)?

F Almost never or never
F About % of the time
F About /of the time
D About 3/4 of the time
F Almost always or always

72. How often did you eat GROUND chicken or
turkey? (DO NOT include soy or vegetable
protein substitutes. We will ask about these
later.)

-- D NEVER (GO TO QUESTION 73)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


F 2 times per week
D 3-4 times per week
E 5-6 times per week
D 1 time per day
D 2 or more times per day


72a. Each time you ate GROUND chicken or
turkey, how much did you usually eat?

D Less than 2 ounces or less than 12 cup
F 2 to 4 ounces or 12 to 1 cup
D More than 4 ounces or more than 1 cup


73. How often did you eat beef hamburgers or
cheeseburgers? (DO NOT include soy or
vegetable protein substitutes. We will ask about
these later.)

l- NEVER (GO TO QUESTION 74)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


D 2 times per week
] 3-4 times per week
D 5-6 times per week
D 1 time per day
D 2 or more times per day


73a. Each time you ate beef hamburgers or
cheeseburgers, how much did you usually
eat?

E Less than 1 patty or less than 2 ounces
E 1 patty or 2 to 4 ounces
D More than 1 patty or more than 4 ounces

73b. How often were the beef hamburgers or
cheeseburgers you ate made with lean
ground beef?

F Almost never or never
F About % of the time
] About / of the time
D About 3/4 of the time
SAlmost always or always

74. How often did you eat ground beef in mixtures
(such as meatballs, casseroles, or meatloaf)?
(DO NOT include soy or vegetable protein
substitutes. We will ask about these later.)

r- NEVER (GO TO QUESTION 75)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


D 2 times per week
E 3-4 times per week
D 5-6 times per week
E 1 time per day
E 2 or more times per day


74a. Each time you ate ground beef in mixtures,
how much did you usually eat?

E Less than 3 ounces or less than 12 cup
D 3 to 8 ounces or 12 to 1 cup
E More than 8 ounces or more than 1 cup










75. How often did you eat hot dogs or frankfurters?
(DO NOT include sausages or vegetarian hot
dogs.)

-- NEVER (GO TO QUESTION 76)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


D 2 times per week
F 3-4 times per week
E 5-6 times per week
D 1 time per day
D 2 or more times per day


75a. Each time you ate hot dogs or frankfurters,
how many did you usually eat?

D Less than 1 hot dog
D 1 to 2 hot dogs
D More than 2 hot dogs

75b. How often were the hot dogs or frankfurters
you ate light or low-fat hot dogs?

D Almost never or never
D About % of the time
D About /of the time
D About 3/4 of the time
D Almost always or always



Over the past 12 months...

76. How often did you eat beef mixtures such as
beef stew, beef pot pie, beef and noodles, or
beef and vegetables? (DO NOTinclude
products made with soy or vegetable protein
substitutes. We will ask about these later.)

j- D NEVER (GO TO QUESTION 77)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


D 2 times per week
] 3-4 times per week
E 5-6 times per week
D 1 time per day
D 2 or more times per day


76a. Each time you ate beef stew, beef pot pie,
beef and noodles, or beef and vegetables,
how much did you usually eat?

D Less than 1 cup
D 1 to 2 cups
D More than 2 cups


77. How often did you eat roast beef or pot roast?
(DO NOT include roast beef or pot roast in
sandwiches, subs or wraps. DO NOT include soy
or vegetable protein substitutes. We will ask
about these later.)

-- D NEVER (GO TO QUESTION 78)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


D 2 times per week
F 3-4 times per week
D 5-6 times per week
F 1 time per day
D 2 or more times per day


77a. Each time you ate roast beef or pot roast
(including in mixtures), how much did you
usually eat?

F Less than 2 ounces
D 2 to 5 ounces
D More than 5 ounces

78. How often did you eat steak (beef)? (DO NOT
include steak in sandwiches, subs or wraps.)

r- D NEVER (GO TO QUESTION 79)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


F 2 times per week
F 3-4 times per week
D 5-6 times per week
E 1 time per day
E 2 or more times per day


78a. Each time you ate steak (beef), how much
did you usually eat?

E Less than 3 ounces
D 3 to 7 ounces
l More than 7 ounces

78b. How often was the steak you ate lean steak?

D Almost never or never
D About % of the time
E About / of the time
D About 3/4 of the time
E Almost always or always

79. How often did you eat pork or beef spareribs?

r- D NEVER (GO TO QUESTION 80)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


] 2 times per week
F 3-4 times per week
D 5-6 times per week
F 1 time per day
F 2 or more times per day










79a. Each time you ate pork or beef spareribs,
how much did you usually eat?

D Less than 4 ribs
D 4 to 12 ribs
D More than 12 ribs

80. How often did you eat roast turkey, turkey
cutlets, or turkey nuggets (including in
sandwiches)? (DO NOT include soy or vegetable
protein substitutes. We will ask about these
later.)

r- NEVER (GO TO QUESTION 81)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


F 2 times per week
D 3-4 times per week
] 5-6 times per week
F 1 time per day
E 2 or more times per day


80a. Each time you ate roast turkey, turkey
cutlets, or turkey nuggets, how much did
you usually eat? (Please note: 4 to 8 turkey
nuggets = 3 ounces.)

D Less than 2 ounces
F 2 to 4 ounces
O More than 4 ounces


81. How often did you eat chicken as part of salads,
sandwiches, casseroles, stews, or other
mixtures? (DO NOT include soy or vegetable
protein substitutes. We will ask about these
later.)

D NEVER (GO TO QUESTION 82)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


D 2 times per week
F 3-4 times per week
D 5-6 times per week
F 1 time per day
D 2 or more times per day


Over the past 12 months...

81a. Each time you ate chicken as part of salads,
sandwiches, casseroles, stews, or other
mixtures, how much did you usually eat?

D Less than 12 cup
D 2 to 11/2 cups
F More than 11/2 cups


82. How often did you eat baked, broiled, roasted,
stewed, or fried chicken (including nuggets)?
(DO NOT include chicken in mixtures or products
made with soy or vegetable protein substitutes.
We will ask about these later.)

r- D NEVER (GO TO QUESTION 83)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


E 2 times per week
D 3-4 times per week
E 5-6 times per week
D 1 time per day
D 2 or more times per day


82a. Each time you ate baked, broiled, roasted,
stewed, or fried chicken (including
nuggets), how much did you usually eat?

D Less than 2 drumsticks or wings, less than 1
breast or thigh, or less than 4 nuggets
D 2 drumsticks or wings, 1 breast or thigh, or 4
to 8 nuggets
E More than 2 drumsticks or wings, more than 1
breast or thigh, or more than 8 nuggets

82b. How often was the chicken you ate fried
chicken (including deep fried) or chicken
nuggets?

F Almost never or never
D About % of the time
D About /of the time
D About 3/4 of the time
F Almost always or always

82c. How often was the chicken you ate WHITE
meat?

D Almost never or never
D About % of the time
D About / of the time
D About 34 of the time
D Almost always or always

82d. How often did you eat chicken WITH skin?

O Almost never or never
F About % of the time
D About / of the time
D About 3/4 of the time
D Almost always or always










83. How often did you eat baked ham or ham
steak?

F- D NEVER (GO TO QUESTION 84)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


D 2 times per week
D 3-4 times per week
E 5-6 times per week
D 1 time per day
D 2 or more times per day


83a. Each time you ate baked ham or ham steak,
how much did you usually eat?

D Less than 1 ounce
F 1 to 3 ounces
D More than 3 ounces


84. How often did you eat pork (including chops,
roasts, sausage and in mixed dishes)? (DO NOT
include ham, or ham steak.)

r- NEVER (GO TO QUESTION 85)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


D 2 times per week
F 3-4 times per week
D 5-6 times per week
E 1 time per day
E 2 or more times per day


84a. Each time you ate pork, how much did you
usually eat?

F Less than 2 ounces or less than 1 chop
D 2 to 5 ounces or 1 chop
F More than 5 ounces or more than 1 chop


85. How often did you eat gravy on meat, chicken,
potatoes, rice, etc.?

D NEVER (GO TO QUESTION 86)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


E 2 times per week
D 3-4 times per week
E 5-6 times per week
D 1 time per day
F 2 or more times per day


85a. Each time you ate gravy on meat, chicken,
potatoes, rice, etc., how much did you
usually eat?

F Less than 1/8 cup
D 1/8 to 12 cup
F More than 12 cup


Over the past 12 months...

86. How often did you eat liver (all kinds) or
liverwurst?

D NEVER (GO TO QUESTION 87)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


D 2 times per week
F 3-4 times per week
D 5-6 times per week
D 1 time per day
D 2 or more times per day


86a. Each time you ate liver or liverwurst, how
much did you usually eat?

D Less than 1 ounce
F 1 to 4 ounces
D More than 4 ounces

87. How often did you eat bacon (including low-fat
but not imitation)? (DO NOT include soy or
vegetable protein substitutes. We will ask about
these later.)

r- NEVER (GO TO QUESTION 88)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


F 2 times per week
F 3-4 times per week
D 5-6 times per week
F 1 time per day
E 2 or more times per day


87a. Each time you ate bacon, how much did you
usually eat?

F Fewer than 2 slices
D 2 to 3 slices
F More than 3 slices

87b. How often was the bacon you ate light, low-
fat, or lean bacon?

F Almost never or never
D About of the time
D About / of the time
D About 3/4 of the time
F Almost always or always





Please continue on next page.










88. How often did you eat beef sausage (including
low-fat)? (DO NOT include soy or vegetable
protein substitutes. We will ask about these
later.)

- D NEVER (GO TO QUESTION 89)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


D 2 times per week
F 3-4 times per week
D 5-6 times per week
D 1 time per day
D 2 or more times per day


88a. Each time you ate beef sausage, how much
did you usually eat?

D Less than 1 patty or 2 links
D 1 to 3 patties or 2 to 5 links
D More than 3 patties or 5 links

88b. How often was the beef sausage you ate
light, low-fat, or lean beef sausage?

D Almost never or never
D About of the time
D About / of the time
D About 3/4 of the time
D Almost always or always

89. How often did you eat fish sticks, fried fish, or
fried seafood (not including clams, shrimp, or
other shellfish or soy or vegetable protein fish
substitutes)?

I- NEVER (GO TO QUESTION 89b)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


F 2 times per week
D 3-4 times per week
F 5-6 times per week
D 1 time per day
D 2 or more times per day


89a. Each time you ate fish sticks, fried fish, or
fried seafood how much did you usually
eat?

D Less than 2 ounces or less than 1 fillet
F 2 to 7 ounces or 1 fillet
D More than 7 ounces or more than 1 fillet

89b. How often did you eat clams, mussels,
oysters, or scallops?

- D NEVER (GO TO QUESTION 89d)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


D 2 times per week
F 3-4 times per week
D 5-6 times per week
F 1 time per day
D 2 or more times per day


89c. Each time you ate clams, mussels, oysters,
or scallops how much did you usually eat?

E Less than 2 ounces
D 2 to 7 ounces
D More than 7 ounces

89d. How often did you eat shrimp, crab, or
lobster? (DO NOT include imitation products
or products made with soy or vegetable
protein substitutes.)

- D NEVER (GO TO QUESTION 90)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


D 2 times per week
D 3-4 times per week
E 5-6 times per week
D 1 time per day
D 2 or more times per day


89e Each time you ate shrimp, crab, or lobster
how much did you usually eat?

D Less than 2 ounces or less than 1 fillet
E 2 to 7 ounces or 1 fillet
D More than 7 ounces or more than 1 fillet


90. How often did you eat fish or seafood (not
including clams, shrimp, or other shellfish or soy
or vegetable protein fish substitutes) that was
NOT FRIED? (Include intake from fish sushi.)

D NEVER (GO TO QUESTION 90b)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


F 2 times per week
D 3-4 times per week
E 5-6 times per week
D 1 time per day
D 2 or more times per day


90a. Each time you ate eat fish or seafood that
was NOT FRIED, how much did you usually
eat?

D Less than 2 ounces or less than 1 fillet
E 2 to 5 ounces or 1 fillet
D More than 5 ounces or more than 1 fillet

90b. How often did you eat shellfish (all kinds) that
was NOT FRIED?

r- D NEVER (GO TO QUESTION 91)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


F 2 times per week
D 3-4 times per week
] 5-6 times per week
E 1 time per day
E 2 or more times per day










90c. Each time you ate shellfish (all kinds) that
was NOT FRIED how much did you usually
eat?

D Less than 2 ounces
D 2 to 7 ounces
D More than 7 ounces


Over the past 12 months...

Now think about all the meat, poultry, and fish
you ate in the past 12 months and how they were
prepared.

91. How often was oil, butter, margarine, or other
fat used to FRY, SAUTE, BASTE, OR
MARINATE any meat, poultry, or fish you ate?
(Please do not include deep frying.)

1--- NEVER (GO TO QUESTION 92)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


2 times per week
3-4 times per week
5-6 times per week
1 time per day
2 or more times per day


91a. Which of the following fats were regularly
used to prepare your meat, poultry, or fish?
(Mark all that apply.)


D Margarine
(including low-fat)
D Butter (including
low-fat)
D Lard, fatback, or
bacon fat
D Olive oil


D Corn oil
F Canola or rapeseed oil
D Oil spray, such as
Pam or others
D Other kinds of oils

D None of the above


92. How often did you eat tofu?

-- D NEVER (GO TO QUESTION 93)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


2 times per week
3-4 times per week
5-6 times per week
1 time per day
2 or more times per day


92a. Each time you ate tofu, how much did you
usually eat?

F Less than % cup or less than 2 ounces
D Y to 1/cup or 2 to 4 ounces
F More than 12 cup or more than 4 ounces


93. Over the past 12 months, did you eat soups?

-D NO (GO TO QUESTION 94)

D YES

93a. How often did you eat soup DURING THE
WINTER?

D NEVER


1-6 times per winter
7-11 times per winter
1 time per month
2-3 times per month
1 time per week


2 times per week
3-4 times per week
5-6 times per week
1 time per day
2 or more times
per day


93b. How often did you eat soup DURING THE
REST OF THE YEAR?

D NEVER


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


2 times per week
3-4 times per week
5-6 times per week
1 time per day
2 or more times
per day


93c. Each time you ate soup, how much did you
usually eat?

D Less than 1 cup
D 1 to 2 cups
D More than 2 cups

93d. How often were the soups you ate bean
soups?

F Almost never or never
E About % of the time
F About /of the time
D About 3/4 of the time
F Almost always or always

93e. How often were the soups you ate cream
soups (including chowders)?

D Almost never or never
D About % of the time
D About / of the time
D About 3/4 of the time
D Almost always or always










Over the past 12 months...

93f. How often were the soups you ate tomato or
vegetable soups?

D Almost never or never
F About % of the time
F About /of the time
D About 3/4 of the time
F Almost always or always

93g. How often were the soups you ate broth
soups (including chicken) with or without
noodles or rice?

F Almost never or never
F About % of the time
D About /of the time
D About 3/4 of the time
D Almost always or always

94. How often did you eat pizza?

-- D NEVER (GO TO QUESTION 95)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


F 2 times per week
D 3-4 times per week
I 5-6 times per week
D 1 time per day
D 2 or more times per day


94a. Each time you ate pizza, how much did you
usually eat?

D Less than 1 slice or less than 1 mini pizza
F 1 to 3 slices or 1 mini pizza
D More than 3 slices or more than 1 mini pizza


94b. How often did you eat pizza with
pepperoni, sausage, or meat other than
beef? (DO NOT include pizza made 11 ith
soy or vegetable protein substitutes. We
will ask about these later.)

D Almost never or never
D About % of the time
D About / of the time
D About 3/4 of the time
D Almost always or always

94c. How often did you eat pizza with ground
beef or beef meatballs? (DO NOT include
pizza made I/ ith soy or vegetable protein
substitutes. We will ask about these later.)

F Almost never or never
D About % of the time
F About / of the time
D About 34 of the time
E Almost always or always
95. How often did you eat crackers?

- D NEVER (GO TO QUESTION 96)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


2 times per week
3-4 times per week
5-6 times per week
1 time per day
2 or more times per day


Please continue on next page.










95a. Each time you ate crackers, how many did
you usually eat?

D Fewer than 4 crackers
D 4 to 10 crackers
D More than 10 crackers

96. How often did you eat corn bread or corn
muffins?

D NEVER (GO TO QUESTION 97)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


2 times per week
3-4 times per week
5-6 times per week
1 time per day
2 or more times per day


96a. Each time you ate corn bread or corn
muffins, how much did you usually eat?

F Less than 1 piece or muffin
D 1 to 2 pieces or muffins
,r More than 2 pieces or muffins

97. How often did you eat biscuits?

I- D NEVER (GO TO QUESTION 98)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


2 times per week
3-4 times per week
5-6 times per week
1 time per day
2 or more times per day


97a. Each time you ate biscuits, how many did
you usually eat?

D Fewer than 1 biscuit
D 1 to 2 biscuits
D More than 2 biscuits

98. How often did you eat potato chips, tortilla
chips, or corn chips (including low-fat, fat-free,
or low-salt)?

F- D NEVER (GO TO QUESTION 99)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


2 times per week
3-4 times per week
5-6 times per week
1 time per day
2 or more times per day


Over the past 12 months...

98a. Each time you ate potato chips, tortilla
chips, or corn chips, how much did you
usually eat?

F Fewer than 10 chips or less than 1 cup
D 10 to 25 chips or 1 to 2 cups
F More than 25 chips or more than 2 cups

98b. How often were the chips you ate Wow
chips or other chips made with fat
substitute (Olean or Olestra)?

F Almost never or never
D About of the time
F About / of the time
F About 3/4 of the time
D Almost always or always

98c. How often were the chips you ate other low-
fat or fat-free chips?

D Almost never or never
F About % of the time
D About / of the time
D About 3/4 of the time
E Almost always or always

99. How often did you eat popcorn (including low-
fat)?

--I NEVER (GO TO QUESTION 100)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


2 times per week
3-4 times per week
5-6 times per week
1 time per day
2 or more times per day


99a. Each time you ate popcorn, how much did
you usually eat?

O Less than 2 cups, popped
D 2 to 5 cups, popped
E More than 5 cups, popped

100. How often did you eat pretzels?

- D NEVER (GO TO QUESTION 101)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


2 times per week
3-4 times per week
5-6 times per week
1 time per day
2 or more times per day










100a. Each time you ate pretzels, how many did
you usually eat?

D Fewer than 5 average twists
D 5 to 20 average twists
D More than 20 average twists

101. How often did you eat peanuts, walnuts,
seeds, or other nuts?

-- D NEVER (GO TO QUESTION 102)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


D 2 times per week
E 3-4 times per week
D 5-6 times per week
E 1 time per day
D 2 or more times per day


101a. Each time you ate peanuts, walnuts, seeds,
or other nuts, how much did you usually eat?

F Less than % cup
D 4 to 1 cup
F More than 1 cup

102. How often did you eat an energy, high-protein,
or breakfast bar?

I-- NEVER (GO TO QUESTION 103)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


E 2 times per week
D 3-4 times per week
F 5-6 times per week
E 1 time per day
E 2 or more times per day


102a. Which type of energy, high-protein or
breakfast bar did you usually eat? (Mark as
many that apply.)

D Nutri-Grain Bar/Kellogg's Granola Bar
D Power Bar
F Power Bar Performance/Luna Bar
D Power Bar Protein Plus
F Power Bar Harvest
D Power Bar Pria
F Balance Bar
D Slimfast Bar
F Zone Bar
D Other

102b. Each time you ate an energy, high-protein,
or breakfast bar, how much did you usually
eat?

F Less than 1 bar
D 1 bar
F More than 1 bar


103. How often did you eat yogurt (NOT including
frozen yogurt)?

r- NEVER (GO TO QUESTION 104)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


2 times per week
3-4 times per week
5-6 times per week
1 time per day
2 or more times per day


Over the past 12 months...

103a. Each time you ate yogurt, how much did
you usually eat?

E Less than 12 cup or less than 1 container
D 1 to 1 cup or 1 container
D More than 1 cup or more than 1 container

104. How often did you eat cottage cheese
(including low-fat)?

D NEVER (GO TO QUESTION 105)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


] 2 times per week
F 3-4 times per week
D 5-6 times per week
F 1 time per day
E 2 or more times per day


104a. Each time you ate cottage cheese, how
much did you usually eat?

E Less than % cup
D to 1 cup
F More than 1 cup

105. How often did you eat cheese (including low-fat;
including on cheeseburgers or in sandwiches,
subs or wraps)? (DO NOTinclude cheese made
from soy or vegetable protein.)

r- D NEVER (GO TO QUESTION 106)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


E 2 times per week
D 3-4 times per week
F 5-6 times per week
D 1 time per day
D 2 or more times per day


105a. Each time you ate cheese, how much did
you usually eat?

D Less than 12 ounce or less than 1 slice
S12 to 11/2 ounces or 1 slice
D More than 11/2 ounces or more than 1 slice










105b. How often was the cheese you ate light or
low-fat cheese?

D Almost never or never
D About % of the time
D About / of the time
D About 3/4 of the time
F Almost always or always


105c. How often was the cheese you ate fat-free
cheese?

F Almost never or never
D About of the time
D About /of the time
D About 3/4 of the time
F Almost always or always


106. How often did you eat frozen yogurt, or
sherbet (including low-fat or fat-free)? Please
do not include Tofutti.

[-- NEVER (GO TO QUESTION 107)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


E 2 times per week
l 3-4 times per week
E 5-6 times per week
l 1 time per day
E 2 or more times per day


106a. Each time you ate frozen yogurt, or
sherbet, how much did you usually eat?

F Less than 12 cup or less than 1 scoop
D 12 to 1 cup or 1 to 2 scoops
F More than 1 cup or more than 2 scoops

107. How often did you eat ice cream or ice cream
bars, (including low-fat or fat-free)?

r- O NEVER (GO TO QUESTION 108)


D 1-6 times per year
F 7-11 times per year
F 1 time per month
D 2-3 times per month
F 1 time per week


D 2 times per week
F 3-4 times per week
F 5-6 times per week
D 1 time per day
D 2 or more times per day


107a. Each time you ate ice cream or ice cream
bars, how much did you usually eat?

D Less than 12 cup or less than 1 scoop
E 12 to 11/2 cups or 1 to 2 scoops
F D More than 11/2 cups or more than 2 scoops


107b. How often was the ice cream you ate light,
low-fat, or fat-free ice cream?

F Almost never or never
E About % of the time
F About /of the time
D About 3/4 of the time
E Almost always or always

Over the past 12 months...

108. How often did you eat cake (including low-fat or
fat-free)?

-- E NEVER (GO TO QUESTION 109)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


E 2 times per week
F 3-4 times per week
D 5-6 times per week
F 1 time per day
E 2 or more times per day


108a. Each time you ate cake, how much did you
usually eat?

F Less than 1 medium piece
D 1 medium piece
F More than 1 medium piece

108b. How often was the cake you ate light, low-
fat, or fat-free cake?

F Almost never or never
F About of the time
F About / of the time
D About 3/4 of the time
E Almost always or always

109. How often did you eat cookies or brownies
(including low-fat or fat-free)?

1--E NEVER (GO TO QUESTION 110)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


D 2 times per week
F 3-4 times per week
E 5-6 times per week
l 1 time per day
E 2 or more times per day


109a. Each time you ate cookies or brownies,
how much did you usually eat?

F Less than 2 cookies or 1 small brownie
D 2 to 4 cookies or 1 medium brownie
E More than 4 cookies or 1 large brownie










109b. How often were the cookies or brownies you
ate light, low-fat, or fat-free cookies or
brownies?

D Almost never or never
D About of the time
D About / of the time
D About 3/4 of the time
F Almost always or always


110. How often did you eat doughnuts, sweet rolls,
Danish, or pop-tarts?

F- D NEVER (GO TO QUESTION 111)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


] 2 times per week
E 3-4 times per week
D 5-6 times per week
E 1 time per day
F 2 or more times per day


110a. Each time you ate doughnuts, sweet rolls,
Danish, or pop-tarts, how much did you
usually eat?

F Less than 1 piece
D 1 to 2 pieces
F More than 2 pieces

111. How often did you eat sweet muffins or
dessert breads (including low-fat or fat-free)?

F- D NEVER (GO TO QUESTION 112)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


E 2 times per week
D 3-4 times per week
F 5-6 times per week
D 1 time per day
D 2 or more times per day


111 a. Each time you ate sweet muffins or dessert
breads, how much did you usually eat?

D Less than 1 medium piece
F 1 medium piece
D More than 1 medium piece

111 b. How often were the sweet muffins or
dessert breads you ate light, low-fat, or fat-
free sweet muffins or dessert breads?

D Almost never or never
D About % of the time
D About / of the time
D About 3/4 of the time
D Almost always or always


112. How often did you eat fruit crisp, cobbler, or
strudel?

r-E NEVER (GO TO QUESTION 113)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


2 times per week
3-4 times per week
5-6 times per week
1 time per day
2 or more times per day


Over the past 12 months...

112a. Each time you ate fruit crisp, cobbler, or
strudel, how much did you usually eat?

F Less than 12 cup
D 1 to 1 cup
D More than 1 cup

113. How often did you eat pie?

r- NEVER (GO TO QUESTION 114)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


D 2 times per week
E 3-4 times per week
D 5-6 times per week
E 1 time per day
F 2 or more times per day


113a. Each time you ate pie, how much did you
usually eat?

F Less than 1/8 of a pie
D About 1/8 of a pie
F More than 1/8 of a pie

The next four questions ask about the kinds of
pie you ate. Please read all four questions
before answering.

113b. How often were the pies you ate fruit pie
(such as apple, blueberry, others)?

E Almost never or never
F About % of the time
D About / of the time
D About 3/4 of the time
D Almost always or always

113c. How often were the pies you ate cream,
pudding, custard, key lime or meringue
pie?

D Almost never or never
D About % of the time
D About /of the time
D About 3/4 of the time
D Almost always or always










113d. How often were the pies you ate pumpkin or
sweet potato pie?

D Almost never or never
D About % of the time
D About / of the time
D About 3/4 of the time
F Almost always or always


113e. How often were the pies you ate pecan pie?

D Almost never or never
D About % of the time
D About / of the time
D About 3/4 of the time
D Almost always or always

114. How often did you eat chocolate candy?

I-D NEVER (GO TO QUESTION 115)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


2 times per week
3-4 times per week
5-6 times per week
1 time per day
2 or more times per day


114a. Each time you ate chocolate candy, how
much did you usually eat?

D Less than 1 average bar or less than 1 ounce
F 1 average bar or 1 to 2 ounces
D More than 1 average bar or more than 2 ounces

115. How often did you eat other candy?

p- NEVER (GO TO QUESTION 116)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


2 times per week
3-4 times per week
5-6 times per week
1 time per day
2 or more times per day


115a. Each time you ate other candy, how much
did you usually eat?

D Fewer than 2 pieces
F 2 to 9 pieces
D More than 9 pieces


116. How often did you eat eggs or egg whites, (NOT
counting egg substitutes or eggs in baked goods
and desserts)? (Please include eggs in salads,
quiche, and souffles.)

rD NEVER (GOTO QUESTION 116b)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


2 times per week
3-4 times per week
5-6 times per week
1 time per day
2 or more times per day


Over the past 12 months...

116a. Each time you ate eggs, how many did you
usually eat?

D 1 egg
D 2 eggs
F 3 or more eggs

116b. Over the past 12 months, how often did you
eat an egg substitute product (NOT
counting egg substitute product used in
baked goods and desserts)? (Please
include egg substitute used in quiche,
souffles, and egg dishes.)

-D NEVER (GO TO QUESTION 116e)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


2 times per week
3-4 times per week
5-6 times per week
1 time per day
2 or more times per day


116c. Which of the following brands do you eat
most often (mark as many that apply)?

E Morningstar Farms Scrambles
E Morningstar Farms Better n' Eggs
D Eggbeaters
E Second Nature
D Other

116d. Each time you ate egg substitute product,
how much did you usually eat?

D Less than 12 cup
D 1 to 1 cup
D More than 1 cup


145










116e. How often were the eggs or egg substitute
product you ate cooked in oil, butter, or
margarine?

D Almost never or never
D About % of the time
D About / of the time
D About 3/4 of the time
F Almost always or always

116f. How often were the eggs you ate part of
egg salad?

D Almost never or never
D About of the time
D About / of the time
D About 3/4 of the time
D Almost always or always






117. How many cups of coffee, caffeinated or
decaffeinated, did you drink?

- D NEVER (GO TO QUESTION 118)


Less than 1 cup per
month
1-3 cups per month
1 cup per week
2-4 cups per week


D 5-6 cups per week
D 1 cup per day
E 2-3 cups per day
D 4-5 cups per day
E 6 or more cups per day


117a. How often was the coffee you drank
decaffeinated?

D Almost never or never
D About of the time
D About / of the time
D About 3/4 of the time
D Almost always or always

118. How many glasses of ICED tea, caffeinated or
decaffeinated, did you drink?

- D NEVER (GO TO QUESTION 119)


Less than 1 cup per
month
1-3 cups per month
1 cup per week
2-4 cups per week


5-6 cups per week
1 cup per day
2-3 cups per day
4-5 cups per day
6 or more cups per day


119a. How often was the iced tea you drank
decaffeinated or herbal tea?

D Almost never or never
E About % of the time
D About / of the time
D About 3/4 of the time
E Almost always or always

119. How many cups of HOT tea, caffeinated or
decaffeinated, did you drink?

- D NEVER (GO TO QUESTION 120)


Less than 1 cup per
month
1-3 cups per month
1 cup per week
2-4 cups per week


E 5-6 cups per week
D 1 cup per day
E 2-3 cups per day
D 4-5 cups per day
E 6 or more cups per day


119a. How often was the hot tea you drank
decaffeinated or herbal tea?

D Almost never or never
E About % of the time
D About / of the time
D About 3/4 of the time
F Almost always or always

Over the past 12 months...

120. How often did you add sugar or honey to your
coffee or tea?

r-E NEVER (GO TO QUESTION 121)


Less than 1 time per
month
1-3 times per month
1 time per week
2-4 times per week


] 5-6 times per week
E 1 time per day
D 2-3 times per day
E 4-5 times per day
D 6 or more times per day


120a. Each time sugar or honey was added to
your coffee or tea, how much was usually
added?

D Less than 1 teaspoon
E 1 to 3 teaspoons
D More than 3 teaspoons

121. How often did you add artificial sweetener to
your coffee or tea?

r- NEVER (GO TO QUESTION 122)


D Less than 1 time per
month
D 1-3 times per month
E 1 time per week
D 2-4 times per week


D 5-6 times per week
F 1 time per day
D 2-3 times per day
F 4-5 times per day
D 6 or more times per day










121a. What kind of artificial sweetener did you
usually use?

F Equal or aspartame
D Sweet N Low or saccharin

122. How often was non-dairy creamer added to
your coffee or tea?

--] NEVER (GO TO QUESTION 123)


Less than 1 time per
month
1-3 times per month
1 time per week
2-4 times per week


D 5-6 times per week
E 1 time per day
E 2-3 times per day
D 4-5 times per day
E 6 or more times per day


122a. Each time non-dairy creamer was added to
your coffee or tea, how much was usually
used?

F Less than 1 teaspoon
D 1 to 3 teaspoons
F More than 3 teaspoons

122b. What kind of non-dairy creamer did you
usually use?

D Regular powdered
F Low-fat or fat-free powdered
D Regular liquid
F Low-fat or fat-free liquid


123. How often was cream or half and
your coffee or tea?

I-- NEVER (GO TO QUESTION 124)


F Less than 1 time per
month
F 1-3 times per month
D 1 time per week
F 2-4 times per week


half added to


F 5-6 times per week
D 1 time per day
F 2-3 times per day
D 4-5 times per day
F 6 or more times per day


123a. Each time cream or half and half was
added to your coffee or tea, how much was
usually added?

F Less than 1 tablespoon
D 1 to 2 tablespoons
F More than 2 tablespoons


124. How often was milk added to your coffee or
tea?

--E NEVER (GO TO QUESTION 125)


Less than 1 time per
month
1-3 times per month
1 time per week
2-4 times per week


D 5-6 times per week
D 1 time per day
D 2-3 times per day
F 4-5 times per day
D 6 or more times per day


124a. Each time milk was added to your coffee or
tea, how much was usually added?

D Less than 1 tablespoon
E 1 to 3 tablespoons
D More than 3 tablespoons

124b. What kind of milk was usually added to your
coffee or tea?

E Whole milk (including lactose-free variety)
D 2% milk (including lactose-free variety)
E 1% milk (including lactose-free variety)
D Skim, nonfat, or /% milk (including lactose-
free variety)
E Evaporated or condensed (canned) milk
D Soy milk
F Rice milk
Other

Over the past 12 months...

125. How often was sugar or honey added to foods
you ate? (Please do not include sugar in coffee,
tea, other beverages, or baked goods.)

r-0 NEVER (GO TO INTRODUCTION TO
QUESTION 126)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


2 times per week
3-4 times per week
5-6 times per week
1 time per day
2 or more times
per day


125a. Each time sugar or honey was added to
foods you ate, how much was usually
added?

F Less than 1 teaspoon
D 1 to 3 teaspoons
F More than 3 teaspoons










The following questions are about the kinds of
margarine, mayonnaise, sour cream, cream
cheese, and salad dressing that you eat. If
possible, please check the labels of these foods
to help you answer.

126. Over the past 12 months, did you eat
margarine?

D NO (GO TO QUESTION 127)

D YES


126a. How often was the margarine you ate
regular-fat margarine (stick or tub)?

D Almost never or never
D About % of the time
D About /of the time
D About 3/4 of the time
D Almost always or always

126b. How often was the margarine you ate light
or low-fat margarine (stick or tub)?

F Almost never or never
F About of the time
F About /of the time
D About 3/4 of the time
D Almost always or always

126c. How often was the margarine you ate fat-
free margarine?

D Almost never or never
D About of the time
D About /of the time
D About 34 of the time
F Almost always or always

127. Over the past 12 months, did you eat butter?

-- NO (GO TO QUESTION 128)

D YES


127a. How often was the butter you ate light or
low-fat butter?

D Almost never or never
D About of the time
D About / of the time
D About 3/4 of the time
D Almost always or always


128. Over the past 12 months, did you eat
mayonnaise or mayonnaise-type dressing?

-D NO (GO TO QUESTION 129)

D YES

128a. How often was the mayonnaise you ate
regular-fat mayonnaise?

F Almost never or never
E About % of the time
D About /of the time
D About 3/4 of the time
D Almost always or always

128b. How often was the mayonnaise you ate light
or low-fat mayonnaise?

D Almost never or never
D About % of the time
D About 2 of the time
D About 3/4 of the time
D Almost always or always

Over the past 12 months...

128c. How often was the mayonnaise you ate fat-
free mayonnaise?

] Almost never or never
D About % of the time
D About 2 of the time
E About 34 of the time
F Almost always or always

129. Over the past 12 months, did you eat sour
cream?

NO (GO TO QUESTION 130)

D YES

129a. How often was the sour cream you ate
regular-fat sour cream?

D Almost never or never
D About % of the time
D About 2 of the time
D About 3/4 of the time
F Almost always or always

129b. How often was the sour cream you ate light,
low-fat, or fat-free sour cream?

] Almost never or never
D About % of the time
D About /2 of the time
D About 3/4 of the time
F Almost always or always










130. Over the past 12 months, did you eat cream
cheese?

-- NO (GO TO QUESTION 131)

D YES

130a. How often was the cream cheese you ate
regular-fat cream cheese?

D Almost never or never
D About of the time
D About / of the time
D About 3/4 of the time
F Almost always or always

130b. How often was the cream cheese you ate
light, low-fat, or fat-free cream cheese?

I Almost never or never
D About of the time
F About / of the time
F About 3/4 of the time
D Almost always or always

131. Over the past 12 months, did you eat salad
dressing?

-- NO (GO TO INTRODUCTION TO QUESTION 132)

D YES

131a. How often was the salad dressing you ate
regular-fat salad dressing (including oil
and vinegar dressing)?

D Almost never or never
F About of the time
F About / of the time
D About 3/4 of the time
D Almost always or always

131 b. How often was the salad dressing you ate
light or low-fat salad dressing?

D Almost never or never
D About % of the time
D About /of the time
D About 3/4 of the time
D Almost always or always

131c. How often was the salad dressing you ate
fat-free salad dressing?

D Almost never or never
F About % of the time
F About /of the time
D About 3/4 of the time
F Almost always or always


The following two questions ask you to
summarize your usual intake of vegetables and
fruits. Please do not include salads, potatoes, or
juices.

132. Over the past 12 months, how many servings of
vegetables (not including salad or potatoes) did
you eat per week or per day?


Less than 1 per week
1-2 per week
3-4 per week
5-6 per week
1 per day


E 2 per day
D 3 per day
E 4 per day
D 5 or more per day


Over the past 12 months...

133. Over the past 12 months, how many servings of
fruit (not including juices) did you eat per week
or per day?


Less than 1 per week
1-2 per week
3-4 per week
5-6 per week
1 per day


2 per day
3 per day
4 per day
5 or more per day


134. Over the past month, which of the following
foods did you eat AT LEAST THREE TIMES?
(Mark all that apply.)


Avocado, guacamole
Cheesecake
Chocolate, fudge, or
butterscotch toppings
or syrups
Chow mein noodles
Croissants
Dried apricots
Egg rolls
Granola bars
Hot peppers
Jello, gelatin
Milkshakes or
ice-cream sodas


D Olives
D Oysters
D Pickles or pickled
vegetables or fruit
D Plantains
E Pork neckbones, hock,
head, feet
E Pudding or custard
D Veal, venison, lamb
E Whipped cream, regular
E Whipped cream,
substitute
D NONE


Please continue on next page.











135. For ALL of the past 12 months, have you
followed any type of vegetarian diet?

-D NO (GO TO INTRODUCTION TO QUESTION 136)

SYES


135a. Which of the following foods did you
TOTALLY EXCLUDE from your diet?
(Mark all that apply.)

D Beef, veal
D Pork, lamb
E Poultry (chicken, turkey, duck)
D Fish and seafood
D Shellfish
D Eggs (please do not include egg substitutes)
E Dairy products (milk, cheese, etc.) (please do
not include milk/dairy substitutes)

136. Over the past 12 months, did you eat any meat
substitute products made with soy or
vegetable protein?

D NO Thank you very much for completing
this questionnaire! Because we want
to be able to use all the information
you have provided, we would greatly
appreciate it if you would please take a
moment to review each page making
sure that you:
Did not skip any pages
Crosssed out the incorrect answer
and circled the correct answer if
you made any changes


YES (GO TO QUESTION 137)


137. How often did you eat breakfast patties or
breakfast links made with soy or vegetable
protein?

- D NEVER (GO TO QUESTION 138)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


2 times per week
3-4 times per week
5-6 times per week
1 time per day
2 or more times per day


137a. Which of the following brands do you eat
most often (mark as many that apply)?

D Morningstar Farms
D Worthington
E Loma Linda
D Other (such as Boca, Quorn, etc.)

137b. Each time you ate a breakfast patty or
breakfast link made with soy or
vegetable protein, how much did you
usually eat?

D Less than 1 patty or 2 links
D 1 to 3 patties or 2 to 5 links
D More than 3 patties or 5 links


138. How often did you eat breakfast strips
(imitation bacon) made with soy or vegetable
protein?

-- D NEVER (GO TO QUESTION 139)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


2 times per week
3-4 times per week
5-6 times per week
1 time per day
2 or more times per day


138a. Which of the following brands do you eat
most often? (Mark as many that apply.)

D Morningstar Farms
E Worthington
D Loma Linda
E Other (such as Boca, Quorn, etc.)


138b. Each time you ate breakfast strips
(imitation bacon) made with soy or
vegetable protein, how much did you
usually eat?

E Fewer than 2 slices
D 2 to 3 slices
F More than 3 slices

139. How often did you eat burgers made with soy
or vegetable protein?

--D NEVER (GO TO QUESTION 140)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


2 times per week
3-4 times per week
5-6 times per week
1 time per day
2 or more times per day










139a. Which of the following brands do you eat
most often? (Mark as many that apply.)

D Morningstar Farms
D Worthington
D Loma Linda
F Other (such as Boca, Quorn, etc.)

139b. Each time you ate a burger made with soy
or vegetable protein, how much did you
usually eat?

F Less than 1 patty or less than 2 ounces
D 1 patty or 2 to 4 ounces
F More than 1 patty or more than 4 ounces

140. How often did you eat imitation meat dinner
entrees (not including imitation chicken or
fish) made with soy or vegetable protein?

r--D NEVER (GO TO QUESTION 141)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


2 times per week
3-4 times per week
5-6 times per week
1 time per day
2 or more times per day


140a. Which of the following brands do you eat
most often (mark as many that apply)?

F Morningstar Farms
D Worthington
F Loma Linda
D Other (such as Boca, Quorn, etc.)


140b. Each time you ate an imitation meat
dinner entree (not including imitation
chicken or fish) made with soy or
vegetable protein, how much did you
usually eat?

D Less than 1 portion or 1 /cup
F One portion or 1 cup
D More than one portion or more than 1 cup

141. How often did you eat imitation chicken/turkey
patties, nuggets, wings or other imitation
chicken/turkey product made with soy or
vegetable protein?

r- NEVER (GO TO QUESTION 142)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


2 times per week
3-4 times per week
5-6 times per week
1 time per day
2 or more times per day


141a. Which of the following brands do you eat
most often? (Mark as many that apply.)

E Morningstar Farms
D Worthington
D Loma Linda
E Other (such as Boca, Quorn, etc.)

141b. Each time you ate an imitation
chicken/turkey patties, nuggets, wings or
other imitation chicken/turkey product
made with soy or vegetable protein, how
much did you usually eat?

D Less than 1 patty or less than 2 ounces
D 1 patty or 2 to 4 ounces
F More than 1 patty or more than 4 ounces


142. How often did you eat entrees like chili, Mexican
foods (tacos, burritos, tostados, enchiladas,
etc) lasagna, manicotti, ravioli, stuffed shells,
tortellini spaghetti with meat sauce, meatballs
or casserole made with soy or vegetable
protein products like Burger Crumbles?

--El NEVER (GO TO QUESTION 143)


1-6 times per year
7-11 times per year
1 time per month
2-3 times per month
1 time per week


2 times per week
3-4 times per week
5-6 times per week
1 time per day
2 or more times per day


142a. Which of the following brands do you eat
most often? (Mark as many that apply.)

D Morningstar Farms
D Worthington
F Loma Linda
D Other (such as Boca, Quorn, etc.)


142b. Each time you ate an entree like chili,
Mexican foods (tacos, burritos, tostados,
enchiladas, etc) lasagna, manicotti, ravioli,
stuffed shells, tortellini spaghetti with meat
sauce, meatballs or casserole made with
soy or vegetable protein products like
Burger Crumbles, how much did you
usually eat?

F Less than 12 cup
D 1 to 1 cup
F More than 1 cup










143. How often did you eat cold cut substitutes
(such as meatless salami, meatless bologna,
meatless chicken roll, meatless smoked
turkey, meatless corned beef) made with soy
or vegetable protein?

NEVER (GO TO END)

D 1-6 times per year D 2 times per week
F 7-11 times per year F 3-4 times per week
D 1 time per month D 5-6 times per week
F 2-3 times per month F 1 time per day
D 1 time per week D 2 or more times per day

143a. Which of the following brands do you eat
most often? (Mark as many that apply.)

F Morningstar Farms
D Worthington
D Loma Linda
D Other (such as Boca, Quorn, etc.)

143b. Each time you ate cold cut substitutes
(such as meatless salami, meatless
bologna, meatless chicken roll, meatless
smoked turkey, meatless corned beef)
made with soy or vegetable protein, how
much did you usually eat?

D Less than 3 slices or less than 2 ounces
D 3 to 6 slices or 2 to 4 ounces
D More than 6 slices or more than 4 ounces


Thank you very much for completing
this questionnaire! Because we want
to be able to use all the information
you have provided, we would greatly
appreciate it if you would please take a
moment to review each page making
sure that you:

* Did not skip any pages
* Crossed out the incorrect answer
and circled the correct answer if
you made any changes









APPENDIX C
DIETARY HISTORY QUESTIONNAIRE INSTRUCTIONS MATERIALS

Please read these instructions prior to beginning the Diet History Questionnaire. These
additional instructions combined with the "General Instructions" (found on the first page of the
Diet History Questionnaire) will guide you while completing the questionnaire.

ADDITIONAL INSTRUCTIONS

1. When answering each question, think about your diet over the past year and NOT the past
few weeks.

2. Several questions refer you to additional handouts that have been provided in your
packet. Please be sure to use these handouts when you get to these questions.


If you have any questions while completing the questionnaire, please contact
Amanda Brown at 352-392-1991 ext 246. Please leave a voice message with your
name, contact number, and best time or way to reach you.


Question Number Handout

8e Caffeinated versus Non-caffeinated
Beverages
13b Fortified Cereal
19a, 21a, 22a, 23a, Seasonal Fruits and Vegetables
24a, 25a, 34a, 42a









Remember when answering these questions, we want you to think
about your diet over the past year and NOT the past few weeks.
Here are a few examples, that you may find helpful when
completing the questionnaire.



1. Please look at question #57 on page 16. I go to IHOP for breakfast on the first Sunday of
every month of the year. I always order one medium size Belgian waffle. I never make
waffles, pancakes, or French toast at home. I only eat waffles at IHOP. Since there are
12 months in a year, I eat 12 waffles a year or 1 waffle a month. If you look at question
#57, I would mark an "X" for 1 time per month. For question #57a, I would answer 1 to 3
medium pieces.


2. Please look at question #107 on page 31. I only eat ice cream during the summer months
of June, July, and August. During that time, I eat 1 cup of ice cream twice a week. That
means I eat ice cream 8 times during each of those months. So that adds up to 24 times.
Since I do not eat ice cream during the other months, I can say that I only have ice cream
24 times a year. If I remember that there are 12 months in a year that means, on average,
I only have ice cream twice a month. If you look at question #107 on page 31, I would
mark an "X" for 2-3 times a month. For question #107a, I would mark 12 to 1 12 cups or
1 to 2 scoops.








Directions on How to Change Your Answer Choice

Over the past 12 months, how often did you drink tomato juice or
vegetable juice?

a. X 1 time per month or less

b. 2-3 times per month You select "a" as your
first answer, but then
c. I 1-2 times per week you change your mind.

d. 3-4 times per week

e. D 5-6 times per week


D 2-3 times per month

D 1-2 times per week

D 3-4 times per week

5-6 times per week


a. 1wi L J LJ %u X X--3,3

b. 2-3 times per month

c. I 1-2 times per week

d. I 3-4 times per week

e. 5-6 times per week


Step #1
Cross out answer "a"


Step #2


Mark an "X" in the box
of your new choice and
circle that answer too.









Please use this list for Question 8e


Caffeinated versus non-caffeinated beverages


Caffeinated
A & W Creme Soda
Barq's Root Beer
Canada Dry Cola
Coffee
Diet A & W Creme Soda
Diet Coke with Lemon
Diet Cherry Coke
Cherry Coke
Cherry Pepsi
Coke
Diet Coke
Diet Barq's Root Beer
Diet Dr. Pepper
Diet Mountain Dew
Diet Pepsi
Diet RC Cola
Diet Shasta Cola
Diet Snapple Flavored teas
Diet Sunkist Orange
Dr. Pepper
Hot Chocolate Mix
Jolt
Lipton Brisk
Mellow Yellow
Mountain Dew
Mountain Dew Code Red
Nestea Sweetened & Unsweetened Iced Teas
Pepsi
Pepsi One
Pepsi Twist
RC Cola
Shasta Cola
Snapple Flavored teas
Sunkist Orange
Surge
Tea: Iced, Brewed, Instant, Green


Non-caffeinated
7-Up
A & W Root Beer
Diet 7-Up
Diet A & W Root Beer
Diet Sprite
Minute Maid Orange
Sprite


I V '








Please use this list for Question 13b





All-Bran Buds
All-Bran Extra Fiber
All-Bran Original
Cheerios Multigrain
Complete Oat Bran Flakes
Complete Wheat Bran Flakes
Just Right Fruit and Nut
KASHI Heart to Heart
Kellogg's Low Fat Granola no raisins "pm"j
Kellogg's Low Fat Granola with raisins
Mueslix
Product 19
Smart Start
Smart Start Soy Protein
Special K
Total Original
Total Whole Grain
Total Brown Sugar and Oat
Total Corn Flakes
Total Raisin Bran






Please use this list for Questions
19a, 21a, 22a, 23a, 24a, 25a, 34a, 42a


Seasonal Fruits and Vegetables


Fruit or Vegetable Months When In Season
Cantaloupe March to July
Corn August to June
Grapefruit September to June
Honeydew Melon June to October
Nectarines July to September
Oranges October to June
Peaches June to September
Plums June to October
Strawberries October to June
Tangelos November to February
Tomatoes September to June


% l


of









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BIOGRAPHICAL SKETCH

Melanie was born in Escondido, California. She received her Bachelor of Science in

nutrition applied sciences from the Pennsylvania State University in 2005. After graduation,

Melanie was accepted into the University of Florida's combined Master of Science Dietetic

Internship Program. During graduate school, she served as the Gainesville District Dietetic

Association Newsletter Co-Editor for 1/2 years, and as the team leader for a school-based

nutrition intervention project that she and her classmates developed and implemented at Talbot

Elementary School during the 2005 to 2006 school year. She and several classmates also had the

opportunity to present their work as part of the 2006 Florida Dietetic Association Annual

Symposium Poster Session. In recognition of her academic accomplishments, Melanie recently

was awarded a scholarship from the Agricultural Women's Club. Melanie plans to move to

Philadelphia after she graduates from the University of Florida. Her immediate career goal is to

pass the Registration Examination for Dietitians and work as a registered dietitian specializing in

pediatrics.





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1 RELATIVE CONTRIBUTION OF FOOD FOLATE AND FOLIC ACID TO INTAKE AND STATUS OF YOUNG MEN AND WOMEN By MELANIE LYN GRABIANOWSKI A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF FOOD SCIENCE AND HUMAN NUTRITION UNIVERSITY OF FLORIDA 2007

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2 2007 Melanie Lyn Grabianowski

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3 ACKNOWLEDGEMENTS I would like to sin cerely thank my supervisory committ ee members Lynn B. Bailey, PhD, Gail P.A. Kauwell, PhD, RD, LDN, and Lind a Young, PhD for the unique contributions that each of them made to my research project. In particular, I would like to thank my committee cochairs, Drs. Lynn Bailey and Gail Kauwell, for guiding and encouraging me through this challenging experience. They ar e both remarkable mentors and ro le models and have been an inspiration from the very begi nning. Without their expertise and support, I could not have accomplished this endeavor. I have learned more in the past few years than I ever could have anticipated, and I know that the kno wledge and wisdom I have gain ed will take me great places in the future. I also would like to thank Dave Maneval for his assistance in the laboratory, James Colee for his assistance with th e statistical analyses of my data, and Karla Shelnutt, PhD, RD, Kristina von-Castel-Roberts, PhD, and Ama nda Wittmann, MS, RD, LDN for be ing great listeners. Each of them played an important role in guiding me through this long journey. My family and friends have been an amazi ng support system to me and have been a constant source of encouragement and sage advice for which I am very gr ateful. Finally, I would like to thank Dominick Savoca. He reminded me everyday how proud he was of my hard work and accomplishments. His love and encouragemen t gave me the strength to complete this program.

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4 TABLE OF CONTENTS Page ACKNOWLEDGMENTS...............................................................................................................3 LIST OF TABLES................................................................................................................. ..........7 LIST OF FIGURES................................................................................................................ .........8 LIST OF ABBREVIATIONS.......................................................................................................... 9 ABSTRACT....................................................................................................................... ............12 CHAPTER 1 INTRODUCTION................................................................................................................. .14 Overall Goals.................................................................................................................. ........16 Specific Objectives............................................................................................................ .....16 2 BACKGROUND AND LI TERATURE REVIEW................................................................17 Folate......................................................................................................................... .............17 Chemistry..................................................................................................................... ...17 Dietary Sources............................................................................................................... 17 Bioavailability............................................................................................................... ..19 Absorption, Transport and Storage.................................................................................21 Metabolism and Excretion..............................................................................................22 Dietary Reference Intakes...............................................................................................23 Biochemical Functions....................................................................................................24 Key-Folate Dependent Reactions in One-Carbon Metabolism......................................24 Methylenetetrahydrofolate Reductase Polymorphism....................................................26 Folate Status Assess ment....................................................................................................... .27 Serum and Red Blood Cell Folate Concentrations.........................................................27 Plasma Homocystei ne Concentration.............................................................................28 Folate Deficiency.............................................................................................................. ......29 Etiology...................................................................................................................... .....29 Megaloblas tic An emia....................................................................................................29 Neural Tube Defects.......................................................................................................31 Vascular Dis ease and Stroke...........................................................................................34 Cancer........................................................................................................................ .....39 Folic Acid Fortification....................................................................................................... ...43 Effect of Fortification on Folate Stat us of Women of Repr oductive Potential...............45 Effect of Fortification on Neural Tube Defects..............................................................47 Folic Acid Recommenda tions and Awareness.......................................................................49 Analytical Methodology......................................................................................................... 50 Blood Folate Analysis.....................................................................................................51

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5 Plasma Homocysteine Analysis......................................................................................53 MTHFR Genotype Determination..................................................................................54 Dietary Intake Assessment..................................................................................................... 55 USDA National Nutr ient Da taba se.................................................................................55 Food Frequency Questionnaire.......................................................................................55 Assessment of Folate/Folic Acid Intake in Women of Reproductive Potential.....................58 3 STUDY DESIGN AND METHODS.....................................................................................63 Study Design and Methods Overview....................................................................................63 Human Subject Procedures....................................................................................................64 Dietary History Questionnaire...............................................................................................65 Processing the Dietary History Questionnaire.......................................................................65 Analysis of Nutrient Intakes................................................................................................... 66 Dietary History Questionnair e Instruction Pretest.................................................................66 Blood Sample Collectio n and Processing..............................................................................67 Analytical Methods for Assessing Folate Status....................................................................68 Identification of Food Groups from th e Dietary History Questionnaire........................68 Serum and RBC Folate Concentrations..........................................................................69 Homocysteine Concentrations........................................................................................69 MTHFR Genotype Determination..................................................................................69 Statistical Methods............................................................................................................ .....69 4 RESULTS...................................................................................................................... .........72 Characteristics of Study Population.......................................................................................72 Dietary Folate In take and Gender..........................................................................................73 Dietary Folate Inta ke and Genotype.......................................................................................73 Dietary Folate Equiva lents, Folic Acid a nd Recommended Intakes......................................74 Contribution of Folate from Food Groups.............................................................................74 Folate Intake from Fortifie d Ready-to-Eat Cereals................................................................76 Folate and Homocysteine Concentrations and Gender..........................................................77 Folate and Homocysteine Co ncentrations and Genotype......................................................78 Serum Folate a nd Homocysteine............................................................................................79 Relationship Between Fola te Intake and Status.....................................................................79 Folate Status and Fortified Read y-to-Eat Cereal Consumption.............................................80 5 DISCUSSION AND CONCLUSIONS..................................................................................95 Strengths and Limitati ons.....................................................................................................1 04 Summary........................................................................................................................ ......105 Practical Application.......................................................................................................... ..106 APPENDIX A SUBJECT DATA COLLECTION FORM...........................................................................108 B DIETARY HISTORY QUESTIONNAIRE.........................................................................113

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6 C DIETARY HISTORY QUESTIONNAIRE INSTRUCTIONS MATERIALS...................153 LIST OF REFERENCES............................................................................................................. 159 BIOGRAPHICAL SKETCH.......................................................................................................172

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7 LIST OF TABLES Table Page 2-1 Definitions of Dietary Re ference Intake recommendations..............................................61 2-2 Folate intake recommendations for men and non-pregnant women 19 years.................61 3-1 Description of foods categor ized within each food group.................................................71 4-1 Demographic characteristi cs of males and females...........................................................81 4-2 Mean intake (g/d) by folate categor y and gender............................................................82 4-3 Mean intake (g/1,000 kcals/d) by folate categ ory and gend er........................................83 4-4 Mean intake (g/d) by folate category and genotype........................................................84 4-5 Mean intake (g/1,000 kcals/d) by folate categor y and genotype.....................................84 4-6 Mean total folate intake (g/d) by food group..................................................................86 4-7 Mean DFE intake (g DFE/d) by food group...................................................................86 4-8 Mean folic acid intake (g/d) by food group.....................................................................87 4-9 Mean food folate in take (g/d) by food group..................................................................87 4-10 Comparison of serum and RBC folate and homocysteine concentrations by gender.......90 4-11 Comparison of mean serum folate concentrations (nmo l/L) by genotype........................92 4-12 Comparison of mean RBC folate concentrations (nmol/L) by genotype..........................92 4-13 Comparison of mean homocysteine concentrations ( mol/L) by genotype......................92

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8 LIST OF FIGURES Figure Page 2-1 Folate/folic acid structures.............................................................................................. ...60 2-2 One-carbon metabolism fo late-dependent pathway...........................................................62 4-1 Mean intake (g/d) by folate categor y and gender............................................................82 4-2 Mean intake (g/1,000 kcals/d) by folate categ ory and gend er.........................................83 4-3 Percentage of subjects who consumed sp ecific ranges of folate intake (g DFE/d).........85 4-4 Percentage of subjects who consumed specific ranges of folic acid (g/d)......................85 4-5 Percentage of total inta ke derived from each food group by folate category for males....88 4-6 Percentage of total inta ke derived from each food group by folate category for females.88 4-7 Mean percent contribution of fortified RT E cereal to total mean intake for each folate category for males............................................................................................................. 89 4-8 Mean percent contribution of fortified RT E cereal to total mean intake for each folate category for females..........................................................................................................8 9 4-9 Mean serum folate concentration (nmol/L) by gender......................................................91 4-10 Mean RBC folate concentr ation (nmol/L) by gender........................................................91 4-11 Mean homocysteine concen tration (mol/L) by gender....................................................91 4-12 Mean serum folate concentration (nmol/L) by genotype...................................................93 4-13 Mean RBC folate concentr ation (nmol/L) by genotype....................................................93 4-14 Mean homocysteine concentr ation (mol/L) by genotype................................................94

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9 LIST OF ABBREVIATIONS 125I iodine-125 1-C one-carbon AI Adequate Intake ASCII American Standard Code for Information Interchange BMI body mass index C cytosine C degrees Celcius CDC Centers for Disease Control and Prevention CH2 methylene CH3 methyl CHD coronary heart disease CRC colorectal cancer CSFII Continuing Survey of Food Intakes by Individuals CVD cardiovascular disease d day DASH™ dynamic allele-specific hybridization DFE dietary folate equivalents DHF dihydrofolic acid DHQ Dietary History Questionnaire DNA deoxyribonucleic acid DRI Dietary Reference Intakes dTMP thymidylate dUMP deoxyuridylate

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10 EAR Estimated Average Requirement F females FA folic acid FAD flavin adenine dinucleotide FDA Food and Drug Administration FF food folate FFQ food frequency questionnaire FIN food identification number GLM generalized linear models kcals kilocalories IOM Institute of Medicine L liter LC-MS/MS liquid chromatographytandem mass spectrometry M males mg milligram ml milliliter MTHFR methylenetetrahydrofolate reductase NCI National Cancer Institute NHANES National Health and Nu trition Examination Surveys nmol nanomole NTD neural tube defect PCR polymerase chain reaction RBC red blood cell

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11 RDA Recommended Dietary Allowance RFC reduced folate carrier RNA ribonucleic acid RTE ready-to-eat SAH S-adenosylhomocysteine SAM S-adenosylmethionine SD standard deviation SE standard error SNP single nucleotid e polymorphism T thymine TF total folate THF tetrahydofolic acid g microgram UL Tolerable Upper Intake Level mol micromole UNC University of North Carolina US United States USDA United States Department of Agriculture

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12 Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of F ood Science and Human Nutrition RELATIVE CONTRIBUTION OF FOOD FOLATE AND FOLIC ACID TO INTAKE AND STATUS OF YOUNG MEN AND WOMEN By Melanie Lyn Grabianowski December 2007 Chair: Gail P.A. Kauwell Major: Food Science and Human Nutrition Folate, a water-soluble vitami n essential for one-carbon metabo lism, is available in the diet as naturally occurrin g food folate or synthetic folic acid (1). Since 1998, the United States (US) Food and Drug Administration has mandated foli c acid fortification of all enriched cerealgrain products with the intent of increasing folic acid intake of women of reproductive potential to help reduce neural tube defect (NTD) risk (2). W ith the introduction of folic acid fortification, researchers have been in terested in determining the impact of fortification on folate status and intake of the population (3-6). The aim of this study was to as sess folate/folic acid intake and folate status of non-supplemen t consuming healthy young men a nd women, and to examine the relative contribution of different food categories to folate/folic acid intake. Furthermore, the relationship between folate st atus and methylenetetrahydrof olate reductase (MTHFR) 677 C T genotype was examined because of previous re ports suggesting a genotype effect on folate status. Folate status was determined for men (n = 140) and women (n = 162) (ages 18 to 49 years). Mean serum and red bl ood cell folate (RBC) concentrati ons for males (39.9 nmol/L; 810 nmol/L, respectively) did not di ffer (P>0.2) from that of females (41.7 nmol/L; 767 nmol/L). The mean plasma homocysteine co ncentration was significantly high er for males (8.0 mol) than

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13 females (6.6 mol/L) (P<0.0001). Compar isons of folate status by MTHFR 677 C T genotype for all subjects revealed that individuals with the CC genotype had higher RBC folate and lower plasma homocysteine concentra tions than individuals with the CT or TT genotypes. To estimate usual folate/folic acid intake, subjects completed a modi fied Dietary History Questionnaire. Mean total intake expressed as dietary folate equivalents (DFE ) for males and females (652 g DFE/d; 512 g DFE/d, resp ectively) exceeded the Recommended Dietary Allowance of 400 g DFE/d. Average intake of folic acid for females was 128 g/d, with only 3% of females consuming at least 400 g/d of folic acid, the amount recommended by the Institute of Medicine to reduce the risk of an NTD-affected pregnancy (7). The largest contributors to folic acid intake for males and females included enriched cereal-grain products (41.1%; 41.9%, respectively), fortif ied ready-to-eat (RTE) cereals and bars (29.3%; 36.0%), and combination foods that included “enriched” ingredients (17. 5%; 14.8%). The food categories that provided the most naturally occurring food folate in the diets of males and females, respectively, were vegetabl es (31.6%; 38.4%) and legumes and nuts (16.2%; 14.4%). The present study is one of the first to use the US Department of Agriculture National Nutrient Database for Standard Reference (Release 17) to assess dietary folate intake expressed as food folate, folic acid, total folate, and DFEs (8). Dietary folic acid from enriched cerealgrain products and fortifie d RTE cereals positively af fected total folate inta ke and status of males and females; however, folic acid intake for females was less th an the level recommended for NTD risk reduction (i.e., 400 g/d ). Daily consumption of fort ified RTE cereals may be an effective intervention strategy for increasing folic acid intake in women of reproductive potential.

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14 CHAPTER 1 INTRODUCTION Folate is a water-soluble B-co mplex vitamin that enables en zymes to transfer one-carbon groups to a variety of target mo lecules that are essential to th e regulation of one-carbon (1-C) reactions involved in py rimidine metabolism and methylation. Folate is an important nutrient that is found in the diet as naturally occurring food folate or synthetic folic acid. Foods naturally rich in food folate include da rk green leafy vegetables, legumes, and orange juice. In 1998, the United States (US) Food and Drug Administration (FDA) mandated folic acid fortification of all enriched cereal-grain products (e.g., bread, pasta, flour, rice, corn meal, and cereals, including ready-to-eat (RTE) breakfast cereals) with th e intent of reducing neural tube defect (NTD) risk by increasing daily foli c acid intake of women of reproductive potential (2). To decrease the risk of having a NTD affected pregnancy, it is currently recommended that women of reproductive age consum e 400 g/d of folic acid from supplements or fortified foods, in addition to dietary food folate (9). Fo r the general population, it is recommended that both males and females 19 years and older consume the Recommended Dietar y Allowance (RDA) of 400 g DFE/d. The RDA was based on controlled st udies on the adequacy of folate intake to maintain normal blood concentrations of folate status indicators, in cluding serum and RBC folate, and plasma homocysteine concentrations ( 10). A continuous public health challenge is to ensure that adults, especially women capable of becoming pr egnant, consume the recommended amount of folic acid intake on a daily basis. Since the FDA mandated folic acid fortifi cation, there has been public interest in determining the folate status a nd intake of adult men and women. Dietary intake of folate has been assessed in adult men and women through a variety of methods, including food frequency questionnaires. Research has c onsistently shown that folic acid fortificat ion has improved the

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15 folate status of the population compared to pre-fortification and dramatically decreased the incidence of NTDs (11-16). Ho wever, more recent studies have suggested that despite folate status improvement, women of reproductive age stil l fail to consume the recommended amount of folic acid daily (13). Furthermore, public heal th educational programs have focused attention on the recommendation that foli c acid supplements be taken daily by women of reproductive age; however, recent surveys i ndicated that only 33% of wo men reported taking a daily supplement containing folic acid (17). The data from this study provide valuable information regarding the relative and total contributions of dietary sources of folic acid to daily folic acid intake, which may help determine improved strategies for helping women meet th e recommended daily leve l of intake for folic acid. In addition, this study adds valuable information concerning the folate status of healthy young men and women. To evaluate folate status this study was also de signed to evaluate plasma homocysteine concentration, which is a known folate status indicator. Blood folate and homocysteine concentrations are inversely correl ated indicating homocys teine concentration is elevated in response to re duced folate status (18). With regard to other factors that may in fluence folate status this study provides validating evidence of a geneti c single-nucleotide polymorphism that has been extensively studied and demonstrated to influence the folate-dependent metabolic pathway of 1-C metabolism. This polymorphism is a cytosine (C) to thymine (T) substitution at base pair 677 in the gene that encodes for 5,10-methylenetetra hydrofolate reductase (MTHFR) (19). The MTHFR 677 C T variant can be characterized as homoz ygous (TT) or hete rozygous (CT) and is prevalent in approximately 12% and 50%, respectively, of th e overall population (20). In individuals who are homozygous (TT) for th e MTHFR 677 C T polymorphism, the impaired

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16 enzyme activity is associated with reduced bl ood folate and elevated plasma homocysteine concentrations, both of which can be significantly improved in res ponse to improved folate status (21). Overall Goal The goal of the pres ent study was to assess folate stat us of non-supplement consuming healthy young men and women and to estimate the relative contribu tion of folate/folic acid from different food categories to overall intake. Specific Objectives 1. To assess folate/folic acid inta ke and folate status of young ma le and female non-supplement users. 2. To evaluate the adequacy of folate intake relative to the RDA for men and women, and to evaluate the adequacy of foli c acid intake for women rela tive to the recommendation for NTD risk reduction. 3. To compare the actual and relative contribution of specific food categories to folate/folic acid intake in young male and fe male non-supplement users. 4. To compare folate status of young adu lts by folate intake and by MTHFR 677C T genotype.

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17 CHAPTER 2 BACKGROUND AND LITERATURE REVIEW Folate Chemistry Folate is a water-soluble B-co mplex vitamin that is consum ed in the diet as either naturally occurring food folate or synthetic folic acid. Folic aci d is the synthetic, monoglutamate form of folate used in fortifie d foods and supplements (1, 22). The chemical structure of folic acid, which has a molecular weight of 441.4, is formed through the co mbination of three structural features that are required for its functional activit y: a para-aminobenzoic acid molecule is linked at one end to a pterid ine ring by a methylene bridge (C -9–N-10), and at the other end joined by peptide linkage to a glutamic acid molecule (1, 23) (Figure 2-1). Naturally occurring food folates exist in various chemical forms, co ntaining a side-chain co mposed of two to ten additional glutamate residues jo ined to the first glutamate re sidue (1, 9) (Figure 2-1). The structure of the folate molecule can change through th e reduction of the pteridine ring to dihydrofolic aci d (DHF) and tetrahydofo lic acid (THF), elonga tion of the chain of glutamate residues, and 1-C subs titutions at the N-5, N-10, or bot h positions of THF (23). The polyglutamyl form of the THF mole cule can be converted to vari ous forms of folate through the transfer of methyl (CH3), methylene (–CH 2–), methenyl (–CH=), formyl (–CH=O), or formimino (–CH=NH) groups (1) (F igure 2-1). These one carbon enti ties can be transferred in diverse pathways including thos e required for the synthesis of deoxyribonucleic acid (DNA), amino acid interconversions (e.g., homocysteine to methionine), and DNA methylation. Dietary Sources Eukaryotic cells are unable to synthesize the folate molecu le in the body. Thus, folate must be consumed through diet ary sources, which include natu rally occurring food folate and

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18 foods fortified with synthetic fo lic acid. Endogenous food folate is found in a variety of foods including orange juice, dark green leafy vege tables, asparagus, stra wberries, peanuts, and legumes such as kidney beans, black beans and lima beans (9). Meat is not considered a good source of food folate, ex cept for liver. Additionally, folate concentrations are higher in raw foods than cooked foods, due to fo late losses that occur through cooking processes, with losses dependent on oxygen exposure, ascorbic acid content, and the quantity of wa ter used. Folate in plant foods can lose up to 40% of thei r folate content du ring cooking (24). Synthetic folic acid is another ma jor source of folate in the di et and is found in enriched cereal-grain products and fortif ied RTE breakfast cereals. Since January 1, 1998, the FDA has mandated folic acid fortif ication of all enriched cereal-grai n products (e.g., bread, pasta, flour, rice, corn meal, and cereals, including RTE breakfast cer eals) and mixed food products containing these grains (9). The target folic acid concentration of cereal-g rain products is 140 g folic acid per 100 g cereal-grai n, with a range in recommended amount to be added to various foods (95 to 309 g per 100 g of product) (2). The majority of RTE breakfast cereals provide 25% of the Daily Value of folic acid (~100 g/serving), with some brands providing up to 100% (400 g/serving) (9, 25). Naturally occurring food sources of folate are considered “excellent” sources of folate if they contain 100 to 200 g DFE/serving, “good” sour ces if they contain 50 to 99 g DFE/serving, “moderate” s ources if they contain 25 to 49 g DFE/serving, and “fair to poor” sources if they contai n <25 g DFE/serving (9). Total folate is the term that refers to the combination of endogenous food folate and/or folic acid from fortificat ion. Total folate intake per day is expressed as g /d. Total folate does not take into account the differen ce in bioavailability between natu rally occurring food folate and

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19 folic acid, whereas the expressi on of folate intake in terms of DFE accounts for the higher bioavailability of folic acid compared to food folate. Bioavailability The bioavailability of folate refers to the de gree to which folate is absorbed and utilized, including difference s in folate absorption, transport, me tabolism, catabolism, enterohepatic circulation and urinary excretion (25). Physiolo gical conditions, variability of food constituents, and pharmaceutical drugs can possibl y affect the efficiency of inte stinal absorption of food folate through a variety of ways: entrap ment of food folate within th e food matrix; the presence of enzyme inhibitors required for th e deconjugation of the polyglutamate form of food folate to the monoglutamate form required for absorption; the instability of THF in the acidi c pH of the stomach before absorption; and the presence of food constituen ts that may inhibit folate deconjugation (1, 25). A great deal of ambiguity ex ists among food folate resear ch with regard to potential factors affecting bioavailability, particularly the difference in bioavailability between the monoglutamate and polygl utamate folate forms, and the degree of bioavailability of food folates. Research findings reported by Mc Killop and colleagues suggest that the extent of conjugation is not a factor affecting the efficiency of absorption of food folate in the intestines (25). Variation in bioavailability reported in pr evious research could be due to differences in fo late digestion, absorption and metabolism, as well as inconsistencies between pr otocols and anal ytical methods used to measure and report bioa vailability. Alcohol and pharmace utical drugs also can inhibit the absorption and metabo lism of folate (26). Estimates of the bioavailability of food folate relative to folic acid have varied in the measured percentage of ab sorption, ranging from 10 to 98% (25, 27-32); however, the best

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20 estimate is provided by data from Sauberlich a nd colleagues indicating that food folate is approximately 50% bioavailable re lative to folic acid (9, 30). Wh en folic acid is consumed as a supplement in the fastin g state or without food, it is 100% bioavailable (33). Folic acid absorption is slightly reduced and estimated at 85% bioavailable when fo lic acid is consumed with food, which is the case with enriched cereal-grain products or fortified foods (10, 34, 35). The physiological requirement fo r folate represents the amoun t the body requires post-folate absorption. Considering th e differences in folate bioavailabilit y, a larger quantity of food folate compared to folic acid must be ingested to meet the physiologica l requirement (9). These data coupled with the estimate that f ood folate is approximately 50% bioavailable were used by the Dietary Reference Intake (DRI) Committee of the Institute of Me dicine (IOM) to develop the concept of DFE, a unit of me asure that acknowledges differe nces in bioavailability and absorption among the various chemical forms of the vitamin (9). The DFE values for folic acid in enriched cereal-grain products and vitami n supplements are calcu lated using conversion factors that account for differences in bioavailability relative to naturally occurring food folate (9). Folic acid in supplemental form taken while fasting is estimat ed to be twice as bioavailable relative to food folate (i.e., 100: 50). Folic acid from supplements taken with food or folic acid from enriched or fortified food sources is estim ated to be 1.7 times more bioavailable than naturally occurring food folate (i .e., 85:50) (9). The total folate content of any food product in DFE units can be calculated by mu ltiplying the micrograms of synt hetic folic acid by 1.7 then adding this value to the micrograms of food folate present (9). No calculation or adjustment is needed when solely determining food folate intake.

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21 Absorption, Transport and Storage Folate must be in the monoglut amate form in order for abso rption to occur. Synthetic folic acid is already a monoglutamate, and thus readily absorbed. However, since food folates are polyglutamates, they must first be hyd rolyzed by the enzymatic action of folypolyglutamate carboxypeptidase II, also known as pteroylpolyglut amate hydrolase or folate conjugase, before absorption can occur (36) Following complete deconjugation of the polyglutamate molecule, folates are transported across the brush border membrane in the jejunum via an acidic pH-depende nt (pH of 6.5 to 7.0) carriermediated mechanism (37, 38). Drugs or diseases that impair jejunal pH may impair folate absorption (1). Prior to entry into the portal system, the majority of fola te is reduced by dihydrofolate reductase to DHF and THF (36). Once folate is absorbed, it travels to the liver via portal circulation, primarily in the form of 5-methylTHF. To allow for folate storage and retention in the liver, folates are reduced a nd conjugated. A small fraction of 5-methylTHF in the plasma can be present as free folate or bound with high affinity to a fola te binding protein; however, the majority of plasma folate is bound with low affinity to albumin (23, 39, 40). Two mechanisms exist for transport of folate into the cells: a fola te transporter and a folate receptor (41, 42). These transport mechanisms are required for absorption of folate in the small intestine, reabsorption of endogenous folate by the kidneys, and uptake of folate through the plasma membrane into a deve loping embryo (41). The reduced folate car rier (RFC) is the folate transporter in the intestinal epithelial cells and is a clas sic facilitated transport protein that carries reduced folate (5-methyl THF) across the membrane; it is encoded by the RFC gene (RFC1), which is expressed in mo st tissues and is capable of mediating bi-directional flux of folate. The second transport mechanism is a fo late receptor-mediated process by which folate

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22 binds with high-affinity to a folate-receptor at the membrane surf ace and is transported into the cells via a unidirectional system. The efficiency of cellular uptake of the carrier-mediated process is much greater than th at of the receptor–mediated pro cess (41, 42). Pharmacological quantities of folic acid are absorbed via diffusi on as absorption by the RFC is a saturable process (38). Once cellular uptake of 5-me thylTHF is complete, it is demethylated by methionine synthase and converted to th e polyglutamyl form through th e action of folylpolyglutamate synthetase. This mechanism allows for intracellu lar folate retention becau se polyglutamates are unable to cross the cell membrane ( 43). Previous measurements of folate concentrations in liver tissue indicate that the liver specifically stores approximately 6 to 14 mg of folate. Based on the assumption that the liver stores 50 % of total body folate, it is es timated that the body stores 15 to 30 mg of folate (44). Metabolism and Excretion When folate polyglutamates are released from tissues into circula tion, the polyglutamate form of folate molecules is rec onverted to the mo noglutmyl form by -glutamylhydrolase. The mechanisms of polyglutamation and monoglutama tion are necessary for the function of 1-C metabolism. The majority of circulating folate is reabsorbed in the pr oximal tubules of the kidney primarily via a folate receptor (45-47). Folate may also be packag ed as part of bile (~100 g/d), concentrated and stored in the gall bladder, and subsequently secreted into the intestinal tract. The majority of folate in bile is recycled during entero hepatic circulation, although some may be excreted in the feces along with unabsorbed dietary folate, folate from endogenous secretions, and folate synthesized by bacteria in the gut (48). Re search findings have determined that folate losses via fecal and urinary excreti on are comparable and represent approximately 0.5

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23 to 1% of folate (48, 49). These loses must be replaced da ily through dietary folate or supplemental folic acid to mainta in normal folate metabolism. Dietary Reference Intakes Dietary Reference Intakes represent the mo st current recommendations for each vitamin and mineral and include for each nutrient the Estimated Average Requirements (EAR), RDA, Adequate Intake (AI), and Tolerable Upper Limit (UL). See Table 1-1 fo r definitions of each DRI (10). The most recent DRI recommendations were published by the National Academy of Sciences IOM in 1998 (10). The previous recommendations for nutri ent intake were established to prevent clinical deficiencies. In establ ishing the 1998 DRI recommend ations, the IOM shifted the focus of recommendations from nutrient quantities needed to prev ent clinical deficiencies to intakes that ensure optimum hea lth (10). Dietary Reference In take recommendations for folate are presented in Table 1-2. The RDA for men and non-pregnant women 19 years and older is 400 g DFE/d. The recommendations are increas ed to 600 g DFE/d and 500 g DFE/d during pregnancy and lactation, respectivel y. Since folate is not associat ed with toxic side-effects, the UL of 1,000 g/d of syntheti c folic acid is based solely on the concern that folic acid supplementation can mask the dia gnosis of a vitamin B12 deficiency, which is prevalent in approximately 10 to 15% of the elderl y population over 60 year s of age (10, 50). The IOM also recommends that all women of childbearing age co nsume 400 g/d of synthetic folic acid from fortified foods and/or supplements in ad dition to consuming food folate from a varied diet to reduce th e risk of having an NTD-affected pregnancy (9). However, the achievement of the latter is problematic as compliance with the supplementation recommendation is often suboptimal It is estimated that ap proximately 33% of women of

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24 reproductive age take a daily foli c acid-containing suppl ement (17). Evidence for an association between folic acid intake and NTD risk reduction provided the basi s for the mandat ed folic acid fortification of cer eal-grain products. Biochemical Functions Interconnected reactions requi ring specific forms of folate to accept and transfer onecarbon units, together referred to as 1-C metabolism, incl ude amino acid metabolism, homocysteine remethylation, pur ine and pyrimidine synthesis, and the generation of Sadenosylmethionine (SAM) (1). One-carbon me tabolism is a series of folate-dependent pathways that requires the don ation of one-carbon units from individual folate coenzymes resulting in the regeneration of THF. An illust ration of the 1-C metabolism pathway is presented in Figure 2-2. In 1-C metabolism, THF is the primary acceptor molecule and is required for continuation of the cycle allowing for nucleotide biosynthesis and meth ylation reactions (1). Key-Folate Dependent Reaction s in One-Carbon Metabolism Nucleotide biosynthesis. Through a reversible reaction, THF is converted to 5,10methyleneTHF in association with the conversion of serine to glycine, both of which are nonessential amino acids, by the vitamin B6-dependent enzyme se rine hydroxymethyltransferase (SHMT). DNA synthesis is dependent upon the availability of 5,10-me thyleneTHF, which can donate a one-carbon unit to deoxyur idylate (dUMP) through the acti on of thymidylate synthase to irreversibly synthesize t hymidylate (dTMP). This rate-l imiting step is essential for DNA synthesis to proceed. During the process of dona ting a one-carbon group, 5,10-methyleneTHF is oxidized to form DHF and reduced back to THF by dihydrofolate reductase. Additional nucleotide production requires 5,10-methyleneTH F for the formation of 10-formylTHF by 10-

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25 formylTHF synthetase, which can be used to donate multiple one-car bon units for purine synthesis and the regene ration of THF for recirculation (1, 51). Methylation reactions. The folate-dependent reactions of 1-C metabolism hinge on the production of 5,10-methyleneTHF, wh ich is also required for severa l other functional outcomes. In order for the remethylation of methionine from homocysteine to occur, 5,10-me thyleneTHF is first reduced by MTHFR to 5-methyl THF. The 5-methylTHF form of folate, in conjunction with methionine synthetase and cobalamin (vitamin B12), irreve rsibly donates a one-carbon group to cobalamin to become methyl cobalamin. The methyl gr oup from methyl cobalamin is transferred to homocysteine to form methionine, and 5-methylTH F is ultimately converted to THF. Regeneration of methi onine and THF is required fo r the 1-C metabolism cycle to continue. During a cobalamin deficiency, a seco ndary folate deficiency often occurs. The “methyl-trap hypothesis” suggests the latter emer ges because there is not enough cobalamin to accept and donate a one-carbon unit to homocysteine (52). Thus, folate is “trapped” within 5methylTHF, and THF is not rege nerated to continue the cycle through the synthesis of dTMP. Consequently, DNA replication and ce llular mitosis are interrupted. Methionine is essential for the formation of S-adenosylmethionine (SAM) by the enzyme SAM synthase and activation by adenosine 5’-triphosphate (ATP). Methionine, an essential amino acid, can be derived from di etary protein intake or by ge neration from homocysteine. SAM is the primary methylating agent that don ates a methyl group, originally accepted from 5methylTHF, that is used in more than 100 meth yltransferase reactions, including the methylation of DNA, ribonucleic acid (RNA), protein, phospholip ids and neurotransmitters (53). Once SAM donates its methyl group, it is converted to S-adenosylhomocys teine (SAH) by various cellular methyltransferases. SAH is hydrolyzed by S AH hydrolase to homocys teine and adenosine

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26 through a reversible reaction in wh ich SAH is favored in the bala nce. SAM also regulates the production of 5-methylTHF. Wh en dietary methionine is ad equate, SAM will inhibit MTHFR from producing 5-methylTHF and thus methionine; whereas, when the availability of methionine is low, the concentration of SAM will be decrease d (54). This releases the inhibition of MTHFR to allow for greater production of 5-methyl THF and regeneration of methionine (54). SAM also regulates the pyri doxal phosphate-dependent re action in which homocysteine is catabolized to cystathionine in combination with serine and the enzyme cystathionine synthase. The transsulfu ration pathway catabolizes excess homocysteine not required for methyl transfer reactions. This transsulfuration pathwa y ultimately leads to the synthesis of cysteine (non-essential amino acid) and gl utathione (antioxidant) (1). Methylenetetrahydrofolate Reductase (MTHFR) Polymorphism Methylenetetrahydrofolate redu ctase, the enzyme required to reduce 5,10-methyleneTHF to 5-methylTHF, is the main circ ulating form of folate and the major methyl donor for the remethylation of homocysteine to methionine. The most extensively st udied polymorphisms related to folate metabolism are MTHFR polymorphi sms. Frosst and collea gues discovered that individuals homoz ygous for the 677C T polymorphism (TT genoty pe) have decreased MTHFR enzyme activity resulting in reduced levels of 5-methylTHF available to donate a methyl group for the remethylation of homocyste ine to methionine (19). This substitution renders the enzyme “thermolabile” and may cause elevated plasma levels of th e amino acid homocysteine. The homozygous TT genotype is termed thermolabile because the enzymatic activity is decreased compared to the CC genotype (55). Kang and colleagues disc overed the TT genotype had a specific enzymatic activity of approximately 50 % of the normal levels seen with the CC genotype (55). Decreases in MTHFR func tion have been asso ciated with mild

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27 hyperhomocysteinemia and may theref ore be a risk factor for the development of cardiovascular disease (CVD) (56). Approxi mately 12% of the population is homozygous for the 677C T variant (i.e., TT genotype), wherea s approximately 50% is heteroz ygous (i.e., CT ge notype) (20). Matthews and colleagues charact erized the decrease in MT HFR enzyme activity through an increased dissociation of flavin adenine dinucleotide (FAD) from thermolabile MTHFR (56). Furthermore, these researcher s found that folic acid supplementation provided protection by increasing the affinity of the enzyme for FAD. These results agreed with the findings by Guenther and colleagues (57) w ho also found that disso ciation of FAD resulted in a loss in MTHFR enzyme activity. These investigator s monitored enzyme activity and found the TT genotype loses activity at a rate ten times faster compared to the enzyme activity as sociated with the CC genotype (57). Folate Status Assessment Serum and Red Blood Cell Folate Concentrations Serum folate concentration best reflects curren t and recent intakes of folate and is an early and sensitive measure of short-term folate st atus (58). Controlled, metabolic studies have shown that serum folate concentr ations decrease quickly within a period of one to three weeks when folate intake is limited (21, 52, 59). Th ese metabolic changes pr ecede decreases in RBC folate concentrations. The lower limit of normal used to define inadequate serum folate status using the microbiol ogical assay is 13.6 nmol/L (60). An alternat ive method for assessing folate status is the radiobinding assay, which has been shown to yield lowe r blood folate values relative to the microbiological as say (61). The lower li mit of normal for the ra diobinding assay is <7 nmol/L.

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28 Red blood cell folate is considered a better indicator of long-te rm folate status than serum folate concentration as this marker reflects tissu e folate storage. A pr evious study using liver tissue biopsies found that RBC folate concentrations correspo nd to liver folate concentrations (62). Uptake of folate into erythrocytes only occurs in the bone marrow during the early stages of erythropoeisis. Since fola te cannot permeate the membrane of a mature RBC during its 120day lifespan, RBC folate reflects folate status over the pr eceding three months. The lower limit of normal used to define inad equate RBC folate status us ing the radiobinding assay is 317 nmol/L (10, 52). Plasma Homocysteine Concentration In addition to measuring blood folate concen trations, other “functiona l” indicators should be used to evaluate folate stat us as these indices may reflect a bnormalities in metabolic function that may or may not be reflect ed in altered blood folate con centrations. The most notable “functional” indicator is to tal plasma homocysteine concen tration (63). An elevated homocysteine concentration reflect s not only a reduction in blood fo late concentration secondary to inadequate folate intake, but also an insufficient concentra tion of 5-methylTHF required to convert homocysteine to methionine in the 1-C metabolism pathway (10, 18). Plasma homocysteine concentration has been shown to be directly and inversely co rrelated with folate status; however, it is not specific to folate deficiency because ot her nutrient deficiencies (i.e., vitamins B12 and B6), genetic a bnormalities, renal insufficiency, a nd dietary and lifestyle factors may influence homocysteine concen tration (64, 65). Although disc repancies exist when defining acceptable homocysteine concentrations, individu als with a plasma homoc ysteine concentration 12 mol/L are often considered to be at higher risk for adve rse health effects, including vascular disease (4).

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29 To assess homocysteine status in the US Ganji and colleagues (66) presented the distribution of plasma homocystei ne concentrations of adults in the US by using data from the National Health and Nutrition Examina tion Surveys (NHANES) 1999-2000 and 2001-2002. These researchers studied the hom ocysteine distribution by age, gender and race-ethnicity in 9,196 subjects. Results showed th at plasma homocysteine concentr ations were higher in men than in women (aged 19-70 years) and in older persons compared to younger persons (66). In a preceding study, these researchers also repor ted that mean circ ulating homocysteine concentrations in the period si nce folic acid fortification we re 7.6 mol/L in 1999-2000 and 7.9 mol/L in 2001-2002, as compared with 9.5 mol/L during the peri od prior to fortification (1994 to 1998) (67). Folate Deficiency Etiology In addition to inadequate fola te in the diet, a folate defi ciency can develop through other causes, such as drug-nutrient interactions, genetic variations impaired intestinal folate absorption or folate metabolism secondary to high alcohol consumpti on, and increased renal folate excretion. High doses of nonsteroidal anti-inf lammatory drugs (i.e., aspirin, ibuprofen, and acetaminophen) have been shown to exert an tifolate activity (68-71). However, impaired folate status has not been reporte d with low doses of these drugs. Folate deficien cy associated with genetic variations, alcohol consumption and dietary defi ciency are discussed in the following sections. Megaloblastic Anemia The development of a folate deficiency is associated with a progression of events that can eventually lead to megaloblastic anemia seen with chronic severe folate deficiency. The

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30 sequence of events begins with a reduction in serum folate concentration, which can rapidly decrease within one to three we eks. Following a long phase of folate deficiency, RBC folate concentration will also decline (58). The most common etiology of megaloblastic anemia is impaired DNA synthesis, linked to abnormalities in the folate path way (1-C metabolism). A decrease in the availability of folate from circulation and tissues will lead to a decline in DNA synthe sis, reducing cell division, and ultimately resulting in the formation of large, immature RBCs characteristic of megaloblastic anemia. Abnormalities in RBC fo rmation will lead to decreases in hemoglobin, hematocrit, and RBC number. Clinical manifestations of megaloblastic anemia can develop after a period of compromised folate status. Folate status ca n be compromised and a folate deficiency can develop through various factors in cluding: inadequate folate in take; malabsorption induced by abnormalities of the small intest ine, drugs or alcohol; altered metabolism; increased requirement (i.e., pregnancy, diseases); increased losses (i.e., dialysis, some sk in diseases); abnormalities of folate metabolism; and inherite d abnormalities of folate ab sorption and metabolism (72). Pregnancy is a common cause of megaloblas tic anemia in women. Folate requirements increase five to ten fold for pr egnant women relative to non-pre gnant women. The higher folate requirement during pregnancy is re lated to increases in cellular proliferation s econdary to the growth and development of the fetus, placenta, and maternal tissues ( 73, 74), rather than an increase in folate catabolism as substantiated by Caudill and coll eagues (75). This elevated demand for folate must be met by adequate dietary intake.

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31 Neural Tube Defects Neural tube defects are c ongenital malformations that o ccur in the brain and spinal column that are caused primarily when closur e of the neural tube fails during embryonic development (76). The development and closure of the neural tube of an embryo occurs within 28 days after conception, often be fore women are aware they are pregnant. The extent of an NTD varies with different degr ees of tissue protrusion and neur al involvement. The two most common forms of NTDs are anencep haly (failure of the brain to develop) and spina bifida (exposure of the spinal co rd due to defective clos ure of the neural tube). In some instances, defects in the orofacial cleft can develop from an abnormal increase in cerebrospinal fluid pressure on the closed neural tu be during the first trimester of embryonic development (76). The incidence of NTDs (anencephaly or spina bifida) is 0.75 cases pe r 10,000 births in the US, with roughly 400,000 cases per year worldwide (77) It has long been suspected th at a cause-and-effect relations hip exists between diet and NTDs. Hibbard was one of the first researchers to address the importance of folate during pregnancy. He proposed that the incidence of megalobl astic anemia in mid to late pregnancy may have resulted from a defect in folate metabolism (78). Shor tly after, Hibbard hypothesized that fetal spontaneous abortions an d placental abruption were associated with folate deficiencies during pregnancy (79). In 1976, Smithells and collea gues further sugges ted that folate deficiency was linked to NTDs because women who gave birth to NTD-affected infants had low blood folate concentrations (80). These investigators reported that supple mentation with folic acid consumed periconceptionally reduced the recurrence of NTD pregnancies in high-risk mothers. These results spawned further research studies that have continued to explore the benefits of folic acid on reducing the incidence of NTDs. The pow erful driving force in folate

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32 research was the report from the Medical Resear ch Council (MRC) Vitamin Study Research Group (81). In 1983, the MRC la unched a large-scale, rando mized, double-blind, prevention trial evaluating the effect of multivitamin supplementation with and without folic acid on the recurrence of NTD-affected pr egnancies. The study was ende d early because 72% of NTDs were prevented with 4 mg/d of supplemental folic acid (81). Moreov er, in 1992, Czeizel and Dudas found that periconceptiona l folic acid supplementation ( 800 g/d) decreased first time occurrence of NTDs in Hungarian women comp ared to subjects receivi ng no folic acid (82). Further research led to the conc lusion that folic acid supplemen tation can prevent up to 70% of NTDs (83). A subsequent intervention study conducted by Berry et al. (84) s upported the work of earlier investigators who observed a relationship between folic ac id and NTDs. Berry and his team investigated the effect of periconceptional use of folic ac id in Chinese women living in northern regions of China where NTDs rates were high and southern regions of China where NTDs rates were low (84). Among the fetuses or infants of wome n who did not take folic acid, NTDs rates were 4.8 and 1.0 per 1,000 pregnancies in the northern and southern regions of China, respectively; wh ereas, 400 g/d of foli c acid reduced the occu rrence of NTDs in the northern and southern regions of China to 1.0 and 0.6 per 1,000 preg nancies, respectively. These NTD rate decreases are equivalent to a respective 79% a nd 40% reduction in the incidence of NTDs in the northern an d southern regions. In a study of 56,000 Irish women, Daly and co lleagues (85) examined the relationship between blood folate concentration and number of NTDs. The investigat ors observed that as RBC folate concentration increased, the risk for NTDs decreased. They al so reported an eightfold difference in the incidenc e of NTDs between women with RBC concentrations <341 nmol/L

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33 and women with concentrations 908 nmol/L (85). It was es timated that a RBC folate concentration of 908 nmol/L could be achieved with 400 g/d of foli c acid. In a case-control study by M oore and colleagues, investigat ors reported an NTD risk of 3.4 cases per 1,000 pregnancies for women who c onsumed <150 g DFE/d. Risk for NTDs significantly decreased by 77% to 0.8 cases per 1,000 pregna ncies for women who consumed 1,200 g DFE/d. These researchers concluded th at the prevalence of NTDs decreased by 0.78 cases per 1,000 pregnancies with folate intake increments of 500 g DF E/d (86). These data suggest that NTD risk d eclined significantly with modest increases of to tal folate intake during early pregnancy. Overall, an overwhelming amount of scient ific evidence supports an association between adequate fola te status/folic acid intake an d NTD risk reduction (31). MTHFR polymorphism and neural tube defects The MTHFR 677C T polymorphism was identified as the first genetic risk factor for NTDs. The MTHFR 677C T mutation, as previously reviewed, causes redu ced activity of the en zyme. The homozygous mutation (TT genotype) is associ ated with an elevated plasma homocysteine concentration and reduced RBC and plasma folate concentrations. It has also been suggeste d that individuals with the TT genotype have higher folate requirements (87). Proposed mechanisms for folate-responsiv e NTDs include decreased rates of DNA synthesis and cell division due to impaired dTMP synthesis, a nd a reduction in DNA methylation (83). Embryonic tissues grow very rapidly, with high requir ements for DNA synthesis and methyl groups from SAM (88). Individuals homozygous for the MTHFR genotype may need more folate in their diets to pr ovide adequate amounts of methyl groups and to provide substrate for the DNA methylation and synthesis via 1-C metabolism. During ea rly pregnancy, a mild deficiency can impair DNA synthesis and met hylation, the latter of wh ich can affect gene

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34 expression. Disordered gene expression may explain how a relatively mild deficiency can initiate the development of severe neural tube defo rmities in an embryo (88). van der Put and colleagues (89) exam ined the frequency of the MTHFR 677C T polymorphism in patients with sp ina bifida (n = 55) and their pa rents (n = 130) and controls who did not have spina bifida or offspring with spin a bifida (n = 207). On ly 5% of the control subjects were homozygous for the TT genotype, whereas 13% of pa tients with spina bifida had the TT genotype, as well as 16 % and 10% of their mothers an d fathers, resp ectively. The researchers found that all subject s and controls with the TT genotype had decreased MTHFR activity, low plasma folate concentrations, a nd high plasma homocysteine and RBC folate concentrations (89). Th ese researchers later repor ted that the risk of ha ving offspring born with spina bifida was strongest when both the mother a nd her child carried the homozygous variant genotype (TT) (90). van der Put a nd colleagues conclu ded that the 677C T polymorphism should be regarded as a genetic ri sk factor for spina bifida. Vascular Disease and Stroke Several epidemiological studies have confir med an inverse association between folate and homocysteine concentrations based on the metabolic role of folate as a coenzyme in the regulation and methylation of homocysteine in 1-C metabolism. Furthermore, studies have indicated the role of an el evated plasma homocysteine concentration, also known as hyperhomocysteinemia, as a signif icant risk factor for atherosc lerotic vascular and coronary heart disease. Homocysteine may increase va scular and coronary h eart disease risk and atherogenesis through direct toxicity to endo thelial cells via lipid peroxidation, increased coagulation and platelet aggregation, decreased endothelial cell reactivity and stimulation of smooth muscle cell pr oliferation (91-94).

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35 To establish if there is a dose-dependent e ffect of folic acid su pplementation on plasma homocysteine concentration, meta-a nalyses of randomized controlle d trials have been conducted (95). Specifically, the Homocysteine Loweri ng Trialists’ Collaborati on reported results from randomized controlled trials in two different meta-analyses (1998 and 20 05) to determine the size of reduction in homocyste ine concentration achieved by di fferent daily supplemental doses of folic acid, with or without vi tamins B12 or B6 (96, 97). The first meta-ana lysis revealed that the effect of folic acid on blood homocysteine concentra tion seemed to depend on the pretreatment blood homocys teine and folate concentrations (9 7). Participants with lower blood folate concentration or higher baseline plasma homocysteine concentration experienced the greatest improvement from supplements containing folic acid. After adju sting for pretreatment blood homocysteine and folate concen trations, results indicated that daily folic acid doses of <1 mg/d (mean = 0.5 mg/d), 1–3 mg/d, and >3 mg /d lowered homocysteine concentration by approximately 25%. The second meta-analysis provided new data regarding a folic acid dose-dependent lowering effect on homocysteine co ncentrations (96). Baseline plasma folate and homocysteine concentrations before treatment with folic acid were standardiz ed at 12 nmol/L and 12 mol/L, respectively. Results suggested that doses 800 g/d of folic acid ar e typically required to achieve maximum reductions in plasma homocys teine concentrations produced by folic acid supplementation. Folic acid doses of 200 and 400 g/d were associat ed with homocysteine reductions of 60% and 90%, re spectively, from baseline levels. The results revealed that 400 g/d of folic acid produced near-maximum lowering effects in plasma homocysteine concentration, which may be signifi cant to public health (96).

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36 Considering the role of supplemental foli c acid in lowering ho mocysteine, the main unanswered question is whether folic acid supple mentation will reduce the overall rate of vascular disease. Cardiovascul ar disease is the leading cause of death in the US, accounting for approximately 37% of all deat hs (98). Between 1980 and 1994, Rimm and colleagues (99) examined the intakes of fola te and vitamin B6 of 80,082 wome n in the Nurses’ Health Study Cohort in relation to the incidence of nonfatal myocardial infarction (MI) and fatal coronary heart disease (CHD). The results revealed an approximate 30% lo wer relative risk of CHD for women with higher folate intake (~700 g/d) compared to intake s at or below the RDA at that time (~160 g/d), after controlling for vascular disease risk factors (i.e., smoking, hypertension, alcohol consumption, fiber, vitamin E, and saturated, polyunsaturated, and trans fat) (99). Because this study was conducted be fore implementation of folic acid fortificati on, the largest contributor to the overall inta ke of folate was multivitamins followed by RTE cereals, orange juice, lettuce, eggs, broccoli, and spinach (99). The resear chers noted that th eir findings were also consistent with previous data linking higher intakes of folate with lower homocysteine concentrations. However, one li mitation to this study was that it was an observational study, and the individuals who fell into the highest range of folate intake might have had overall “healthier diets” and were taking supplements that might have co ntained other nutrient s that could have affected CHD risk. In addition, Refsum and colleagues (100) c onducted a meta-analysi s in 1998 including data from 80 clinical and epidem iological studies (~10,000 pa tients). The results of this analysis provided unequivocal evidence th at hyperhomocysteinemia is a common, independent CVD risk factor in the general population, and may also enhance the effect of other conventional risk factors (100). Because supplementation with B vita mins, in particular foli c acid, is an efficient,

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37 safe, and inexpensive means to re duce an elevated homocysteine level, studies have tried to establish whether such therapy will reduce CVD risk. A meta-analysis conducted by the Homocysteine Studies Collaboration assessed the relationship of homoc ysteine concentrations with primary prevention of vasc ular disease and determined that an estimated 25% lower usual homocysteine concentr ation was associated with approximately 11% lower ischemic heart disease risk and 19% lower stroke risk (101). Moreover, a meta-analysis by Bazzano and co lleagues (102) summarized the results of twelve randomized, controlled c linical trials on dietary supplem entation with folic acid on CVD risk reduction. These studies in cluded data from 16,958 participan ts with preexisting vascular disease. Folic acid supplementa tion was not shown to reduce re currence risk of CVD or allcause mortality among subjects with a prior history of vasc ular disease (102). It is possible that folic acid supplementation may ha ve a protective effect on risk reduction and prevention of a primary rather than secondary occurrence of a vascular even t. Several ongoing randomized, controlled, clinical trials in th e US, Canada, Europe and Australia with large sample sizes as part of the B-Vitamin Treatment Triali sts’ Collaboration mi ght provide a more de finitive answer to the role of folic acid on primary risk reduction (103). Although the results of these studies are uncertain, subjects enrolled in tr ials conducted in the US and Cana da are expected to have lower baseline homocysteine concentrations because folic acid fortification is mandatory in these regions and has previously been associated with significant re ductions in plasma homocysteine concentrations. These ongoing intervention trials in the US and Canada may not observe the same decreases in homocysteine that might appear in populations not exposed to fortification (11). Furthermore, it is diffic ult to detect a cause-and-effect relationship between folic acid supplementation and vascular dis ease risk secondary to difficul tly in controlling for other

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38 nutrients in the diet (i.e., vita mins B12 and B6) that may signifi cantly lower homocysteine, as well as the fact that the underlying causes of vascular disease are multif actorial (99, 100, 102). Research studies have also be en conducted to investigate th e efficacy of folate therapy on stroke prevention. Wang and coll eagues conducted a meta-analysis of eight randomized trials of folic acid that reported stroke as an endpoint (104). The data indi cated that folic acid supplementation significantly reduced the risk of stroke by 18%, a nd an even greater beneficial effect was seen in those trials in which homoc ysteine concentration wa s reduced by more than 20%, treatment duration was more than 36 m onths, no fortification program or partial fortification of grains had been introduced, and there was no hist ory of stroke. Investigators concluded that the inverse relationship det ected between the duration of folic acid supplementation and the risk of stroke suggests that folic acid supplem entation can effectively reduce the risk of stroke in primary prevention (104). MTHFR polymorphism and vascular disease Studies have also indicated that individuals with the homozygous variant (TT) of the MTHFR 677 C T polymorphism have elevated plasma homocys teine concentrations compared to individuals who ex press either the heterozygous (CT) or normal (CC) genotypes when folate status is low. Jacques and colleagues (105) assessed the poten tial interaction between the MTHFR 677 C T polymorphism and folate status in homocysteine metabo lism. This study in cluded 365 individu als from the National Heart, Lung, and Blood Institute Family Hear t Study. The investigators found that among individuals with lower plasma folate concentrations (<15.4 nmol/L), those with the TT genotype had a mean total fasting homocysteine concentration (12.1 mol/L) that was 24% greater than individuals with the CC genotype (9.8 mol/L). A differ ence in homocystein e concentrations between the TT and CC genoty pes (7.9 and 7.8 mol/L, respec tively) was not seen for

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39 individuals with a fo late concentration 15.4 nmol/L (105). A meta-a nalysis by Brattstrom and colleagues (106) combined the re sults of studies that have do cumented plasma homocysteine concentrations in relation to the MTHFR genotypes (C C, CT and TT). Similar to previous findings, the meta-analysis reveal ed that individuals with the TT genotype had higher mean homocysteine concentration (25% or 2.6 mol/L increase) than su bjects with the CC genotype when both populations had suboptimal folate status. A differ ence in homocysteine concentrations among the MTHFR genotypes was not detected when folate status was high. Ultimately, these studies concluded that the MTHFR 677C T mutation is a ma jor cause of mild hyperhomocysteinemia and that i ndividuals are at greater risk for hyperhomocysteinemia when they have the MTHFR 677 TT genotype coupled with low serum folate concentrations. However, there appears to be littl e evidence that individuals with the TT genotype are at greater risk for vascular disease. Similarly, Kauwell and colleagu es reported data suggesting that older women with the TT genotype may be at gr eater risk for elevated plasma homoc ysteine in response to low dietary intake of folate in comparison to older women with the CC or CT genotypes (59). Studies investigating the eff ects of the MTHFR 677 C T polymorphism on homocysteine metabolism suggest that individuals with the TT genotype may have higher folate requirements to maintain adequate folate status and pr event hyperhomocysteinemia. Cancer Compelling evidence from epidemiologic, animal and human studies suggests that folate status is associated with risk reduction for developing several ty pes of cancers, including breast, cervical, pancreatic, brain, lung, and colorectal cancer (CRC) (107), al though more recent evidence is contradictory to the latter. Research also suggests that fo late depletion enhances

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40 carcinogenesis, whereas large dos es of supplemental folic aci d, above what is presently recommended for the general popula tion, appear to have a protecti ve effect. Although the exact mechanism is still uncertain, the beneficial effect of folate on carcinogenesis is speculated to come from the metabolic role of folate in DNA methylation and st ability (107). When there is a deficiency of folate in circula tion, dTMP synthesis is impaired and results in nucleotide errors that incorporate uracil in to DNA stands. This may lead to th e instability of DNA strands that are associated with increased cancer risk. Moreover, folate can a ffect cellular SAM levels, which regulate DNA methylation and gene transcrip tion by methylating specific cytosines in DNA (108). As a consequence of fo late deficiency, SAM is deplet ed, which in turn induces DNA hypomethylation and potentially indu ces expression of cancer-prone genes (109). Research has shown that folate administrati on can repair DNA synthesis by reversing excessive DNA uracil misincorporation thereby reduci ng the number of chromosomal br eaks, and supplying adequate methyl groups for DNA methylati on and reducing expression of cancer-related genes (110). Significant evidence from population cohorts from the Nurses’ Health Study (15,984 women) and Health Professional Follow-Up St udy (9,490 men) supports the association between folate and CRC risk (111). Thes e epidemiological studies revealed a 30 to 40% reduction in the risk for CRC and adenocarcinomas in participants with the highest folate intake (>700 g/d), the majority of who were supplement us ers, compared to those with th e lowest intake of folate (166 and 241 g/d for women and men, respectively) (111). In the Nurses’ Health Study, a reduction in the risk for CR C was not associated with folate intake from food sources alone (111). Ho wever, when total folate intake exceeded 400 g/d, a 30% lower risk was observed compared to the gr oup of women who consumed 200 g/d. It was noted that of the group of women who consumed 400 g/d of folate, 86%

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41 consumed multivitamins. An appa rent association between length of supplement use and risk for cancer was also noted. Women who had been consuming supplem ents containing folic acid for at least 15 years had a 75% decr eased risk for CRC co mpared to non-supplement users; whereas no significant reductions in risk were observed in women who had been taking multivitamins for shorter lengths of time (111). In contrast, more recent population-based obser vations from two data sets have suggested that the incidence of CRC in North America increased as a result of folic acid fortification. Mason and colleagues (1 12) noted a surge in absolute rate s of CRC in 1996 to 1998 (US) and 1998 to 2000 (Canada), despit e a downward trend in CRC incide nce in both countries during the decade prior to fortification. These investigators attributed this inci dence to the role of folate in nucleotide synthesis, includi ng the vitamin’s role as a potential gr owth factor for neoplastic cells. Evidence indicating that high doses of folic acid can accelerat e the growth of established neoplasms has been documented in previous research (113, 114). Subsequent research also noted that supplemental folic acid is only protective before neoplastic cells appear in the intestine. Once these proliferative cells ar e established, microscopic and macroscopic tumors develop faster as more folic acid is administered (115, 116). The observations by Mason and colleagues that rates in CRC spiked as a resu lt of fortification suggest that fortification “unveiled” a significant numbe r of suppressed neoplasms that would have otherwise never transformed into cancers or woul d have gradually evolved into ca ncer over a l onger period of time (112). In a double-blind, placebocontrolled, randomized clinical tr ial (the Aspirin/Folate Polyp Prevention Study) conduced by Cole and coll eagues (117), 1,021 participants with recently removed colorectal adenomas were randomized to receive either 1 mg of fo lic acid or a placebo

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42 over a period of three to five y ears. Analysis of the final foll ow-up colonoscopy did not reveal a protective effect of foli c acid supplementation; ra ther, incidence of at least 1 recurrent advanced lesion was significantly higher for folic acid ( 11.6%) compared to the placebo (6.9%) (P = 0.05) (117). Further research is need ed to investigate th e potential of folic acid supplementation to accelerate the growth of existing neoplasms and in crease risk of CRC. In contract to the studies by Mason et al. (112) and Cole et al. (117), the American Cancer Society, the Centers for Disease Control and Prevention (CDC), the National Cancer Institute, and the North American Association of Central Cancer Registries found that for all races/ethnicities combined in th e US, favorable trends in lower incidence and mortality were noted for lung and CRC in men and women and for breast cancer in women (118). Cancer incidence data were available for up to 82% of the US population, and cancer deaths were available for the entire US population. Overa ll cancer death rates d ecreased by 2.1% per year from 2002 through 2004, nearly twice the annual decrease of 1.1% per year from 1993 through 2002. Among men and women, death rates declined for most cancers. Breast cancer incidence rates in women decreased 3.5% per year from 2001 to 2004, the first d ecrease observed in 20 years. Colorectal cancer incidence and death rate s and prostate cancer d eath rates declined, with CRC death rates dropping more sharply from 2002 through 2004. The role of folic acid supplementation in risk reduction for other cancers also is supported by evidence of the role of adequate folate intake in th e reduction of br east cancer in relation to alcohol consumption. Folate status may be compromi sed in chronic alcohol abusers secondary to inadequate folate intake. Ethanol may also ag gravate folate deficiency by impairing intestinal folate absorption, interferi ng with folate metabolism, or increasing renal folate excretion (119). Excessive alcohol cons umption is defined as 14 to 15 g/d, which is

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43 equivalent to 5 to 6 ounces of wi ne and 13 to 14 ounces of beer. Data from the Nurses’ Health Study revealed that for women consuming 15 g/d of alcohol, risk for breast cancer was lowest in those women consuming 600 g/d of folate from both food sources and supplements compared to women who consumed 150 to 200 g /d of folate (61). Furthermore, a 26% reduction in risk for breast cancer was observed in wome n consuming alcohol and taking multivitamins compared to similar women who were not taking supplements. Additional data from the Canadian National Breast Screening Study (56,837 women) showed a 43% reduction in breast cancer risk associated with folate consumption (> 300 g/d) in women consuming 14 g/d of alcohol compared to women with equivalent alcohol intake who consumed <225 g/d of folate (120). These re sults suggest that a diet high in folate may co mpensate for the negative effects on folate metabolism cau sed by alcohol intake and may consequently reduce breast cancer risks (11). Ongoi ng research is still being conducted with the hope of providing better insight into the potential role of folate in cancer prevention. Folic Acid Fortification Following reports from clinical intervention studies identifying a direct association between periconceptional folic acid intake and NTD risk reduction, the US Public Health Service issued a recommendation in Sept ember 1992 that women of child bearing age (i.e., 15 to 44 years) capable of becoming preg nant should consume 400 g of th e folic acid dail y to reduce the number of cases of NTD. Since then, an ongoing national public health campaign has encouraged women to consume dietary supplements containing folic acid. In addition, the FDA mandated in 1996 that al l “enriched” cereal-grain products be fortified with folic acid by 1998 (2). Although the eff ective date was January 1, 1998, the ma jority of food manufacturers had implemented folic acid fortification by mid-1997. The predicted increas e of daily folic acid

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44 consumption attributable to fort ification of enriched cereal-grain products was estimated to be approximately 100 g/d Originally, the FDA considered several options that included fortif ication of enriched cereal-grain products, RTE breakfast cereals, dairy products, and fru it juices. However, analysis of folic acid intake by consumers in some non-target groups (i.e., men and elderly) showed that when fruit juices and dairy produ cts were fortified with folic acid, in addition to cereal-grain products, RTE breakfast cereals an d dietary supplements, folic aci d intake exceeded the UL of 1,000 g/d even at the lowest leve l of fortification. As a re sult of FDA’s analysis, the FDA determined that fortification s hould be limited to cereal-grain products, RTE breakfast cereals and dietary supplements (2). The FDA reviewed informati on showing that cereal-grain products and RTE breakfast cereals are consumed on a daily routine basis by 90% of the ta rget population (i.e., women of childbearing age). Furthermore, representatives of manufacturers in th e cereal-grain industry stated that these products could be easily fortified with folic acid and that a fortification mandate would not be unfair to the i ndustry. Thus, the FDA determin ed that mandatory folic acid fortification of cereal-grain products with 140 g per 100 g, along with voluntary fortification of RTE breakfast cereals up to 400 g per serving and dietary supplem ents up to 400 g per unit or per serving, would provide increas ed intakes of folic acid for wo men in reproductive years while keeping intakes for the n on-target population below the UL (2 ). In addition to the US, other countries that have implemente d a mandated folic acid fortifi cation program include Canada (121), Chile (122), and some La tin American countries (123).

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45 Effect of Fortification on Folate Stat us of Women of Reproductive Potential The initiation of fortifica tion of the US food supply with folic acid has had a positive effect on the folate status of the entire population and has d ecreased the incidence of NTDaffected pregnancies in the US by approximate ly 26% (13). Dietrich and colleagues have assessed the benefit of fortification by comparing serum and RBC folate concentrations for women of reproductive age from two separate NHANES data sets. NHANES III, conducted during 1988 to 1994, reflects the time prior to folic acid fortifi cation, and NHANES 1999-2000, reflects the time period post-fort ification. Compared to pre-fo rtification concentrations, both serum and RBC folate concentr ations increased significantly post-fortification. For women between the ages of 20 to 39 years and 40 to 59 years who did not use supplements, the mean serum folate concentration incr eased more than two-fold fr om 10.3 to 26.0 nmol/L and 11.4 to 27.1 nmol/L, respectively. These increases represent a respectiv e 153% and 137% increase in serum folate concentrations (13). The mean RBC folate concentration increase d for women 20 to 39 years old and 40 to 59 years old from 341 to 556 nmol/L and 386 to 629 nmol/L, respectively, between NHANES III to NHANES 1999-2000. A 63% increase in RBC folate concentrati on was observed for both age groups of women. These increases appear to be associated with the consumption of folic acid in enriched and fortified foods (i.e., enriched cer eal-grain products and fo rtified RTE breakfast cereals) because data for supplement user s were evaluated se parately (11, 17). Jacques and colleagues (4) evaluated the effect of fortification on indices of folate status in a cross-sectional st udy with participants in the Fram ingham Offspring Cohort (men and women, ages 30 to 59). These researchers found that among nonusers of folic acid supplements, mean plasma folate concentra tion among individuals examined af ter fortification increased 46%

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46 (10.4 to 22.7 nmol/L; P <0.001) relative to indi viduals examined before fortification. Furthermore, the prevalence of low folate concentrations (<7 nmol/L) significantly decreased from 22.0 to 1.7% (P <0.001). C houmenkovitch and colleagues also evaluated folate status of participants in the Framingham Offspring Cohort and observed a 38% increase in mean RBC folate concentration of subjects compar ing preto post-fo rtification (3). In addition, Lawrence and colleagues re viewed pre-existing data on serum folate concentrations in more than 98,000 blood sa mples submitted to Kaiser Permanente’s Southern California Endocrinology Laboratory between 1994 and 1998 to evaluate chan ges in serum folate concentrations since implementati on of folic acid fortification of cereal-grain products (5). These researchers observed a median pre-fortification serum folate value of 28.6 nmol/L in 1994, which gradually increased by 48% during the transition period betw een initiation of fortification in 1996 and full implementati on in 1998 to 42.4 nmol/L. Recently the CDC assessed the trends in folate status of women and reported that blood folate concentrations among non-pregnant US women of ch ildbearing age declined from NHANES 1999-2000 through NHANES 2003-2004 ( 124). Based on data from NHANES 19992000, 2001-2002, and 2003-2004, the median serum fola te concentrations among women 15 to 44 years old were 28.6 nmol/L, 25.8 nmol/L and 24.0 nmol/L, respec tively. This reduction in medium serum folate concentrations repor ted from 1999-2000 through 2003-2004 represented a statistically significant reduc tion of 16% (P <0.001). Simila rly, RBC folate concentration decreased 8% (P = 0.03) from 578 nmol/L re ported during NHANES 1999-2000 to 533 nmol/L reported from NHANES 2003-2004 data. Although a d ecline has been noted, the majority of the women participating in NHA NES 2003-2004, particularly nonHispanic Caucasian women, achieved the 2010 national health objective of a median RBC fola te concentration of 500 nmol/L

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47 (124). Nevertheless, this recent decline in folate status emphasiz es the need for enhanced folic acid awareness for women of reproductive age. Effect of Fortification on Neural Tube Defects Folic acid fortification has a direct positive and significant effect on the reduction of NTD birth prevalence. Honein a nd colleagues (14) estimated redu ctions in NTDs in the US by evaluating data from birth certificates from 45 states and Washington, DC and reporting the number of infant birth certificates reporting births affected by eith er spina bifida or anencephaly. Data post-fortification revealed a decline in the prevalence of spina bifida and anencephaly by 23% and 11%, respectively. Howe ver, the data included only live births, since NTD-affected infants who were either miscarried or stillbor n were not reported (14). The CDC reported that rates of NTDs have decreased 26% in the US si nce folic acid fortificat ion was mandated. The prevalence of anencephaly reported on birt h certificates declined from 18.38 cases per 100,000 live births in 1991 to 9.40 in 2001. The prevalence of spina bifida reported on birth certificates declined from 24.88 cases per 100,000 live births in 1991 to 20.09 in 2001 (15). Although the level NTD risk reduction observed in the US was not as high as originally expected, research has shown that the apparent positive reduction in NTD incidence has been asso ciated with improved folate status post-fortification and supports food fortification as an effective intervention strategy for individuals (11). In 2006, Botto and colleagues (12) assessed two crucia l issues relative to the benefits and impact of folic acid in the prevention of birth defects: whet her folic acid supplementation alone, without fortification, is effectiv e in reducing the population-wide rates of NTDs, and whether the current policies can reduce the o ccurrence of other birth defects. These investigators used data from 15 birth registries from areas with either official supplementati on recommendations of 400

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48 g/d of folic acid or a folic acid fortification program to assess the effectiveness of both on the rates and trends of major birth de fects, including NTDs. The results revealed significant changes in the incidence of NTDs in areas with folic acid fortification, but not in areas with folic acid supplementation recommendations and no fortifi cation program. The investigators concluded that recommending folic acid s upplementation alone remains an ineffective approach in translating the known protective effect of folic acid in reducing NTD rate s. In contrast, fortification appears to be ef fective in reducing NTDs (12). When folic acid fortification was first implemented, the estima ted increase of folic acid intake in the US food supply that resulted from consumption of folic acid enriched and fortified products was approximately 200 g/d or two times the quantity or iginally predic ted at 100 g/d (16). Actual measurements of total folate cont ent in enriched cereal-gra in products have shown that many of these products cont ained total folate levels that were higher than the amounts required by federal regulation (125127). Although ther e is a lack of c onsistent research evaluating the folic acid content of enriched cer eal-grain products, recent research groups have reported data suggesting a declin e in the amount of folic acid adde d to foods since the initiation of fortification. Johnston and Tamura (128) measured the fola te content of white sandwich breads containing enriched flour during 2001 to 2003 and fo und that the mean folate content in enriched white bread significantly declined from 2001 to 2002 or 2003. In addition, Po-Prieto and colleagues (129) analyzed the folic acid content of numer ous enriched foods and found no evidence of folic acid overages in enriched produ cts contrary to previous reports. In fact, many of the fortified foods te sted tended to contain less folic acid than required by law (129).

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49 Folic Acid Recommendations and Awareness Despite significant reductions in the in cidence of NTDs observed since folic acid fortification, the majority of wo men capable of becoming pregnant have not achieved the level of intake associated with NTD ri sk reduction (13). Therefore, increasing the number of women who consume 400 g of folic acid daily from dietary supplements or folic acid fortified foods remains an important public h ealth goal for NTD prevention. Numerous public health polic ies have been implemented worldwide based on the strength of the evidence relating pericon ceptional folic acid supplementati on to NTD risk reduction. In 2005, the CDC and the March of Dimes sponsored the first Na tional Summit on Preconception Care and launched the Preconcepti on Health and Health Care Initia tive, the goal of which is to improve both the hea lth of mothers before pregnancy and ma ternal and infant health outcomes (130). The recommendations from this summ it were published on April 21, 2006 (131). The recommendations noted that the ti me has come to ensure that e fforts promoting adequate folic acid intake and improved pregnancy outcomes, in cluding NTD risk reducti on, are not limited to prenatal care but should be expanded to include preconcept ion health and health care. The panel, which included an array of heal th care providers, public health pr actitioners, and researchers, developed strategies to implement the recommend ations across three area s: clinical practice, consumer roles, and public health practice (130). Current and futu re plans to promote folic acid include developing and distribut ing clinical guidelines and t ools, educating consumers, integrating preconception care activ ities into clinical and public health programs, educating and training clinical and pub lic health care providers, developi ng a research agenda, identifying, documenting, and promoting best practices, and supporting state and local initiatives (130).

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50 However, despite these recommendations and other folic acid awareness campaigns, there does not appear to be a significant in crease in the number of women taking folic acid supplements. A survey conducted by the March of Dimes recently provided evidence that only 33% of women of reproductive ag e (18 to 45 years) take a fo lic acid containing supplement daily, compared to 40% in 2004 and 28% in 1995 (17). Furtherm ore, reports have indicated that women not contemplating pregnancy, women between the ages of 18 to 24 years, non-white women, those less educated, and thos e of lower socioeconomic status have both lower folic acid knowledge and lower folic acid supplement use (132-134). The March of Dimes has also reported that only 24% of women aged 18 to 24 years took a supplement with folic acid daily, yet this population accounts for at least one third of all US births (135). In addition, although 84% of women reported awareness of folic acid, up from 78% in 2004, only 25% of women reported knowing that folic acid pr events birth defects, and only 7% of women reported knowing that folic acid should be taken pr econception (17). Comparable da ta were reported in a survey conducted in Puerto Rico, wher e nationwide campaigns had been promoting folic acid for four years prior to the study. Despite the majority of pregnant wo men surveyed who understood the importance of folic acid (88%), re gardless of whether or not they planned their pregnancy, only 32% actually took a foli c acid supplement prio r to conception (136). Analytical Methodology for Assessing Folate Status There are several analytical methods used to assess blood folate status. Not all researchers have used the same methodology for detecting folate concentrations, and even when research groups have used the same method, there may be differen ces in protocols between labs. This section reviews the two most commonly used methods.

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51 Blood Folate Analysis Serum and RBC folate concentr ations are most often measur ed using the radiobinding or the microbiological assays. The microbiological assay is consid ered the gold standard method for determining folate concentrations in blood, urine, tissue, and food samples (137, 138). The microbiological assay us es the test organism Lactobacillus rhamnosis as it metabolizes the highest number of folate deriva tives, including 5-methylTHF, th e predominant folate form in plasma and RBC (22). Ascorbic acid and a phosphate buffer (p H 6.1) are added to the test sample and micro-organism to offer better stability and pr event the oxidative loss of labile reduced folates. Each 96-well fl at bottom microtiter plate contains serial dilutions of a control sample, samples of folic acid st andard, and subject samples. Samples are inoculated with L. rhamnosis and incubated at approximately 37C to allow for growth of the organism. Growth of the folate-dependent microorganism is assessed by comparing the degr ee of turbidity of the sample compared to the turbidity of known concentrations of folic ac id standard. Cell growth is determined by absorbance at 650 nanometers using a computer-inter faced microtiter plate reader and data reduction software (137). A standard curve is generated by plotting the log-linear absorbance against the folic acid standard concentration to interpolate unknown folate concentrations of samples. The radiobinding assay also is used for blood folate anal ysis (139). Th e radiobinding assay uses a competitive fola te binding protein attached to microbeads and iodine-125 [125I]labeled folic acid, which are used to quantify seru m or RBC folate concentr ations (140). In the assay, the folate binding protein ha s an equal affinity for the stan dard and the folate present in the serum or plasma. The unlabe led folate competes with the labeled folate for the limited

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52 number of available binding sites on the folate binding protein. T hus, the level of radioactivity bound is inversely related to the concentr ation in the sample or standard. The radiobinding assay is conducted by adding dithiothreitol solution to the folate tracer (125I, borate buffer with human serum albumin, dextran, po tassium cyanide, dye and preservative). This mixture is added to serum or RBC samples and heated in a water bath at 100oC for 15 minutes. Once cooled, the folate binder is added to the mixture, which is protected from exposure to light, and in cubated for one hour. During incubation, endogenous folate and 125I compete for binding sites to the folate binder. Samples ar e centrifuged and bound folates and microbeads precipitated. The bound folate (lab eled and unlabeled) acc umulates in a pellet, while unbound folate is in the supernatant, which is gently discarded. Th e radioactivity of the pellet is measured using a sc intillation gamma counter. The microbiological assay has been the preferred me thod for measuring folate concentrations. However, the method is te dious and time consuming, requires microbiological expertise, and growth of the te st organism may be inhibited by non-folate substances, such as prescription medications (e.g., antibi otics or methotrexate) taken by subjects. The advantages of using the radiobinding assay include the fact that it is relati vely inexpensive, easy to perform, and has a high specificity towards folate isomer s. One limitation of the radiobinding assay is that it gives lower folate values than the microbiological assay since this method underestimates certain folate forms as evidence d by recovery analysis (61). Another method used to assess folate concentrations in bi ological sample s uses liquid chromatography-tandem mass spect rometry (LC-MS/MS). Fazili and colleagues (61) compared serum folate species analyzed using LC-M S/MS with total folate measured by the microbiological and Bi o-Rad radiobinding assays. Adva ntages of the LC-MS/MS method

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53 compared to the microbiological assay are that it provides information on the different folate species in addition to total folate and is less prone to interferences such as antibiotics. Mean and median total folate concentra tions measured by LC-MS/MS a nd microbiological assays were generally in agreement, but the radiobinding assay values were much lower (-29% relative to LC-MS/MS values). Fazili and colleagues concl uded that the radiobindi ng assay produces much lower results, on average, probab ly due to underrecovery of 5methylTHF, which is the main circulating form of folate (61). The Bio-Rad QuantaPhas e II radiobinding assa y has been used to measure blood folate in NHANES fo r 25 years. Due to the fact that the radiobinding assay is being discontinued in 20 07, NHANES is switching to the micr obiological assay for all samples, and will use the LC-MS/ MS method for a subset of the population. Plasma Homocysteine Analysis Determination of plasma homocysteine conc entration is generally measured as total homocysteine concentration. Th e most commonly used approach for quantifyi ng homocysteine concentration is capillary gas chromatography-mass spectrometry with selected ion monitoring (141). The term “total homocyst eine” as applied to biological samples (i.e., serum or plasma) refers to the sum of homocyste ine “that is linked via disulfid e bond formation in a variety of compounds that include homocystein e, homocysteine-cysteine mixed disulfide, prot eins via their cysteine moieties, and peptides su ch as glutathione via their cystei ne moieties (141).” The assay is conducted by adding 2-mercapto ethanol to the sample in order to chemically reduce and release endogenous homocysteine fro m proteins and other disulfides The samples are analyzed using a gas chromatograph-mass spectrometer eq uipped with a falli ng needle injector. Quantitation is based on the ratio of the areas of the base peak ion for homocysteine to the areas of the base peak ion for the deri vative of the resp ective stable isotope inte rnal standards (141).

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54 MTHFR Genotype Determination Since the discovery of single nucleotide pol ymorphisms (SNP), tec hnological advances have been made in methods for determining subject genotypes for these SNPs. A common method for genotype determination involves poly merase chain reaction (PCR) to amplify the desired region or target sequen ce of the DNA strand. Once the preferred region is magnified, specific restriction enzymes are added depending on the SNP being studied. The SNP of greatest interest with regard to fola te metabolism is the MTHFR 677 C T that results in the substitution of the amino acid alanin e with valine in the gene product (19). In order to achieve unambiguous determin ation of all SNP variations, Howell and colleagues (142) created a new SNP scori ng method known as dynamic allele-specific hybridization (DASHTM). Using this approach, the target sequence is amplified by PCR with one biotinylated primer. The biotin ylated product strand is bound to a streptavidin-coated microtiter plate well, and the non-biotinylated, unbound strand is removed with alkali. An oliogonucleotide probe, specific for the desired allele, is hybridized to th e bound DNA at low temperature. This forms a probe-target duple x that interacts with a double strand-specific in tercalating dye. The temperature is increased and the denaturation temperature and fluorescence emitted from the dye, which is proportional to the amount of double-strande d DNA present, are recorded. For analysis, the negative derivatives of the melting curves are plotted to sh ow a lower temperature peak for a homozygous allele mi smatch, a higher temperature pe ak for a homozygous match, or both peaks for a heteroz ygous sample (142).

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55 Dietary Intake Assessment USDA National Nutrient Database The USDA National Nutrient Database for St andard Reference is the major source of food composition data in the US and provides the foundation for most public and private sector databases. The 2007 release (Rel ease 20) contains nutrient data for 7,517 food items for up to 140 food components including vitami ns, minerals, amino acids, and fatty acids (143). Major applications of the USDA Nationa l Nutrient Database include th e development of the Food and Nutrient Database for Dietary Surveys by the Fo od Surveys Research Group, which is used to process dietary data records from the survey “W hat We Eat in America”, the dietary intake component of NHANES. The version of the USDA Na tional Nutrient Database used to estimate nutrient intakes for subjects in the present study was Release 17 (2004), which contains nutrient data for 6,839 food items for up to 128 food components (8). In contrast to older database releases, a unique component of the Release 17 nu trient composition database is that it provides data for four categories of dietar y folate including folic acid ( g/d), natural food folate (g/d), total folate (g/d), and DFE (g DFE/d). Until recently, rese archers reporting folate intake from NHANES data used an ol der version of the nutrient databa se that only provided nutrient composition data for total folate (g/d). Food Frequency Questionnaire A food frequency questionnaire (FFQ) is ofte n used as one method for determining usual dietary intake. The FFQ is the most practical and economi cal method for collection of comprehensive dietary data and is most commonly used in large ep idemiological studies (144). Compared with other dietary assessment methods, such as 24-hour dietary recalls or multiple-day food records, the FFQ obtains less detailed info rmation regarding food ty pe or portion size.

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56 Although alternative methods are more detailed, FFQ is self-administered and is designed to capture usual dietary intake, unlik e records or recalls. The FFQ often generalizes food intake into food groupings, such as fortified cereal, wh ereas a 24-hour recall method or multiple-day food record provides more detail ed information such as brand names of foods consumed. The FFQ has predetermined questions and portion sizes to which an i ndividual responds. This type of questionnaire can be electr onically scanned and th e information upload ed by software for further analysis (144). The limitation with this dietary assessment meth od is that there is little opportunity for individuals to list di etary intake data not included as part of the questionnaire. Concern regarding measurement error has stim ulated numerous validation studies comparing nutrient intakes estimated from FFQs with those estimated by other methods. Validation of this method, whic h is the process of determinin g if estimated nutrient intake is true to actual intake, is esse ntial to providing valid estimates of dietary intake for observational epidemiological studies and clini cal trials. The National Cancer Institute (NCI) Diet History Questionnaire (DHQ) is an FFQ developed by staff at the Risk Factor Monitoring and Methods Branch (144). The DHQ captures data on freque ncy of consumption and estimated portion size from a list 124 individual f ood and beverage items over the past year. The 124 food items currently included in the DHQ we re selected based on the work of Subar and colleagues (145) who originally categorized 5,261 individuals foods with 170 food groups. The DHQ has been refined over the years based on results from intens ive cognitive interviewing. Subar and colleages (146) conducted cognitive evaluation of various approaches to asking about usual dietary intake and identified ways to improve the FFQ so completing th e FFQ would be easier while the accuracy of responses would be enhanced. Numerous cognitive issues in FFQs have been addressed in the DHQ, incl uding comprehension, order of f ood items, intake of seasonal

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57 foods (e.g., fruits and vegetables), specificity of low-fa t or fat-free food item s, intake averages from multiple food items, and format. Other im provements to the DHQ in clude modification of portion size categories based on an alysis of data and responses from 10,019 adults in the CSFII (1994-1996) study (147). Rather than categorizing portion sizes as small, medium, or large, portion size choices were change d to provide more detail about specific intake of a food item, such as “less than 1 cup” “1 to 2 cups”, and “m ore than 2 cups”. Thes e changes have ultimately improved the validity and accuracy of nutrient intake estimated by the DHQ (146, 148). In 2001, Subar and colleagues (144) evaluated the NCI DHQ against repeated 24-hour recalls and compared to the Block and Wille tt FFQ, and ultimately validated the DHQ as a suitable method for estimating nutrient intake. The Block FFQ is an ei ght-page questionnaire that inquires about 106 food items, and categorizes portion size choices as small, medium, or large (144). This questi onnaire also has eight ques tions concerning use of added fats or low-fat foods, 13 dietary supplement questions, five su mmary questions, and six questions on eating at restaurants. The Willett FFQ asks about 126 foods over four pa ges, including ten questions each on supplement fat intake. This questionnaire do es not include separate portion size questions, but rather categorizes responses in to intake frequency of a reference portion si ze. This validation study consisted of 1,301 men and women who comp leted four 24-hour reca lls during one session over the telephone. The subjects were then randomized to comp lete two FFQs, either the DHQ and Block FFQ or the DHQ and Willett FFQ. Researchers found th at the DHQ was more accurate compared to the 24-hour recall method th an the Block or Willett FFQ in determining nutrient intake (144).

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58 Assessment of Folate/Folic Acid Intake in Women of Reproductive Potential Assessment of folate/folic acid intake among men and women typically has been evaluated using data from NHANE S or the Framingham Offspri ng Cohort Studies. However, there are few studies that have evaluated folate /folic acid intake for women of reproductive potential who do not consume suppl ements containing folic acid. Choumenkovitch and colleagu es (125) examined food a nd nutrient intake of 1,480 individuals who participated in the Framingham Offspring Cohor t Studies to assess folic acid intake from fortification in the US. Particip ants completed a 126-item semi-quantitative FFQ developed by Willett et al. (149) that allowed for estimation of usual nutrient intakes during the previous year. Among the 186 women (ages 30 to 80 years) who did not us e supplements, intake of folic acid after exposure to fo rtification increased by a mean of 192 g/d (125). Furthermore, 9% of non-supplement consuming women post-fort ification compared to 55% of women prefortification consumed less th an the EAR (<320 g DFE/d) (1 25). A study by Dietrich and colleagues (13) who also used data from NHANE S 1999-2000 estimated mean total folate intake for women between the ages of 20 to 39 years an d 40 to 59 years to be 294 g/d and 302 g/d, respectively. A study by Yang and collea gues (6) used NHANES 2001-2002 data to compare differences in folic acid intake in women of childbearing age (ages 15 to 49 years) in the US after folic acid fortifi cation. Information on folic acid intake from fortified foods and intake of food folate was obtained from a si ngle 24-hour food recall questionnaire. Nutrient values were calculated using the USDA National Nutrient Database (Release 17). Yang and colleagues reported that estimated mean consumption of folic acid from fortifie d foods in women of childbearing age was 128 g/d. Approximate ly 8% of non-supplement consuming women

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59 reported consuming 400 g/d of folic acid from fortified foods alone, and 26% of the women surveyed reported taking 400 g/d of folic acid from supplemen ts in the previous month. The mean food folate intake from di etary sources was 151 g/d. Yang and colleagues concluded that at the present level of folic aci d fortification, most women need to take a supplement containing folic acid to achieve the IOM recommendation for 400 g/d of folic acid (6). However, a limitation to this study is that it did not characterize sources of folate/folic acid. Further research is needed to identify which com ponents of the diet c ontribute to folic acid intake in women of reproductive potential who do not consume a folic acid supplement or supplements containing this nutrient.

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60 Figure 2-1. Folate/folic acid structures adapted from Present Knowledge in Nutrition (1). Folic acid consists of a para-a minobenzoic acid molecule li nked on one side by a methylene bridge to a pteridine ring, and joined by peptide linka ge to a glutamic acid molecule on the other side. Naturall y occurring food folates exist in various chemical forms, containing a side-chain composed of two to ten additional glutamate residues ( n ) joined to the first glutamic acid. The pterid ine ring of the folate/folic acid structure can be re duced to form dihydrofolic acid and tetrahydrofolic acid (THF). Folate coenzymes are formed by substitution of onecarbon units at the N5, N10, or both positions ( R ) to the polyglutamyl form of THF.

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61 Table 2-1. Definitions of Dietary Reference Intake (DRI) recommendations. DRI recommendation Definition Estimated Average Requirement (EAR) A daily nutrient intake value th at is estimated to meet the requirement of half the h ealthy individuals in a group Recommended Dietary Allowance (RDA) The average daily dietary intake le vel that is sufficient to meet the nutrient requirement of ne arly all (97 to 98%) healthy individuals in a pa rticular life stage and gender group. Individuals should aim fo r this intake level. Adequate Intake (AI) A recommended da ily intake value based on observed or experimentally determined appr oximations of nutrient intake by a group (or groups) of healthy people that are assumed to be adequate—used when an RDA cannot be determined. Tolerable Upper Intake Level (UL) The highest level of daily nutrient intake that is likely to pose no risk of adverse health effects to almost all individuals in the general population. As intake increases above the UL, the risk for adverse health effects increases. Adapted from the Institute of Medicine (10) Table 2-2. Folate intake recommenda tions for men and non-pregnant women 19 years. DRI recommendation Physiological folate requirement Estimated Average Requirement (EAR) 320 g DFE/d Recommended Dietary Allowance (RDA) 400 g DFE/d Adequate Intake (AI) Not applicable Tolerable Upper Intake Level (U L) 1,000 g synthetic folic acid/d Adapted from the Institute of Medicine (10)

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62 Figure 2-2. One-carbon metabolism folate-d ependent pathway adapted from Present Knowledge in Nutrition (1).

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63 CHAPTER 3 MATERIALS AND METHODS Study Design and Methods Overview Approximately 1,000 healthy male and female adult volunteer s were recruited through newspaper advertisements, flyers and radio broadcasts. Prosp ective subjects were screened by phone using a screening que stionnaire (Appendix A) to determ ine their eligibil ity. Individuals were included in the study if they met the follow ing criteria: (a) age 18 to 49 years, (b) currently not using prescription medications (birth control medication wa s permitted), (c) low alcohol intake (< one drink per day), (d ) non-smoker, (e) no history of ch ronic disease, (f) non-pregnant, (g) non-lactating, (h) no use of supplements within the last 6 months, and (i) no major dietary changes within the last 3 years. Individuals also were scr eened for consumption of highly fortified RTE cereals containing 400 g/d of folic acid (e.g. Total), and were not eligible to participate if they indicated they consumed these highly fortified RTE cereals. Eligible male and female volunteers (n = 388) were chosen to participate in the study. Subjects were scheduled to ha ve a fasting blood sample drawn by a certified phlebotomist and to receive comprehensive deta iled verbal and written instruc tions lasting 15 to 20 minutes regarding the completion of the DHQ (Appendix B). Subj ects took the DHQ and prepaid mailing envelopes home with them and were as ked to mail the completed DHQ to the primary investigator within 2 weeks of recei ving the in-person instructions. Fasting blood samples were collected and proc essed for multiple anal yses, including serum and RBC folate concentrations plasma homocysteine concentr ation, and MTHFR genotype. Folate intake (total folate, DFE, folic acid, and food folate), in addition to other nutrients were assessed using the DHQ. If subjects completed a ll aspects of the study th ey were compensated

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64 $50 for their participation. The University of Florida Instituti onal Review Board approved this protocol, and all subjects sign ed informed consent forms. Human Subject Procedures Eligible subjects enrolled in the study were scheduled for an a ppointment and were instructed to report to the Food Science and Human Nutriti on building on the University of Florida campus following an overnight fast Upon arrival, the purpose of the study was discussed with each subject who wa s then asked to sign the approved informed consent form. To ensure the privacy of personal health informati on, subjects who granted co nsent were assigned a subject identification number. As a follow up to questions asked during the phone screening, subjects were again asked a bout supplement use during th e past 6 months. Subjects acknowledging that they had ta ken supplements were asked to provide more detailed information. This information was recorded on each subject’s data form. Blood samples were collected by a phlebotomist for analysis of biochemical indices. Following the blood draw, subjects we re given a snack and were in structed on how to complete the DHQ. During the 20 minute DHQ instruction session, each sm all group of participants received detailed verbal and wri tten descriptions of how to comp lete the DHQ. Subjects were also taught how to report accurate portion sizes by reviewing how to read food labels and how to use measuring cups and spoons to visua lly interpret portion sizes correctly. Additional handouts were reviewed and given to subjects to assist them in properly completing the DHQ. These handou ts included a detailed drawi ng illustrating the proper method for marking or changing an answer (to ensure that the forms w ould scan correctly) and handouts on “Caffeinated versus Non-caffe inated Beverages,” “Fortified Cereals”, and “Seasonal Fruits

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65 and Vegetables” (Appendix C). Subjects were gi ven the opportunity to ask questions regarding the study and the DHQ. Dietary History Questionnaire This study was a collaborative effort between re searchers at the Univer sity of Florida and the University of North Caro lina (UNC), Chapel Hill. The original DHQ from NCI was modified with the addition of questions pertaining to beef c onsumption, beef-containing food products, and vitamin B12 fortifie d foods since the primary object ive of this study was to assess vitamin B12 intake in the diet. The DHQ data also were used to evaluate the contributions of folate and folic acid containing foods to overall folate intake. The total intake of each nutrient was analyzed using the Diet*Cal c Analysis program provided by NC I. This software program can be downloaded free of char ge from the NCI website ( www.riskfactor.cancer.gov ). The nutrient database within the Diet*Calc software is from the USDA Nation al Nutrient Database Standard Reference, Release 17 (8). The Diet*Calc Analysis program nutrient database had been updated in August 2004. Processing the Dietary History Questionnaire Subjects were instructed to mail the comp leted DHQ within 2 we eks of the initial instruction. Subjects who failed to return th e DHQ within this time frame were contacted by phone or email. All returned DHQs were reviewed to ensure they were completed entirely and that no questions were missing a re sponse. Marked responses were not reviewed at this time to prevent bias. Subjects who neglected to respond to one or more DHQ questions were contacted to obtain the missing informati on. The completed paper versi ons of the DHQ were mailed to Optimal Solutions Corporation, Lynbrook, New Yor k, in groups of 100 wher e they were scanned electronically. After the scanni ng process was finished, Optima l Solutions Corporation mailed

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66 the DHQs and electronic data in the form of an American St andard Code for Information Interchange (ASCII) text file to collaborators at UNC. Investig ators at UNC upl oaded the files into the Diet*Calc Analysis prog ram to analyze the nutrient inta ke of each subject based on their DHQ responses. Analysis of Nutrient Intakes After the paper version of th e DHQ was electronically scanned, each food item or question included in the DHQ was linked to a predetermi ned Food Identification Number (FIN). Each FIN had defined nutrients for gender and servi ng size. The FINs corre sponding to the DHQ food items and questions were linke d to the Diet*Calc Nutrient and Food Group database. The Diet*Calc Analysis program used the FIN associated with each response on the DHQ to evaluate the nutrient intake for each participant based on gender and serving size. When Diet*Calc identified a FIN from the ASCII text file, the software used the database to calculate an individual’s nutrient intake. The FINs and corres ponding nutrient values estimated using each individual’s responses to questi ons on the DHQ were entered into a Microsoft Excel spreadsheet. The Diet*Calc software produced da ta files that were available fo r analysis: the details.txt file and the results.txt file. The deta ils.txt file was an expanded ve rsion of the data calculated from the ASCII text file and allowed investigators to compare FINs to individual nutrient intake and daily frequency of intake. The results.txt file was a condensed version that prov ided combined daily nutrient intake values for each subject regardless of the food source. Dietary History Questionnaire Instruction Pretest To ensure that the in structions and handouts on how to complete the DHQ would be clear to the subjects, a pret est of the DHQ instructions was condu cted with a group of 20 graduate students in the Food Science a nd Human Nutrition Department a nd five vegetarian and vegan

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67 consumers prior to starting the st udy. This pretest group was instru cted using the same script to be used during the study. The st udents were asked to follow the instructions and complete the DHQ as if they were participants in the study. They were also given an additional survey that asked questions regarding the le ngth of time needed to comple te the DHQ, if any regularly consumed foods were missing from the DHQ, and if they had any othe r comments to improve the instructions and/or the DHQ instrument. Feedback from this group was reviewed and incorporated into the DHQ and DHQ instructions. Blood Sample Collection and Processing Fasting blood samples were collected for each particip ant by a phlebotomist (Vacutainer Blood Collection Set; Becton Dickinson, Vacutainer Systems; Franklin La kes, NJ). All blood samples were processed within on e hour of collection. A total of 70 ml of blood were obtained from each subject. Blood for serum samples was collected in 8.3 ml serum separator gel clot activator tubes (Vacutainer, Becton Dickinson, Rutherford, NJ) and ke pt at room temper ature for 30 to 60 minutes to allow time for clo tting. Serum was obtained by ce ntrifuging the tubes at 650 x g for 15 minutes at 21oC (International Equipment Compant; Model HN-S II Centrifuge, Needham Heights, MA). Supernatant sera were mixed w ith sodium ascorbate (1 mg/ml), aliquoted into 200 l samples, and stored at -30oC until analysis. Whole blood was collected in 7 ml tubes containing K3 ethylenediaminetraacetic acid (Vacutainer, Becton Dickinson, Rutherford, NJ). Bl ood for plasma homocys teine was kept on ice prior to processing. A small aliquot of whole blood held at room temperature was diluted 20fold in 1 mg/ml ascorbic acid and aliquoted into 200 l samp les and frozen for measurement of RBC folate concentrat ion. The iced blood wa s centrifuged at 2000 x g at 4oC for 30 minutes.

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68 The plasma from these samples was frozen and used to measure the plasma homocysteine concentration. Following removal of the plasma, the samples were used to extract DNA to be used to determine geno type for the MTHFR 677 C T polymorphism. Aliquot s were frozen at -30oC for subsequent analysis of serum and RBC folate concentrations. Analytical Methods Identification of Food Groups from the Dietary History Questionnaire All foods included in the DHQ were cat egorized into specific food groups: beef, pork/other meat, poultry, dairy, eg gs, seafood, cereal, soy, meal repl acements, mixed dishes with meat type unknown, non-dairy fats beans, rice/pasta, soups/sau ces, breads/crackers/cakes/pies, nuts/seeds, vegetables, fruit, syrup/honey/gelatin/candy, and be verages. A new set of food categories were derived including the following: (1) enriched cer eal-grains, (2) fortified cereals and bars, (3) vegetables, (4) fru it, (5) juice (only orange and gr apefruit), (6) legumes and nuts, (7) dairy, (8) eggs, (9) meat, (10) combination foods, (11) snacks, (12) miscellaneous, (13) other. Foods that were fortified with folic acid but consumed minimally were grouped together as (12) miscellaneous, and foods that were not fortified and contained mini mal levels of food folate were grouped together in the (13) other category. Table 3-1 descri bes the types of food included in each food category. Although we attempted to sc reen out individuals who consumed highly fortified RTE cereals, indi viduals that later reporte d consumption of these cereals were included in the final analysis. Intake of highly fortified RTE cereals was included in the fortified cereals and bars food category. The nutr ient composition database used to calculate nut rient estimates from the DHQ was the USDA National Nutrient Database Standard Reference Release 17 (Diet*Calc Nutrient and Food Gr oup database). The nutrient da tabase provided data for four

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69 categories of dietary folate inta ke, including folic acid (g/d), food folate ( g/d), total folate (g/d) and DFE (g DFE/d) from foods in each of these new food categories. Serum and RBC Folate Concentrations The serum and RBC folate conc entrations of all subjects we re determined using the MP Biomedicals, Inc. SimulTRAC-S Radioassay Kit (Orangebur y, New York), a competitive protein binding assay previously described in detail. Folate concentrations were inversely related to the measured radioac tivity. Serum and RBC folate concentrations >12 nmol/L and >317 nmol/L, respectively, repres ented the lower limit of norm al values for this study. Homocysteine Concentrations Samples to be analyzed for homocysteine were sent to Dr. Sally Stabler, University of Colorado Health Sciences Center, Division of He matology (Denver, CO). Total homocysteine concentration was quantified us ing a gas chromatograph-mass sp ectrometer (141). A plasma homocysteine concentration 12 mol/L was considered elevated when interpreting the results for this study. MTHFR Genotype Determination Samples were sent to DynaMetrix Limited, University of Leicester ( United Kingdom, where primers and probes were designed for the MTHFR 677C T polymorphism and analyses were performed using the DASH™ met hod with DynaScore Software v. 0.7.7 (http://www.dynametrix-ltd.com). Statistical Methods The initial statistical analys is determined basic means and standard deviations for demographic data, folate inta ke, and blood folate and homocys teine concentrations for the overall population. Another initial statistical analysis was conducte d to determine if there was a

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70 relationship among any of the covari ates of interest, which includ ed gender, genotype, age, and body mass index (BMI). Fo r the continuous variables (i.e., BMI and age), a one-way analysis of variance was used to determine whether the mean BMI and mean age differed between males and females. A Chi-square test was used to determine whether the propo rtion of responses in each of the categories differed among groups when an alyzing categorical response variables. All possible pairings were run, and only two pair s were found to be corr elated. Significant differences in BMI were detect ed between males and females w ith males having a higher mean BMI compared to females. BMI also was found to be positively correlated with age. Since BMI and gender and BMI and age were both highly correlated, BMI was not used in the analysis as correlations could lead to conf using results. Therefore, only gender and age were used as covariates in the analysis. Linear regression anal ysis was used to test for relationships between folate/folic acid intake and folate and homocysteine concentr ations. Generalized linear models (GLM) were used to conduct the re st of the analyses to determin e correlations for given response variables. The data used in the analyses were natu rally bound at zero, beca use individuals cannot have either negative intake or bl ood concentrations. This result ed in data that were non-normal and positively skewed to the right. To correct fo r this, the data were transformed to the natural log scale. The GLM were used on the transfor med data. The estimate d least squares means obtained from the GLM were retransformed to original scale along w ith their appropriate confidence bounds. The estimated l east squares means also are the means that are corrected for the covariates of interest (age and gender). Normalized data were used consistently in reporting the results of the analyses.

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71 Table 3-1. Description of foods categorized within each food group. Food group Type of foods included Enriched cerealgrains Breads (enriched breads, rolls, bage ls, muffins, croissants, dumplings, biscuits, dessert breads, pan cakes, waffles, French toast) Cakes, cookies, doughnuts, Danish, swee t rolls, pies, cobblers, crisps, enriched with cereal-grain Enriched rice Enriched pasta, noodles, pasta with meatless red sauce Fortified cereals and bars Fortified ready-to-eat cer eals, hot breakfast cereal s, cereal bars, granola bars, power bars, slim fast bars Vegetable Vegetables, vegetable juice, tomato juice, tomato sauce without meat, vegetable medley Fruit Fruit, fruit juice, fruit drinks (other than orange/grapefruit juice) Juice (orange and grapefruit) Orange and grapefruit juice Legumes and nuts Legumes, soup with legumes, chili without meat, nuts/seeds, nut butters Dairy Dairy products (cheese, yogurt, milk, milkshakes, ice cream, frozen yogurt, cheesecake, sour cream, puddings/custards) Eggs Whole eggs, egg salad, egg whites, egg substitute Meat Meat, fish, poultry, meat/fish sauces, liver (all m eats except products prepared with enriched cereal-gra ins [e.g., breaded, batter dipped, and meatloaf]) Combination Combination foods (e.g., Mexican food, pizza, macaroni and cheese, chili with meat, lasagna with and without meat, pot pies, egg rolls, pasta salad, pasta with meat sauce) Breaded meats (meats and fish, breade d, batter-fried or meatloaf with enriched flour) Soups (creamed, noodles/rice, vegetables, no beans or meat) Snacks Snacks (chips, pretzels, crackers, popcorn) Miscellaneous Other fortified foods eaten minimally (beer, fortified vegetarian meals (e.g., Boca entre, burger, breakfast sausage, chicken, ground), tofu/soy products, soymilk, liquid m eal replacements, fortified egg substitute) Other Other foods with negligible folate (fats/oils, alcoho l [except beer], dressings, sugars, gelatins/jam s/jelly, candy, soda, tea, coffee without cream, rice milk)

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72 CHAPTER 4 RESULTS Characteristics of Study Population Three hundred eighty-eight subjects were enro lled in this study and 302 subjects (140 males, 162 females) were eligible for inclusion in th e final analyses. Su bjects excluded after enrollment included 62 subjects who reported supple ment use within the pa st six months and six who failed to return their DHQ. Data from another 18 subjects we re excluded because estimates of their total caloric intake derived from their DHQ data was less than 600 calories (n = 9 females) or greater than 5,000 cal ories (n = 9 males). These ca lorie levels are considered improbable, so these subjects we re excluded from the study. Of the 302 eligible subjects, the mean daily caloric intake (mean SD) fo r males and females was 2,382 958 and 1,744 805, respectively; males consumed si gnificantly more calories per day than females (P <0.0001). The demographic characteristics of males and females in this study population are presented in Table 4-1. The me an age (years) and BMI (kg/m2) (mean SD) for all study participants were 26.0 7.7 and 23.5 4.1, respectively. The education level of all subjects was 15.0 2.4 years, which is equivalent to status as a ju nior at the university level. Body mass index was the only variable that was significantly di fferent between genders with males having a higher BMI (P <0.0001) than females. All eligible participants were categor ized by their genotyp e for the MTHFR 677C T single nucleotide polymorphism. The number of subjects within each genotype group was as follows: CC genotype (n = 151) CT genotype (n = 118), and TT genotype (n = 31). Two subjects had unknown genotypes. The subjects with known genotype data included 140 men (68 CC, 56 CT, 16 TT) and 160 wome n (83 CC, 62 CT, 15 TT).

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73 Dietary Folate Intake and Gender In relation to objective 1, four cat egories of folate/fol ic acid intake were analyzed from the DHQ data. These categories incl uded total folate, DFE, folic acid, and food folate. After adjusting for differences in age, the mean in take for each folate category (g/d and g/1,000 kcals/d) was evaluated for males and females. Mean daily folate /folic acid intake (g/d) for males and females among the four fo late categories is presented in Table 4-2 and illustrated in Figure 4-1. Unadjusted for calorie s, mean total folate, DFE, foli c acid and food folate intake were significantly (P <0.01) high er for males than females. Mean folate/folic acid intake for males and females was further analyzed after adjusting for calories (g/1,000 kcals/d), the data for which are presented in Table 4-3 and illustrated in Figure 4-2. After adjusting for caloric intake, females had significan tly higher mean intakes (g/1,000 kcals/d) of total folate (P = 0.02) DFE (P = 0.05), and food folate (P <0.01) than males. There was no statistically significant difference between males and females for me an folic acid intake (g/1,000 kcals/d) (P = 0.9). Dietary Folate Intake and Genotype Mean folate intake (g/ d) of subjects by MTHFR 677C T genotype was compared for each folate category (Table 4-4) and adjusted for age and gender. As expected, no significant differences were detected among ge notype groups for intakes of tota l folate (P = 0.7), DFE (P = 0.9), folic acid (P = 0.9), or food folate (P = 0.8). Similarly, wh en intake was expressed as g/1,000 kcals/d significant differen ces among genotype groups were not detected for total folate (P = 0.6), DFE (P = 0.9), folic ac id (P = 0.9), or food folate (P = 0.9) intake (Table 4-5).

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74 Dietary Folate Equivalents, Foli c Acid and Recommended Intakes Relative to objective 2, total da ily folate intake of males and females expressed as DFE was compared to the Reco mmended Dietary Allowance (RDA) of 400 g DFE/d. A significantly higher pr oportion of males (81.4%) consumed the RDA for folate compared to females (67.9%) (P <0.01) (Figure 4.3 ) In addition, female subjects’ intake of diet -derived folic acid, including folic acid from fortified ready-to-eat (RTE ) cereals, was compared to the recommended folic acid intake fo r women of reproductive potentia l (i.e., 400 g/d). Only 3.1% of females (n = 5) met this recommendation (Fi gure 4-4). Of the rema ining female subjects, 24.7% (n = 40) consumed 200 to 399 g/d folic acid, and 72.2% (n = 117) consumed <200 g/d folic acid. Consumption of fo lic acid did not exceed the UL ( 1,000 g/d) for any of the male or female participants. The highes t estimated intake of folic acid for any male or female subject was 947 g/d and 665 g/d, respectively. Contribution of Folate from Food Groups In relation to objective 3, to an alyze the contribution of vari ous foods to dietary folate intake, foods included in the DHQ were categorized into thir teen categories. A comprehensive descriptive list of foods within each food cate gory is presented in Table 3-1. Foods were categorized based on type, folic acid enrichment/f ortification status, and the amount of naturally occurring food folate. Mean dail y total folate intake (g/d) contributed by various food sources is presented in Table 4-6. Both genders cons umed the greatest amount of total folate from enriched grain products, and fo rtified cereals and bars including fortified RT E breakfast cereals, vegetables, and legumes and nuts. Mean daily DFE intake (g DFE/d) contributed from various food categories is presented in Table 4-7. The largest contribu tors of DFE for both genders included fortified cereals and bars, enriched grain products, vegetables, and combination foods.

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75 Mean daily folic acid intake ( g/d) contributed from various food categories is presented in Table 4-8. Both males and females primarily cons umed folic acid in their diet from enriched grain products, fortified cereals a nd bars, and combination foods. Lastly, mean daily food folate intake (g/d) contribut ed from various food categories is pr esented in Table 4-9. Vegetables were the largest contributor of food folate, two-fold higher than any other food source. However, other good sources of food folate fo r both genders included legumes and nuts, and fruits. Within each folate category, no significant di fferences between male s and females were detected in the folate/folic acid contribution from various food categories except for the snack category. The folate/folic acid contribution from snack foods was significantly higher (P <0.02) for females than males; however, snack foods were not a large contributor to the overall intake of folic acid or food folate. The proportion of total folate, DFE, folic acid and food folate intake provided by different food categories to total intake is illustrated for males and fe males in Figures 4-5 and 4-6, respectively. Enriched grains (24.4%), vegetables (21.8%), and fortified RTE cereals and bars (12.5%) provided the majority of total folate for males, whereas vegetables (27.6%) provided the most total folate in females, followed by enri ched grains (20.4%) and fo rtified RTE cereals and bars (13.3%). Enriched grains contributed the most to DFE in take for both males (26.2%) and females (22.0%), followed by vege tables (17.0%) and fortified RT E cereals and bars (16.4%) for males, and similarly, vege tables (21.6%) and fortif ied RTE cereals and bars (19.0%) for females. The food categories that supplied the most folic acid for males a nd females included enriched grains (41.1%; 41.9%, respect ively), fortified RTE cereal s and bars (29.3%; 36.0%, respectively), and combination f oods (17.5%; 14.8%, respectively). The largest contributors of

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76 food folate for males and female s included vegetables (31.6%; 38.4%, respectively) and legumes and nuts (16.2%; 14.4%, respectively). With the exception of vegetabl es, the greatest contributors of folic acid intake (i.e., enriched cereal-grain products, fortified cereals and bars, and co mbination foods that included “enriched” ingredients) were also the greatest contributors to DFE inta ke. Without folic acid fortification of these products, DFE intake would have been sole ly provided by food folate intake from dietary sources (e.g., vegetables, legumes and nuts, and fruit). Furthe rmore, DFE intake for males and females would have been 293 g DFE/d and 249 g DFE/d, respectively, which would have been insuffic ient to meet the RDA. Folate Intake from Fortified Ready-to-Eat Cereals Intake of DFE and folic acid consumed from fortified RTE cereals wa s compared to the total daily DFE and folic acid intake to determ ine what proportion of DF E and folic acid in the diet comes from fortified RTE cereals. The percentage of ma les and females who consumed fortified RTE cereals was 91.4% an d 96.9%, respectively. Of thes e subjects, 50% and 58% of males and females, respectively, consumed highly fort ified RTE cereals. For males, 41.6% of total DFE intake and 23.0% of to tal folic acid intake came from consumption of fortified RTE cereals (Figure 4-7); for females, 37.8% of total DFE intake and 20.9% of total folic acid intake came from consumption of fortified RTE cereal s (Figure 4-8). The mean DFE intake from fortified RTE cereals alone for males and females was 103 g DFE/d and 97 g DFE/d, respectively. The mean folic acid intake from fortified RTE cereals for males and females was 59 g/d and 56 g/d, respectively.

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77 Folate and Homocysteine Concentrations and Gender Relative to objective 1, mean serum folate, RBC folate, and homocysteine concentrations were determined for males and females, adjusted for age, and are presen ted in Table 4-10. No significant differences in serum folate (P = 0.3) or RBC folate (P = 0.2) concentrations were detected between males and females. Mean seru m folate and RBC folate concentrations for both genders were above the limits considered normal for the radi obinding assay (>13.6 nmol/L; >317 nmol/L, respectively) (Figures 4-9 and 4-10). Although males had a significantly higher mean homocysteine concentration compared to females (P <0.0001), mean homocysteine concentrations for males and females were in an acceptable range (<12.0 mol/L) (Figure 4-11). Each subjects’ serum folate, RB C folate, and homocysteine co ncentrations were compared against the limits considered normal for each meta bolite. Only one male subject fell below the recommended limit for serum folate (>13.6 nmol/L ) with a concentratio n of 11.7 nmol/L. The participants whose values were below the mini mum recommended concentr ation for RBC folate (>317 nmol/L) included 1 male subject (289 nmol/L) and 3 female subjects (179, 259, and 278 nmol/L). Nine male subjects had elevated (>12.0 mol/L) homocysteine concentrations (12.4 to 45.7 mol/L). Of these males, 4 subjects had homocysteine concentrat ions >14 mol/L (21.6, 27.0, 29.6, 45.7 mol/L). Three female subjects had high homocysteine concentrations (12.4, 12.9, 13.0 mol/L). The one male subject with a low serum folate concentration (11.7 nmol/L) had a high homocysteine concentration (29.6 mol /L) and normal RBC fola te concentration (460 nmol/L). The male subject w ith the highest homoc ysteine concentrati on (45.7 mol/L) had a borderline low serum folate co ncentration (14.9 nmol/L) and nor mal RBC folate concentration (589 nmol/L).

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78 Folate and Homocysteine Concentrations and Genotype Relative to objective 4, the re lationship between MTHFR 677 C T genotype and serum folate concentration and MTHFR 677 C T genotype and RBC folate concentration also were determined. Mean serum and RBC folate concentrations by geno type for all subjects and for males and females are presented in Tables 4-11 and 4-12, respectively, and illustrated in Figures 4-12 and 4-13. Significant differe nces were not detected in se rum folate concentrations among the genotype groups for all subject s or for males or females. However, for all participants, individuals with the CT or TT genotypes had significan tly lower mean RBC folate concentrations (P <0.01) relative to the mean RBC folate of i ndividuals with the CC genotype (Table 4-12). No significant difference was detected for mean RBC folate concentration between individuals with the CT and TT genotypes (P = 0.5 ). When mean RBC folate concentrations among genotype gr oups were compared within each gender, the trend for RBC folate concentrations by genotype for males was similar to the tr end for all subjects. However, the same genotype effect on RBC folate was not detect ed for female subjects. When mean homocysteine concentrations were compared among the MTHFR 677 C T genotype groups for all subjects, significant diff erences (P <0.05) were detected between the CC versus CT genotypes and the CC versus TT genotypes. No signi ficant differences were detected between individuals with the CT and TT genotypes (P = 0.4) (Table 4-13). As presented in Table 4-13, the genotype effect on homocysteine concentrations observed for all subjects was different when data were separated by gender Significant differences in mean homocysteine concentrations were detected among genotype groups for males, but not for females. Male subjects with the TT or CT genot ypes had the highest mean hom ocysteine concentrations (9.2

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79 mol/L; 8.5 mol/L, respectively), whereas, male subjects with th e CC genotype had the lowest mean homocysteine concentration (7 .5 mol/L). These data are also illustrated in Figure 4-14. Serum Folate and Homocysteine When the association between serum folate and homocysteine concentrations was evaluated, a significant inverse association was found for all par ticipants (P <0.001), as well as within gender groups (i.e., males, P = 0.02; females, P = 0.02). Th ese associations indicate that as the concentration of serum folate increased, homocysteine concentration decreased. This association was further tested within each gender and by genotype group. For males, a significant association between serum folate and homocysteine co ncentration was not detected for either the CC or TT genotype groups (P = 0.5; P = 0.3, respectively). Th ere was a trend toward a significant association for CT genotype group (P = 0.06). A significant association also was not detected for females within the CC ge notype group (P = 0.4) or the TT genotype group (P = 0.1). However, a significant inverse asso ciation between serum folate and homocysteine concentration was detected for females within the CT genotype group (P = 0.03). Relationship Between Fola te Intake and Status Relative to objective 4, the a ssociation between serum or RBC folate concentrations and folate/folic acid intake (g DF E/d) was assessed using linear re gression analysis to determine whether the level of DFE intake impacted serum or RBC folate concentra tions. As hypothesized, a significant positive association between serum folate concentr ation and DFE intake for all subjects was detected (P = 0.03). When this relationship was analyzed by gender, there was insufficient power to detect whet her significant differences existe d for males or females (P = 0.2; P = 0.4, respectively). No relationship was detect ed between RBC folate and DFE intake for all subjects (P = 0.5) or by gender (P = 0.07).

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80 The association between homocys teine concentration and fola te/folic acid intake (g DFE/d) also was examined to determine if the level of folate/folic acid intake impacted homocysteine concentration. A significant inverse associ ation between homocysteine concentration and DFE intake wa s found for all participants (P <0.0001), as well as for males (P = 0.02). The behavior of this re lationship was not constant betw een genders as an association was not detected in females. Folate Status and Fortified Re ady-to-Eat Cereal Consumption Although no significant difference was detected, serum folate concentration tended to be different (P = 0.08) between consumers and nonconsumers of fortified RTE cereals for all subjects. Gender differences in serum folate and RBC folate concentrations between consumers and non-consumers of fortifie d RTE cereals were not detected Comparison of homocysteine concentrations of consumers and non-consumers of fortified RTE cereals identified a significant difference in mean homocystein e concentration between female consumers (6.5 mol/L) and non-consumers (8.4 mol/L; P <0.05 ). A significant difference in mean homocysteine concentration between male c onsumers (8.0 mol/L) and non-c onsumers (8.4 mol/L) was not detected (P >0.5).

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81 Table 4-1. Demographic characteristics of males and females.a Demographic variable Males (n = 140) Females (n = 162) P-value Age (years)b,c 26.0 8.2 25.3 7.10.6 BMI (kg/m2)b,c 24.5 4.322.6 3.6<0.0001 Education level (years)b,c 16.0 0.2 15.2 2.50.4 Ethnicity (%)d 0.4 White 5863 Asian 1712 African American 74 Indian 23 Hispanic 1115 Other 41 Unknown 11 Marital Status (%)d 0.7 Single 7977 Married 1517 Divorced 43 Separated 12 Refused 21 Student Status (%)d 0.2 Full-Time 6765 Part-Time 47 Not a student 915 Unknown 1913 Employed (%)d 0.2 Yes 5559 No 3436 Unknown 115 aDue to rounding, percentages may not always sum to 100. bMean standard deviation (SD). cOne-way ANOVA was used for statistical comparisons between genders. dChi-square test was used for statistical comparisons betwee n genders and demographic variables.

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82 Table 4-2. Mean intake (g/d) by folate category and gender.a,b,c Folate group Males (n = 140) Females (n = 162) P-value Total folate 462 (426, 501)371 (344, 400)0.0001 DFE 652 (603, 705)512 (476, 551)<0.0001 Folic acid 175 (156, 195)128 (115, 142)<0.0001 Food folate 293 (268, 321)249 (229, 270)0.01 aMean (5%, 95% CI). bOne-way ANOVA was used for statis tical comparisons between genders after adjusting for age. cP-values were based on normalized (l og transformed) data. The results have been back-transformed to original scale. TF DFE FA FF 0 250 500 750 Males Females *Folate CategoryMean Folate Intake (g/d) Figure 4-1. Mean intake (g/d) by folate category and gender. TF = total folate; DFE = dietary folate equivalents; FA = folic acid; FF = food folate. *Significant difference (P <0.01) in mean intake (g/ d) by folate category between males and females after adjusting for age.

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83 Table 4-3. Mean intake (g/1,000 kcal s/d) by folate category and gender.a,b,c Folate group Males (n = 140) Females (n = 162) P-value Total folate 210 (197, 224)233 (219, 247)0.02 DFE 296 (279, 314)321 (304, 340)0.05 Folic acid 79 (72, 87)80 (73, 88)0.9 Food folate 133 (124, 143)156 (146, 167)<0.01 aMean (5%, 95% CI). bOne-way ANOVA was used for statis tical comparisons between genders after adjusting for age. cP-values were based on normalized (l og transformed) data. The results have been back-transformed to original scale. TF DFE FA FF 0 50 100 150 200 250 300 350 Males Females *Folate GroupMean Folate Intake (g/1,000 kcals/d) Figure 4-2. Mean intake (g/1,000 kcals/d) by folate category and gender. TF = total folate; DFE = dietary folate equivalents; FA = fo lic acid; FF = food folate. *Significant difference (P 0.05) in mean intake (g/1,000 kcals/d) by fola te category between males and females after adjusting for age.

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84 Table 4-4. Mean intake (g/d) by folate category and genotype.a,b,c Folate group CC genotype (n = 151) CT genotype (n = 118) TT genotype (n = 31) P-value Total folate 423 (391, 456)395 (361, 429)410 (344, 477) 0.7 DFE 581 (538, 625)559 (512, 606)583 (490, 675) 0.9 Folic acid 151 (135, 167)141 (125, 158)161 (126, 196) 0.9 Food folate 273 (250, 296)262 (238, 287)265 (218, 311) 0.8 aMean (5%, 95% CI). bOne-way ANOVA was used for statis tical comparisons between genders after adjusting for age. cP-values were based on normalized (l og transformed) data. The results have been back-transformed to original scale. Table 4-5. Mean intake (g/1,000 kcals/d) by folate category and genotype.a,b,c Folate group CC genotype (n = 151) CT genotype (n = 118) TT genotype (n = 31) P-value Total folate 227 (213, 241)215 (200, 229)224 (195, 252) 0.6 DFE 312 (294, 330)304 (285, 323)317 (279, 356) 0.9 Folic acid 81 (74, 88)77 (69, 84)88 (71, 104) 0.9 Food folate 146 (136, 157)143 (132, 154)144 (123, 165) 0.9 aMean (5%, 95% CI). bOne-way ANOVA was used for statis tical comparisons between genders after adjusting for age. cP-values were based on normalized (l og transformed) data. The results have been back-transformed to original scale.

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85 0% 25% 50% 75% 100% Females Males <200 g/d 200-399 g/d 400-999 g/d 1,000 g/d Percentage of males/females consuming specific ranges of folate expressed as g DFE/d Figure 4-3. Percentage of subj ects who consumed speci fic ranges of folate expressed as g DFE/d. 0% 25% 50% 75% 100% Females Males <200 g/d 200-399 g/d 400-999 g/d Percentage of males/females consuming specific ranges of folic acid (g/d) Figure 4-4. Percentage of su bjects who consumed specific ra nges of folic acid (g/d).

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86 Table 4-6. Mean total fola te contribution ( g/d) by dietary source and gender.a Dietary source Males (n = 140) Females (n = 162) Enriched cereal-grains 104 694 5 Fortified cereals and bars 66 963 8 Vegetable 117 9127 9 Fruit 25 227 2 Juice (orange and grap efruit) 16 214 2 Legumes and nuts 58 651 5 Meat 4 03 0 Combination 18 219 2 Snacks 7 2b13 2 Dairy 14 115 1 Eggs 5 15 1 Miscellaneous 24 425 4 Other 9 19 1 aMean SE. One-way ANOVA was used for stat istical comparisons between genders after adjusting for age and calories. bSignificantly lower than females (P = 0.01). Table 4-7. Mean DFE contri bution (g DFE/d) by diet ary source and gender.a Dietary source Males (n = 140) Females (n = 162) Enriched cereal-grains 158 9143 9 Fortified cereals and bars 128 17129 15 Vegetable 119 10130 9 Fruit 25 2 27 2 Juice (orange and grap efruit) 16 214 2 Legumes and nuts 59 652 5 Meat 5 14 1 Combination 66 459 4 Snacks 10 2b18 2 Dairy 14 115 1 Eggs 5 15 1 Miscellaneous 35 536 5 Other 9 110 1 aMean SE. One-way ANOVA was used for stat istical comparisons between genders after adjusting for age and calories. bSignificantly lower than females (P = 0.01).

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87 Table 4-8. Mean folic acid contribution (g/d) by di etary source and gender.a Dietary source Males (n = 140) Females (n = 162) Enriched cereal-grains 78 571 5 Fortified cereals and bars 73 1073 9 Vegetable 0 00 0 Fruit 0 00 0 Juice (orange and grap efruit) 0 00 0 Legumes and nuts 0 00 0 Meat 0 00 0 Combination 26 224 2 Snacks 4 1b7 1 Dairy 0 00 0 Eggs 0 00 0 Miscellaneous 7 26 2 Other 0 00 0 aMean SE. One-way ANOVA was used for stat istical comparisons between genders after adjusting for age and calories. bSignificantly lower than females (P = 0.02). Table 4-9. Mean food folate cont ribution (g/d) by dietary source and gender.a Dietary source Males (n = 140) Females (n = 162) Enriched cereal-grains 25 123 1 Fortified cereals and bars 4 14 0 Vegetable 117 9127 9 Fruit 25 227 2 Juice (orange and grap efruit) 16 214 2 Legumes and nuts 58 651 5 Meat 4 03 0 Combination 8 18 1 Snacks 3 1b6 1 Dairy 14 115 1 Eggs 5 1 5 1 Miscellaneous 23 327 3 Other 9 19 1 aMean SE. One-way ANOVA was used for stat istical comparisons between genders after adjusting for age and calories. bSignificantly lower than females (P = 0.02).

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88 0% 20% 40% 60% 80% 100% FF FA DFE TF Enriched cereal grains Fortified cereals and bars Vegetables Fruit Juice (orange and grapefruit) Legumes and nuts Dairy Eggs Meat Combination Snacks Miscellaneous Other Food groups Percent of folate and folic acid intake by food group (males) Figure 4-5. Percentage of tota l intake derived from each food group by folate category for males. TF = total folate, DFE = dietary folate equivalents, FA = folic acid, FF = food folate. The relative co ntribution of each food gr oup to TF, DFE, FA and FF intake was calculated by dividing the mean intake of each food group by total intake for each folate category (e.g., mean DFE inta ke from enriched cereal grains divided by total mean DFE intake). 0% 20% 40% 60% 80% 100% FF FA DFE TF Enriched cereal grains Fortified cereals and bars Vegetables Fruit Juice (orange and grapefruit) Legumes and nuts Dairy Eggs Meat Combination Snacks Miscellaneous Other Food groups Percent of folate and folic acid intake by food group (females) Figure 4-6. Percentage of to tal intake derived from each f ood group by folate category for females. TF = total folate, DFE = dietar y folate equivalents, FA = folic acid, FF = food folate. The relative co ntribution of each food gr oup to TF, DFE, FA and FF intake was calculated by dividing the mean intake of each food group by total intake for each folate category (e.g., mean DFE inta ke from enriched cereal grains divided by total mean DFE intake).

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89 0% 20% 40% 60% 80% 100% FF FA DFE TF Fortified RTE cereals All other dietary sources Percent contribution of enriched RTE cereal to the folate/folic acid intake (males) Figure 4-7. Mean percent contribu tion of fortified RTE cereal to the total me an intake for each folate category for males. TF = total fola te, DFE = dietary folate equivalents, FA = folic acid, FF = food folate. 0% 20% 40% 60% 80% 100% FF FA DFE TF Fortified RTE cereals All other dietary sources Percent contribution of enriched RTE cereal to the folate/folic acid intake (females) Figure 4-8. Mean percent contribu tion of fortified RTE cereal to the total me an intake for each folate category for females. TF = total fo late, DFE = dietary folate equivalents, FA = folic acid, FF = food folate.

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90 Table 4-10. Comparison of serum and RBC folate, and homocysteine concentrations by gendera,b,c MalesFemalesP-value Serum folate (nmol/L) 39.9 (37.8, 42.0)41.7 (39.6, 43.8)0.3 RBC folate (nmol/L) 810 (766, 857)767 (727, 809)0.2 Homocysteine (mol/L) 8.0 (7.7, 8.4)6.6 (6.3, 6.9)<0.0001 aConcentrations expressed as mean (5%, 95% CI). bOne-way ANOVA was used for statistical comparisons between genders after adjusting for age. cP-values were based on normalized (log transformed) data. The results have been back-transformed to original scale.

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91 Males Females 0 5 10 15 20 25 30 35 40 45 Normal (>13.6 nmol/L)Serum Folate (nmol/L) Figure 4-9. Mean serum folate concentration (n mol/L) by gender after adjusting for age. Normal serum folate status was define d as a serum folate concentration >13.6 nmol/L. Males Females 0 100 200 300 400 500 600 700 800 900 Normal (>317 nmol/L)RBC Folate (nmol/L) Figure 4-10. Mean RBC folate concentration (n mol/L) by gender after adjusting for age. Normal RBC folate status was defined as a RBC folate concentration >317 nmol/L. Males Females 0 2 4 6 8 10 12 Acceptable cutoff (<12mol/L)*Homocysteine (mol/L) Figure 4-11. Mean homocystein e concentration (mol/L) by gend er after adjusting for age. Normal homocysteine concentration de fined as <12 mol /L. *Significant difference (P <0.0001) in mean homocyste ine concentration (mol/L) between males and females.

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92 Table 4-11. Comparison of mean serum fola te concentrations (nmol/L) by genotype.a,b Gender CC genotypeCT genotypeTT genotype All subjectsc 42.5 (40.3, 44.7)39.3 (37.1, 41.6)38.5 (34.3, 42.6) Malesd 41.7 (38.6, 44.9)38.6 (35.4, 41.8)36.9 (31.3, 42.4) Femalesd 43.3 (40.2, 46.3)40.0 (36.8, 43.2)40.2 (34.1, 46.3) aMean (5%, 95% CI). bGLM procedure was used for statis tical comparisons of mean serum folate concentrations between ge notypes for each gender after adju sting for age. P-values were based on normalized (log transforme d) data. The results have been back-transformed to original scale. cUnable to detect significant differences in serum folate co ncentrations among genotype groups (P >0.05). dNo significant differences detected in serum folate among genotype groups for males or females (P >0.1). Table 4-12. Comparison of mean RBC folate concentra tions (nmol/L) by genotype.a,b Gender CC genotypeCT genotypeTT genotype All subjectsc,d 848 (803, 895)744 (700, 790)713 (634, 801) Males e,f 878 (811, 944) 756 (693, 819)733 (622, 845) Femalesg,h 821 (762, 880)732 (673, 790)691 (584, 798) aMean (5%, 95% CI). bGLM procedure was used for statistical comparisons of mean RBC folate concentrations between genotype groups after adjusting for ag e. P-values were based on normalized (log transformed) data. The results have been back-tra nsformed to original scale. cCC genotype significantly higher than CT genotype (P = 0.002). dCC genotype significantly higher than TT ge notype (P <0.01). eCC genotype significantly high er than CT genotype (P = 0.01). fCC genotype significantly higher than TT genotype (P = 0.05). gCC genotype significantly high er than CT genot ype (P = 0.04). hUnable to detect a si gnificant difference for CC versus TT genotype (P = 0.07). Table 4-13. Comparison of mean homocystei ne concentrations ( mol/L) by genotype.a,b Gender CC genotypeCT genotypeTT genotype All subjectsc 6.9 (6.6, 7.2)7.5 (7.1, 7.8)7.8 (7.1, 8.6) Malesd,e 7.5 (7.0, 7.9)8.5 (7.9, 9.0)9.2 (8.0, 10.3) Femalesfg 6.5 (6.1, 6.8)6.6 (6.2, 7.1)6.6 (5.8, 7.5) aMean (5%, 95% CI). bGLM procedure was used for stat istical comparisons of mean homocysteine concentrations betw een genotypes for each gender after adjusting for age. P-values were based on normali zed (log transformed) data. The results have been backtransformed to or iginal scale. cCT and TT genotypes significantly higher than CC genotype (P = 0.03). dCT genotype significantly highe r than CC genotype (P = 0.01). eTT genotype significantly high er than CC genotype (P <0.01). fNo significant differ ences detected in homocysteine concentrations among geno type groups for females (P >0.05).

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93 CC CT TT 0 10 20 30 40 50 Females Males All subjects MTHFR 677C T GenotypeSerum Folate (nmol/L) Figure 4-12. Mean serum folate concentration (n mol/L) by genotype after adjusting for age. CC CT TT 0 250 500 750 1000 Females Males ***All subjects ** **MTHFR 677C T GenotypeRBC Folate (nmol/L) Figure 4-13. Mean RBC folate concentration (nmo l/L) by genotype after adjusting for age. *Significant difference (P <0.01) in mean RBC folate concentration compared to all subjects with the CC genotype **Significant difference (P 0.05) in mean RBC folate concentration compared to males with the CC genotype. ***Significant difference (P = 0.04) in mean RBC folate concentration compared to females with the CC genotype.

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94 CC CT TT 0 1 2 3 4 5 6 7 8 9 10 11 Females Males **All subjects **MTHFR 677C T GenotypeHomocysteine (mol/L) Figure 4-14. Mean homocys teine concentration (mol/L) by geno type after adjusting for age. *Significant difference (P = 0.03) in mean homocysteine concentration compared to all subjects with th e CC genotype. **Signi ficant difference (P 0.01) in mean homocysteine concentration compared to males with the CC genotype.

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95 CHAPTER 5 DISCUSSION AND CONCLUSIONS The focus of this study was to assess folate intake a nd status of non-supplement consuming healthy young men and women and to estimate the rela tive contribution of folate/folic acid from different f ood categories to overall intake. Previous research studies have addressed the impact of folic acid enrichment of cereal-grain products and fo lic acid fortification of breakfast cereals on folate status of men and women (36, 13, 125, 150), but the present study is the first to include an estimat e of the relative contri bution of folate/folic acid from different food categories to overall intake Information related to the contribution of different food categories to folate/folic acid in take can be used to develop ta rgeted intervention strategies directed at improving fola te/folic acid intake. Recent updates of the USDA National Nu trient Database for Standard Reference (Release 17), which reflects folic acid fortification of foods, separa tes dietary folate intake into four categories: food folate, fo lic acid, total folate, and DFEs (8). Unlike most previous investigations that only reported total folate intake (i.e., g of food folate plus g of folic acid without adjustment for di fferences in bioavailability), our st udy provides separate data on folic acid and food folate intake enab ling a comparison with the reco mmended level of folic acid for women of reproductive potential, as well as intake expressed as DFE, a calculated value essential for comparison to the RDA for males and females. The majority of reports in th e literature have used data from NHANES to evaluate the adequacy of folate intake in th e US post-fortification (4, 13, 151). One of the limi tations of that database is that nutrient inta ke data are estimated for each individual from a single 24-hour dietary recall. In the presen t study, the previously validated DHQ was used to assess dietary intake based on recall of food frequency over a 12 month period (144). Another limitation

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96 associated with using the NHANES da tabase is the fact that intake analysis data are reported as total folate intake (g/d) and do not provide folate intake expr essed as DFEs; consequently, the researchers using that database were unable to compare inta ke relative to the RDA. A study by Dietrich et al (13), in which total folate inta ke (g/d) of males and females was compared before and after fort ification, reported that the abso lute increase in total folate (g/d) were larger among men than women. These investigators attr ibuted this to the fact that men, in general, have higher dietary intakes of folate than women. Alth ough our analysis also indicated that males consumed more folate per day than females, the average total folate intake (g/1,000 kcals/d) was significantly higher for females. Adjusti ng for calories allows for more equal comparison between genders as the DHQ revealed that males consume more daily calories, and for this reason, males are more likely to consume more folate /folic acid than females on any given day. Based on data from NHANES 1999-2000, Briefel et al. (152) reported that among women of childbearing age (20 to 39 years), mean total folate in take was 327 g/d. Dietrich et al. (13) also evalua ted folate intake of NHANES 1999-2000 participants and found that the mean total folate intake of ma les and females combined (aged 20 years) was 351 g/d. In comparison, our data revealed a slightly higher average total folate intake of 371 g/d for women. The average total folate intake for men was 462 g/d. Ho wever, since these data do not account for the form of the vita min consumed (i.e., food folate versus folic acid from enriched and fortified products), it is not pos sible to compare these intake le vels fairly, nor is there a way to determine if they would meet the current RDA for folate. Our da ta, however, revealed that the mean total intake expressed as DFEs for ma les and females (652 g DFE/d; 512 g DFE/d, respectively) exceeded the RDA of 400 g DFE/d, with the majority of males (81%) and females

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97 (68%) meeting the RDA. Interestingly, after ad justing for caloric inta ke, females in our study had a significantly higher averag e intake of food folate compared to males, but no difference was detected for folic acid intake between genders. The present study estimated th e actual and relative cont ribution of different food categories to overall folate/folic acid intake. This unique aspect of the present study identified the major contributors of daily folate intake (i.e., g DFE/d) to be enriched cereal-grains products, fortified cereals and ba rs, and combination foods that include “enriched” ingredients for both males and females. When comparing th e actual contribution of food categories to total folate intake, vegetables contri buted the highest amount of tota l folate, followed by enriched cereal-grains products for men and women alike. The differe nces noted in average intake between total folate and DFE are due to the wa y DFEs are calculated, where more weight is given to folic acid than endogenous food folate (g DFE/d = 1.7 x g/d folic acid + g/d food folate). The IOM recommends that all women of repr oductive potential cons ume 400 g of folic acid daily from vitamin supplements and/or fortif ied foods in addition to consuming food folate from a varied diet to reduce the risk of having an NTD-affected pregnancy (7). Yang et al. (6) examined folic acid in take in women of childbearing age in the US using NHANES 2001-2002 data and nutrient intakes estimated from th e USDA National Nutrient Database Standard Reference, Release 16. Mean c onsumption of folic acid from fo rtified foods was 128 g/d in non-supplement consuming, non-pregnant women, and only 8% of the women reported consuming 400 g/d of folic acid from fortified foods Similar to the findings by Yang et al., the average intake of folic acid from fortified foods for women in the present study who were non-supplement users was 128 g/d or approximate ly one-third of the amount recommended for

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98 NTD risk reduction. Furthermore, only 3% of female subjects consumed 400 g/d of folic acid from enriched and fortified foods. This falls well short of the Healthy People 2010 goal of 80% of women of reproductive potentia l achieving a daily folic acid inta ke of 400 g. However, even though folic acid intake was inad equate relative to the amount recommended for NTD risk reduction, and only two-thirds of the women in our study ex ceeded the folate RDA, the estimated levels of folate/folic acid intake reported in our study were associated with normal blood folate status. Whether or not the level of intake that was associated with normal blood folate status in our study is adeq uate to achieve the level of NTD risk reduction associated with 400 g of folic acid is not known. Since folic acid enrichment of cereal-grain products was de signed to augment folic acid intake from supplements, it is understandable that the majority of women would not be consuming the recommended 400 g/d of folic acid from di etary sources alone. What the data from this investigation illustrate so clearly is that when women do not take supplements, which is the case for the majority of wo men, dependence on dietary sour ces of folic acid to meet the recommended intake is totally inadequate. The CDC, the Marc h of Dimes, and the National Council on Folic Acid have organized the Natio nal Folic Acid Campaign for the prevention of NTDs, with the goal of teach ing all women about the importa nce of folic acid and ensuring women consume 400 g of folic acid daily ( 17). The campaign ta rgets all women of reproductive age, the health care professi onals who serve these women, and community advocacy groups (153). However, despite all of these major folic acid awareness campaigns, these data illustrate that more attention needs to be focused on increasing folic acid intake from dietary sources for women of childbear ing age who do not take supplements.

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99 In 2005, the CDC and the March of Dimes launched the Prec onception Health and Health Care Initiative, the aims of wh ich are to improve th e health of mothers before pregnancy and maternal and infant health outcome s (130). As a result of this summit and initiative, strategies for improving precon ception health incorporating folic acid awareness have been incorporated into numerous programs throughout th e US, including areas in clinical practice, consumer roles, and public health (130, 154). However, many of th ese campaigns have prov en to be ineffective as recent data from the 2005 March of Dimes Gallup Survey reported by the CDC suggested only 33% of the general population of women of reproductive age ta ke a folic acid supplement daily (17). Therefore, the sole sour ce of folic acid in the diets of the majority of th is target group is enriched and fortified foods. Because only 3% of women in our study consumed 400 g/d of folic acid from fortified foods al one, it can be concluded that nonsupplement users are at risk for not meeting the recommended leve l of folic acid intake shown to result in NTD risk reduction. Women of reproductive potentia l who do not take supplement s should be encouraged to consume adequate amounts of fo rtified or enriched cereal grai n products daily to achieve the recommended level of folic acid intake. The present study also evaluated th e impact of folate intake on folate status of male and female non-supplement users. A significant positiv e association was detected between intake of DFE and serum folate concentrations for all su bjects, and a significant inverse association was found between intake of DFE and homocysteine concentrations for all subjects and for male subjects only. No relationship was detected between intake of DFE and RBC folate concentrations for all subjects or by gender. The mean folate/folic acid intake observed in this study was associated with adequate mean seru m folate concentrati ons for both males (39.9 nmol/L) and females (41.7 nmol/L). These concentrations are high er than reported by Jacques et

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100 al. (4), who reported that the m ean plasma folate concentratio n for both males and females who were non-supplement users in the Framingham Offspring Study (aged 32 to 80 years) was 23 nmol/L. Lawrence et al. (5) re ported a median serum folate concentration of 42.4 nmol/L postfortification; however, no inform ation was given on subjects’ foli c acid supplement use. Caudill et al. (155) investigated 135 women (aged 18 to 45 years) who were non-supplement users postimplementation of folic acid fortification. These women were reported to have a mean serum folate concentration of 50 nmol/L. Possible explanations for differences in blood folate concentrations among studies could be due to various ages of st udy participants, differences in analytical methodology for measurin g blood folate, and the fact that folic acid fort ification had been in effect longer when Caudill et al. comp leted their study and when blood samples used for folate analysis were collect ed in the current study. Based on data from the NHANES, Pfeiffer et al. (156) reported that the prevalence of low serum folate concentrations (<7 nmol/L) for wo men of childbearing age (15 to 45 years) was <1%. Serum folate samples collected as part of the NHANES study were analyzed using the Bio-Rad QuantaPhase II radiobindi ng assay. This method yields lo wer serum folate values than the microbiological method, the latter of whic h uses a cutoff value of 13.6 nmol/L (61). Although the cutoff value for serum folate concentration of 13.6 nm ol/L was used in the present study, none of the women in this investigation fell below this le vel. If the Bi o-Rad norm for serum folate that was establishe d with the radiobinding assay had b een used in th is study, then none of the male or female subj ects would have had a low serum folate concentration. Taken together, the data examining folate status pr ovide convincing evidence to indicate that a significant proportion of the population, particularly women of reproductive age, have more than adequate folate status.

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101 With respect to RBC folate, mean RBC fola te concentrations in the present study for males (810 nmol/L) and females (767 nmol/L) we re well above the lower limit of normal (i.e., 317 nmol/L) based on the value established for us e with the radiobinding assay RBC folate concentration is a better indicator of long-term folate status than seru m folate concentration. Only 2% of females and <1% of males had RBC folate concentrations <317 nmol/L. Our data are similar to those reported by Choumenkovitch et al. (3) indicating th at 96.1% of women >45 years who were non-supplement users had adequate RBC folate concentrations ( 362.6 nmol/L). Despite the fact that the major ity of our female subjects did no t meet the recommended level of intake for NTD risk reduction, 98 % of them had adequate RBC fola te concentrations. However, whether or not their status is sufficient to re duce NTD risk is unknown. Research from an Irish population indicated that R BC folate concentrations 908 nmol/L were associated with an eightfold difference in NTD incide nce compared to concentrations <341 nmol/L (85). However, these results may not be appli cable to a US population so furt her research is warranted. The benefits of adequate bl ood folate status may confer benefits related to chronic disease risk reduction. Mild fo late deficiency has been associ ated with an increased plasma homocysteine concentration, an in dependent risk factor for va scular disease (100, 157, 158). Homocysteine is also recognized to be a “functional indicator of folate status” (63). The data from our study confirm that se rum folate concentration is inversely related to plasma homocysteine concentrations. A lthough no significant differences in mean serum or RBC folate concentrations were detected be tween males and females, males had a significantly higher mean homocysteine concentration (8.0 m ol/L) compared to females (6.6 mol/L). These data agree with findings reported by Ganji et al. (66) that plasma homocys teine concentrations are often higher in males than females. Of the 6.5% of males (n = 9) with elevated homocysteine ( 12

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102 mol/L), five subjects fell below the mean serum folate concentration for males, six were below the mean RBC folate concentration for males, and four subjects consumed <400 g DFE/d. Although not evaluated as part of this study, a pos sible explanation for the elevated homocysteine concentrations of these subjects could be related to vitamin B12 deficiency (159, 160). Because elevated plasma homocysteine concentrations are considered a risk factor for cardiovascular disease, the association between homocysteine and folate broadens the healthrelated consequences of low fo late status and has implica tions for both men and women. Therefore, marketing folic acid consumption should not be limited to prevention of NTDs as folate status plays a role in various health-related problems. Our study also investigated the relationship between folate status, homocysteine and the MTHFR 677C T polymorphism. Comparab le to the findings of Br attstrom et al. (106), Kauwell et al.(59), and Shelnutt et al. (21), subjects with the TT genotype had higher homocysteine concentrations and lower RBC folate concentrations than individu als with the CC genotype. However, in contrast to these reports, serum folate co ncentrations in the present study population did not differ among geno type groups. One explanation fo r this could be due to the fact that the majority of our population consumed sufficient diet ary folate to maintain normal serum folate concentrations. Previous resear ch by Shelnutt et al. ( 21) determined that a predisposition for reduced blood fo late and elevated plasma hom ocysteine concentration could be significantly improved in re sponse to improved folate stat us. Other research has also confirmed the ability of increased food folate an d folic acid intake to positively modify the negative effects of the MTHFR polymorphism on folate status (161, 162). Ashfield-Watt and colleagues (161), however, determin ed that individuals with the TT genotype require higher

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103 intakes of folic acid from fortified foods or s upplements than individu als with the CC or CT genotype to achieve a similar lowering effect on homocystein e concentrations. Another aim of our study wa s to characterize the relative contribution of specific food categories to food folate/folic acid intake. The findings from this investigation in dicate that the intake of folic acid from fortif ied RTE cereals alone represented 20% of the total folic acid intake for women of reproductive age who consumed fortified cereal s. Seventy-eight percent of total folic acid in their diets was provided by a combination of enriched cereal-grain products and fortified cereals and bars, in cluding RTE cereals. For females, RTE cereal consumption also accounted for 13.3% of total folate intake and 19.0% of DFE intake Our findings are similar to those of Dietrich et al. (13) who reported that fortified RTE breakfast cereals pr ovided 12.1% of total folate intake. However, estimates of fo lic acid intake from our study population may not be representative of women of reproductive potentia l because we used the da ta collected for a study designed to assess vitamin B12 in take from dietary sources. The pr otocol for that study excluded women who consumed highly fortified RTE cer eals on a daily basis, but not those who consumed these products less frequen tly. These results may not be observed in other parts of the world where fortified RTE cereals are not a prevalent component of the diet. Although actual intake of fortified cereals mi ght be greater than re ported in the present study, our study identified fortified RTE cereal as an important source of folic acid in the diet. Most RTE cereals contain 100 g of folic acid per serving, and some highly fortified cereals contain as much as 400 g per serving. Examples of specific brands of highly fortified RTE cereal include All-Bran, Multigrain Cheerios, Complete, KASHI Heart-to-Heart, Mueslix, Product 19, Smart Start, Special K, and Total (all varieties). In this study, 58% of women who consumed fortified cereals re ported consuming highly fortif ied RTE cereals. A note of

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104 caution is that cereals are of ten consumed in amounts twice th e labeled serving size (163); therefore, individuals w ho consume highly fortified breakfast ce reals in large quantities have the potential to exceed the UL (1,000 g/d of folic acid). Nevertheless, no subjects in this study were found to consume the UL for folic acid. Since the majority of women of childbeari ng age are not taking fo lic acid supplements, promoting folic acid consumption from fortified RTE cereals and enriched cereal-grain products represents an important approach to the delive ry of folic acid in th e diet. Ensuring women consume the recommended qu antities of folic acid daily is fund amental as more than half of all pregnancies are unplanned and beca use the development of NTDs o ccurs during the first 28 days of pregnancy, often before many wo men realize they are pregnant. Strengths and Limitations Weaknesses of the current investigation incl ude limitations associated with the use of a FFQ to assess dietary folate inta ke, including the fact that there is no opti on for individuals to add personal responses related to food intake on the questionnaire. More accurate approaches to assess dietary intake include weighed food records or multiple-day food records. However, these methods are time consuming and te dious and are generally discourag ed for larger studies. The DHQ used in this study has been validated as an appr opriate method of as sessing usual dietary intake (144, 164). Nevert heless, this subgroup may not be repr esentative of men and women in the US population in contrast to NHANES, a population based study designed to be representative of the US population. Furthermore, the criteria used for the study from which our data were obtained attempted to exclude wome n who consumed highly fo rtified RTE cereals on a daily basis. This may have resulted in an underestimation of folic acid intake from fortified RTE cereals.

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105 Another limitation of this st udy was the lack of statistical power due to variability and limited observations when comparing folate status of subjects by MTHFR 677C T genotype within the same gender group. Anot her limitation of this study is that differences in measured blood folate concentrations have been noted between the radiobi nding assay, the method used in this study, and the microbiological as say method used by many other in vestigators. Fazili et al. (61) reported that the vitami n B12/folate radioassay kit ma y underestimate serum folate concentrations compared to the LC-MS/MS and microbiological assay. An advantage of using the radioassay in this st udy is the fact that this was the method used for analysis of blood folate concentrations in the most r ecent NHANES, making comparisons between the data in this study and data reported in the NHANES study possible. Strengths of the present study included the ability to estima te DFE intake, thus enabling us to compare intake to the RDA, and the abilit y to compare the relative contribution of specific food categories to total folate/folic acid intake in non-supplement us ers. Moreover, the fact that supplement users were excluded fr om the study made it possible to assess th e impact of dietary folate/folic acid intake on folate status. It would have b een ideal to compare folate status between consumers and non-consumers of RTE ce reals; however, since 90% of our subjects consumed these products, it was not f easible to make this comparison. Summary This study supports previous research that has shown a positive relationship between folate/folic acid intake an d folate status. The estimated average dietary folate/folic acid intake of our study population exceeded the current RDA, and average blood folate concentrations were well within the acceptable range. Although ma le subjects had a significantly higher mean homocysteine concentration compar ed to females, the majority of all subjects had plasma

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106 homocysteine concentrations w ithin the acceptable limit. In relation to the MTHFR 677 C T polymorphism, the data from all subjects are consistent with pr evious studies in which the TT genotype was associated with significantly lower RBC fola te and higher homocysteine concentrations compared to the CC genotype. Based on the findings from this study, it appears that fo lic acid fortif ied foods are important contributors to the diet in terms of meeting the folate RDA of non-supplement users. The major food categories that c ontributed toward meeting the R DA for males and females were enriched cereal-grain products, fortified cereals and bars and co mbination foods that include “enriched” ingredients. This highlights the integral role of folic acid fortif ication in achieving an adequate intake (based on th e RDA) and status of this v itamin in non-supplement users; however, despite the relative cont ribution of folic acid fortified foods toward meeting the RDA, intake of these foods was not sufficient among our study population to meet the IOM recommendation (400 g/d of foli c acid) for women of reproductiv e potential. The extremely low proportion of females (3%) who met this r ecommendation is disappoin ting considering the extensive efforts to promote awareness and behavior change among women of reproductive potential. Practical Application Since public health educational programs a nd strategies designed to market daily folic acid supplementation to women of reproductive age have not been effective, women might be more responsive to food advertis ements, including promotion of fo rtified cereals and snack bars as good to excellent dietary sour ces of folic acid. Rather th an solely promoting folic acid supplementation, researchers, dietitians, health educators and food industr y manufacturers should forge efforts to create and market educational outreach progr ams promoting inta ke of fortified

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107 RTE cereals, breakfast and snack bars, and enriched cereal-grain products as easy alternatives to help women consume 100% of the RDA, m eet the IOM recommendation for folic acid and maintain adequate folate status. However, folic acid fortificati on will not ensure adequate folic acid intake in target women unl ess women are educated to chan ge the way they eat. Health professionals and health educators could promot e eating one serving of cereal that has been fortified with 100% of the daily value of folic acid as a means of c onsuming the recommended amount of folic acid daily. Health professionals and researchers should make an effort to ex plore the reasons why women of reproductive age are no t consuming sufficient levels of folic acid to prevent NTDs. These future findings may allo w researchers to better under stand barriers to folic acid consumption in this population. Previous surv eys have shown that the biggest barrier to overcome with women is the lack of knowledge about folic acid usage. Many women who have heard of the vitamin do not know that they need to take it before pregnancy to reduce NTD risk. Therefore, educational awareness about the im portance of consuming supplemental folic acid and/or folic acid-fortif ied foods every day is another obj ective that should be taken into consideration. Targeted messages aimed at younger women, regardless of pregnancy plans, should include campaigns focused on promoting consumption of fortified RTE cereals to help increase the number of women of childbearing age who consume adequate folic acid daily.

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108 APPENDIX A SUBJECT DATA COLLECTION FORM Introduction I am calling in regard to your interest in our nutrition study; do you have a few minutes right now? This is a UF Nutrition depart ment study and involves coming in one morning for about 1 hour for a fasting blood sample, we take about 1 ounces of blood, and you only need to fast 8 hours. We will give you a breakfast snack right afterward, and then give a brief ex planation of a food frequency questionnaire you will be taking home. You will be asked to mail it back in the provided envelope, and once we receive the questi onnaire you would get paid the $50. I just have to ask you some questions to see if you are eligible fo r our study and to get background information, OK? How old are you? 18-49 Do you smoke? no Are you pregnant or breastfeeding ? no Do you take any prescription medications other than oral contraceptives ? no If not within the age range or if they answer yes to any question: I am very sorry, but you do not meet our exclusion criteria, but thank you for your interest. Now I just have a few questions about your diet to see what specific category of our study you would fit in to. Please answer as best you can, estimates are ok and consider all instances of when you might eat the items I will ask about, even if only occasionally. Do you take a multi-vitamin, complex, red star nutritional yeast, or any other supplement or additive ever? If they take a multivitamin, B complex, red star nutritional yeast, complete the session through all diet info but do not record. Conc lude by confirming their name and saying “This has been a preliminary screening call, your in formation will be reviewed by th e principal investigator based on need, and our selection criteri a at this time. If you are chos en you will be called again to schedule an appointment over the next two weeks. Thank you very much for your interest and your time. Do you eat breakfast cereals? ( If so) What Kind do you eat mostly? If they eat a 100% fortified cer eal or eats a 50% cereal daily complete the through all diet info but do not record. Conclude by confirming their name and saying “This has been a preliminary screening call, your information will be reviewed by the principal investigator based on need, and our selection criteria at this time. If you ar e chosen you will be called again to schedule an appointment over the next two we eks. Thank you very much for your interest and your time.

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109 If the interviewee fulfills all se lection criteria continue with th e questionnaire, record info on moderate/non-fortified cereal consumption below. Do you eat breakfast cereals? o Yes o No Name/Brand Quantity Frequency Are you a vegan, vegetarian or meat eater? Vegan – this means you eat NO animal derived fo ods intentionally (if they eat small amount like in cake then OK) Vegetarian – this means you eat NO b eef, chicken, turkey, pork, or fish How often do you eat … Never Rarely (<1 x/mo) Occasionally (1-4 x/mo) Frequently (2-4 x/wk) Always (5-7 x/wk) Beef Chicken Turkey Pork Fish Eggs Cheese Cow’s Milk Yogurt Other Dairy Do you follow a restricted diet such as: o No red meat o Lactose-free o Kosher o Weight loss o Weight gain o Low salt o Low fat o Low cholesterol o Low carbohydrate o Hypoallergenic (If so) How long have you consumed this type of diet? ________________________________________________________________________ Have you made any major dietary ch anges within the last 3 years? o No o Yes; How long ago did you make cha nges and what changes did you make? ________________________________________________________________________ NO YES Do you consume alcoholic beverages? How often/quantity

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110 Health Information I am going to ask you a few questions about your health to determine if you are eligible for our study. I will be recording this information, but it will be kept confidential and is this ok with you? _______ Height: Weight: Have you do you currently have any of the following?NO YES Alcoholism Anemia Blood clots Bronchitis Cystic Fibrosis Dermatitis Diabetes Eating disorders/Chronic nausea or vomiting Food allergy Gall bladder disease GI problems/ Lactose intolerance Gout Migraines Hemorrhoids Hepatitis/Liver disease Heart disease/High cholestero l/High blood pressure HIV Kidney disease Neurological disorder Obesity Seizures/Stroke Thyroid problem Tumors/Cancer Ulcers Other Have you been hospitalized with in the last 5 years? Cause Do you have a history of more than 1 miscarriage? o Yes o No If you are selected to participate in this st udy are you willing to sign an informed consent understanding we have access to medical information on you? o Yes o No

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111 Demographic Information What is your birth date? _______/_______/_________ Month Day Year How would you describe your race or ethnic background? o White o Black or African American o American Indian or Alaska Native o Hispanic or Latino o Asian o Native Hawaiian or Ot her Pacific Islander o Other _________________________________________________ What is the highest level of school or training that you have completed? [Circle only one response] Grade school 01 02 03 04 05 06 07 08 High school 09 10 11 12 Technical school or college 13 14 15 16 Graduate or professional 17 18 19 20+ Don’t know X Marital status? o Single/never married o Married o Separated o Divorced o Widowed Are you a full-time or part-t ime student? Are you employed? o Full time o Yes o Part time o No o Not a student o Student employee

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112 Contact Information Name of person and phone number to call in case of an emergency if you are invited to participate in this study: ______________________________________________________________________________ If we need to contact you, and can not reach you where/with who can a message be left? ______________________________________________________________________________ How did you hear about our study? __________________________________________________ Name M / F Last First Middle Street Apt. # Address City Zip code Phone Day Evening Cell E-mail

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113 APPENDIX B DIETARY HISTORY QUESTIONNAIRE NATIONAL INSTITUTES OF HEALTH Diet History Questionnaire Today's date: MONTH DAY YEAR |___|___| 0 0 1 1 2 2 3 3 4 5 6 7 8 01 Jan 02 Feb 03 Mar 04 Apr 05 May 06 Jun 07 Jul 08 Aug 09 Sep 10 Oct 11 Nov 12 Dec 9 2002 2003 2004 2005 2006 In what month were you born? 01 Jan 02 Feb 03 Mar 04 Apr 05 May 06 Jun 07 Jul 08 Aug 09 Sep 10 Oct 11 Nov 12 Dec In what year were you born? 19 |___|___| 0 0 1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9 Are you male or female? Male Female GENERAL INSTRUCTIONS Answer each question as best you can. Estimate if you are not sure. A guess is better than leaving a blank. Use only a black ball-point pen. Do no t use a pencil or felt-tip pen. Do not fold, staple, or tear the pages. Put an X in the box next to your answer. If you make any changes, cross out the incorrect answer and put an X in the box next to the correct answer. Also draw a circle around the correct answer. If you mark NEVER, NO, or DON’T KNOW for a question, please follow any arrows or instructions that direct you to the next question. BEFORE TURNING THE PAGE, PLEASE CO MPLETE THE FOLLOWING QUESTIONS. BAR CODE LABEL OR SUBJECT ID HERE |___|___|___|___|___|___|___|___|

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1141. Over the past 12 months how often did you drink tomato juice or vegetable juice ? NEVER (GO TO QUESTION 2) 1 time per month or less 1 time per day 2–3 times per month 2–3 times per day 1–2 times per week 4–5 times per day 3–4 times per week 6 or more times per day 5–6 times per week 1a. Each time you drank tomato juice or vegetable juice how much did you usually drink? Less than cup (6 ounces) to 1 cups (6 to 10 ounces) More than 1 cups (10 ounces) 2. Over the past 12 months how often did you drink orange juice? NEVER (GO TO QUESTION 3) 1 time per month or less 1 time per day 2–3 times per month 2–3 times per day 1–2 times per week 4–5 times per day 3–4 times per week 6 or more times per day 5–6 times per week 2a. Each time you drank orange juice how much did you usually drink? Less than cup (6 ounces) to 1 cups (6 to 10 ounces) More than 1 cups (10 ounces) 3. Over the past 12 months how often did you drink other 100% fruit juice or 100% fruit juice mixtures (such as apple, grape, pineapple, grapefruit or others)? NEVER (GO TO QUESTION 4) 1 time per month or less 1 time per day 2–3 times per month 2–3 times per day 1–2 times per week 4–5 times per day 3–4 times per week 6 or more times per day 5–6 times per week 3a. Each time you drank other fruit juice or fruit juice mixtures how much did you usually drink? Less than cup (6 ounces) to 1 cups (6 to 12 ounces) More than 1 cups (12 ounces) Over the past 12 months … 4. How often did you drink other fruit drinks (such as cranberry cocktail, Hi-C, lemonade, or KoolAid, diet or regular)? NEVER (GO TO QUESTION 5) 1 time per month or less 1 time per day 2–3 times per month 2–3 times per day 1–2 times per week 4–5 times per day 3–4 times per week 6 or more times per day 5–6 times per week 4a. Each time you drank fruit drinks how much did you usually drink? Less than 1 cup (8 ounces) 1 to 2 cups (8 to 16 ounces) More than 2 cups (16 ounces) 4b. How often were your fruit drinks diet or sugar-free drinks ? Almost never or never About of the time About of the time About of the time Almost always or always 5. How often did you drink milk, including lactosefree milk (but NOT milk substitutes) as a beverage (NOT in coffee, NOT in cereal)? (Please include chocolate milk, flavored milk like Ovaltine or Quick, and hot chocolate.) NEVER (GO TO QUESTION 6) 1 time per month or less 1 time per day 2–3 times per month 2–3 times per day 1–2 times per week 4–5 times per day 3–4 times per week 6 or more times per day 5–6 times per week 5a. Each time you drank milk as a beverage how much did you usually drink? Less than 1 cup (8 ounces) 1 to 1 cups (8 to 12 ounces) More than 1 cups (12 ounces) Please continue on next page. 5b. What kind of milk did you usually drink?

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115 Whole milk (includi ng lactose-free variety) 2% fat milk (including lactose-free variety) 1 % fat milk (includi ng lactose-free variety) Skim, nonfat, or % fat milk (including lactose-free variety) 6. How often did you drink a milk substitute such as soy or rice milk as a beverage (NOT in coffee, NOT in cereal)? (Please include milk substitute used to make other beverages such as chocolate “milk”, Ovaltine, Quick and hot chocolate.) NEVER (GO TO QUESTION 7) 1 time per month or less 1 time per day 2–3 times per month 2–3 times per day 1–2 times per week 4–5 times per day 3–4 times per week 6 or more times 5–6 times per week per day 6a. Each time you drank a milk substitute as a beverage, how much did you usually drink? Less than 1 cup (8 ounces) 1 to 1 cups (8 to 12 ounces) More than 1 cups (12 ounces) 6b. What kind of milk substitute did you usually drink? Soy milk (8th Continent) Soy milk (Whitewave Silk) Soy milk (VitaSoy) Soy milk (other brand) Rice milk Over the past 12 months … 7. How often did you drink meal replacement, energy, or high-protein beverages such as Instant Breakfast, Ensure, Slimfast, AdvantEdge, Boost or others? NEVER (GO TO QUESTION 8) 1 time per month or less 1 time per day 2–3 times per month 2–3 times per day 1–2 times per week 4–5 times per day 3–4 times per week 6 or more times per day 5–6 times per week 7a. Each time you drank meal replacement, energy, or high protein beverages how much did you usually drink? Less than 1 cup (8 ounces) 1 to 1 cups (8 to 12 ounces) More than 1 cups (12 ounces) 8. Over the past 12 months, did you drink soft drinks, soda, or pop ? NO (GO TO QUESTION 9) YES 8a. How often did you drink soft drinks, soda, or pop IN THE SUMMER ? NEVER 1 time per month or less 1 time per day 2–3 times per month 2–3 times per day 1–2 times per week 4–5 times per day 3–4 times per week 6 or more times 5–6 times per week per day 8b. How often did you drink soft drinks, soda, or pop DURING THE REST OF THE YEAR ? NEVER 1 time per month or less 1 time per day 2–3 times per month 2–3 times per day 1–2 times per week 4–5 times per day 3–4 times per week 6 or more times 5–6 times per week per day 8c. Each time you drank soft drinks, soda, or pop, how much did you usually drink? Less than 12 ounces or less than 1 can or bottle 12 to 16 ounces or 1 can or bottle More than 16 ounces or more than 1 can or bottle 8d. How often were these soft drinks, soda, or pop diet or sugar-free ? Almost never or never About of the time About of the time About of the time Almost always or always

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1168e. How often were these soft drinks, soda, or pop caffeine-free ? (See list of caffeine and caffeine-free sodas provided by the researcher.) Almost never or never About of the time About of the time About of the time Almost always or always 9. Over the past 12 months did you drink beer ? NO (GO TO QUESTION 10) YES 9a. How often did you drink beer IN THE SUMMER ? NEVER 1 time per month or less 1 time per day 2–3 times per month 2–3 times per day 1–2 times per week 4–5 times per day 3–4 times per week 6 or more times 5–6 times per week per day 9b. How often did you drink beer DURING THE REST OF THE YEAR ? NEVER 1 time per month or less 1 time per day 2–3 times per month 2–3 times per day 1–2 times per week 4–5 times per day 3–4 times per week 6 or more times 5–6 times per week per day 9c. Each time you drank beer how much did you usually drink? Less than a 12-ounce can or bottle 1 to 3 12-ounce cans or bottles More than 3 12-ounce cans or bottles Over the past 12 months … 10. How often did you drink wine or wine coolers ? NEVER (GO TO QUESTION 11) 1 time per month or less 1 time per day 2–3 times per month 2–3 times per day 1–2 times per week 4–5 times per day 3–4 times per week 6 or more times 5–6 times per week per day 10a. Each time you drank wine or wine coolers, how much did you usually drink? Less than 5 ounces or less than 1 glass 5 to 12 ounces or 1 to 2 glasses More than 12 ounces or more than 2 glasses 11. How often did you drink liquor or mixed drinks ? NEVER (GO TO QUESTION 12) 1 time per month or less 1 time per day 2–3 times per month 2–3 times per day 1–2 times per week 4–5 times per day 3–4 times per week 6 or more times 5–6 times per week per day 11a. Each time you drank liquor or mixed drinks, how much did you usually drink? Less than 1 shot of liquor 1 to 3 shots of liquor More than 3 shots of liquor 12. Over the past 12 months did you eat oatmeal, grits, or other cooked cereal? NO (GO TO QUESTION 13) YES 12a. How often did you eat oatmeal, grits, or other cooked cereal IN THE WINTER ? NEVER 1–6 times per winter 2 times per week 7–11 times per winter 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day

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11712b. How often did you eat oatmeal, grits, or other cooked cereal DURING THE REST OF THE YEAR ? NEVER 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 12c. Each time you ate oatmeal, grits, or other cooked cereal, how much did you usually eat? Less than cup to 1 cups More than 1 cups 13. How often did you eat cold cereal ? NEVER (GO TO QUESTION 14) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 13a. Each time you ate cold cereal how much did you usually eat? Less than 1 cup 1 to 2 cups More than 2 cups 13b. How often was the cold cereal you ate a fortified cereal? (See list of fortified cereals provided by the researcher.) Almost never or never About of the time About of the time About of the time Almost always or always 13c. How often was the cold cereal you ate any other type of cold cereal (such as Corn Flakes, Rice Krispies, Frosted Flakes, Fruit Loops, Cap'n Crunch, or others)? Almost never or never About of the time About of the time About of the time Almost always or always 13d. Was milk or a milk substitute added to your cold cereal? NO (GO TO QUESTION 14) YES 13e. What kind of milk or milk substitute was usually added? Whole milk (including lactose-free variety) 2% fat milk (including lactose-free variety) 1% fat milk (including lactose-free variety) Skim, nonfat, or % fat milk (including lactose-free variety) Soy milk (8th Continent) Soy milk (Whitewave Silk) Soy milk (VitaSoy) Soy milk (other brand) Rice milk Other 13f. Each time milk or a milk substitute was added to your cold cereal how much was usually added? Less than cup to 1 cup More than 1 cup 14. How often did you eat applesauce ? NEVER (GO TO QUESTION 15) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day Please continue on next page.

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11814a. Each time you ate applesauce how much did you usually eat? Less than cup to 1 cup More than 1 cup 15. How often did you eat apples ? NEVER (GO TO QUESTION 16) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 15a. Each time you ate apples how many did you usually eat? Less than 1 apple 1 apple More than 1 apple 16. How often did you eat pears (fresh, canned, or frozen)? NEVER (GO TO QUESTION 17) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 16a. Each time you ate pears how many did you usually eat? Less than 1 pear 1 pear More than 1 pear 17. How often did you eat bananas? NEVER (GO TO QUESTION 18) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day Over the past 12 months … 17a. Each time you ate bananas how many did you usually eat? Less than 1 banana 1 banana More than 1 banana 18. How often did you eat dried fruit such as prunes or raisins (not including dried apricots)? NEVER (GO TO QUESTION 19) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 18a. Each time you ate dried fruit how much did you usually eat (not including dried apricots)? Less than 2 tablespoons 2 to 5 tablespoons More than 5 tablespoons 19. Over the past 12 months did you eat peaches, nectarines, or plums ? NO (GO TO QUESTION 20) YES 19a. How often did you eat fresh peaches nectarines or plums WHEN IN SEASON ? (See list for description of “in season”.) NEVER 1–6 times per season 2 times per week 7–11 times per season 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 19b. How often did you eat peaches, nectarines, or plums (fresh, canned, or frozen) DURING THE REST OF THE YEAR ? NEVER 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day

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11919c. Each time you ate peaches, nectarines, or plums, how much did you usually eat? Less than 1 fruit or less than cup 1 to 2 fruits or to cup More than 2 fruits or more than cup 20. How often did you eat grapes ? NEVER (GO TO QUESTION 21) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 20a. Each time you ate grapes how much did you usually eat? Less than cup or less than 10 grapes to 1 cup or 10 to 30 grapes More than 1 cup or more than 30 grapes 21. Over the past 12 months did you eat cantaloupe ? NO (GO TO QUESTION 22) YES 21a. How often did you eat fresh cantaloupe WHEN IN SEASON ? (See list for description of “in season”.) NEVER 1–6 times per season 2 times per week 7–11 times per season 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 21b. How often did you eat fresh or frozen cantaloupe DURING THE REST OF THE YEAR ? NEVER 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day Over the past 12 months … 21c. Each time you ate cantaloupe how much did you usually eat? Less than melon or less than cup melon or to 1 cup More than melon or more than 1 cup 22. Over the past 12 months did you eat melon, other than cantaloupe (such as watermelon or honeydew)? NO (GO TO QUESTION 23 YES 22a. How often did you eat fresh melon, other than cantaloupe (such as watermelon or honeydew) WHEN IN SEASON ? (See list for description of “in season”.) NEVER 1–6 times per season 2 times per week 7–11 times per season 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 22b. How often did you eat fresh or frozen melon, other than cantaloupe (such as watermelon or honeydew) DURING THE REST OF THE YEAR ? NEVER 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 22c. Each time you ate melon other than cantaloupe how much did you usually eat? Less than cup or 1 small wedge to 2 cups or 1 medium wedge More than 2 cups or 1 large wedge

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12023. Over the past 12 months did you eat strawberries ? NO (GO TO QUESTION 24) YES 23a. How often did you eat fresh strawberries WHEN IN SEASON ? (See list for description of “in season”.) NEVER 1–6 times per season 2 times per week 7–11 times per season 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 23b. How often did you eat fresh or frozen strawberries DURING THE REST OF THE YEAR ? NEVER 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 23c. Each time you ate strawberries how much did you usually eat? Less than cup or less than 3 berries to cup or 3 to 8 berries More than cup or more than 8 berries 24. Over the past 12 months did you eat oranges, tangerines, or tangelos ? NO (GO TO QUESTION 25) YES 24a. How often did you eat fresh oranges, tangerines, or tangelos WHEN IN SEASON ? (See list for description of “in season”.) NEVER 1–6 times per season 2 times per week 7–11 times per season 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day Over the past 12 months … 24b. How often did you eat oranges, tangerines, or tangelos (fresh or canned) DURING THE REST OF THE YEAR ? NEVER 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 24c. Each time you ate oranges, tangerines, or tangelos, how many did you usually eat? Less than 1 fruit 1 fruit More than 1 fruit 25. Over the past 12 months did you eat grapefruit ? NO (GO TO QUESTION 26) YES 25a. How often did you eat fresh grapefruit WHEN IN SEASON ? (See list for description of “in season”.) NEVER 1–6 times per season 2 times per week 7–11 times per season 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 25b. How often did you eat grapefruit (fresh or canned) DURING THE REST OF THE YEAR? NEVER 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 25c. Each time you ate grapefruit how much did you usually eat? Less than grapefruit grapefruit More than grapefruit

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12126. How often did you eat other kinds of fruit such as pineapple, mangoes, blueberries, or others? NEVER (GO TO QUESTION 27) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 26a. Each time you ate other kinds of fruit how much did you usually eat? Less than cup to cup More than cup 27. How often did you eat COOKED greens (such as spinach, turnip, collard, mustard, chard, or kale)? NEVER (GO TO QUESTION 28) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 27a. Each time you ate COOKED greens how much did you usually eat? Less than cup to 1 cup More than 1 cup 28. How often did you eat RAW greens (such as spinach, turnip, collard, mustard, chard, or kale)? ( We will ask about lettuce later.) NEVER (GO TO QUESTION 29) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 28a. Each time you ate RAW greens how much did you usually eat? Less than cup to 1 cup More than 1 cup Over the past 12 months … 29. How often did you eat coleslaw ? NEVER (GO TO QUESTION 30) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 29a. Each time you ate coleslaw how much did you usually eat? Less than cup to cup More than cup 30. How often did you eat sauerkraut or cabbage (other than coleslaw)? NEVER (GO TO QUESTION 31) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 30a. Each time you ate sauerkraut or cabbage how much did you usually eat? Less than cup to 1 cup More than 1 cup 31. How often did you eat carrots (fresh, canned, or frozen)? NEVER (GO TO QUESTION 32) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 31a. Each time you ate carrots how much did you usually eat? Less than cup or less than 2 baby carrots to cup or 2 to 5 baby carrots More than cup or more than 5 baby carrots

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12232. How often did you eat string beans or green beans (fresh, canned, or frozen)? NEVER (GO TO QUESTION 33) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 32a. Each time you ate string beans or green beans how much did you usually eat? Less than cup to 1 cup More than 1 cup 33. How often did you eat peas (fresh, canned, or frozen)? NEVER (GO TO QUESTION 34) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 33a. Each time you ate peas how much did you usually eat? Less than cup to cup More than cup 34. Over the past 12 months did you eat corn ? NO (GO TO QUESTION 35) YES 34a. How often did you eat fresh corn WHEN IN SEASON ? (See list for description of “in season”.) NEVER 1–6 times per season 2 times per week 7–11 times per season 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day Over the past 12 months … 34b. How often did you eat corn (fresh, canned, or frozen) DURING THE REST OF THE YEAR ? NEVER 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 34c. Each time you ate corn how much did you usually eat? Less than 1 ear or less than cup 1 ear or to 1 cup More than 1 ear or more than 1 cup 35. Over the past 12 months how often did you eat broccoli (fresh or frozen)? NEVER (GO TO QUESTION 36) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 35a. Each time you ate broccoli how much did you usually eat? Less than cup to 1 cup More than 1 cup 36. How often did you eat cauliflower or Brussels sprouts (fresh or frozen)? NEVER (GO TO QUESTION 37) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 36a. Each time you ate cauliflower or Brussels sprouts how much did you usually eat? Less than cup to cup More than cup

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12337. How often did you eat mixed vegetables ? NEVER (GO TO QUESTION 38) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 37a. Each time you ate mixed vegetables how much did you usually eat? Less than cup to 1 cup More than 1 cup 38. How often did you eat onions ? NEVER (GO TO QUESTION 39) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 38a. Each time you ate onions how much did you usually eat? Less than 1 slice or less than 1 tablespoon 1 slice or 1 to 4 tablespoons More than 1 slice or more than 4 tablespoons 39. Now think about all the cooked vegetables you ate in the past 12 months and how they were prepared. How often were your vegetables COOKED WITH some sort of fat including oil spray? (Please do not include potatoes.) NEVER (GO TO QUESTION 40) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day Over the past 12 months … 39a. Which fats were usually added to your vegetables DURING COOKING ? (Please do not include potatoes. Mark all that apply. ) Margarine (including low-fat) Canola or rapeseed oil Butter (including low-fat) Oil spray, such as Pam or others Lard, fatback, or bacon fat Other kinds of oils Olive oil None of the above Corn oil 40. Now, thinking again about all the cooked vegetables you ate in the past 12 months how often was some sort of fat, sauce, or dressing added AFTER COOKING OR AT THE TABLE ? (Please do not include potatoes.) NEVER (GO TO QUESTION 41) 1–6 times per year 3–4 times per week 7–11 times per year 5–6 times per week 1 time per month 1 time per day 2–3 times per month 2 times per day 1–2 times per week 3 or more times per day 40a. Which fats, sauces, or dressings were usually added AFTER COOKING OR AT THE TABLE ? (Please do not include potatoes. Mark all that apply. ) Margarine including low-fat) Cheese sauce Butter (including low-fat) White sauce Lard, fatback, or bacon fat Other Salad dressing 40b. If margarine, butter, lard, fatback, or bacon fat was added to your cooked vegetables AFTER COOKING OR AT THE TABLE how much did you usually add? Did not usually add these Less than 1 teaspoon 1 to 3 teaspoons More than 3 teaspoons 40c. If salad dressing, cheese sauce, or white sauce was added to your cooked vegetables AFTER COOKING OR AT THE TABLE how much did you usually add? Did not usually add these Less than 1 tablespoon 1 to 3 tablespoons More than 3 tablespoons

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12441. Over the past 12 months how often did you eat sweet peppers (green, red, or yellow)? NEVER (GO TO QUESTION 42) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 41a. Each time you ate sweet peppers how much did you usually eat? Less than 1/8 pepper 1/8 to pepper More than pepper 42. Over the past 12 months did you eat fresh tomatoes (including those in salads)? NO (GO TO QUESTION 43) YES 42a. How often did you eat fresh tomatoes (including those in salads) WHEN IN SEASON ? (See description of “in season”.) NEVER 1–6 times per season 2 times per week 7–11 times per season 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 42b. How often did you eat fresh tomatoes (including those in salads) DURING THE REST OF THE YEAR ? NEVER 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 42c. Each time you ate fresh tomatoes how much did you usually eat? Less than tomato to tomato More than tomato Over the past 12 months … 43. How often did you eat lettuce salads (with or without other vegetables)? NEVER (GO TO QUESTION 44) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 43a. Each time you ate lettuce salads how much did you usually eat? Less than cup to 11/4 cups More than 11/4 cups 44. How often did you eat salad dressing (including low-fat) on salads? NEVER (GO TO QUESTION 45) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 44a. Each time you ate salad dressing on salads, how much did you usually eat? Less than 2 tablespoons 2 to 4 tablespoons More than 4 tablespoons 45. How often did you eat sweet potatoes or yams ? NEVER (GO TO QUESTION 46) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 45a. Each time you ate sweet potatoes or yams how much did you usually eat? 1 small potato or less than cup 1 medium potato or to cup 1 large potato or more than cup

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12546. How often did you eat French fries, home fries, hash browned potatoes or tater tots ? NEVER (GO TO QUESTION 47) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 46a. Each time you ate French fries home fries hash browned potatoes or tater tots how much did you usually eat? Less than 10 fries or less than cup 10 to 25 fries or to 1 cup More than 25 fries or more than 1 cup 47. How often did you eat potato salad ? NEVER (GO TO QUESTION 48) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 47a. Each time you ate potato salad how much did you usually eat? Less than cup to 1 cup More than 1 cup 48. How often did you eat baked, boiled, or mashed potatoes ? NEVER (GO TO QUESTION 49) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 48a. Each time you ate baked, boiled, or mashed potatoes how much did you usually eat? 1 small potato or less than cup 1 medium potato or to 1 cup 1 large potato or more than 1 cup Over the past 12 months … 48b. How often was sour cream (including lowfat) added to your potatoes, EITHER IN COOKING OR AT THE TABLE ? Almost never or ne ver (GO TO QUESTION 48d) About of the time About of the time About of the time Almost always or always 48c. Each time sour cream was added to your potatoes, how much was usually added? Less than 1 tablespoon 1 to 3 tablespoons More than 3 tablespoons 48d. How often was margarine (including low-fat) added to your potatoes, EITHER IN COOKING OR AT THE TABLE ? Almost never or never About of the time About of the time About of the time Almost always or always 48e. How often was butter (including low-fat) added to your potatoes, EITHER IN COOKING OR AT THE TABLE ? Almost never or never About of the time About of the time About of the time Almost always or always 48f. Each time margarine or butter was added to your potatoes, how much was usually added? Never added Less than 1 teaspoon 1 to 3 teaspoons More than 3 teaspoons 48g. How often was cheese or cheese sauce added to your potatoes, EITHER IN COOKING OR AT THE TABLE? Almost never or ne ver (GO TO QUESTION 49) About of the time About of the time About of the time Almost always or always

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12648h. Each time cheese or cheese sauce was added to your potatoes, how much was usually added? Less than 1 tablespoon 1 to 3 tablespoons More than 3 tablespoons 49. How often did you eat salsa ? NEVER (GO TO QUESTION 50) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 49a. Each time you ate salsa how much did you usually eat? Less than 1 tablespoon 1 to 5 tablespoons More than 5 tablespoons 50. How often did you eat catsup ? NEVER (GO TO QUESTION 51) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 50a. Each time you ate catsup how much did you usually eat? Less than 1 teaspoon 1 to 6 teaspoons More than 6 teaspoons 51. How often did you eat stuffing, dressing, or dumplings ? NEVER (GO TO QUESTION 52) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 51a. Each time you ate stuffing, dressing, or dumplings how much did you usually eat? Less than cup to 1 cup More than 1 cup Over the past 12 months … 52. How often did you eat chili made with beef ? (DO NOT include chili made with soy or vegetable protein substitute. We will ask about this later.) NEVER (GO TO QUESTION 52b) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 52a. Each time you ate chili made with beef how much did you usually eat? Less than cup to 13/4 cups More than 13/4 cups 52b. How often did you eat chili made with meat other than beef ? (DO NOT include chili made with soy or vegetable protein substitutes. We will ask about these later.) NEVER (GO TO QUESTION 52d) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 52c. Each time you ate chili made with meat other than beef how much did you usually eat? Less than cup to 13/4 cups More than 13/4 cups 52d. How often did you eat chili without meat ? (DO NOT include chili made with a soy or vegetable protein meat substitute. We will ask about meat substitutes later.) NEVER (GO TO QUESTION 53) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day

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127 52e. Each time you ate chili made without meat how much did you usually eat? Less than cup to 13/4 cups More than 13/4 cups 53. How often did you eat Mexican foods (such as tacos, tostados, burritos, tamales, fajitas, enchiladas, q uesadillas, and chimichangas) made with beef ? (DO NOT include Mexican foods made with soy or vegetable protein substitutes. We will ask about these later.) NEVER (GO TO QUESTION 53b) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 53a. Each time you ate Mexican foods made with beef how much did you usually eat? Less than 1 taco, burrito, etc. 1 to 2 tacos, burritos, etc. More than 2 tacos, burritos, etc. 53b. How often did you eat Mexican foods (such as tacos, tostados, burritos, tamales, fajitas, enchiladas, q uesadillas, and chimichangas) made with meat other than beef ? (DO NOT include Mexican foods made with soy or vegetable protein substitutes. We will ask about these later.) NEVER (GO TO QUESTION 53d) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 53c. Each time you ate Mexican foods made with meat other than beef how much did you usually eat? Less than cup to 13/4 cups More than 13/4 cups 53d. How often did you eat Mexican foods (such as tacos, tostados, burritos, tamales, fajitas, enchiladas, q uesadillas, and chimichangas) made without meat ? (DO NOT include Mexican foods made with a soy or vegetable protein meat substitute.) NEVER (GO TO QUESTION 54) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 53e. Each time you ate Mexican foods made without meat, how much did you usually eat? Less than cup to 13/4 cups More than 13/4 cups 54. How often did you eat cooked dried beans (such as baked beans, pintos, kidney, blackeyed peas, lima, lentils, soybeans, refried beans, or chick peas/garbanzo beans, including hummus)? (Please don't include bean soups or chili.) NEVER (GO TO QUESTION 55) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 54a. Each time you ate beans how much did you usually eat? Less than cup to 1 cup More than 1 cup 54b. How often were the beans you ate refried beans, beans prepared with any type of fat or with meat added ? Almost never or never About of the time About of the time About of the time Almost always or always

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12855. How often did you eat other kinds of vegetables such as asparagus, mushrooms, zucchini or others ? NEVER (GO TO QUESTION 56) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 55a. Each time you ate other kinds of vegetables how much did you usually eat? Less than cup to cup More than cup 56. How often did you eat rice or other cooked grains (such as bulgur, cracked wheat, or millet)? NEVER (GO TO QUESTION 57) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 56a. Each time you ate rice or other cooked grains how much did you usually eat? Less than cup to 11/2 cups More than 11/2 cups 56b. How often was butter, margarine or oil added to your rice IN COOKING OR AT THE TABLE ? Almost never or never About of the time About of the time About of the time Almost always or always Over the past 12 months … 57. How often did you eat pancakes, waffles, or French toast ? NEVER (GO TO QUESTION 58) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 57a. Each time you ate pancakes, waffles, or French toast how much did you usually eat? Less than 1 medium piece 1 to 3 medium pieces More than 3 medium pieces 57b. How often was margarine (including low-fat) added to your pancakes, waffles, or French toast AFTER COOKING OR AT THE TABLE ? Almost never or never About of the time About of the time About of the time Almost always or always 57c. How often was butter (including low-fat) added to your pancakes, waffles, or French toast AFTER COOKING OR AT THE TABLE ? Almost never or never About of the time About of the time About of the time Almost always or always 57d. Each time margarine or butter was added to your pancakes, waffles, or French toast, how much was usually added? Never added Less than 1 teaspoon 1 to 3 teaspoons More than 3 teaspoons 57e. How often was syrup added to your pancakes, waffles, or French toast? Almost never or ne ver (GO TO QUESTION 58) About of the time About of the time About of the time Almost always or always 57f. Each time syrup was added to your pancakes, waffles, or French toast, how much was usually added? Less than 1 tablespoon 1 to 4 tablespoons More than 4 tablespoons

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12958. How often did you eat lasagna, stuffed shells, stuffed manicotti, ravioli, or tortellini made with beef ? (DO NOT include spaghetti or other pasta or products made with soy or vegetable protein substitutes. We will ask about these later.) NEVER (GO TO QUESTION 58b) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 58a. Each time you ate lasagna, stuffed shells, stuffed manicotti, ravioli, or tortellini made with beef how much did you usually eat? Less than 1 cup 1 to 2 cups More than 2 cups 58b. How often did you eat lasagna, stuffed shells, stuffed manicotti, ravioli, or tortellini made with meat other than beef ? (DO NOT include spaghetti or other pasta or products made with soy or vegetable protein substitutes. We will ask about these later.) NEVER (GO TO QUESTION 58d) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 58c. Each time you ate lasagna, stuffed shells, stuffed manicotti, ravioli, or tortellini made with meat other than beef how much did you usually eat? Less than 1 cup 1 to 2 cups More than 2 cups 58d. How often did you eat lasagna, stuffed shells, stuffed manicotti, ravioli, or tortellini made without meat ? (DO NOT include spaghetti or other pasta or products made with a soy or vegetable protein substitutes. We will ask about these later.) NEVER (GO TO QUESTION 59) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 58e. Each time you ate lasagna, stuffed shells, stuffed manicotti, ravioli, or tortellini made without meat how much did you usually eat? Less than 1 cup 1 to 2 cups More than 2 cups 59. How often did you eat macaroni and cheese ? NEVER (GO TO QUESTION 60) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 59a. Each time you ate macaroni and cheese how much did you usually eat? Less than 1 cup 1 to 11/2 cups More than 11/2 cups 60. How often did you eat pasta salad or macaroni salad ? NEVER (GO TO QUESTION 61) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day Over the past 12 months … 60a. Each time you ate pasta salad or macaroni salad how much did you usually eat? Less than cup to 1 cup More than 1 cup

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13061. Other than the pastas listed in Questions 58, 59, and 60, how often did you eat pasta, spaghetti or other noodles ? NEVER (GO TO QUESTION 62) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 61a. Each time you ate pasta, spaghetti or other noodles how much did you usually eat? Less than 1 cup 1 to 3 cups More than 3 cups 61b. How often did you eat your pasta, spaghetti, or other noodles with tomato sauce or spaghetti sauce made with beef ? (DO NOT include tomato or spaghetti sauce made with soy or vegetable protein substitutes. We will ask about these later.) Almost never or never About of the time About of the time About of the time Almost always or always 61c. How often did you eat your pasta, spaghetti, or other noodles with tomato sauce or spaghetti sauce made with meat other than beef ? (DO NOT include tomato or spaghetti sauce made with soy or vegetable protein substitutes. We will ask about these later.) Almost never or never About of the time About of the time About of the time Almost always or always 61d. How often did you eat your pasta, spaghetti, or other noodles with tomato sauce or spaghetti sauce made WITHOUT meat ? (DO NOT include tomato sauce or spaghetti sauce made with a soy or vegetable protein meat substitute. We will ask about these later.) Almost never or never About of the time About of the time About of the time Almost always or always 61e. How often did you eat your pasta, spaghetti, or other noodles with margarine, butter, oil, or cream sauce ? 62. How often did you eat bagels or English muffins ? NEVER (GO TO INTRODUCTION TO QUESTION 63) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 62a. Each time you ate bagels or English muffins how many did you usually eat? Less than 1 bagel or English muffin 1 bagel or English muffin More than 1 bagel or English muffin 62b. How often was margarine (including low-fat) added to your bagels or English muffins? Almost never or never About of the time About of the time About of the time Almost always or always 62c. How often was butter (including low-fat) added to your bagels or English muffins? Almost never or never About of the time About of the time About of the time Almost always or always 62d. Each time margarine or butter was added to your bagels or English muffins, how much was usually added? Never added Less than 1 teaspoon 1 to 2 teaspoons More than 2 teaspoons Almost never or never About of the time About of the time About of the time Almost always or always

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13162e. How often was cream cheese (including lowfat) spread on your bagels or English muffins? Almost never or neve r (GO TO INTRODUCTION TO QUESTION 63) About of the time About of the time About of the time Almost always or always Over the past 12 months … 62f. Each time cream cheese was added to your bagels or English muffins, how much was usually added? Less than 1 tablespoon 1 to 2 tablespoons More than 2 tablespoons The next questions ask about your intake of breads other than bagels or English muffins. First, we will ask about bread you ate as part of sandwiches only. Then we will ask about all other bread you ate. 63. How often did you eat breads or rolls AS PART OF SANDWICHES (including burger and hot dog rolls)? NEVER (GO TO QUESTION 64) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 63a. Each time you ate breads or rolls AS PART OF SANDWICHES how many did you usually eat? 1 slice or roll 2 slices or 1 roll More than 2 slices or more than 1 roll 63b. How often were the breads or rolls that you used for your sandwiches white bread (including burger and hot dog rolls)? Almost never or never About of the time About of the time About of the time Almost always or always 63c. How often was mayonnaise or mayonnaise-type dressing (including lowfat) added to your sandwich bread or rolls? Almost never or neve r (GO TO QUESTION 63e) About of the time About of the time About of the time Almost always or always 63d. Each time mayonnaise or mayonnaise-type dressing was added to your sandwich breads or rolls, how much was usually added? Less than 1 teaspoon 1 to 3 teaspoons More than 3 teaspoons 63e. How often was margarine (including low-fat) added to your sandwich bread or rolls? Almost never or never About of the time About of the time About of the time Almost always or always 63f. How often was butter (including low-fat) added to your sandwich bread or rolls? Almost never or never About of the time About of the time About of the time Almost always or always 63g. Each time margarine or butter was added to your sandwich breads or rolls, how much was usually added? Never added Less than 1 teaspoon 1 to 2 teaspoons More than 2 teaspoons 64. How often did you eat breads or dinner rolls, NOT AS PART OF SANDWICHES ? NEVER (GO TO QUESTION 65) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day

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13264a. Each time you ate breads or dinner rolls, NOT AS PART OF SANDWICHES how much did you usually eat? 1 slice or 1 dinner roll 2 slices or 2 dinner rolls More than 2 slices or 2 dinner rolls Over the past 12 months … 64b. How often were the breads or rolls you ate white bread ? Almost never or never About of the time About of the time About of the time Almost always or always 64c. How often was margarine (including low-fat) added to your breads or rolls? Almost never or never About of the time About of the time About of the time Almost always or always 64d. How often was butter (including low-fat) added to your breads or rolls? Almost never or never About of the time About of the time About of the time Almost always or always 64e. Each time margarine or butter was added to your breads or rolls, how much was usually added? Never added Less than 1 teaspoon 1 to 2 teaspoons More than 2 teaspoons 64f. How often was cream cheese (including lowfat) added to your breads or rolls? Almost never or ne ver (GO TO QUESTION 65) About of the time About of the time About of the time Almost always or always 64g. Each time cream cheese was added to your breads or rolls, how much was usually added? Less than 1 tablespoon 1 to 2 tablespoons More than 2 tablespoons 65. How often did you eat jam, jelly, or honey on bagels, muffins, bread, rolls, or crackers? NEVER (GO TO QUESTION 66) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 65a. Each time you ate jam, jelly, or honey how much did you usually eat? Less than 1 teaspoon 1 to 3 teaspoons More than 3 teaspoons 66. How often did you eat peanut butter or other nut butter ? NEVER (GO TO QUESTION 67) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 66a. Each time you ate peanut butter or other nut butter how much did you usually eat? Less than 1 tablespoon 1 to 2 tablespoons More than 2 tablespoons 67. How often did you eat roast beef or steak IN SANDWICHES, SUBS or WRAPS ? NEVER (GO TO QUESTION 68) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day

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13367a. Each time you ate roast beef or steak IN SANDWICHES, SUBS or WRAPS how much did you usually eat? Less than 1 slice or less than 2 ounces 1 to 2 slices or 2 to 4 ounces More than 2 slices or more than 4 ounces Over the past 12 months … 68. How often did you eat turkey or chicken COLD CUTS (such as loaf, luncheon meat, turkey ham, turkey salami, or turkey pastrami), including those used in sandwiches, subs or wraps? (DO NOT include turkey or chicken cold cuts made with soy or vegetable protein substitutes. We will ask about these later.) NEVER (GO TO QUESTION 69) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 68a. Each time you ate turkey or chicken COLD CUTS how much did you usually eat? Less than 1 slice 1 to 3 slices More than 3 slices 69. How often did you eat luncheon or deli-style ham including luncheon or deli-style ham used in sandwiches, subs or wraps? (DO NOT include luncheon or deli-style ham made with soy or vegetable protein substitutes. We will ask about these later.) NEVER (GO TO QUESTION 70) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 69a. Each time you ate luncheon or deli-style ham how much did you usually eat? Less than 1 slice 1 to 3 slices More than 3 slices 69b. How often was the luncheon or deli-style ham you ate light low-fat or fat-free ? Almost never or never About of the time About of the time About of the time Almost always or always 70. How often did you eat other cold cuts or luncheon meats (such as beef bologna, corned beef, pastrami, or others, including low-fat)? Include other cold cuts and luncheon meats used in sandwiches, subs or wraps. ( DO NOT include ham, turkey, salami, chicken cold cuts or cold cuts made with soy or vegetable protein.) NEVER (GO TO QUESTION 71) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 70a. Each time you ate other cold cuts or luncheon meats how much did you usually eat? Less than 1 slice 1 to 3 slices More than 3 slices 70b. How often were the other cold cuts or luncheon meats you ate light, low-fat or fatfree cold cuts or luncheon meats ? (Please do not include ham, turkey, or chicken cold cuts.) Almost never or never About of the time About of the time About of the time Almost always or always Please continue on next page.

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13471. How often did you eat canned tuna (including in salads, sandwiches, or casseroles)? NEVER (GO TO QUESTION 72) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 71a. Each time you ate canned tuna how much did you usually eat? Less than cup or less than 2 ounces to cup or 2 to 3 ounces More than cup or more than 3 ounces 71b. How often was the canned tuna you ate water-packed tuna ? Almost never or never About of the time About of the time About of the time Almost always or always Over the past 12 months … 71c. How often was the canned tuna you ate prepared with mayonnaise or other dressing (including low-fat)? Almost never or never About of the time About of the time About of the time Almost always or always 72. How often did you eat GROUND chicken or turkey ? (DO NOT include soy or vegetable protein substitutes. We will ask about these later.) NEVER (GO TO QUESTION 73) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 72a. Each time you ate GROUND chicken or turkey how much did you usually eat? Less than 2 ounces or less than cup 2 to 4 ounces or to 1 cup More than 4 ounces or more than 1 cup 73. How often did you eat beef hamburgers or cheeseburgers ? (DO NOT include soy or vegetable protein substitutes. We will ask about these later.) NEVER (GO TO QUESTION 74) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 73a. Each time you ate beef hamburgers or cheeseburgers how much did you usually eat? Less than 1 patty or less than 2 ounces 1 patty or 2 to 4 ounces More than 1 patty or more than 4 ounces 73b. How often were the beef hamburgers or cheeseburgers you ate made with lean ground beef ? Almost never or never About of the time About of the time About of the time Almost always or always 74. How often did you eat ground beef in mixtures (such as meatballs, casseroles, or meatloaf)? (DO NOT include soy or vegetable protein substitutes. We will ask about these later.) NEVER (GO TO QUESTION 75) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 74a. Each time you ate ground beef in mixtures how much did you usually eat? Less than 3 ounces or less than cup 3 to 8 ounces or to 1 cup More than 8 ounces or more than 1 cup

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13575. How often did you eat hot dogs or frankfurters ? (DO NOT include sausages or vegetarian hot dogs.) NEVER (GO TO QUESTION 76) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 75a. Each time you ate hot dogs or frankfurters how many did you usually eat? Less than 1 hot dog 1 to 2 hot dogs More than 2 hot dogs 75b. How often were the hot dogs or frankfurters you ate light or low-fat hot dogs ? Almost never or never About of the time About of the time About of the time Almost always or always Over the past 12 months … 76. How often did you eat beef mixtures such as beef stew, beef pot pie, beef and noodles, or beef and vegetables ? (DO NOT include products made with soy or vegetable protein substitutes. We will ask about these later.) NEVER (GO TO QUESTION 77) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 76a. Each time you ate beef stew, beef pot pie, beef and noodles, or beef and vegetables, how much did you usually eat? Less than 1 cup 1 to 2 cups More than 2 cups 77. How often did you eat roast beef or pot roast ? (DO NOT include roast beef or pot roast in sandwiches, subs or wraps. DO NOT include soy or vegetable protein substitutes. We will ask about these later.) NEVER (GO TO QUESTION 78) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 77a. Each time you ate roast beef or pot roast (including in mixtures), how much did you usually eat? Less than 2 ounces 2 to 5 ounces More than 5 ounces 78. How often did you eat steak (beef)? ( DO NOT include steak in sandwiches, subs or wraps. ) NEVER (GO TO QUESTION 79) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 78a. Each time you ate steak (beef), how much did you usually eat? Less than 3 ounces 3 to 7 ounces More than 7 ounces 78b. How often was the steak you ate lean steak ? Almost never or never About of the time About of the time About of the time Almost always or always 79. How often did you eat pork or beef spareribs ? NEVER (GO TO QUESTION 80) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day

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13679a. Each time you ate pork or beef spareribs how much did you usually eat? Less than 4 ribs 4 to 12 ribs More than 12 ribs 80. How often did you eat roast turkey, turkey cutlets, or turkey nuggets (including in sandwiches)? (DO NOT include soy or vegetable protein substitutes. We will ask about these later.) NEVER (GO TO QUESTION 81) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 80a. Each time you ate roast turkey, turkey cutlets, or turkey nuggets how much did you usually eat? (Please note: 4 to 8 turkey nuggets = 3 ounces.) Less than 2 ounces 2 to 4 ounces More than 4 ounces 81. How often did you eat chicken as part of salads, sandwiches, casseroles, stews, or other mixtures ? (DO NOT include soy or vegetable protein substitutes. We will ask about these later.) NEVER (GO TO QUESTION 82) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day Over the past 12 months … 81a. Each time you ate chicken as part of salads, sandwiches, casseroles, stews, or other mixtures, how much did you usually eat? Less than cup to 11/2 cups More than 11/2 cups 82. How often did you eat baked, broiled, roasted, stewed, or fried chicken (including nuggets)? (DO NOT include chicken in mixtures or products made with soy or vegetable protein substitutes. We will ask about these later.) NEVER (GO TO QUESTION 83) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 82a. Each time you ate baked, broiled, roasted, stewed, or fried chicken (including nuggets), how much did you usually eat? Less than 2 drumsticks or wings, less than 1 breast or thigh, or less than 4 nuggets 2 drumsticks or wings, 1 breast or thigh, or 4 to 8 nuggets More than 2 drumsticks or wings, more than 1 breast or thigh, or more than 8 nuggets 82b. How often was the chicken you ate fried chicken (including deep fried) or chicken nuggets ? Almost never or never About of the time About of the time About of the time Almost always or always 82c. How often was the chicken you ate WHITE meat ? Almost never or never About of the time About of the time About of the time Almost always or always 82d. How often did you eat chicken WITH skin ? Almost never or never About of the time About of the time About of the time Almost always or always

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13783. How often did you eat baked ham or ham steak ? NEVER (GO TO QUESTION 84) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 83a. Each time you ate baked ham or ham steak how much did you usually eat? Less than 1 ounce 1 to 3 ounces More than 3 ounces 84. How often did you eat pork (including chops, roasts, sausage and in mixed dishes)? (DO NOT include ham, or ham steak.) NEVER (GO TO QUESTION 85) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 84a. Each time you ate pork how much did you usually eat? Less than 2 ounces or less than 1 chop 2 to 5 ounces or 1 chop More than 5 ounces or more than 1 chop 85. How often did you eat gravy on meat, chicken, potatoes, rice, etc.? NEVER (GO TO QUESTION 86) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 85a. Each time you ate gravy on meat, chicken, potatoes, rice, etc., how much did you usually eat? Less than 1/8 cup 1/8 to cup More than cup Over the past 12 months … 86. How often did you eat liver (all kinds) or liverwurst ? NEVER (GO TO QUESTION 87) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 86a. Each time you ate liver or liverwurst how much did you usually eat? Less than 1 ounce 1 to 4 ounces More than 4 ounces 87. How often did you eat bacon (including low-fat but not imitation)? (DO NOT include soy or vegetable protein substitutes. We will ask about these later.) NEVER (GO TO QUESTION 88) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 87a. Each time you ate bacon how much did you usually eat? Fewer than 2 slices 2 to 3 slices More than 3 slices 87b. How often was the bacon you ate light, lowfat, or lean bacon ? Almost never or never About of the time About of the time About of the time Almost always or always Please continue on next page.

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13888. How often did you eat beef sausage (including low-fat)? (DO NOT include soy or vegetable protein substitutes. We will ask about these later.) NEVER (GO TO QUESTION 89) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 88a. Each time you ate beef sausage how much did you usually eat? Less than 1 patty or 2 links 1 to 3 patties or 2 to 5 links More than 3 patties or 5 links 88b. How often was the beef sausage you ate light, low-fat or lean beef sausage ? Almost never or never About of the time About of the time About of the time Almost always or always 89. How often did you eat fish sticks, fried fish, or fried seafood (not including clams, shrimp, or other shellfish or soy or vegetable protein fish substitutes)? NEVER (GO TO QUESTION 89b) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 89a. Each time you ate fish sticks, fried fish or fried seafood how much did you usually eat? Less than 2 ounces or less than 1 fillet 2 to 7 ounces or 1 fillet More than 7 ounces or more than 1 fillet 89b. How often did you eat clams, mussels, oysters, or scallops ? NEVER (GO TO QUESTION 89d) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 89c. Each time you ate clams, mussels, oysters, or scallops how much did you usually eat? Less than 2 ounces 2 to 7 ounces More than 7 ounces 89d. How often did you eat shrimp, crab, or lobster ? (DO NOT include imitation products or products made with soy or vegetable protein substitutes.) NEVER (GO TO QUESTION 90) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 89e Each time you ate shrimp, crab, or lobster how much did you usually eat? Less than 2 ounces or less than 1 fillet 2 to 7 ounces or 1 fillet More than 7 ounces or more than 1 fillet 90. How often did you eat fish or seafood ( not including clams, shrimp, or other shellfish or soy or vegetable protein fish substitutes) that was NOT FRIED ? (Include intake from fish sushi .) NEVER (GO TO QUESTION 90b) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 90a. Each time you ate eat fish or seafood that was NOT FRIED how much did you usually eat? Less than 2 ounces or less than 1 fillet 2 to 5 ounces or 1 fillet More than 5 ounces or more than 1 fillet 90b. How often did you eat shellfish (all kinds) that was NOT FRIED ? NEVER (GO TO QUESTION 91) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day

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13990c. Each time you ate shellfish (all kinds) that was NOT FRIED how much did you usually eat? Less than 2 ounces 2 to 7 ounces More than 7 ounces Over the past 12 months … Now think about all the meat, poultry, and fish you ate in the past 12 months and how they were prepared. 91. How often was oil, butter, margarine or other fat used to FRY, SAUTE, BASTE, OR MARINATE any meat, poultry, or fish you ate? (Please do not include deep frying.) NEVER (GO TO QUESTION 92) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 91a. Which of the following fats were regularly used to prepare your meat, poultry, or fish? (Mark all that apply.) Margarine (including low-fat) Corn oil Canola or rapeseed oil Butter (including low-fat) Oil spray, such as Pam or others Lard, fatback, or bacon fat Other kinds of oils Olive oil None of the above 92. How often did you eat tofu ? NEVER (GO TO QUESTION 93) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 92a. Each time you ate tofu, how much did you usually eat? Less than cup or less than 2 ounces to cup or 2 to 4 ounces More than cup or more than 4 ounces 93. Over the past 12 months did you eat soups ? NO (GO TO QUESTION 94) YES 93a. How often did you eat soup DURING THE WINTER ? NEVER 1–6 times per winter 2 times per week 7–11 times per winter 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 93b. How often did you eat soup DURING THE REST OF THE YEAR ? NEVER 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 93c. Each time you ate soup how much did you usually eat? Less than 1 cup 1 to 2 cups More than 2 cups 93d. How often were the soups you ate bean soups ? Almost never or never About of the time About of the time About of the time Almost always or always 93e. How often were the soups you ate cream soups (including chowders)? Almost never or never About of the time About of the time About of the time Almost always or always

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140Over the past 12 months … 93f. How often were the soups you ate tomato or vegetable soups ? Almost never or never About of the time About of the time About of the time Almost always or always 93g. How often were the soups you ate broth soups (including chicken) with or without noodles or rice ? Almost never or never About of the time About of the time About of the time Almost always or always 94. How often did you eat pizza ? NEVER (GO TO QUESTION 95) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 94a. Each time you ate pizza how much did you usually eat? Less than 1 slice or less than 1 mini pizza 1 to 3 slices or 1 mini pizza More than 3 slices or more than 1 mini pizza 94b. How often did you eat pizza with pepperoni, sausage or meat other than beef ? (DO NOT include pizza made with soy or vegetable protein substitutes. We will ask about these later.) Almost never or never About of the time About of the time About of the time Almost always or always 94c. How often did you eat pizza with ground beef or beef meatballs ? (DO NOT include pizza made with soy or vegetable protein substitutes. We will ask about these later.) Almost never or never About of the time About of the time About of the time Almost always or always 95. How often did you eat crackers ? NEVER (GO TO QUESTION 96) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day Please continue on next page.

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14195a. Each time you ate crackers how many did you usually eat? Fewer than 4 crackers 4 to 10 crackers More than 10 crackers 96. How often did you eat corn bread or corn muffins ? NEVER (GO TO QUESTION 97) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 96a. Each time you ate corn bread or corn muffins, how much did you usually eat? Less than 1 piece or muffin 1 to 2 pieces or muffins More than 2 pieces or muffins 97. How often did you eat biscuits ? NEVER (GO TO QUESTION 98) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 97a. Each time you ate biscuits how many did you usually eat? Fewer than 1 biscuit 1 to 2 biscuits More than 2 biscuits 98. How often did you eat potato chips, tortilla chips, or corn chips (including low-fat, fat-free, or low-salt)? NEVER (GO TO QUESTION 99) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day Over the past 12 months … 98a. Each time you ate potato chips, tortilla chips, or corn chips how much did you usually eat? Fewer than 10 chips or less than 1 cup 10 to 25 chips or 1 to 2 cups More than 25 chips or more than 2 cups 98b. How often were the chips you ate Wow chips or other chips made with fat substitute (Olean or Olestra)? Almost never or never About of the time About of the time About of the time Almost always or always 98c. How often were the chips you ate other lowfat or fat-free chips ? Almost never or never About of the time About of the time About of the time Almost always or always 99. How often did you eat popcorn (including lowfat)? NEVER (GO TO QUESTION 100) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 99a. Each time you ate popcorn how much did you usually eat? Less than 2 cups, popped 2 to 5 cups, popped More than 5 cups, popped 100. How often did you eat pretzels ? NEVER (GO TO QUESTION 101) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day

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142100a. Each time you ate pretzels how many did you usually eat? Fewer than 5 average twists 5 to 20 average twists More than 20 average twists 101. How often did you eat peanuts, walnuts, seeds, or other nuts ? NEVER (GO TO QUESTION 102) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 101a. Each time you ate peanuts, walnuts, seeds, or other nuts how much did you usually eat? Less than cup to cup More than cup 102. How often did you eat an energy, high-protein or breakfast bar ? NEVER (GO TO QUESTION 103) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 102a. Which type of energy, high-protein or breakfast bar did you usually eat? (Mark as many that apply.) Nutri-Grain Bar/Kellogg’s Granola Bar Power Bar Power Bar Performance/Luna Bar Power Bar Protein Plus Power Bar Harvest Power Bar Pria Balance Bar Slimfast Bar Zone Bar Other 102b. Each time you ate an energy, high-protein or breakfast bar how much did you usually eat? Less than 1 bar 1 bar More than 1 bar 103. How often did you eat yogurt (NOT including frozen yogurt)? NEVER (GO TO QUESTION 104) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day Over the past 12 months … 103a. Each time you ate yogur t, how much did you usually eat? Less than cup or less than 1 container to 1 cup or 1 container More than 1 cup or more than 1 container 104. How often did you eat cottage cheese (including low-fat)? NEVER (GO TO QUESTION 105) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 104a. Each time you ate cottage cheese how much did you usually eat? Less than cup to 1 cup More than 1 cup 105. How often did you eat cheese (including low-fat; including on cheeseburgers or in sandwiches, subs or wraps)? (DO NOT include cheese made from soy or vegetable protein.) NEVER (GO TO QUESTION 106) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 105a. Each time you ate cheese how much did you usually eat? Less than ounce or less than 1 slice to 11/2 ounces or 1 slice More than 11/2 ounces or more than 1 slice

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143105b. How often was the cheese you ate light or low-fat cheese ? Almost never or never About of the time About of the time About of the time Almost always or always 105c. How often was the cheese you ate fat-free cheese ? Almost never or never About of the time About of the time About of the time Almost always or always 106. How often did you eat frozen yogurt, or sherbet (including low-fat or fat-free)? Please do not include Tofutti. NEVER (GO TO QUESTION 107) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 106a. Each time you ate frozen yogurt, or sherbet how much did you usually eat? Less than cup or less than 1 scoop to 1 cup or 1 to 2 scoops More than 1 cup or more than 2 scoops 107. How often did you eat ice cream or ice cream bars, (including low-fat or fat-free)? NEVER (GO TO QUESTION 108) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 107a. Each time you ate ice cream or ice cream bars how much did you usually eat? Less than cup or less than 1 scoop to 11/2 cups or 1 to 2 scoops More than 11/2 cups or more than 2 scoops 107b. How often was the ice cream you ate light, low-fat, or fat-free ice cream ? Almost never or never About of the time About of the time About of the time Almost always or always Over the past 12 months … 108. How often did you eat cake (including low-fat or fat-free)? NEVER (GO TO QUESTION 109) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 108a. Each time you ate cake how much did you usually eat? Less than 1 medium piece 1 medium piece More than 1 medium piece 108b. How often was the cake you ate light, lowfat, or fat-free cake ? Almost never or never About of the time About of the time About of the time Almost always or always 109. How often did you eat cookies or brownies (including low-fat or fat-free)? NEVER (GO TO QUESTION 110) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 109a. Each time you ate cookies or brownies how much did you usually eat? Less than 2 cookies or 1 small brownie 2 to 4 cookies or 1 medium brownie More than 4 cookies or 1 large brownie

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144109b. How often were the cookies or brownies you ate light, low-fat, or fat-free cookies or brownies ? Almost never or never About of the time About of the time About of the time Almost always or always 110. How often did you eat doughnuts, sweet rolls, Danish, or pop-tarts ? NEVER (GO TO QUESTION 111) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 110a. Each time you ate doughnuts, sweet rolls, Danish, or pop-tarts how much did you usually eat? Less than 1 piece 1 to 2 pieces More than 2 pieces 111. How often did you eat sweet muffins or dessert breads (including low-fat or fat-free)? NEVER (GO TO QUESTION 112) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 111a. Each time you ate sweet muffins or dessert breads how much did you usually eat? Less than 1 medium piece 1 medium piece More than 1 medium piece 111b. How often were the sweet muffins or dessert breads you ate light, low-fat, or fatfree sweet muffins or dessert breads ? Almost never or never About of the time About of the time About of the time Almost always or always 112. How often did you eat fruit crisp, cobbler, or strudel ? NEVER (GO TO QUESTION 113) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day Over the past 12 months … 112a. Each time you ate fruit crisp, cobbler, or strudel, how much did you usually eat? Less than cup to 1 cup More than 1 cup 113. How often did you eat pie ? NEVER (GO TO QUESTION 114) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 113a. Each time you ate pie how much did you usually eat? Less than 1/8 of a pie About 1/8 of a pie More than 1/8 of a pie The next four questions ask about the kinds of pie you ate. Please read all four questions before answering. 113b. How often were the pies you ate fruit pie (such as apple, blueberry, others)? Almost never or never About of the time About of the time About of the time Almost always or always 113c. How often were the pies you ate cream, pudding, custard, key lime or meringue pie ? Almost never or never About of the time About of the time About of the time Almost always or always

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145113d. How often were the pies you ate pumpkin or sweet potato pie ? Almost never or never About of the time About of the time About of the time Almost always or always 113e. How often were the pies you ate pecan pie ? Almost never or never About of the time About of the time About of the time Almost always or always 114. How often did you eat chocolate candy ? NEVER (GO TO QUESTION 115) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 114a. Each time you ate chocolate candy how much did you usually eat? Less than 1 average bar or less than 1 ounce 1 average bar or 1 to 2 ounces More than 1 average bar or more than 2 ounces 115. How often did you eat other candy ? NEVER (GO TO QUESTION 116) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 115a. Each time you ate other candy how much did you usually eat? Fewer than 2 pieces 2 to 9 pieces More than 9 pieces 116. How often did you eat eggs or egg whites, (NOT counting egg substitutes or eggs in baked goods and desserts)? (Please include eggs in salads, quiche, and souffls.) NEVER (GO TO QUESTION 116b) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day Over the past 12 months … 116a. Each time you ate eggs how many did you usually eat? 1 egg 2 eggs 3 or more eggs 116b. Over the past 12 months, how often did you eat an egg substitute product (NOT counting egg substitute product used in baked goods and desserts)? (Please include egg substitute used in quiche, souffls, and egg dishes.) NEVER (GO TO QUESTION 116e) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 116c. Which of the following brands do you eat most often (mark as many that apply)? Morningstar Farms Scrambles Morningstar Farms Better n’ Eggs Eggbeaters Second Nature Other 116d. Each time you ate egg substitute product, how much did you usually eat? Less than cup to 1 cup More than 1 cup

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146116e. How often were the eggs or egg substitute product you ate cooked in oil, butter, or margarine ? Almost never or never About of the time About of the time About of the time Almost always or always 116f. How often were the eggs you ate part of egg salad ? Almost never or never About of the time About of the time About of the time Almost always or always 117. How many cups of coffee caffeinated or decaffeinated, did you drink? NEVER (GO TO QUESTION 118) Less than 1 cup per 5–6 cups per week month 1 cup per day 1–3 cups per month 2–3 cups per day 1 cup per week 4–5 cups per day 2–4 cups per week 6 or more cups per day 117a. How often was the coffee you drank decaffeinated ? Almost never or never About of the time About of the time About of the time Almost always or always 118. How many glasses of ICED tea, caffeinated or decaffeinated, did you drink? NEVER (GO TO QUESTION 119) Less than 1 cup per 5–6 cups per week month 1 cup per day 1–3 cups per month 2–3 cups per day 1 cup per week 4–5 cups per day 2–4 cups per week 6 or more cups per day 119a. How often was the iced tea you drank decaffeinated or herbal tea ? Almost never or never About of the time About of the time About of the time Almost always or always 119. How many cups of HOT tea caffeinated or decaffeinated, did you drink? NEVER (GO TO QUESTION 120) Less than 1 cup per 5–6 cups per week month 1 cup per day 1–3 cups per month 2–3 cups per day 1 cup per week 4–5 cups per day 2–4 cups per week 6 or more cups per day 119a. How often was the hot tea you drank decaffeinated or herbal tea ? Almost never or never About of the time About of the time About of the time Almost always or always Over the past 12 months … 120. How often did you add sugar or honey to your coffee or tea? NEVER (GO TO QUESTION 121) Less than 1 time per 5–6 times per week month 1 time per day 1–3 times per month 2–3 times per day 1 time per week 4–5 times per day 2–4 times per week 6 or more times per day 120a. Each time sugar or honey was added to your coffee or tea, how much was usually added? Less than 1 teaspoon 1 to 3 teaspoons More than 3 teaspoons 121. How often did you add artificial sweetener to your coffee or tea? NEVER (GO TO QUESTION 122) Less than 1 time per 5–6 times per week month 1 time per day 1–3 times per month 2–3 times per day 1 time per week 4–5 times per day 2–4 times per week 6 or more times per day

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147121a. What kind of artificial sweetener did you usually use? Equal or aspartame Sweet N Low or saccharin 122. How often was non-dairy creamer added to your coffee or tea? NEVER (GO TO QUESTION 123) Less than 1 time per 5–6 times per week month 1 time per day 1–3 times per month 2–3 times per day 1 time per week 4–5 times per day 2–4 times per week 6 or more times per day 122a. Each time non-dairy creamer was added to your coffee or tea, how much was usually used? Less than 1 teaspoon 1 to 3 teaspoons More than 3 teaspoons 122b. What kind of non-dairy creamer did you usually use? Regular powdered Low-fat or fat-free powdered Regular liquid Low-fat or fat-free liquid 123. How often was cream or half and half added to your coffee or tea? NEVER (GO TO QUESTION 124) Less than 1 time per 5–6 times per week month 1 time per day 1–3 times per month 2–3 times per day 1 time per week 4–5 times per day 2–4 times per week 6 or more times per day 123a. Each time cream or half and half was added to your coffee or tea, how much was usually added? Less than 1 tablespoon 1 to 2 tablespoons More than 2 tablespoons 124. How often was milk added to your coffee or tea? NEVER (GO TO QUESTION 125) Less than 1 time per 5–6 times per week month 1 time per day 1–3 times per month 2–3 times per day 1 time per week 4–5 times per day 2–4 times per week 6 or more times per day 124a. Each time milk was added to your coffee or tea, how much was usually added? Less than 1 tablespoon 1 to 3 tablespoons More than 3 tablespoons 124b. What kind of milk was usually added to your coffee or tea? Whole milk (including lactose-free variety) 2% milk (including lactose-free variety) 1% milk (including lactose-free variety) Skim, nonfat, or % milk (including lactosefree variety) Evaporated or condensed (canned) milk Soy milk Rice milk Other Over the past 12 months … 125. How often was sugar or honey added to foods you ate? (Please do not in clude sugar in coffee, tea, other beverages, or baked goods.) NEVER (GO TO INTRODUCTION TO QUESTION 126) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 125a. Each time sugar or honey was added to foods you ate, how much was usually added? Less than 1 teaspoon 1 to 3 teaspoons More than 3 teaspoons

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148The following questions are about the kinds of margarine, mayonnaise, sour cream, cream cheese, and salad dressing that you eat. If possible, please check the labels of these foods to help you answer. 126. Over the past 12 months did you eat margarine ? NO (GO TO QUESTION 127) YES 126a. How often was the margarine you ate regular-fat margarine (stick or tub)? Almost never or never About of the time About of the time About of the time Almost always or always 126b. How often was the margarine you ate light or low-fat margarine (stick or tub)? Almost never or never About of the time About of the time About of the time Almost always or always 126c. How often was the margarine you ate fatfree margarine ? Almost never or never About of the time About of the time About of the time Almost always or always 127. Over the past 12 months, did you eat butter ? NO (GO TO QUESTION 128) YES 127a. How often was the butter you ate light or low-fat butter ? Almost never or never About of the time About of the time About of the time Almost always or always 128. Over the past 12 months did you eat mayonnaise or mayonnaise-type dressing ? NO (GO TO QUESTION 129) YES 128a. How often was the mayonnaise you ate regular-fat mayonnaise ? Almost never or never About of the time About of the time About of the time Almost always or always 128b. How often was the mayonnaise you ate light or low-fat mayonnaise ? Almost never or never About of the time About of the time About of the time Almost always or always Over the past 12 months … 128c. How often was the mayonnaise you ate fatfree mayonnaise ? Almost never or never About of the time About of the time About of the time Almost always or always 129. Over the past 12 months did you eat sour cream ? NO (GO TO QUESTION 130) YES 129a. How often was the sour cream you ate regular-fat sour cream ? Almost never or never About of the time About of the time About of the time Almost always or always 129b. How often was the sour cream you ate light, low-fat, or fat-free sour cream ? Almost never or never About of the time About of the time About of the time Almost always or always

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149130. Over the past 12 months did you eat cream cheese ? NO (GO TO QUESTION 131) YES 130a. How often was the cream cheese you ate regular-fat cream cheese ? Almost never or never About of the time About of the time About of the time Almost always or always 130b. How often was the cream cheese you ate light, low-fat, or fat-free cream cheese ? Almost never or never About of the time About of the time About of the time Almost always or always 131. Over the past 12 months did you eat salad dressing ? NO (GO TO INTRODUCTION TO QUESTION 132) YES 131a. How often was the salad dressing you ate regular-fat salad dressing (including oil and vinegar dressing)? Almost never or never About of the time About of the time About of the time Almost always or always 131b. How often was the salad dressing you ate light or low-fat salad dressing ? Almost never or never About of the time About of the time About of the time Almost always or always 131c. How often was the salad dressing you ate fat-free salad dressing ? Almost never or never About of the time About of the time About of the time Almost always or always The following two questions ask you to summarize your usual intake of vegetables and fruits. Please do not include salads, potatoes, or juices. 132. Over the past 12 months how many servings of vegetables (not including salad or potatoes) did you eat per week or per day? Less than 1 per week 2 per day 1–2 per week 3 per day 3–4 per week 4 per day 5–6 per week 5 or more per day 1 per day Over the past 12 months … 133. Over the past 12 months how many servings of fruit (not including juices) did you eat per week or per day? Less than 1 per week 2 per day 1–2 per week 3 per day 3–4 per week 4 per day 5–6 per week 5 or more per day 1 per day 134. Over the past month which of the following foods did you eat AT LEAST THREE TIMES? (Mark all that apply.) Avocado, guacamole Olives Cheesecake Oysters Chocolate, fudge, or Pickles or pickled butterscotch toppings vegetables or fruit or syrups Plantains Chow mein noodles Pork neckbones, hock, Croissants head, feet Dried apricots Pudding or custard Egg rolls Veal, venison, lamb Granola bars Whipped cream, regular Hot peppers Whipped cream, Jello, gelatin substitute Milkshakes or NONE ice-cream sodas Please continue on next page.

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150 135. For ALL of the past 12 months have you followed any type of vegetarian diet ? NO (GO TO INTRODUCTIO N TO QUESTION 136) YES 135a. Which of the following foods did you TOTALLY EXCLUDE from your diet? (Mark all that apply.) Beef, veal Pork, lamb Poultry (chicken, turkey, duck) Fish and seafood Shellfish Eggs ( please do not include egg substitutes) Dairy products (milk, cheese, etc.) ( please do not include milk/dairy substitutes) 136. Over the past 12 months, did you eat any meat substitute products made with soy or vegetable protein ? NO Thank you very much for completing this questionnaire! Because we want to be able to use all the information you have provided, we would greatly appreciate it if you would please take a moment to review each page making sure that you: Did not skip any pages Crosssed out the incorrect answer and circled the correct answer if you made any changes YES (GO TO QUESTION 137) 137. How often did you eat breakfast patties or breakfast links made with soy or vegetable protein ? NEVER (GO TO QUESTION 138) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 137a. Which of the following brands do you eat most often (mark as many that apply)? Morningstar Farms Worthington Loma Linda Other (such as Boca, Quorn, etc.) 137b. Each time you ate a breakfast patty or breakfast link made with soy or vegetable protein how much did you usually eat? Less than 1 patty or 2 links 1 to 3 patties or 2 to 5 links More than 3 patties or 5 links 138. How often did you eat breakfast strips (imitation bacon) made with soy or vegetable protein ? NEVER (GO TO QUESTION 139) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 138a. Which of the following brands do you eat most often? (Mark as many that apply.) Morningstar Farms Worthington Loma Linda Other (such as Boca, Quorn, etc.) 138b. Each time you ate breakfast strips (imitation bacon) made with soy or vegetable protein how much did you usually eat? Fewer than 2 slices 2 to 3 slices More than 3 slices 139. How often did you eat burgers made with soy or vegetable protein ? NEVER (GO TO QUESTION 140) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day

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151139a. Which of the following brands do you eat most often? (Mark as many that apply.) Morningstar Farms Worthington Loma Linda Other (such as Boca, Quorn, etc.) 139b. Each time you ate a burger made with soy or vegetable protein how much did you usually eat? Less than 1 patty or less than 2 ounces 1 patty or 2 to 4 ounces More than 1 patty or more than 4 ounces 140. How often did you eat imitation meat dinner entrees (not including imitation chicken or fish) made with soy or vegetable protein ? NEVER (GO TO QUESTION 141) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 140a. Which of the following brands do you eat most often (mark as many that apply)? Morningstar Farms Worthington Loma Linda Other (such as Boca, Quorn, etc.) 140b. Each time you ate an imitation meat dinner entree (not including imitation chicken or fish) made with soy or vegetable protein how much did you usually eat? Less than 1 portion or cup One portion or 1 cup More than one portion or more than 1 cup 141. How often did you eat imitation chicken/turkey patties, nuggets, wings or other imitation chicken/turkey product made with soy or vegetable protein ? NEVER (GO TO QUESTION 142) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 141a. Which of the following brands do you eat most often? (Mark as many that apply.) Morningstar Farms Worthington Loma Linda Other (such as Boca, Quorn, etc.) 141b. Each time you ate an imitation chicken/turkey patties, nuggets, wings or other imitation chicken/turkey product made with soy or vegetable protein how much did you usually eat? Less than 1 patty or less than 2 ounces 1 patty or 2 to 4 ounces More than 1 patty or more than 4 ounces 142. How often did you eat entrees like chili, Mexican foods (tacos, burritos, tostados, enchiladas, etc) lasagna, manicotti, ravioli, stuffed shells, tortellini spaghetti with meat sauce, meatballs or casserole made with soy or vegetable protein products like Burger Crumbles? NEVER (GO TO QUESTION 143) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 142a. Which of the following brands do you eat most often? (Mark as many that apply.) Morningstar Farms Worthington Loma Linda Other (such as Boca, Quorn, etc.) 142b. Each time you ate an entre like chili, Mexican foods (tacos, burritos, tostados, enchiladas, etc) lasagna, manicotti, ravioli, stuffed shells, tortellini spaghetti with meat sauce, meatballs or casserole made with soy or vegetable protein products like Burger Crumbles how much did you usually eat? Less than cup to 1 cup More than 1 cup

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152143. How often did you eat cold cut substitutes (such as meatless salami, meatless bologna, meatless chicken roll, meatless smoked turkey, meatless corned beef) made with soy or vegetable protein? NEVER (GO TO END) 1–6 times per year 2 times per week 7–11 times per year 3–4 times per week 1 time per month 5–6 times per week 2–3 times per month 1 time per day 1 time per week 2 or more times per day 143a. Which of the following brands do you eat most often? (Mark as many that apply.) Morningstar Farms Worthington Loma Linda Other (such as Boca, Quorn, etc.) 143b. Each time you ate cold cut substitutes (such as meatless salami, meatless bologna, meatless chicken roll, meatless smoked turkey, meatless corned beef) made with soy or vegetable protein how much did you usually eat? Less than 3 slices or less than 2 ounces 3 to 6 slices or 2 to 4 ounces More than 6 slices or more than 4 ounces Thank you very much for completing this questionnaire! Because we want to be able to use all the information you have provided, we would greatly appreciate it if you would please take a moment to review each page making sure that you: Did not skip any pages Crossed out the incorrect answer and circled the correct answer if you made any changes

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153 APPENDIX C DIETARY HISTORY QUESTIONNAIRE INSTRUCTIONS MATERIALS Please read these instructions prior to begi nning the Diet History Questionnaire. These additional instructions combined with the “General Instructions ” (found on the first page of the Diet History Questionnaire ) will guide you while comp leting the questionnaire. ADDITIONAL INSTRUCTIONS 1. When answering each question, think about your di et over the past year and NOT the past few weeks. 2. Several questions refer you to additional handouts that ha ve been provided in your packet. Please be sure to use these handouts when you get to these questions. If you have any questions while comple ting the questionnaire, please contact Amanda Brown at 352-392-1991 ext 246 Please leave a voice message with your name, contact number, and best time or way to reach you. Question Number Handout 8e Caffeinated versus Non-caffeinated Beverages 13b Fortified Cereal 19a, 21a, 22a, 23a, 24a, 25a, 34a, 42a Seasonal Fruits and Vegetables

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154Remember when answering these que stions, we want you to think about your diet over the past year and NOT the past few weeks. Here are a few examples, that you may find helpful when completing the questionnaire 1. Please look at question # 57 on page 16. I go to IHOP for br eakfast on the fi rst Sunday of every month of the year. I al ways order one medium size Belgian waffle. I never make waffles, pancakes, or French toast at home. I only eat waffles at IHOP. Since there are 12 months in a year, I eat 12 waffles a year or 1 waffle a month. If you look at question #57, I would mark an "X" for 1 time per month. For question #57a, I would answer 1 to 3 medium pieces. 2. Please look at questi on #107 on page 31. I only eat ice cream during the summer months of June, July, and August. During that time, I eat 1 cup of ice cream twice a week. That means I eat ice cream 8 times during each of those months. So that adds up to 24 times. Since I do not eat ice cream dur ing the other months, I can sa y that I only have ice cream 24 times a year. If I remember that there are 12 months in a year th at means, on average, I only have ice cream twice a month. If you look at question #107 on page 31, I would mark an “X” for 2-3 times a month. For que stion #107a, I would mark to 1 cups or 1 to 2 scoops.

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155 Directions on How to Change Your Answer Choice Over the past 12 months how often did you drink tomato juice or vegetable juice ? a. 1 time per month or less b. 2-3 times per month c. 1-2 times per week d. 3-4 times per week e. 5-6 times per week a. 1 time per month or less b. 2-3 times per month c. 1-2 times per week d. 3-4 times per week e. 5-6 times per week a. 1 time per month or less b. 2-3 times per month c. 1-2 times per week d. 3-4 times per week e. 5-6 times per week You select “a” as your first answer, but then you change your mind. Step #1 Cross out answer “a” Step #2 Mark an “X” in the box of your new choice and circle that answer too

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156Please use this list for Question 8e Caffeinated Non-caffeinated A & W Creme Soda 7-Up Barq’s Root Beer A & W Root Beer Canada Dry Cola Diet 7-Up Coffee Diet A & W Root Beer Diet A & W Creme Soda Diet Sprite Diet Coke with Lem on Minute Maid Orange Diet Cherry Coke Sprite Cherry Coke Cherry Pepsi Coke Diet Coke Diet Barq’s Root Beer Diet Dr. Pepper Diet Mountain Dew Diet Pepsi Diet RC Cola Diet Shasta Cola Diet Snapple Flavored teas Diet Sunkist Orange Dr. Pepper Hot Chocolate Mix Jolt Lipton Brisk Mellow Yellow Mountain Dew Mountain Dew Code Red Nestea Sweetened & Unsw eetened Iced Teas Pepsi Pepsi One Pepsi Twist RC Cola Shasta Cola Snapple Flavored teas Sunkist Orange Surge Tea: Iced, Brewed, Instant, Green

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157Please use this list for Question 13b All-Bran – Buds All-Bran – Extra Fiber All-Bran – Original Cheerios – Multigrain Complete Oat Bran Flakes Complete Wheat Bran Flakes Just Right Fruit and Nut KASHI Heart to Heart Kellogg’s Low Fat Granola no raisins Kellogg’s Low Fat Granola with raisins Mueslix Product 19 Smart Start Smart Start Soy Protein Special K Total Original Total Whole Grain Total Brown Sugar and Oat Total Corn Flakes Total Raisin Bran

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158Please use this list for Questions 19a, 21a, 22a, 23a, 24a, 25a, 34a, 42a Fruit or Vegetable Months When In Season Cantaloupe March to July Corn August to June Grapefruit September to June Honeydew Melon June to October Nectarines July to September Oranges October to June Peaches June to September Plums June to October Strawberries October to June Tangelos November to February Tomatoes September to June

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159 LIST OF REFERENCES 1. Bailey LB, Gregory JF. Folate. In : Bowman BA, Russell RM, eds. Present Knowledge in Nutrition. Vol I. 9th ed; 2006:278-301. 2. Food and Drug Administration. Food standards: amendment of standards of identity for enriched grain products to re quire addition of folic acid. Fed Regist. 1996;61(44):87818797. 3. Choumenkovitch SF, Jacques PF, Nadeau MR, Wilson PW, Rosenberg IH, Selhub J. Folic acid fortification increases red blood cell folate co ncentrations in the Framingham study. J Nutr. Dec 2001;131(12):3277-3280. 4. Jacques PF, Selhub J, Bostom AG, Wilson PW Rosenberg IH. The ef fect of folic acid fortification on plasma folate and total homocysteine concentrations. N Engl J Med. May 13 1999;340(19):1449-1454. 5. Lawrence JM, Petitti DB, Watkins M, Umekubo MA. Trends in serum folate after food fortification. Lancet. Sep 11 1999;354(9182):915-916. 6. Yang QH, Carter HK, Mulinare J, Berry RJ Friedman JM, Erickson JD. Race-ethnicity differences in folic acid intake in women of childbearing age in the United States after folic acid fortification: fi ndings from the National Health and Nutrition Examination Survey, 2001-2002. Am J Clin Nutr. May 2007;85(5):1409-1416. 7. Institute of Medicine. Dietary reference intakes for Th iamin, Riboflavin, Niacin, Vitamin B6, Folate, Vitamin B12, Pantothenic Aci d, Biotin, and Choline: Standing Committee on the Scientific Evaluation of Dietary Reference Inta kes, Food and Nutrition Board Washington, D.C.: Nationa l Academy Press; 1998. 8. US Department of Agriculture, Agricultural Research Service. USDA National Nutrient Database for Standard Refe rence, Release 17. 2004; Nutr ient Data Laboratory Home Page, http://www.nal.usda .gov/fnic/foodcomp Accessed October 17, 2007. 9. Suitor CW, Bailey LB. Dietary folate equi valents: interpreta tion and application. J Am Diet Assoc. Jan 2000;100(1):88-94. 10. Food and Nutrition Board IOM. Folate. Dietary Reference Inta kes: Thiamin, Riboflavin, Niacin, Vitamin B6, Folate, Vitamin B 12, Pantothenic Acid, Biotin, and Choline Washington DC: National Ac ademy Press; 1998:196-305. 11. Bailey LB, Rampersaud GC, Kauwell GP. Foli c acid supplements and fortification affect the risk for neural tube defects, vasc ular disease and cancer: evolving science. J Nutr. Jun 2003;133(6):1961S-1968S. 12. Botto LD, Lisi A, Bower C, Canfield MA, Da ttani N, De Vigan C, De Walle H, Erickson DJ, Halliday J, Irgens LM, Lowr y RB, McDonnell R, Metneki J, Poetzsch S, Ritvanen A, Robert-Gnansia E, Siffel C, Stoll C, Mastroiaco vo P. Trends of selected malformations in

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163 52. Herbert V, Zalusky R. Interr elations of vitamin B12 and fo lic acid metabolism: folic acid clearance studies. J Clin Invest. Jun 1962;41:1263-1276. 53. Bailey LB, Gregory JF, 3rd. Fola te metabolism and requirements. J Nutr. Apr 1999;129(4):779-782. 54. Cook RJ. Folate metabolism. In: Carmel R, Jacobsen D, eds. Homocysteine in Health and Disease Cambridge, Uk: Cambridge University Press; 2001:113-134. 55. Kang SS, Zhou J, Wong PW, Kowalisyn J, St rokosch G. Intermediate homocysteinemia: a thermolabile variant of meth ylenetetrahydrofolate reductase. Am J Hum Genet. Oct 1988;43(4):414-421. 56. Matthews RG. Methylenetetrahydrofolate re ductase: a common human polymorphism and its biochemical implications. Chem Rec. 2002;2(1):4-12. 57. Guenther BD, Sheppard CA, Tran P, Roze n R, Matthews RG, Ludw ig ML. The structure and properties of methylenetetrahydrofolate reductase from Escherichia coli suggest how folate ameliorates human hyperhomocysteinemia. Nat Struct Biol. Apr 1999;6(4):359365. 58. Herbert V. The 1986 Herman aw ard lecture. Nutrition scienc e as a continually unfolding story: the folate and vitamin B-12 paradigm. Am J Clin Nutr. Sep 1987;46(3):387-402. 59. Kauwell GP, Wilsky CE, Cerda JJ, Herrlin ger-Garcia K, Hutson AD, Theriaque DW, Boddie A, Rampersaud GC, Ba iley LB. Methylenetetrahydrof olate reductase mutation (677C-->T) negatively in fluences plasma homocysteine response to marginal folate intake in elderly women. Metabolism. Nov 2000;49(11):1440-1443. 60. Sauberlich HE, Dowdy RP, Skala JH. Laboratory Tests for the Assessment of Nutritional Status: Folacin Cleveland, OH: CRC Press; 1974. 61. Fazili Z, Pfeiffer CM, Zhang M. Comparis on of serum folate sp ecies analyzed by LCMS/MS with total folate measured by microbiologic assay and Bio-Rad radioassay. Clin Chem. Apr 2007;53(4):781-784. 62. Wu A, Chanarin I, Slavin G, Levi AJ. Folate deficiency in the alc oholic--its relationship to clinical and haematological abnormal ities, liver disease and folate stores. Br J Haematol. Mar 1975;29(3):469-478. 63. Kang SS, Wong PW, Norusis M. Homocyst einemia due to folate deficiency. Metabolism. May 1987;36(5):458-462. 64. Rozen R. Polymorphisms of folate and cobala min metabolism. In: Carmel R, Jacobsen D, eds. Homocysteine in Health and Disease Cambridge, UK: Camb ridge Press; 2001:259270.

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164 65. Carmel R. Cobalamin deficiency. In: Carmel R, Jacobsen D, eds. Homocysteine in Health and Disease Cambridge, UK: Camb ridge Press; 2001:289-305. 66. Ganji V, Kafai MR. Population referen ce values for plasma total homocysteine concentrations in US adults after the fo rtification of cereal s with folic acid. Am J Clin Nutr. Nov 2006;84(5):989-994. 67. Ganji V, Kafai MR. Trends in serum folate, RBC folate, and circulating total homocysteine concentrations in the United States: analysis of data from National Health and Nutrition Examination Su rveys, 1988-1994, 1999-2000, and 2001-2002. J Nutr. Jan 2006;136(1):153-158. 68. Willard JE, Lange RA, Hillis LD. The use of aspirin in ischemic heart disease. N Engl J Med. Jul 16 1992;327(3):175-181. 69. Baggott JE, Morgan SL, Ha T, Vaughn WH, Hine RJ. Inhi bition of folate-dependent enzymes by non-steroidal an ti-inflammatory drugs. Biochem J. Feb 15 1992;282 ( Pt 1):197-202. 70. Eichner ER, Loewenstein JE, McDonald CR Dickson VL. Effect of common drugs on serum level and binding of folate in man. In: Kis liuk RL, Brown GM, eds. Chemistry and Biology of Pteridines New York: Elsevier North Holland; 1979:537-542. 71. Lawrence VA, Loewenstein JE, Eichner ER. As pirin and folate bind ing: in vivo and in vitro studies of serum binding and urin ary excretion of endogenous folate. J Lab Clin Med. Jun 1984;103(6):944-948. 72. Wickramasinghe SN. Diagnosis of megaloblastic anaemias. Blood Rev. May 19 2006. 73. Blackburn ST. The hematologic and hemostat ic systems. In: Blackburn ST, Loper DL, eds. Maternal, Fetal, and Neonatal Ph ysiology: A Clinical Perspective Philadelphia, PA: W. B. Saunders; 1992:159-200. 74. Cunningham FG, MacDonald PC, Gant NF, Le veno KJ, Gilstrap LC. Prenatal Care. In: Cunningham FG, MacDonald PC, Gant NF, Leveno KJ, Gilstrap LC, eds. Williams Obstetrics Norwalk, CN: Applet on and Lange; 1993:249. 75. Caudill MA, Gregory JF, Hutson AD, Bailey LB. Folate catabolism in pregnant and nonpregnant women with cont rolled folate intakes. J Nutr. Feb 1998;128(2):204-208. 76. Myers T. Mosby's Medical Dictionary 7th ed. St. Louis: Mosby/Elsevier; 2006. 77. Botto LD, Moore CA, Khoury MJ, Er ickson JD. Neural-Tube Defects. N Engl J Med. 1999;341(20):1509-1519. 78. Hibbard BM. The Role of Folic Acid in Pregnancy; with Part icular Reference to Anaemia, Abruption and Abortion. J Obstet Gynaecol Br Commonw. Aug 1964;71:529542.

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166 92. Ueland PM, Refsum H, Brattstrom L. Plasma homocyst(e)ine and cardiovascular disease. In: Francis RBJ, ed. Atherosclerotic Cardiovascul ar Disease Homeostasis and Endothelial Function New York, NY: Marcel Dekker Inc; 1992:183-236. 93. Stamler JS, Osborne JA, Jaraki O, Rabb ani LE, Mullins M, Singel D, Loscalzo J. Adverse vascular effects of homocystein e are modulated by e ndothelium-derived relaxing factor and rela ted oxides of nitrogen. J Clin Invest. Jan 1993;91(1):308-318. 94. Tawakol A, Omland T, Gerhard M, Wu JT Creager MA. Hyperhom ocyst(e)inemia is associated with impaired endotheliu m-dependent vasodilation in humans. Circulation. Mar 4 1997;95(5):1119-1121. 95. Bailey LB. Do low doses of folic acid resu lt in maximum lowering of homocysteine? Am J Clin Nutr. Oct 2005;82(4):717-718. 96. Homocysteine Lowering Trialists' Collaborat ion. Dose-dependent e ffects of folic acid on blood concentrations of homocysteine: a meta-analysis of th e randomized trials. Am J Clin Nutr. Oct 2005;82(4):806-812. 97. Lowering blood homocysteine with folic aci d based supplements: meta-analysis of randomised trials. Homocysteine Lowering Trialists' Collaboration. BMJ. Mar 21 1998;316(7135):894-898. 98. Thom T, Haase N, Rosamond W, Howard VJ, Rumsfeld J, Manolio T, Zheng ZJ, Flegal K, O'Donnell C, Kittner S, Lloyd-Jones D, Goff DC, Jr., Hong Y, Adams R, Friday G, Furie K, Gorelick P, Kissela B, Marler J, Meigs J, Roger V, Sidney S, Sorlie P, Steinberger J, Wasserthiel-Smoller S, Wils on M, Wolf P. Heart disease and stroke statistics--2006 update: a re port from the American Hear t Association Statistics Committee and Stroke St atistics Subcommittee. Circulation. Feb 14 2006;113(6):e85151. 99. Rimm EB, Willett WC, Hu FB, Sampson L, Colditz GA, Manson JE, Hennekens C, Stampfer MJ. Folate and vita min B6 from diet and supplemen ts in relation to risk of coronary heart disease among women. JAMA. Feb 4 1998;279(5):359-364. 100. Refsum H, Ueland PM, Nygard O, Vollset SE Homocysteine and cardiovascular disease. Annu Rev Med. 1998;49:31-62. 101. Homocysteine Studies Collaboration. Homocy steine and risk of is chemic heart disease and stroke: a meta-analysis. JAMA. Oct 23-30 2002;288(16):2015-2022. 102. Bazzano LA, Reynolds K, Holder KN, He J. Effect of folic acid supplementation on risk of cardiovascular diseases: a meta-ana lysis of randomized controlled trials. JAMA. Dec 13 2006;296(22):2720-2726. 103. B-Vitamin Treatment Trialists' Collabor ation. Homocysteine-lowering trials for prevention of cardiovascular events: a revi ew of the design and power of the large randomized trials. Am Heart J. Feb 2006;151(2):282-287.

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172 BIOGRAPHICAL SKETCH Melanie was born in Escondido, California. She received her Bachelor of Science in nutrition applied sciences from the Pennsylvania State Universi ty in 2005. After graduation, Melanie was accepted into the University of Flor ida’s combined Master of Science – Dietetic Internship Program. During graduate school, she served as the Gainesv ille District Dietetic Association Newsletter Co-Edito r for 1 years, and as the te am leader for a school-based nutrition intervention project that she and her classmates develo ped and implemented at Talbot Elementary School during the 2005 to 2006 school year. She and seve ral classmates also had the opportunity to present their work as part of the 2006 Florid a Dietetic Association Annual Symposium Poster Session. In recognition of her academic accomplishments, Melanie recently was awarded a scholarship from the Agricultural Women’s Club. Melanie plans to move to Philadelphia after she graduates from the University of Florida. He r immediate career goal is to pass the Registration Examination fo r Dietitians and work as a registered dietitian specializing in pediatrics.