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Zinc status response to folic acid supplementation and the effect of level of zinc intake on folate utilization in human subjects

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Zinc status response to folic acid supplementation and the effect of level of zinc intake on folate utilization in human subjects
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Kauwell, Gail P. Abbott, 1952-
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xvi, 287 leaves : ill. ; 29 cm.

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Infants ( jstor )
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Dissertations, Academic -- Food Science and Human Nutrition -- UF
Food Science and Human Nutrition thesis Ph. D
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Thesis (Ph. D.)--University of Florida, 1993.
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Includes bibliographical references (leaves 253-285).
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Typescript.
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Vita.
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by Gail P. Abbott Kauwell.

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ZINC STATUS RESPONSE TO FOLIC ACID SUPPLEMENTATION
AND THE EFFECT OF LEVEL OF ZINC INTAKE ON
FOLATE UTILIZATION IN HUMAN SUBJECTS


















By

GAIL P. ABBOTT KAUWELL


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


1993

































Copyright 1993

by

Gail P. Abbott Kauwell
































I dedicate this dissertation to all of the people who

were instrumental in helping me achieve this goal and to my

steadfast, loyal and loving companions, Chelsea and Curly.














ACKNOWLEDGMENTS


It is with great difficulty that I write this section of

my dissertation because it is impossible to properly

acknowledge everyone who deserves to be recognized and

thanked. Your support has come in many forms: encouragement,

ideas, storage space, equipment, supplies, technical

assistance, helping hands, friendly smiles and more. I do

remember and sincerely appreciate all the ways that you have

supported me through the trials and tribulations of completing

this research project and dissertation.

Although I do not wish to slight anyone's contributions

by not including their name in this acknowledgment, I would be

remiss if I did not take this opportunity to highlight some of

the people to whom I am most thankful. I am most grateful to

Dr. Lynn Bailey, the chairman of my supervisory committee,

whose encouragement, support, guidance and friendship has been

limitless. I also appreciate the guidance and support

provided by my committee members, Dr. Robert Cousins, Dr.

Jesse Gregory, Dr. Claudia Probart and Dr. Rachel Shireman.

In addition to my committee members, I want to recognize and

thank Dr. Susan Percival, who provided me with the opportunity

to work in her lab, and Peter Johnson for helping me smile

along the way. The subjects who participated in this study

iv








also deserve to be recognized for the dedication, interest and

enthusiasm they displayed throughout the duration of this

research project. Last, but certainly not least, I am

thankful for the support, encouragement and thoughtful advice

that has always been graciously provided to me by Dr. Richard

Gutekunst, Dean of the College of Health Related Professions.

















TABLE OF CONTENTS


page


ACKNOWLEDGMENTS .

LIST OF TABLES .

LIST OF FIGURES .

LIST OF ABBREVIATIONS .

ABSTRACT .


CHAPTERS


1 INTRODUCTION .

2 REVIEW OF THE LITERA

Folate .
Chemistry .
Metabolism .
Biochemical Fun
Recommended D
Folate .


TURE 5

. 5
. 5
. 7
actions .. .. 17
dietary Allowances for
. 21


Effects of High Doses of Folic Acid .
Sources/Distribution and Stability of
Folate in Foods .
Assessment of Folate Status .
Folate Status in Special Population
Groups .
Effects of Environmental Factors on
Folate Status .
Consequences of Compromised Folate
Status .
Methods Used to Assess Folate Status and
Metabolism in Humans .
Zinc . .
Chemistry .
Absorption .
Transport, Distribution and Metabolism .
Excretion .
Zinc Homeostasis .
Biochemical Functions .


. iv

. ix

. .. x

. .. xii

. xv









Recommended Dietary Allowances for Zinc 88
Effects of High Doses of Zinc ... 92
Food Sources of Zinc, Usual Intakes and
Bioavailability ... 94
Factors Affecting Zinc Status ..... 99
Assessment of Zinc Status .. 101
Metallothionein .. 110
Zinc Deficiency 116
Folate-Zinc Interactions .. 118
Effect of Impaired Zinc Status on Folate
Absorption and Metabolism 118
Effect of Supplemental Folic Acid on Zinc
Status .. .. 122

3 RATIONALE FOR RESEARCH PROTOCOL .. ... 140

4 SUBJECTS, EXPERIMENTAL DESIGN, MATERIALS
AND METHODS .. 145

Subjects .. 145
Recruitment and Selection 145
Description of Subjects 146
Experimental Design .. 147
Materials and Methods .. 151
Description of Diet and Supplements 151
Procedures Used to Foster, Monitor and
Assess Compliance 156
Procedure Used to Assess Adequacy of
Blinding .157
Procedures Used to Prevent Zinc
Contamination ... 157
Urine Collection and Processing
Procedures. 160
Blood Collection and Processing
Procedures ... 160
Preparation of Diet Composites .. 163
Determination of Dietary Zinc Content 164
Biochemical Analyses .. 167
Plasma and Urine Zinc Concentrations 167
Erythrocyte Lysate Zinc Concentration 168
Protein Determination .. 169
Erythrocyte Metallothionein
Concentration .......... 170
Determination of Serum Ferritin
Concentration 175
Determination of Serum, Whole Blood and
Urinary Folate Concentrations 176
Determination of Urinary Deuterium-
Labeled Folate 180
Statistical Analysis .. 184


vii










5 RESULTS . .

Effect of Supplemental Folic Acid on
Zinc Status .
Plasma Zinc .
Erythrocyte Zinc .
Serum Alkaline Phosphatase .
Erythrocyte Metallothionein .
Serum Ferritin .
Urinary Zinc .
Effect of Level of Zinc Intake on Folate
Utilization .
Serum Folate .
Erythrocyte Folate .
Urinary Folate .

6 DISCUSSION .

Effect of Supplemental Folic Acid on
Zinc Status .
Effect of Level of Zinc Intake on Folate
Utilization .

7 SUMMARY AND CONCLUSIONS .

APPENDICES

A SUBJECT SELECTION SCREENING TOOL .

B ZINC RESTRICTED METABOLIC DIET .

C CONTRACT .

REFERENCE LIST . .

BIOGRAPHICAL SKETCH .


186


189
190
193
195
S 198
S 201
S 203

S 206
S 207
207
211

219


219

S 227

S 231



235

246

251

S 253

286


viii














LIST OF TABLES


Table page

2-1 Comparison of 1980 and 1989 Recommended Dietary
Allowances (RDA) for folate ... .21

2-2 Physiologic functions of zinc ... 88

4-1 Average nutrient content of three-day diet with and
without supplements .. ..... 152

4-2 Composition of protein shakes ... 153

5-1 Mean weights ( SD) of subjects on zinc-restricted
and zinc-adequate diets .. 187

5-2 Mean ( SD) usual calorie, protein, folate and zinc
intakes of subjects on zinc-restricted or zinc-
adequate diets ... .187

5-3 Molar ratios of zinc to folic acid for each
treatment combination 189

5-4 Overall mean ( SD) values of response variables
used to assess the effect of supplemental folic
acid on zinc status in subjects consuming zinc-
restricted or zinc-adequate diets 191

5-5 Overall mean ( SD) values of response variables
used to assess the response to folic acid
supplementation in subjects consuming zinc-
restricted or zinc-adequate diets ... .209

5-6 Overall mean ( SD) deuterium-labeled folate
excreted expressed as a percentage of total folate
intake, total urinary folate and oral dose 215














LIST OF FIGURES


Figure page

2-1 Structure of folic acid and folic acid
derivatives . 6

2-2 Folate-mediated one-carbon metabolism 8

4-1 Experimental design .. 148

4-2 Schematic of enzyme-linked immunosorbent assay used
for determination of erythrocyte metallothionein
concentrations . 173

5-1 Effect of supplemental folic acid on plasma zinc in
subjects consuming zinc-restricted or zinc-adequate
diets . ... .. 192

5-2 Effect of supplemental folic acid on erythrocyte
zinc in subjects consuming zinc-restricted or zinc-
adequate diets ... 194

5-3 Effect of supplemental folic acid on serum alkaline
phosphatase in subjects consuming zinc-restricted
or zinc-adequate diets ... .197

5-4 Effect of supplemental folic acid on erythrocyte
metallothionein in subjects consuming zinc-
restricted or zinc-adequate diets .. 200

5-5 Effect of supplemental folic acid on serum ferritin
in subjects consuming zinc-restricted or zinc-
adequate diets ... 202

5-6 Effect of supplemental folic acid on urinary zinc
in subjects consuming zinc-restricted or zinc-
adequate diets ... 205

5-7 Serum folate response to folic acid supplementation
in subjects consuming zinc-restricted or zinc-
adequate diets .. 208








5-8 Erythrocyte folate response to folic acid
supplementation in subjects consuming zinc-
restricted or zinc-adequate diets .. 210

5-9 Urinary folate response to folic acid
supplementation in subjects consuming zinc-
restricted or zinc-adequate diets ... .212

5-10 Urinary deuterium-labeled folate response to folic
acid supplementation in subjects consuming zinc-
restricted or zinc-adequate diets 214

5-11 Percent of oral labeled folic acid plus dietary
folate excreted as deuterium-labeled folate in
subjects consuming zinc-restricted or zinc-adequate
diets .... ... 216

5-12 Percent of total urinary folate excreted as
deuterium-labeled folate in subjects consuming
zinc-restricted or zinc-adequate diets ... 217

5-13 Percent of oral deuterium-labeled folic acid
excreted as deuterium-labeled folate in subjects
consuming zinc-restricted or zinc-adequate diets 218















LIST OF ABBREVIATIONS


Abbreviation

AAS

BSA

C


cm

[14C]-PteGlu7


d

DPBS

D2FA

dL

DNA

EDTA

ELISA

F

FA

FBP

FIGLU

FDA

g

g


Meaning

atomic absorption spectrophotometry

bovine serum albumin

Centigrade

carbon 14

centimeter

carbon 14-labeled
pteroylglutamylhexaglutamate

day

Dulbecco's phosphate-buffered saline

deuterium-labeled folic acid

deciliter

deoxyribonucleic acid

ethylenediamine tetraacetic acid

enzyme-linked immunosorbent assay

Fahrenheit

folic acid

folate-binding protein

formimino-glutamic acid

Food and Drug Administration

gram

gravity


xii









GCMS

h

HC1

HPLC

[3H]PteGlu

HPV-16

IU

kcal

kg

L

M

mg

mL

mol

mmol

MRC

MT

N

NaC1

NaOH

NaN3

ng

nm

oz

P

pABG


gas chromatography-mass spectrometry

hour

hydrochloric acid

high-performance liquid chromatography

tritiated pteroylmonoglutamic acid

human papillomavirus 16

International Units

kilocalories

kilogram

liter

molar

milligram

milliliter

mole

millimole

Medical Research Council

metallothionein

normal

sodium chloride

sodium hydroxide

sodium azide

nanogram

nanometer

ounce

probability

para-aminobenzoyl glutamate


xiii









PBS phosphate-buffered saline

ppm parts per million

RDA Recommended Dietary Allowances

RNA ribonucleic acid

rpm revolutions per minute

SD standard deviation

THF tetrahydrofolate

MCi microcurie

/g microgram

pL microliter

AM micromolar

Mmol micromole

v/v volume/volume

y year

Zn zinc


xiv














Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

ZINC STATUS RESPONSE TO FOLIC ACID SUPPLEMENTATION
AND THE EFFECT OF LEVEL OF ZINC INTAKE ON
FOLATE UTILIZATION IN HUMAN SUBJECTS

By

Gail P. Abbott Kauwell

August 1993

Chairperson: Dr. Lynn B. Bailey
Major Department: Food Science and Human Nutrition

Changes in zinc status in response to folic acid

supplementation and the effect of level of zinc intake on

folate utilization were evaluated in human subjects. Twelve

healthy men were randomly assigned to consume a zinc-

restricted diet (3.5 mg zinc/d) or an identical diet

supplemented with 11 mg of zinc as zinc sulfate (14.5 mg

zinc/d total) for 25 days. Half of the subjects in each group

received 800 gg/d deuterium-labeled folic acid. The remaining

subjects received a placebo. After an 80-day washout period,

the study was repeated with the folic acid/placebo treatments

reversed. Fasting blood samples and 24-hour urine collections

were obtained at corresponding time points during both study

periods.

Plasma zinc, erythrocyte zinc, urinary zinc, serum

alkaline phosphatase and erythrocyte metallothionein








concentrations were measured to determine zinc status. No

differences in mean values within or between the zinc diet

groups were detected (P20.05) for these response variables.

A significant (P<0.001) time by supplement by diet interaction

was detected for erythrocyte metallothionein; however,

significant differences in the change in erythrocyte

metallothionein concentrations due to the supplemental effect

were not detected (P>0.05) within the zinc diet groups. No

significant differences due to the effect of the supplement

were detected (P!0.05) within or between the zinc diet groups

for the remaining response variables. These data suggest that

short-term supplementation with 800 pg folic acid/d does not

adversely affect zinc status in healthy men.

To determine the effect of zinc intake on folate

utilization, total urinary, serum and erythrocyte folate

concentrations, and urinary excretion of deuterium-labeled and

unlabeled folate were determined. Differences in mean serum,

erythrocyte, total urinary or urinary deuterium-labeled folate

concentrations between subjects fed 3.5 or 14.5 mg zinc/d were

not detected (P20.05). Similarly, significant differences in

the percent of total folate ingested or the percent of total

urinary folate excreted as deuterium-labeled folate were not

detected (P>O.05) between the groups. These data suggest that

utilization of supplemental folic acid in healthy men is not

influenced by the level of zinc intake.


xvi














CHAPTER 1
INTRODUCTION


The crucial roles of folate and zinc in protein and

nucleic acid metabolism and genetic expression have

contributed to the intense research interest centered around

these two nutrients. Changes in intake, bioavailability

and/or metabolism of either or both of these nutrients can

have deleterious effects in humans and animals. Understanding

the specific mechanisms of action and relationships between

zinc and folate is essential to improving our ability to make

efficacious nutritional recommendations and policies that will

enhance the health and well-being of the human race.

Zinc is an integral constituent of at least 60 different

enzymes (Cousins and Hempe, 1990) including deoxyribonucleic

acid (DNA) transferase, ribonucleic acid (RNA) polymerases,

alkaline phosphatase, gamma aminolevulinic acid dehydratase,

carbonic anhydrase, carboxypeptidase, alcohol dehydrogenase

and glutamic, lactic and malic acid dehydrogenases (Vallee and

Galdes, 1984). Of particular interest with regard to studies

investigating the relationship between zinc and folate

nutriture was the discovery (Silink et al., 1975) that bovine

hepatic folate conjugase is a zinc metalloenzyme. The fact

that zinc is a component of so many metalloenzymes explains








2

the multiplicity of physiologic functions attributed to this

nutrient. Examples of these functions include development and

maintenance of the body's immune system, prevention of lipid

peroxidation, metabolism of energy containing nutrients,

hormonal interactions, bone formation and the replication and

differentiation of cells (Cousins, 1985; Cunnane, 1988;

Hambidge et al., 1986).

"Folate" is the generic term used in reference to the

many different naturally occurring forms of pteroyl-

monoglutamic acid and pteroylpolyglutamic acid. These

compounds have nutritional properties and chemical structures

similar to their parent compound, folic acid. Folate is a

coenzyme for many one carbon reactions, and like zinc, is

required for cell replication. Specifically, folate coenzymes

are essential for the synthesis of the pyrimidine,

thymidylate, which is required for DNA synthesis. Other

metabolic processes requiring folate coenzymes include the

interconversion of serine and glycine, methionine synthesis,

histidine degradation, methylation of biogenic amines,

generation of format and purine biosynthesis (Brody, 1991).

Recent studies suggest that supplemental folic acid may be

important in reducing the incidence of neural tube defects

(Bower and Stanley, 1989; Czeizel and DudAs, 1992; Medical

Research Council Vitamin Study Research Group, 1991; Milunsky

et al., 1989; Mulinare et al., 1988; Smithells et al., 1981);

modulating cancer risk in a variety of tissues including the








3

lung (Heimburger et al., 1987; 1988), cervix (Butterworth et

al., 1982; 1992a; 1992b), esophagus (Jaskiewicz et al., 1988)

and colon (Lashner et al., 1989); and reducing elevated

homocysteine levels, an emerging independent risk factor for

coronary heart disease (Kang et al., 1986; 1987).

Despite the potential benefits associated with folic acid

supplementation, and the fact that it is widely believed to be

nontoxic to humans under normal circumstances (DiPalma and

Ritchie, 1977), concern has been expressed regarding the

relative safety of ingesting additional amounts of this

nutrient. The major safety issues as outlined by Butterworth

and Tamura (1989) focus on: the potential harm to users of

anticonvulsant medications; interference with the diagnosis of

vitamin B12 deficiency; the possibility of other unexpected

adverse health effects; and interference with zinc absorption

or metabolism. For the most part, all but the latter safety

issue have been adequately addressed.

The question of whether supplemental folic acid disturbs

zinc absorption or metabolism, and thus zinc status, has been

examined by numerous researchers (Butterworth et al., 1988;

Fuller et al., 1987; Ghishan et al., 1986; Keating et al.,

1987; Krebs et al., 1988; Milne, 1989; Milne et al., 1984;

Mukherjee et al., 1984; Simmer et al., 1987; Tamura et al.,

1992; Wilson et al., 1983). The results from these studies

have been equivocal. Of these studies, dietary intake was

controlled in only one research design (Milne et al., 1984);








4

consequently, the results and conclusions of these studies may

vary depending on the adequacy and comparability of intake

among subjects. Discrepant results may also be attributed to

the diversity of research designs and protocols used, as well

as the lack of a satisfactory index of zinc nutriture.

The present study was designed to overcome some of the

limitations of previous investigations by evaluating the

response of male human subjects to 0 Ag/d and 800 ig/d of

stable-isotopically labeled (deuterium) folic acid under

controlled dietary conditions. The objective was to determine

if supplemental folic acid affected zinc status in subjects

consuming zinc-adequate (i.e. 14.5 mg/d) or zinc-restricted

(i.e. 3.5 mg/d) diets. Additionally, the use of deuterium-

labeled folic acid provided the opportunity to study folate

utilization under conditions of marginal and adequate zinc

intakes. This is the first time that folate utilization using

a stable isotope has been studied under controlled dietary

conditions. Another significant aspect of this study was the

determination of erythrocyte metallothionein concentrations in

addition to traditional measures of zinc status. Unlike other

indices of zinc status, the concentration of erythrocyte

metallothionein has been shown (Grider et al., 1990) to

respond quickly to acute dietary zinc deficiency and to

supplementation and may therefore be a more reliable and

sensitive indicator of zinc status.














CHAPTER 2
REVIEW OF THE LITERATURE


Folate


Chemistry


Folic acid, or pteroylglutamic acid (2-amino-4-hydroxy-6-

methyleneaminobenzoyl-L-glutamic acid pteridine), consists of

three distinct subunits: a pteridine moiety, para-aminobenzoic

acid and glutamic acid (Figure 2-1). The pteridine moiety is

linked by a methylene bridge to para-aminobenzoic acid, which

is then joined by peptide linkage to glutamic acid. Although

mammals can synthesize all the components of this vitamin,

they are not capable of de novo biosynthesis because they lack

the enzyme needed for coupling the pteridine molecule to para-

aminobenzoic acid (Cooper, 1984). De novo synthesis of

folates does occur in plants and bacteria.

Folic acid is yellow and has a molecular weight of 441.4.

It is only slightly soluble in water in the acid form, but is

quite soluble in the salt form (Brody, 1991). Folic acid

occurs only rarely in nature, although it is the form most

commonly found in vitamin supplements and is the parent

compound of the naturally occurring folate vitamin forms.
















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Compounds having nutritional properties and chemical

structures similar to those of folic acid have been assigned

the generic descriptor "folate." Natural folates occur in the

reduced 7,8-dihydro- and 5,6,7,8-tetrahydro- forms. Folates

may also contain one-carbon substituent groups (Figure 2-1).

These include: 5-methyl-, 10-formyl-, 5-formyl-, 5,10-

methenyl-, 5,10-methylene-, and 5-formimino-tetrahydrofolate

(Brody, 1991). Most of these naturally occurring folates can

be degraded by heat, oxidation and/or ultraviolet light,

although some tetrahydrofolate derivatives are more stable

than others. For example, N5-methyl-tetrahydrofolate is

relatively heat stable, but is destroyed by acid, as is N5'10-

methylene- tetrahydrofolate (Krumdieck, 1990; O'Brion et al.,

1975). For the most part, dietary folates occur in the form

of pteroylpolyglutamates (Halsted, 1979) containing three to

seven glutamic acid residues (Figure 2-1). Although the

principal pteroylpolyglutamate in food is N5-methyl-

tetrahydrofolate, over 150 different forms of folate have been

reported to exist (Sauberlich, 1987).


Metabolism


Absorption. The first stage of intestinal folate

absorption involves the hydrolysis of pteroylpolyglutamates to

pteroylmonoglutatmes (Butterworth et al., 1969). Hydrolysis

is performed by the gamma glutamylcarboxypeptidases, commonly

grouped together and referred to as "folate conjugase".










8








SI2

= I '-4x
000000




= I


0 Z4 3>1
> 0 0

0 4 o oos4)
0 2 -)ELL --
O 0 u. u. u.'5 s
-u-z 3 .


o=0 o-=U a =
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Sz Oz I


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0 7Z 0I^u -Z r

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9

Folate conjugase successively cleaves the gamma glutamyl

peptide bonds of pteroylpolyglutamates to the monoglutamate

form. Reisenauer and Halsted (1987) have estimated that the

activity of human jejunal brush border folate conjugase is

sufficient enough to preclude this step from limiting the rate

of absorption. However, research showing that the

bioavailability of folate polyglutamates is considerably less

than that of folate monoglutamates (Gregory et al., 1991)

suggests that hydrolysis of dietary polyglutamyl folates is a

rate-limiting step in absorption.

Two separate folate conjugases have been identified in

the human jejunum (Reisenauer et al., 1977). One of these is

soluble and located intracellularly, while the other is

membrane-bound and concentrated in the brush border. These

folate conjugases differ with respect to molecular weight, pH

optima and inhibition characteristics.

Brush border folate conjugase is a zinc-dependent

exopeptidase that sequentially cleaves polyglutamates to

monoglutamates and has a pH optimum near neutrality (Chandler

et al., 1986; Reisenauer et al., 1977). Chronic alcohol

consumption (Naughton et al., 1989; Reisenauer et al., 1989),

zinc deficiency (Tamura et al., 1978), mucosal damage

(Halsted, et al., 1986) or exposure to naturally occurring

inhibitors in food (Bhandari and Gregory, 1990) may exert a

negative effect on brush border folate conjugase activity and

subsequent folate absorption. Interestingly, brush border








10

folate conjugase activity does not appear to be affected by

the aging process (Bailey et al., 1984a). In contrast to

brush border folate conjugase, intracellular folate conjugase

is found in the lysosomes of intestinal cells and functions as

an endopeptidase with an acidic pH optimum (Wang et al.,

1986). The role of intracellular folate conjugase is unknown;

however, Wang et al. (1986) have proposed that its role in

cellular folate metabolism is unrelated to the digestion of

dietary folates.

Transport of folate across the brush border membrane is

the second stage of folate absorption. This is a complex

process that has not been entirely elucidated. Transport

occurs mainly in the jejunum and is believed to involve a

carrier system that is saturable, pH dependent (i.e. pH

optimum of 6.0), energy dependent and sodium dependent (Rose

et al., 1978; Said et al., 1987; Selhub et al., 1983). This

carrier-mediated system is thought to include a folate-binding

protein located in the jejunal brush border membrane. This

folate-binding protein is either the transport protein or is

an important component of the intestinal transport system

(Reisenauer, 1980). Data from competitive inhibition studies

(Said, et al., 1987; Selhub et al., 1984) support the

conclusion that the transport system may be the same for all

monoglutamate forms of folate. The carrier-mediated system

becomes saturated at luminal concentrations of 10-20 AM

(Selhub et al., 1984). In addition to carrier-mediated








11

transport, nonsaturable absorption involving passive diffusion

may also occur (Selhub et al., 1983). Absorption of folate by

this process occurs linearly at much higher folate

concentrations.

Decreased hydrolysis of folate polyglutamates and/or

interference with the transport of folate mononglutamates

across the intestinal brush border membrane can inhibit folate

absorption. Studies concerning the impact of dietary

components (i.e. conjugase inhibitors, dietary fiber, etc.);

dietary composition; the actual forms of folate ingested;

nutritional status; alcohol consumption; and the effect of

nutrient interactions on the extent of folate absorption have

been reviewed by Bailey (1988). The conclusions drawn from

these studies are often conflicting, confirming the need for

further research employing more consistent and appropriate

experimental designs and methodologies. A case in point is

the nutrient interaction that is hypothesized to occur between

zinc and folic acid.

Nondietary factors that may interfere with folate

absorption include altered gastrointestinal function and the

use of medications. Damage to the intestinal epithelial

cells, such as that which occurs in Crohn's disease (Hoffbrand

et al., 1968), celiac disease (Halsted et al., 1977; 1978;

Hoffbrand et al., 1970) and tropical sprue (Corcino et al.,

1976; Halsted, 1980b), or changes in the intraluminal

environment due to achlorhydria (Russell et al., 1986), may








12

adversely affect folate absorption. Medications that may

interfere with folate absorption include sulfasalazine

(Franklin and Rosenberg, 1973) and diphenylhydantoin (Gerson

et al., 1972; Hoffbrand and Necheles, 1968; Rosenberg et al.,

1968). Antacids and histamine receptor antagonists may also

adversely affect folate absorption by increasing the

intestinal pH to levels that exceed the optimum for folate

conjugase activity and carrier-mediated transport (Russell et

al., 1979). Folate analogs such as methotrexate, trimethoprim

and pyrimethamine, which are used primarily as dihydrofolate

reductase inhibitors, also suppress intestinal folate

absorption (Selhub et al., 1983).

Transport. Upon entry into the intestinal mucosal cells,

reduced dietary folates are converted, for the most part, to

N5-methyl-tetrahyrofolate monoglutamate (Cooper, 1984; Pratt

and Cooper, 1971). This is the predominant form in the portal

circulation under normal physiologic conditions. Reduction

and methylation of folic acid can also occur, although much of

this form of the vitamin appears in the portal blood unchanged

(Cooper, 1984).

Serum folate binding proteins are involved with the

transport and distribution of folates to the liver and other

tissues. Two types of serum folate-binding proteins have been

identified (Wagner, 1985), one is specific with a high-

affinity binding capacity; the other, which is thought to be

albumin (Soliman and Olesen, 1976), is nonspecific, with a








13

low-affinity binding capacity. Approximately two-thirds of

serum folate is protein-bound, with the majority bound to

albumin.

A small amount (10 to 20%) of absorbed folates in reduced

substituted form is taken up by the liver on the first pass,

while the majority is distributed to other tissues (Steinberg,

1984). Since the liver is capable of secreting Ns-methyl-

tetrahydrofolate monoglutamate into the bile, the potential

exists for continual enterohepatic circulation of folate

(Steinberg, 1984). This idea is not supported, however, by

the results of an isotopic labeling study (Krumdieck et al.,

1978) that examined the routes of folate excretion following

administration of [14C]folic acid to a human subject. The

results of this study suggested that a significant portion of

biliary folate was not reabsorbed.

Contrary to the fate of reduced substituted forms of

folate entering the portal hepatic vein, folic acid entering

this vessel is taken up almost exclusively by the liver. Some

of this folic acid is used for polyglutamate synthesis, and

the rest is converted to N5-methyl-tetrahydrofolate and

secreted into the bile (Lavoie and Cooper, 1974). When a

large oral dose of folic acid is administered, most of it is

recovered in the urine in its original form (Brody, 1991).

Tissue deposition and storage. Transport of folate

across cellular membranes is an energy requiring process,

where anion gradients may serve as the energy source








14

(Yang et al., 1984). The N5-methyl-tetrahydrofolate

transported across cell membranes must be converted to the

polyglutamate form to assume its role as a functional

coenzyme. Polyglutamates cannot cross biological membranes,

so polyglutamation also serves to trap folates inside cells at

concentrations that are one to two orders of magnitude greater

than those of the extracellular fluid. Conversion to the

polyglutamate form requires methionine synthetase, a vitamin

B12-dependent enzyme, and pteroylpolyglutamate synthetase.

Methionine synthetase is responsible for removing the methyl

group, and pteroylpolyglutamate synthetase is responsible for

the addition of glutamyl residues. The resulting

tetrahydrofolate polyglutamates usually contain between four

to seven glutamyl residues.

The total body folate content is in the range of 5 to 10

mg. The liver is considered to be the primary storage organ

containing about 50% of the total body folate (Herbert and

Colman, 1988). A variety of folate derivatives with various

chain lengths is present in the liver and other tissues, with

one particular species usually dominating (Shane, 1990).

Alterations in the distribution of folate polyglutamates have

been observed under certain physiological and nutritional

conditions. For example, longer chain length folates tend to

accumulate under circumstances whereby cellular folate

concentrations are diminished (i.e. folate deficiency;

methionine deficiency; etc.) (Cook et al., 1987). The








15

significance of this particular change has not been

determined, although it has been reasoned that extending the

chain length does not affect the short-term regulation of one-

carbon metabolism since this change occurs very slowly (Shane,

1990).

Utilization of endogenous folate pools may provide a

mechanism for maintaining normal folate supplies to critical

tissues during acute or chronic folate deprivation. A small

pool of intracellular folate monoglutamates is thought to be

available for this purpose. As deprivation continues, the

liver and other "storage tissues" (i.e. the kidney) may

generate folate monoglutamates through the action of

folylpolyglutamyl hydrolase on folate polyglutamates.

Additionally, the amount of folate presented to the liver is

thought to decrease, thereby reducing hepatic monoglutamate

uptake and new polyglutamate synthesis. Thus, over time,

hepatic folate stores decline.

Folate derived from dying cells may be another potential

source of this nutrient during periods of prolonged

deprivation (Steinberg, 1984). For example, erythrocytes

could make an important contribution to folate homeostasis

because of their high folate content and limited life span. A

study (Hillman et al., 1982) of heat-treated, senescent

erythrocytes has shown that labeled folate can be recovered in

the liver and bile. Therefore, folate salvaged from expired








16

erythrocytes may be redistributed through the enterohepatic

circulation.

Catabolism and excretion. Urinary excretion of intact

folates in well-nourished humans consuming a nutritionally

adequate diet is approximately 5-40 Ag per day (Herbert,

1987). The folate compounds identified in the urine include

Ns-methyl-tetrahydrofolate, N10-formyl-tetrahydrofolate, N5'10-

methenyl-tetrahydrofolate and N5-formyl-tetrahyrofolate

(Chanarin, 1979). Excretion of intact folates is limited due

to renal tubular reabsorption of this nutrient and the degree

of catabolism that occurs in vivo. Products of folate

catabolism occurring in the urine include pteridines, para-

acetamidobenzoyl glutamate and para-acetamidobenzoate (Anon.,

Nutrition Reviews, 1990; Chanarin, 1979). The latter two

catabolites appear to be the major excretory products,

suggesting that the principal route of catabolism occurs by

cleavage of the C9-N10 bond. The cleavage mechanism has been

the subject of much controversy. Current in vitro studies

suggest the existence of more than one mechanism for folate

cleavage (Anon., Nutrition Reviews, 1990).

Folate can also be excreted in the feces. The amount

excreted in the feces has been reported, in some cases, to be

higher than the estimated dietary intake, presumably because

of folate synthesis by colonic bacteria (Brody, 1991).

Consequently, fecal folate excretion is not considered a

reliable index of folate metabolism.










Biochemical Functions


Reduction of the pteridine ring to the tetrahydro-form,

elongation of the glutamyl side chain and acquisition of one-

carbon units at the N5 and/or N10 positions of the pteridine

ring system must occur in order to produce the intracellular,

metabolically active forms of folate. Pteridine ring

reduction is accomplished by the cytosolic enzyme, 7,8-

dihydrofolate reductase. Folic acid and dihydrofolic acid can

serve as substrates for this enzyme. Reduction to the

tetrahydrofolate form, and demethylation, in the case of N5-

methyl-tetrahydrofolate, must occur before elongation of the

side-chain or acquisition of one-carbon substituent groups can

proceed. Side-chain elongation is achieved by the action of

folate polyglutamate synthetase. This is an adenosine

triphosphate (ATP)-dependent enzyme which joins glutamyl

residues to the vitamin by peptide bonds in an oligo-gamma-

glutamyl linkage. Once these steps have been accomplished,

one-carbon units at the oxidation level of format,

formaldehyde or methanol can be added. The major contributor

of one-carbon groups is serine, although formimino-glutamate

(FIGLU), formylglutamate and format may also serve as the

single-carbon source. The resulting tetrahyrofolate

derivatives (NS-methyl-, N5-formyl-, N10-formyl-, N5'10-

methylene-, N5'10-methenyl- and Ns-formimino-) are used as

coenzymes and serve as donors and acceptors of one-carbon

units in a host of reactions involved in amino acid and








18

nucleotide metabolism. Examples of specific biochemical

functions of folate include: purine and pyrimidine

biosynthesis; the generation and utilization of format; and

amino acid interconversions including the catabolism of

histidine to glutamic acid, the interconversion of serine and

glycine and the conversion of homocysteine to methionine.

These reactions are collectively referred to as one-carbon

metabolism (Brody, 1991; Krumdieck, 1990).

The cyclical nature of intracellular folate metabolism is

exemplified by tracing the potential fates of tetrahydrofolate

polyglutamates (see Figure 2-2). In mammalian tissues, it

appears that the major cycle of one-carbon metabolism involves

the conversion of tetrahydrofolate polyglutamate to N5'10-

methylene-tetrahydrofolate by serine hydroxymethyl-

transferase. This one-carbon derivative is a key intermediate

that can be: oxidized to NS' 1-methenyl-tetrahydrofolate (by

N5,10-methylene-tetrahydrofolate dehydrogenase) for use in de

novo synthesis of purines; used for the synthesis of

thymidylate (by thymidylate synthetase); or reduced to N5-

methyl-tetrahydrofolate (by N5' 10-methylene-tetrahydrofolate

reductase) for use in the biosynthesis of methionine. The

latter two reactions are irreversible reactions that compete

for the N5'10-methylene-tetrahydrofolate intermediate.

Regardless of the metabolic path into which the N5'10-

methylene-tetrahydrofolate is directed, its eventual fate

(under normal physiologic conditions) is the loss of its one-








19

carbon substituent group, and its return to either dihydro- or

tetrahyro-folate polyglutamate (Brody, 1991; Krumdieck, 1990).

Regulation of one-carbon transfer reactions is highly

complex. Changes in the concentration of substrates, products

and cofactors serve as a means for quickly activating or

inhibiting certain folate-requiring reactions. An example of

this complex regulatory process is one that has been given

much attention and involves the fate of N5'10-methylene-

tetrahydrofolate. As noted above and depicted in Figure 2-2,

this intermediate can be used to synthesize several different

products. Methylene-tetrahydrofolate reductase, the enzyme

catalyzing the conversion of N5'10-methylene-tetrahydrofolate

to N5-methyl-tetrahydrofolate, is very highly regulated. When

insufficient amounts of methionine are available, inhibition

of the reductase enzyme is relaxed allowing for increased

production of methionine from homocysteine. The methionine

produced by this reaction is converted to S-adenosyl

methionine, which then serves as a methyl donor to form

various methylated products. However, when S-adenosyl

methionine accumulates, methylene- tetrahydrofolate reductase

activity is curtailed through feedback inhibition. Methylene-

tetrahydrofolate reductase is also inhibited by the

accumulation of dihydrofolate polyglutamates, a situation that

occurs when thymidylate synthesis increases. Inhibition of

this enzyme under these circumstances allows for continued








20

commitment of folate to nucleotide biosynthesis (Brody, 1991;

Krumdieck, 1990).

In addition to the regulatory influences posed by changes

in the concentration of substrates, products and cofactors,

evidence for a second type of regulation has been reviewed by

Krumdieck (1990). This newly proposed regulatory process is

thought to be a slow-response mechanism based on covalent

modification of the polyglutamyl chain length. The idea that

a second form of regulation existed was generated from in vivo

studies demonstrating that the glutamyl chain length of

cellular folates varies in response to physiological or

pathological stimuli (i.e. developmental age, tissue

regeneration, infection, starvation, alcohol ingestion and

methionine-choline deficiency) that alter the steady-state

equilibrium of one-carbon metabolism. Additionally, the

finding that chain-length distribution of folate

polyglutamates differs from organ to organ in the same species

adds credibility to this concept because it is unlikely that

the requirements for one-carbon transfer reactions are the

same for all organs. Since changes in polyglutamate chain

length are slow to develop and only respond to persistent

stimuli, it is thought that the purpose of this regulatory

mechanism is to correct prolonged deviations rather than brief

fluctuations that may occur in an otherwise steady-state

(Krumdieck, 1990).











Table 2-1. Comparison of 1980 and 1989 Recommended Dietary
Allowances (RDA) for folate.



Category/age 1980 RDA* 1989 RDA**

gg/d Ag/d
---------------------------------------------------
Infants
0.0-0.5 30 25
0.5-1.0 45 35
---------------------------------------------------
Children
1-3 100 50
4-6 200 75
7-10 300 100
---------------------------------------------------
Men
11-14 400 150
15-18 400 200
19-24 400 200
25-50 400 200
51+ 400 200
---------------------------------------------------
Women
11-14 400 150
15-18 400 180
19-24 400 180
25-50 400 180
51+ 400 180
---------------------------------------------------
Pregnant women 800 400
-----------------------------------------------
Lactating women
First 6 months 500 280
Second 6 months 500 260
*Data from Food and Nutrition Board, 1980.
**Data from Food and Nutrition Board, 1989.






Recommended Dietary Allowances for Folate


The 1989 Recommended Dietary Allowances (RDA) for folate


(Food and Nutrition Board, 1989a) represent a significant








22

reduction from the previously recommended amounts (Food and

Nutrition Board, 1980) for all age, sex and special population

categories (see Table 2-1). The rationale for the reduction

of the RDA for folate was based on two types of data: the

quantity of folate required to invoke established physiologic

responses or replace daily losses, after adjusting for

bioavailability and individual variability; and estimates of

dietary folate consumption related to the prevalence of

deficiency in population groups (Bailey, 1990a; Bailey, 1992).

Although some researchers (Herbert, 1987; Reisenauer and

Halsted, 1987) support the view that the existing data are

sufficient to warrant a reduction in the recommended level of

folate intake, others (Bailey, 1992; Sauberlich et al., 1987)

cast doubt on the appropriateness of the new RDA. In a

critique of the research used to establish the 1989 RDA,

Bailey (1992) submits that this new RDA may be insufficient to

provide an adequate margin of safety for specific populations.

The rationale for her conclusion is based on the following

facts: 1) in some cases, the population surveys used to make

conclusions about folate requirements were not specifically

designed to assess folate status; 2) the studies used to

support the 1989 RDA were not always comparable in terms of

the forms of folate used (i.e. synthetic folic acid versus

dietary folate); 3) the correction factor used to account for

folate bioavailability is only an estimate based on limited

data from studies employing different experimental








23

methodologies; and 4) the food composition tables for folate

are incomplete and may not accurately reflect the actual

amount of folate ingested or available. This final problem is

due to the fact that estimates of the folate content of foods

vary due to the method of analysis; the type of food consumed;

the method of food processing, preparation, storage and

handling; and the effect of nutrient interactions.


Effects of High Doses of Folic Acid


Folate is considered nontoxic in small doses as well as

in doses that exceed the RDA several hundredfold (Brody, 1991;

Butterworth and Tamura, 1989; DiPalma and Ritchie, 1977;

Herbert and Colman, 1988). The water soluble nature of

folate, and the apparent requirement for attachment to

saturable folate-binding proteins as a condition for storage,

probably account for the relative nontoxicity of this vitamin.

These features provide a mechanism for rapid excretion of

folate when the serum- and tissue-binding capacity are

exceeded (Herbert and Colman, 1988).

Adverse effects of supplemental folic acid were not noted

in adult humans receiving 400 mg/d for 5 months, or after 10

mg/d for 5 years (Brody, 1991). Similarly, no adverse effects

were reported in a group of well-nourished women ingesting an

oral folic acid supplement (10 mg/d) for 4 months (Butterworth

et al., 1982). Contrary to these reports, insomnia and

irritability were noted in subjects consuming folic acid in








24

the amount of 15 mg/d (Hunter et al., 1970). This effect has

not been confirmed in subsequent studies (Alhadeff et al.,

1984).

Despite the apparent relative safety of supplemental

doses of folic acid in normal adult subjects, large doses of

this nutrient (5 mg/d) (Brody, 1991) can have deleterious

effects when administered to individuals with undiagnosed

and/or untreated pernicious anemia. Administration of folic

acid supplements obscures the diagnosis of pernicious anemia

by correcting the macrocytic anemia associated with this

condition, but fails to alleviate the concurrent neurologic

lesions. Consequently, neurologic damage progresses

unchecked. Assessment of vitamin B12 status prior to the

initiation of folic acid supplementation can prevent this

potentially harmful outcome.

Large doses of folic acid (100 or more times the RDA) may

also be harmful to individuals with epilepsy who are receiving

continuous phenytoin therapy. Supplementation at this level

may precipitate convulsions (Herbert, 1987). Lower doses of

folic acid (0.1 to 1 mg/d) have not been shown to impair

seizure control (Roe, 1989).

It has been suggested that high intakes of folic acid may

interfere with zinc absorption and/or metabolism (Ghishan et

al., 1986; Milne, 1989; Milne et al., 1984; Mukherjee et al.,

1984; Simmer, et al., 1987; Wilson et al., 1983). This topic








25

will be addressed in more detail in a subsequent section of

this dissertation.

In contrast to some of the potentially harmful effects of

supplemental folic acid, there is evidence to suggest that

additional amounts of this vitamin may be beneficial in

modulating cancer risk in a variety of tissues including

cervical dysplasia (Butterworth et al., 1982; Butterworth et

al., 1992a; Butterworth et al., 1992b), bronchial metaplasia

(Heimburger et al., 1987; Heimburger et al., 1988), and

neoplasms associated with the esophagus (Jaskiewicz et al.,

1988) and colon (Lashner, et al., 1989). Supplemental folic

acid has also been associated with a reduction in the risk of

occurrence/recurrence of neural tube defects (Bower and

Stanley, 1989; Czeizel and Dudds, 1992; Medical Research

Council Vitamin Study Research Group, 1991; Milunsky et al.,

1989; Mulinare et al., 1988; Smithells et al., 1981).


Sources/Distribution and Stability of Folate in Foods


Although there is a need for more complete data

concerning the folate content and bioavailability of foods, a

considerable amount of information concerning food sources of

folate and the forms of the vitamin present in these foods has

been generated. The most concentrated sources of folate

include liver, citrus fruits, raw broccoli and dark green

leafy vegetables such as raw spinach. Cooked greens,

including spinach, turnip and mustard greens also contain








26

folate, but in lower amounts. The reduction in folate content

due to cooking occurs primarily due to leaching of the vitamin

into the cooking water, although thermal and oxidative

destruction may also occur. Legumes are another highly

concentrated source of folate; however, it must be recognized

that they contain heat-activated conjugase inhibitors which

may decrease the availability of folate polyglutamates. This

is true for other folate containing foods such as cooked

cabbage. Other good sources of folate include fortified

breakfast cereals. Most fortified cereals supply at least 25%

of the United States Recommended Daily Allowance for this

nutrient. In addition to the more concentrated sources of

dietary folate, it is important to consider the potential

contribution that foods containing modest amounts of folate

can have on total intake if these foods are consumed

frequently and/or in large quantities. Examples of these

include ground beef and whole-grain breads and cereals, as

well as tea (Bailey, 1990a; Bailey, 1992).

Very little folic acid is naturally present in food,

although this is the form used in food fortification because

of its exceptional stability characteristics (Gregory, 1989).

Folates in food occur almost exclusively in reduced

polyglutamate forms, with the predominant forms being N5-

methyl-tetrahydrofolate, N10-formyl-tetrahydrofolate and

unsubstituted tetrahydrofolate polyglutamates of varying

chain-lengths (Gregory, 1989). Reduced folates, with the








27

exception of N5-formyl- tetrahydrofolate, are potentially more

labile and subject to oxidation under aerobic conditions,

especially in the presence of heat, light and/or metal ions

(Cooper et al., 1978; Gregory, 1989). The actual stability of

folate in foods has been difficult to determine, with reported

losses of folate activity being highly variable. This

variability may be due to differences in oxygen exposure

during cooking, the amount of cooking water present and/or

intrinsic differences in ascorbic acid content of different

foods (Gregory, 1989). When partial or full oxidation of

tetrahydrofolate derivatives occurs, they may be further

catabolized to yield compounds that are physiologically

inactive with respect to human nutrition. If these compounds

are capable of supporting growth responses in microorganisms

used to measure the folate content of foods, overestimation of

the biologically useful folate content of foods could occur.


Assessment of Folate Status


The nutritional status of an individual indicates the

degree to which physiological needs for nutrients are being

met. Nutritional status is often evaluated using dietary

history and intake data, and biochemical and clinical

parameters.

Dietary intake. Estimation of the dietary folate content

of foods and the evaluation of folate intake by individuals

and population groups is complicated by many factors.








28

Researchers and practitioners need to recognize the

limitations of the available folate databases and the problems

inherent in collecting dietary intake and food frequency data

when using this information to evaluate nutritional status.

Examples of problems associated with folate food composition

data include: the use of different analytical techniques to

determine the folate content of foods; missing information for

foods that have not been analyzed; failure to incorporate

information on the bioavailability of folate from various

foods; and the effect of different methods of food

preparation, storage and handling (Bailey, 1990a; Bailey,

1992; Gregory, 1989).

The accurate recording and evaluation of individual

dietary intake and food frequency data is also problematic.

For example, it is often difficult for individuals to

correctly remember the types and/or amounts of foods they have

consumed. Food selection may also be affected as a result of

being asked to record food intake. Factors such as age, mood,

intelligence, attention span, frequency of exposure to the

process and perceived importance of the information can also

affect the ability to recall and/or record food intake

information (Blake et al., 1989; Karvetti and Knuts, 1985;

Lissner et al., 1989).

Biochemical and clinical measures of folate status. The

progressive changes in biochemical and clinical parameters

occurring during folate depletion were determined and








29

described by Herbert (1962) in a depletion study in which he

consumed a folate-deficient diet for four months. As a result

of this experiment, Herbert categorized folate depletion into

four stages: early negative folate balance; nutrient

depletion; biochemical nutrient deficiency; and clinical

nutrient deficiency.

During the initial stage of folate depletion, serum

folate values become depressed. Serum folate concentrations

are very responsive to recent dietary intake, with levels

becoming low after consuming a folate deficient diet for only

two to three weeks. A value of less than 3 ng/mL is

indicative of negative folate balance. Values from 3 to 6

ng/mL represent marginally negative folate balance, with

normal serum folate values ranging from 6 to 25 ng/mL. The

sensitivity of serum folate to recency of dietary folate

intake make it a poor indicator of the degree of folate

deficiency. To determine the severity of folate depletion,

parameters indicative of body stores and changes in metabolic

function need to be measured concurrently (Herbert, 1987;

1990).

The second stage of folate depletion is characterized by

a decline in body folate stores. The largest amount of folate

is stored in the liver. When folate intake is deficient,

normal liver folate stores can be maintained for approximately

four months. Coincidentally, the average life span of normal

erythrocytes is four months as well. Since the erythrocyte








30

folate concentration is actually a measure of folate status at

the time the erythrocyte was synthesized, erythrocyte folate

concentrations usually parallel liver folate stores. This

relationship, combined with the greater accessibility of

erythrocytes, has resulted in the routine use of erythrocyte

folate concentration as a means for determining tissue stores.

Thus, stage two is identified by measuring erythrocyte folate

concentrations, which decrease to less than 140 ng/mL when

tissue stores are low. Erythrocyte folate values between 140

to 160 ng/mL suggest marginal depletion, and concentrations

above 160 ng/mL indicate normal folate status (Herbert, 1987;

1990).

Severe depletion of folate stores, characterized by

impaired folate-dependent metabolism, represents the third

stage of depletion. Folate coenzymes are required for many

metabolic functions including the synthesis of thymidylate.

A severe folate deficiency retards the synthesis of this

nucleotide, and thus interferes with DNA synthesis.

Subsequently, deranged DNA synthesis results in morphologic

changes in erythrocytes and neutrophils. Neutrophils become

hypersegmented because more constriction bands are formed,

constricting the DNA into more lobes. Hypersegmentation of

neutrophils is identifiable early in the course of impaired

metabolism because of the short half-life of these cells. The

criterion used to define hypersegmentation of neutrophils is

a lobe average equal to or greater than 3.5 lobes per cell








31

(Herbert, 1987; 1990). This hematologic alteration is thought

to be a sensitive screening tool although it is an unreliable

indicator of folate status during pregnancy (Herbert et al.,

1975) and in a small percentage (1%) of otherwise normal

adults with congenital polymorphonuclear leukocyte

segmentation (Herbert, 1964).

The morphologic changes manifested in erythrocytes during

the third stage of depletion include an increase in size and

conversion to an oval shape. At this stage, morphologic

damage is confined to the youngest erythrocytes, which are not

yet in the majority because of their longer life span, so

macroovalocytosis and an increase in the mean corpuscular

hemoglobin concentration are not evident. Other changes that

occur during stage three of depletion include a further

decline in liver and erythrocyte folate concentration

(Herbert, 1990).

The final depletion stage is clinically manifested as

normochromic, macrocytic anemia. At this point, the majority

of the erythrocytes are larger than normal resulting in an

increase in the mean corpuscular volume. The hemoglobin level

also declines due to decreased erythropoiesis. Further

decreases in liver and erythrocyte folate concentrations may

also be noted (Herbert, 1990).

The type of anemia caused by a folate deficiency is

clinically indistinguishable from that caused by a deficiency

of vitamin B12. Anemia due to vitamin B12 deficiency is








32

thought to be caused by a secondary deficiency of folate. It

has been hypothesized that this secondary deficiency develops

as a result of trapping folate in the N5-methyl-

tetrahydrofolate form.

The predominant form of folate in the serum, liver, and

most likely, other body storage depots, is N5-methyl-

tetrahydrofolate. Another source of this form of folate is

that which is synthesized from NS10-methylene-tetrahydrofolate

when control of Ns'10-methylene-tetrahydrofolate reductase is

relaxed. Conversion of N5-methyl-tetrahydrofolate to

tetrahydrofolate is catalyzed by the vitamin B12-dependent

enzyme methionine synthetase. When vitamin B12 is present in

adequate amounts, the methyl group from N5-methyl-

tetrahydrofolate is removed resulting in the regeneration of

tetrahydrofolate. This reaction is important for two reasons:

1) there is no other mechanism for regenerating

tetrahydrofolate in human cells; and 2) tetrahydrofolate is a

precursor to many other folate coenzymes, including N5'10-

methylene-tetrahydrofolate, which is ultimately used for DNA

synthesis. According to the "methyl-folate trap" hypothesis

(Herbert and Zalusky, 1962), folate gets "trapped" in the N5-

methyl-tetrahydrofolate form when vitamin B12 is deficient.

This form of folate is not usable for any other folate-

requiring reactions, including the production of thymidylate.

Inadequate production of thymidylate interferes with the

synthesis of DNA and eventually causes the development of a








33

macrocytic anemia that is hematologically identical to that

caused by folate deficiency (Herbert, 1990).

The need to properly identify the underlying cause of

macrocytic anemia prior to initiating treatment is essential

in order to avoid deleterious consequences. For this reason,

concurrent assessment of vitamin B12 and folate status is

recommended. As an alternative, the deoxyuridine suppression

test can be used to distinguish between macrocytic anemia

caused by a deficiency of folate, vitamin B12 or both of these

nutrients. As previously discussed, in the absence of folate

and/or B12 deficiency, deoxyuridine is converted to thymidine

by thymidine synthetase. Thymidine is subsequently

incorporated into DNA. The deoxyuridine suppression test is

an in vitro test that measures the activity of thymidine

synthetase by comparing the amount of unlabeled versus labeled

thymidine incorporated into DNA, when labeled thymidine is

added to bone marrow cells or phytohemagglutinin-stimulated

lymphocytes. Labeled thymidine incorporation is suppressed

when folate and vitamin B12 are present in adequate amounts.

Conversely, when a deficiency of folate or vitamin B12 is

present, the conversion of deoxyuridine to thymidine is

reduced and more labeled thymidine is incorporated into DNA.

When suppression of labeled thymidine is low, the underlying

cause can be determined by adding Ns-methyl-tetrahydrofolate

to the medium. If folate is deficient, the suppression rate

will be increased when this coenzyme form is added. If the








34

cause is due to a vitamin B12 deficiency, the addition of N5-

methyl-tetrahydrofolate will have no effect (Brody, 1991;

Herbert, 1990).

In the past, problems associated with sample collection

and processing techniques have limited the application of the

deoxyuridine suppression test to laboratory research settings.

The development of a method using a whole blood (0.1mL)

lymphocyte culture instead of cultures of separated

lymphocytes has made this test more suitable for use in

clinical laboratories, and perhaps, survey studies (Das et

al., 1980); however, as a measure of folate status, it is no

more informative than erythrocyte folate concentration (Tamura

et al., 1990).

The histidine load test is another method that can be

used to assess folate status. Formimino-glutamate, a product

of histidine catabolism, is further catabolized to glutamate,

ammonia and carbon dioxide by tetrahydrofolate formimino-

transferase, a folate-requiring enzyme. When folate is

deficient, urinary excretion of FIGLU is elevated, with

excretion being particularly high after an oral dose of L-

histidine. Despite the sensitivity of this test to folate

deficiency, it is not specific for folate deficiency since a

deficiency of vitamin B12 will also cause an increase in FIGLU

excretion. Additionally, FIGLU excretion is affected by other

diseases and physiological conditions, so its application is

usually limited to scientific studies (Brody, 1991).








35

Measurement of plasma homocysteine has recently been

proposed as a new method for assessing folate status. Under

normal conditions, approximately 50% of the available

homocysteine is converted to methionine by the folate-vitamin

B12-requiring remethylation reaction catalyzed by methionine

synthetase (see Figure 2-2). Inhibition of this reaction due

to vitamin B12 deficiency or inborn errors of folate or

vitamin B12 metabolism, results in an accumulation of

homocysteine in the blood (Krumdieck, 1990). Kang et al.

(1987) and Stabler et al. (1988) investigated the potential

association of folate deficiency with homocysteinemia and

found a negative correlation between serum folate

concentrations and protein-bound homocysteine. Stabler et al.

(1988), reported elevated serum homocysteine concentrations in

18 of 19 folate-deficient patients. Compromised folate status

was attributed to nutritional inadequacy in 17 of these

subjects. However, hyperhomocysteinemia was also found to

occur in patients with a deficiency of vitamin B12 (Stabler et

al., 1988), so the total serum homocysteine concentration must

be used in combination with other parameters in order to

distinguish folate deficiency from a deficiency of vitamin

B12. Determination of serum methylmalonic acid concentration,

which is normal in patients with folate deficiency, has been

recommended for this purpose (Stabler et al., 1988).










Folate Status in Special Population Groups


Folate status is affected by physiological changes

occurring at different stages of the life cycle. The

potential impact of growth, development and maturation on

folate requirements, metabolism, and subsequently, status, in

selected population groups, is summarized below.

Infants. The rapid rate of growth that occurs during the

first year of life influences folate requirements. Folate

requirements are also affected by the developmental immaturity

of the infant. For example, low secretion of pancreatic

proteases and gastric and biliary secretions during the first

months of life may significantly affect the bioavailability of

food folate and thereby influence folate requirements and

status (Picciano, 1990).

It has been reported that blood folate values of infants

at birth are higher than values for pregnant and lactating

mothers (Ek and Magnus, 1979) or normal adults (Smith et al.,

1985; Vanier and Tyas, 1966), and that these values decline

when solid foods are introduced (Smith et al., 1985). A study

(Smith et al., 1985) designed to assess the folate status of

infants supports the concept that milk alone (human or

proprietary formulas) is an important dietary source of folate

during the first year of life, and inclusion of this food can

provide sufficient folate to maintain blood folate

concentrations within an acceptable range.








37

Preterm, low-birth-weight infants have greater folate

requirements than term infants (Rodriguez, 1978). Depressed

serum and erythrocyte folate concentrations and megaloblastic

anemia have been observed in this population subgroup (Gray

and Butler, 1965; Roberts et al., 1969; Strelling et al.,

1966). The efficacy of supplemental folate given to low-

birth-weight infants was demonstrated by Burland et al.

(1971). After comparing the serum and erythrocyte folate

values of supplemented infants to those of unsupplemented

infants during the first nine months of life, these

researchers concluded that folate supplementation should be

provided to all low-birth-weight infants, but that more

research was needed to determine the optimal route, dose and

duration of therapy. Dallman (1974) has stated that folate

supplementation at the level of 50 jg/d for well infants

weighing less than 2,000 g at birth is warranted. The

Subcommittee on Pediatric Parenteral Nutrient Requirements

(Greene et al., 1988) of the American Society for Clinical

Nutrition has recommended 56 4g/kg/d of folic acid for

preterm infants receiving parenteral nutrition.

Adolescents. The rapid growth experienced by adolescents

is characterized by increases in lean body mass, skeletal

tissue and blood volume. Folate requirements are elevated

during this accelerated growth period because of the role

folate plays in cell division. Superimposed on the extra

demands for folate due to growth, are the effects of other








38

factors that may adversely affect folate status. These

include poor diet, alcohol and drug use, smoking and the

possibility of pregnancy in the sexually mature female.

Compromised folate status is prevalent among adolescents,

particularly those living in low-income households. Bailey et

al. (1982a) reported that 45% of rural black and white

adolescent males and females living in low-income households

had erythrocyte folate concentrations below 140 ng/mL. Serum

folate concentrations were less than 6 ng/mL in 56% of these

subjects. A similar trend was found in adolescents from urban

low-income households, where 42% of the subjects had

erythrocyte folate concentrations less than 140 ng/mL, and 45%

had serum folate concentrations below 6 ng/mL (Bailey et al.,

1982b). When assessed as a function of sexual maturation,

these researchers (Bailey et al., 1982 a; 1982 b) found that

serum folate concentrations declined as sexual maturation

progressed. A similar relationship was reported by Daniel et

al. (1975). Based on erythrocyte folate concentration, Tsui

and Nordstrom (1990) found the prevalence of folate deficiency

to be 13% among males and 40% among females. In the same

study, analysis of seven day food records revealed that for

all race, sex and age groups, subjects who were folate

deficient had significantly lower folate intakes than those

with normal folate concentrations. The type of dietary

pattern that contributed to inadequate folate consumption in

this study was not addressed, but a study by Bailey et al.








39

(1984b) suggests that poor folate status among adolescents may

be due in part to their limited consumption of vegetables and

fruit.

Pregnancy. The RDA for folate is doubled during

pregnancy. This large increase in the recommended intake

reflects the fact that cell division and multiplication is

occurring rapidly. Increased dietary folate, along with

increased amounts of other nutrients, are needed to support

the physiological and compositional changes occurring at this

time. Increased maternal erythropoiesis, uterine and mammary

tissue expansion, placental and fetal growth and greater

urinary folate losses all contribute to the increased demand

for folate during pregnancy (National Academy of Sciences,

1990). During the final weeks of the normal gestational

period, the rate of active transport of folate across the

placenta is elevated. This places further demands on maternal

folate stores and may explain why serum and erythrocyte folate

values are usually several-fold higher in neonates than in

pregnant and lactating mothers (Bailey, 1990b).

As with other population subgroups, estimates of dietary

folate intake during pregnancy by women residing in the United

States are limited. A recent study (Huber et al., 1988) of

566 pregnant women who were primarily white, middle class and

at least 20 years old, found that only 8.5% of the women

derived their folate intake entirely from the diet. The mean

folate intake of this group was 257 Ag/d. The remaining women








40

(91.5%) consumed folic acid supplements and had a mean intake

of 1087 Ag/d. The women who did not take folic acid

supplements had significantly lower serum and erythrocyte

folate concentrations compared to those who used supplements.

Data from the 1985 Continuing Survey of Food Intake (United

States Department of Agriculture, 1987) indicated that the

mean folate intake by women (nonpregnant) between the ages of

19 and 34 (all income levels) was 217 gg/d. These data

suggest that unless women are motivated to make dietary

changes, or are instructed or knowledgeable enough to

recognize the need to take a folic acid supplement during

pregnancy, the recommended allowance for folate may not be

met. This is particularly disconcerting in light of the

protective effect of folate against neural tube defects

(Anon., Morbidity and Mortality Weekly, 1992).

A high prevalence of folate deficiency has been suggested

by population studies of pregnant women in whom blood folate

concentrations were measured. Herbert et al. (1975) studied

110 low-income, predominantly black or Puerto Rican women

living in New York City. These researchers reported that 20%

of the subjects had serum folate values below 3 ng/mL, and 16%

of the subjects had erythrocyte folate values below 150 ng/mL.

Bailey et al. (1980) analyzed blood samples from low-income

women in Florida and found that 29% had erythrocyte folate

values below 140 ng/mL. Bailey did not attribute the low

erythrocyte folate concentrations found in these studies to








41

hemodilution because erythrocyte folate concentrations are

indicative of the folate available to precursor red cells in

the bone marrow at the time the currently circulating cells

were developed (Bailey, 1990b).

The value of folic acid supplementation in the prevention

of folate deficiency during pregnancy was demonstrated in a

study of African women (Colman et al., 1975). Pregnant women

receiving folic acid supplemented cereals experienced

significant increases in serum and erythrocyte folate

concentrations, whereas the erythrocyte concentrations in

unsupplemented women decreased by an average of 42 ng/mL

during the last month of gestation (33 days). Despite a

hemoglobin concentration of 11 g/dL or more at the start of

the study, the supplemented women experienced an increase in

hemoglobin concentration suggesting that hematopoiesis was

limited by folate deficiency. These researchers suggested

that this finding gives further credence to the need for folic

acid supplementation in this population.

Not all researchers agree that low serum folate

concentrations during pregnancy are associated with maternal

complications or congenital malformations of the fetus (Hall

et al., 1976). For example, Hall et al. (1976) examined over

2700 women at four time points and found progressive

reductions in serum folate values at each stage. Maternal

complications and fetal outcome were not reported for any of

these pregnancies. Women from low socioeconomic groups,








42

smokers, multigravidae and women with twin pregnancies had

greater declines in serum folate. With the exception of the

smokers, however, significant reductions in mean serum folate

concentrations were not detected. It was concluded that this

decline was due to plasma volume expansion and did not warrant

routine folate supplementation.

Lactation. The effect of lactation on maternal and

infant status and milk folate content in unsupplemented women

has been described by several researchers. Smith et al.

(1983) found that blood folate concentrations were lower in

well-nourished, unsupplemented lactating women compared to

supplemented lactating women and normal nonlactating controls.

While the erythrocyte folate concentration of the

unsupplemented women declined, the folate content of their

milk was comparable to that of the supplemented women. There

was no difference in the blood folate concentrations of

infants of supplemented or unsupplemented mothers. Similar

results were found in a study described by Metz (1970). In

this study, lactating women fed a controlled low-folate diet

experienced rapid reductions in serum folate concentrations,

while the folate content of their milk remained constant.

When folate status during pregnancy and lactation is

severe enough to cause megaloblastic anemia, oral

administration of supplemental folic acid to lactating mothers

has been shown to improve their milk folate content (Cooperman

et al., 1982). Milk folate concentrations have also been








43

increased in lactating women of low socioeconomic status when

folic acid supplements were administered (Sneed et al., 1981).

However, serum folate concentration appears to increase, while

milk folate content remains constant when maternal blood

folate levels are already within an acceptable range (Tamura

et al., 1980). These findings suggest that a regulatory

mechanism controls the level of milk folate secretion (Tamura

et al., 1980) with the concentration of folate in breast milk

being maintained at the expense of maternal reserves and

status (Smith et al., 1983). Folic acid supplementation

during pregnancy and lactation may help to protect the folate

status of lactating women (Smith et al., 1983).

Elderly. While some researchers (Baker et al., 1978)

have suggested that age related changes adversely affect

folate absorption, other investigators (Bailey et al., 1984a)

have concluded that folate absorption is not affected by the

aging process. Although alterations in other aspects of

folate metabolism may exist, as intimated by data showing

depressed erythrocyte folate uptake in elderly subjects

(Ettinger and Colman, 1985), assessment studies of population

subgroups of the elderly suggest that socioeconomic and

environmental factors are probably the most important

contributors to the development of compromised folate status.

The potential effect of socioeconomic level on folate

status in the elderly population is apparent when comparing

assessment studies conducted in population subgroups with








44

different income levels. The majority of high-income elderly

subjects participating in studies conducted in New Mexico

(Garry et al., 1984) and Florida (Wagner et al., 1981) had

normal serum and erythrocyte folate concentrations. Only 3%

and 6% of the participants in the New Mexico and Florida

studies, respectively, had erythrocyte folate values below 140

ng/mL. Conversely, Bailey et al. (1979) found that 60% of

elderly Floridians from very poor socioeconomic backgrounds

had erythrocyte folate concentrations below 140 ng/mL, as well

as evidence of macrocytic anemia.

Environmental factors that may place the elderly at

higher risk for the development of compromised folate status

have been reviewed by Bailey (1990b) and Sauberlich (1990).

These factors include: institutionalization; chronic use of

prescription and/or nonprescription medications; the presence

of disease; and the consumption of alcohol.

Although not specifically addressed for each stage of the

life cycle covered above, it is important to consider the

impact that cultural food habits and customs, food

preferences, educational level, state of mental and physical

health, socioeconomic factors and food availability have on

food selection and nutrient intake. While physiological

changes occurring throughout the life cycle can have a

profound affect on nutrient needs and metabolism, Sauberlich

(1990) has concluded that the most common cause of compromised

folate status is inadequate dietary intake.










Effects of Environmental Factors on Folate Status


Prescription and nonprescription medications. Certain

prescription and nonprescription medications interfere with

folate absorption and/or metabolism, and depending on the dose

and duration of use, may result in compromised folate status.

Examples of these drugs include folate antagonists,

anticonvulsants, histamine receptor blockers, antacids, anti-

inflammatory agents, aspirin and possibly oral contraceptive

agents.

Folate antagonists are used in cancer chemotherapy or to

treat infections such as malaria. Methotrexate is an example

of a folate antagonist. This drug interferes with folate

metabolism by inhibiting dihydrofolate reductase, resulting in

a functional folate deficiency and anemia. Methotrexate can

also cause reversible mucosal ulceration (Roe, 1989). The

resulting damage to enterocytes can impair the absorption of

folate and other nutrients.

Rosenberg et al. (1982) have noted that the frequent use

of antacids and histamine receptor blockers among the elderly

poses a theoretical risk to this population in terms of their

folate status. Histamine receptor blockers such as

cimetidine, and antacids like sodium bicarbonate, may impair

folate status by increasing the intestinal pH to levels that

exceed the optimum for folate conjugase activity and carrier-

mediated transport and/or passive diffusion (MacKenzie and

Russell, 1976; Russell et al., 1979). However, folate








46

bioavailability was not reduced by chronic bicarbonate

administration in a rat bioassay (Hoppner and Lampi, 1988).

Furthermore, evidence of folate deficiency due to use of these

medications is lacking (Rosenberg et al., 1982).

Sulfasalazine is an anti-inflammatory agent frequently

used in the treatment of inflammatory bowel disease. Franklin

and Rosenberg (1973) have demonstrated that this drug

interferes with folate absorption. It appears that

sulfasalazine interferes with the absorptive process through

competitive inhibition (Strum, 1981), and by inhibition of

jejunal brush border folate conjugase activity (Reisenauer and

Halsted, 1981). Sulfasalazine also has the potential to

disturb folate metabolism since it can inhibit several hepatic

folate-dependent enzymes (Selhub et al., 1978). Patients

taking daily therapeutic doses of sulfasalazine are at risk of

developing folate deficiency and should be provided with

therapeutic doses of folic acid in order to prevent a

deficiency of this nutrient.

The interaction between folate and diphenylhydantoin, an

anti-convulsant medication used for treatment of epilepsy, may

actually be a two-way interaction (Reynolds, 1973; Rivey et

al., 1984). Chronic use of this drug has been associated with

folate deficiency and anemia, although the progression of the

deficiency to megaloblastic anemia is rare (Gerson et al.,

1972; Hoffbrand and Necheles, 1968; Reynolds, 1973; Rivey et

al., 1984; Rosenberg et al., 1968) and administration of








47

phenytoin has not been shown to affect the kinetics of folate

excretion (Krumdieck et al., 1978). Conversely, long-term,

high-dose folic acid supplementation of folate-deficient

patients taking diphenylhydantoin may result in lowered serum

concentrations of this drug in selected patients, with the

potential for loss of control of the seizure disorder

(Reynolds, 1973; Rivey et al., 1984).

The mechanism responsible for the development of

compromised folate status in patients receiving

diphenylhydantoin is unclear, but several hypotheses have been

espoused. These hypotheses have been reviewed by Rivey et al.

(1984). They include: drug inhibition of brush border folate

conjugase (Hoffbrand and Necheles, 1968; Rosenberg et al.,

1968); elevation of the intraluminal pH resulting in

malabsorption of dietary folate (Benn et al., 1971); drug-

induced impairment of folate transport into tissues (Krumdieck

et al., 1978); and induction of folate-requiring metabolic

processes in the liver (Maxwell et al., 1972). Studies

employed to prove and/or disprove each of these hypotheses

have provided seemingly contradictory results. Rivey et al.

(1984) have suggested that diphenylhydantoin may affect folate

homeostasis by multiple mechanisms.

Alter et al. (1971) and Lawrence et al. (1984) have

reported that aspirin in therapeutic doses can reduce serum

folate concentrations. After aspirin is discontinued, serum

folate values rapidly increase. Individuals with rheumatoid








48

arthritis, as well as those taking therapeutic doses of

aspirin for the prevention of heart attacks, may be

particularly vulnerable to the effects of chronic aspirin

ingestion. Elderly individuals are more likely to use

aspirin for these purposes, which may make them more

susceptible to folate depletion depending on the amount and

frequency of use.

Several investigators have noted an association between

the use of oral contraceptive agents and low serum and

erythrocyte folate values (Butterworth et al., 1982;

Pietarinen et al., 1977; Smith et al., 1975). Other

researchers have found no difference in folate status of oral

contraceptive users versus nonusers (Paine et al., 1975; Ross

et al., 1976; Whitehead et al., 1973). Results of studies

comparing blood folate concentrations of women using oral

contraceptive agents within six months of conception to those

of nonusers have also been equivocal (Bailey, 1980; Martinez

and Roe, 1977). Factors such as the specific formulation of

the pill (i.e. level of estrogen), duration of pill use and/or

dietary intake may affect the body's response to oral

contraceptive agents. Based on observations that

megaloblastic changes in the cervical epithelium of women

taking oral contraceptive agents were reversed with oral folic

acid supplements (Whitehead et al., 1973), some researchers

have speculated that oral contraceptive use may lead to a










localized folate deficiency in the cervix (Butterworth et al.,

1982).

Smoking. The effects of smoking in nonpregnant women

were investigated by Wittier et al. (1982). The mean serum

and erythrocyte folate concentrations of the smokers who

participated in this study were lower than those of the

nonsmokers. No differences between smokers were detected,

however, with regard to the number of cigarettes smoked per

day.

Heimburger et al. (1987; 1988) compared the folate status

of male smokers to that of a control group of nonsmokers and

found significantly lower serum and erythrocyte folate

concentrations in the smokers. Smokers with metaplasia had

lower blood folate concentrations than did smokers without

metaplasia, and folate concentrations appeared to decrease

with increasing severity of metaplasia. These studies suggest

that smoking may adversely affect folate status.

Alcohol. Compromised folate status is common in chronic

alcoholics and is probably caused by a combination of factors

including: poor dietary habits; intestinal malabsorption;

decreased hepatic uptake; and increased urinary folate

excretion (Halsted, 1980). The effects of binge drinking on

folate absorption have been illustrated by studies using

orally administered radio-labeled folic acid. Evidence of

folic acid malabsorption was provided by decreased plasma

concentrations of radioactivity after oral doses of tritiated








50

pteroylmonoglutamic acid ([3H]PteGlu) (Halsted et al., 1967)

and by reduced luminal disappearance of [3H]PteGlu during

jejunal perfusion (Halsted et al., 1971). The underlying

defect responsible for the limited jejunal uptake of folic

acid was thought to be due to inadequate intake of dietary

folate during chronic alcohol ingestion (Halsted et al.,

1971). As discussed by Halsted (1990), a subsequent human

study that examined the effects of a folate deficient diet on

the absorption of [3H]PteGlu (Halsted et al., 1973), and an

absorption study conducted with monkeys fed diets containing

50% of their calories as ethanol (Romero et al., 1981),

prompted researchers to hypothesize that folate malabsorption

in chronic alcoholism results from the combined effects of

folate deficiency and ethanol exposure.

Recent research has attempted to delineate the

pathogenesis of folate malabsorption occurring in alcoholics.

To separate the effects of poor diet from those of alcohol

exposure, studies using miniature pigs exposed to alcohol for

a short period of time and in the absence of folate deficiency

have been performed (Naughton et al., 1989; Reisenauer et al.,

1989). These studies suggest that inhibition of brush border

folate conjugase, resulting in decreased hydrolysis of folate

polyglutamates, may be the earliest functional lesion

contributing to folate malabsorption and deficiency in

alcoholism (Halsted, 1990).








51

Disease. Folate deficiency has been associated with a

variety of diseases including certain hemolytic diseases

(Brody, 1991), cancer of the head and neck (Brody, 1991),

inborn errors of metabolism (Brody, 1991) and diseases of the

intestinal mucosa (Corcino et al., 1976; Halsted et al., 1977;

1978; Hoffbrand et al., 1968; 1970). The proposed underlying

etiology of folate deficiency in these diseases is defective

absorption and/or altered folate metabolism. These defects

lead to an increased folate requirement. For example, the

increased damage to red blood cells occurring in hemolytic

diseases results in increased cell division in the bone

marrow. This increase in cell division is thought to elevate

the requirement for folate (Brody, 1991). Altered metabolism

may also be responsible for the folate deficiency that

develops in patients with certain types of cancer,

particularly cancer of the head and neck (Brody, 1991). The

effects of altered folate metabolism in these individuals is

independent of the effects of anticancer drugs such as

methotrexate which can also adversely affect folate status.

Patients with inborn errors of folate metabolism may have

lower levels of folate-dependent enzymes, as well as defective

folate absorption, thereby increasing their folate requirement

(Brody, 1991).

Malabsorption is thought to be the major defect

contributing to compromised folate status associated with

untreated gastrointestinal diseases such as celiac sprue and








52

tropical sprue. Luminal disappearance of [3H]PteGlu and

pteroyl [14C]-glutamylhexaglutamate ([14C]-PteGlu7) in patients

with celiac sprue (Halsted et al., 1977; 1978) and tropical

sprue (Corcino et al., 1976) is significantly less than

luminal disappearance in normal subjects. When appropriate

medical and nutritional management of these diseases is

instituted, luminal disappearance increases significantly

(Corcino et al., 1976; Halsted et al., 1977; 1978). Further

evidence corroborating the idea that malabsorption adversely

affects folate status, at least in terms of celiac sprue,

comes from two studies. A significant decrease in the

hydrolysis of perfused [14C]-PteGlu7 in patients with celiac

sprue was noted in one study (Halsted et al., 1977). The

other experiment showed that brush border folate conjugase

activity in jejunal biopsy specimens taken from patients with

celiac sprue was significantly lower than the activity of this

enzyme in specimens from normal subjects (Halsted et al.,

1986).


Consequences of Compromised Folate Status


Megaloblastic anemia is probably the most commonly

recognized clinical problem associated with folate deficiency,

but research is beginning to identify many new potential

consequences of compromised folate status. Negative outcomes

more recently associated with folate deficiency include neural

tube defects, compromised infant birth weight, increased








53

potential for neoplastic changes and altered immune function.

A brief review of the evidence linking folate deficiency to

these problems is presented in this section.

Neural tube defects. Neural tube defects, which include

spina bifida, anencephaly and encephalocele, are among the

most common severe congenital malformations. The idea that

folate deficiency contributed to the causation of fetal

malformations was introduced almost thirty years ago by

Hibbard (1964) and Hibbard and Smithells (1965). Smithells

et al. (1980; 1981) conducted the first intervention trial

which suggested that supplementation with folic acid (0.36

mg/d) or other vitamins near the time of conception might

reduce the risk of recurrence of birth defects categorized as

neural tube defects. The results of this study could not be

taken as definitive proof of a protective effect for folate,

however, due to the co-administration of folic acid with other

vitamins, the lack of randomization of subjects to the control

and treatment groups and the absence of a double-blind design.

Another early intervention trial was conducted by

Laurence et al. (1981). This was a small study that employed

a controlled, randomized, double-blind design to test the

effects of folic acid supplementation (4 mg/d) alone. The

data provided inconclusive results when analyzed using the

original randomization scheme; however, when the data for

women who did not take their supplements were transferred to

the control group, the supplemented subjects had a








54

significantly lower recurrence rate. Preliminary results of

a study using a similar design and a small number of subjects,

found a reduction in the recurrence rate after a

supplementation program had been initiated, compared to the

recurrence rate before a supplementation program had been

established (Holmes-Siedle et al. 1982).

These early studies supported the concept of a protective

role for folic acid, but their flawed designs precluded the

ability to make any definitive conclusions and

recommendations. The results of a recent study (Medical

Research Council (MRC) Vitamin Study Research Group, 1991),

however, have established that supplementation with folic acid

around the time of conception can decrease the risk of neural

tube defects in women who have previously given birth to an

affected infant. This study was a large, multi-center,

double-blind intervention trial that randomized participants

to one of four groups: 1) folic acid alone; 2) folic acid plus

other vitamins; 3) other vitamins alone; and 4) no

supplements. This design allowed the researchers to determine

if vitamins other than folic acid conferred a protective

effect. The results demonstrated that folic acid, rather than

the combination of other vitamins, was responsible for the

improved outcome.

While the MRC Vitamin Study Research Group (1991)

findings are positive with respect to folate's role in

reducing the risk of recurrent neural tube defects, they do








55

not address the association of folate intake with the

occurrence of neural tube defects. In the United States, most

(approximately 95%) of the neural tube defect-affected infants

and fetuses occur in pregnancies of women who have not

previously given birth to an infant with a neural tube defect.

Thus, in order to reduce the overall prevalence of neural tube

defects, folic acid supplementation must exert a protective

effect on the developing embryos of women who have no history

of a neural tube defect-affected pregnancy. The MRC study

also leaves open to question the minimum dose and form of

folate needed to confer a protective effect, since subjects

were given daily supplements containing 4 mg of folic acid.

Over the last five years, several observational studies

have been conducted to evaluate the impact of periconceptional

folic acid/vitamin supplementation on the occurrence of neural

tube defects. Three of these studies were case-control

studies (Bower and Stanley, 1989; Mills et al., 1989; Mulinare

et al., 1988) and one was a prospective cohort study (Milunsky

et al., 1989). All of these studies involved women who had no

history of a neural tube defect-affected infant/fetus. The

occurrence of neural tube defects in women who reported taking

multivitamins containing folic acid (approximately 0.4-0.8 mg

folic acid per day) for at least one month prior to conception

through the first trimester of pregnancy were compared to

women who did not take supplements. Dietary intake of folate

was also considered in some studies. A protective effect was








56

associated with multivitamin supplement use and/or higher

levels of dietary folate intake in three (Bower and Stanley,

1989; Milunsky et al., 1989; Mulinare et al., 1988) of the

four studies.

Two new studies have provided additional support in favor

of a protective effect of folate. One study was a randomized

controlled trial conducted in Hungary (Czeizel and Dudds,

1992). Subjects were 18-35 years old, were not pregnant at

the time of recruitment and had no history of infertility or

fetal death. The volunteers were randomized to receive either

a placebo or a multivitamin supplement (including 0.8 mg folic

acid). There were no cases of neural tube defects in the 2104

participants receiving the multivitamin, whereas, the placebo

group had six occurrences and 2046 unaffected pregnancies.

Although the criteria for inclusion did not specifically

exclude women with a history of a neural tube defect-affected

pregnancy, it is likely that most women falling into this

category would have already been recruited for the MRC study

and would not have been available to participate in the study

just described. Under this assumption, these results have

been interpreted as providing evidence that folic acid can

reduce the risk of occurrence of neural tube defects; and that

the quantity of folic acid needed to produce such an effect is

much less than the 4 mg daily dose used in the MRC trial.

The results of another study examining the rate of

occurrence of neural tube defects in folate supplemented and








57

unsupplemented women has recently been published (Werler et

al., 1993). This large case-control study was conducted in

Boston, Philadelphia and Toronto. The control group consisted

of 2615 infants with birth defects other than neural tube

defects and oral clefts. This group was compared to 443 cases

with neural tube defects. The prevalence of use of folic

acid-containing multivitamins during the periconceptional

period was compared between mothers of cases and controls.

Daily folic acid supplementation was found to reduce the risk

of occurrent neural tube defects by 60%. Since the most

commonly used dose of folic acid was 0.4 mg, these data were

considered to be consistent with the hypothesis that 0.4 mg

supplemental folic acid per day is sufficient to decrease the

risk of neural tube defects among pregnancies of women in the

general population.

In the wake of the findings of the MRC trial (1991) and

the studies by Czeizel and Dudas (1992) and Werler et al.

(1993) the data from observational studies have been

reevaluated. Taken together, the results have been

interpreted as providing support for the hypothesis that folic

acid will decrease the risk of occurrence, as well as the

recurrence, of neural tube defects. Accordingly, the United

States Public Health Service has recently recommended that:

All women of childbearing age in the United States who
are capable of becoming pregnant should consume 0.4 mg of
folic acid per day for the purpose of reducing their risk
of having a pregnancy affected with spina bifida or other
NTDs. Because the effects of higher intakes are not well
known but include complicating the diagnosis of vitamin








58

B12 deficiency, care should be taken to keep total folate
consumption at <1 mg per day, except under the
supervision of a physician. Women who have had a prior
NTD-affected pregnancy are at high risk of having a
subsequent affected pregnancy. When these women are
planning to become pregnant, they should consult their
physicians for advice. (Anon., Morbidity and Mortality
Weekly, 1992, p. 1)

Recognition of the importance of folate in reducing the

risk of bearing a child with a neural tube defect is certain

to stimulate research focused on understanding the mechanism

whereby folate exerts its protective effect. Particular

attention is likely to be directed at identifying the

existence of underlying defects in folate metabolism.

Compromised birth weight. Folate deficiency occurring

during pregnancy has been associated with low infant birth

weight. Several researchers have reported increased infant

birth weights when folic acid supplements were given to

folate-deficient pregnant women (Baumslag et al., 1970;

Iyengar and Rajalakshmi et al., 1975; and Rolschau et al.,

1979). Placental weights were measured in two of these

studies (Iyengar and Rajalakshmi et al., 1975; Rolschau et

al., 1979), and a positive association with birth weight was

noted. These findings suggest that folic acid supplementation

improves fetal outcome by improving nutrition via increased

placental size (Bailey, 1990b). Additional evidence

suggesting a positive relationship between folic acid

supplementation and infant birth weight has recently been

published by Goldenberg et al. (1992). These researchers

found that maternal serum folate concentrations, occurring








59

within a range suggestive of compliance with the

supplementation regimen, were associated with higher infant

birth weight and a decreased rate of fetal growth retardation.

Neoplastic changes. The potential relationship between

folate and cancer was identified as early as 1944 when several

researchers noted that large doses of folic acid directly

interfered with the growth of certain tumors. Subsequently,

it was found that administration of folate antagonists

produced beneficial effects in patients with certain forms of

cancer. This discovery, although it revolutionized the

treatment of childhood leukemia and other forms of cancer,

probably contributed to the paucity of further research

regarding the potential anticancer effect of folate

(Butterworth, 1991). Renewed interest in the anticancer

potential of folate has been sparked by recent findings

suggesting that inadequate dietary intake of folate and/or

compromised folate status may be associated with an increased

risk or prevalence of certain forms of cancer; and that

optimal folate intake may provide a protective effect.

The initiation of carcinogenic activity is thought to

occur due to altered regulation of genetic expression of both

endogenous and exogenous oncogenes. Although the role of

folate deficiency in carcinogenesis has not been completely

elucidated, the co-carcinogenic effect of a deficiency of this

nutrient is thought to be related to folate's role in the

maintenance of chromosome structure and function. It has been








60

well established that folate is important in the regulation of

methyl groups used for DNA methylation. A deficiency of

methyl groups due to folate deficiency results in

undermethylated DNA and substitution of uridylate for

thymidylate. Subsequently, this interferes with histone

binding and results in the transcription of genetic sequences

that would ordinarily be suppressed. Additionally, improper

histone binding results in increased exposure of DNA to attack

by endogenous nucleases, thereby raising the risk of

chromosome breaks and incorporation of viral genomes.

Chromosome breaks associated with folate deficiency occur at

specific heritable fragile sites and many folate-sensitive

breaks occur at positions known to be associated with

translocations seen in cancer. It is thought that these

translocations disturb regulatory patterns of contiguous

segments of genetic information (Butterworth, 1991; Eto and

Krumdieck, 1986).

Compromised folate status has been associated with

increased risk of dysplasia or cancer of the cervix, colon,

bronchus and esophagus. The research documenting these

associations is briefly reviewed in the following paragraphs.

The concept of "localized folate deficiency" was

introduced as a result of a study conducted by Whitehead et

al. (1973). These researchers reported the occurrence of

megaloblastic features in cervical epithelial cells from women

taking oral contraceptive agents. These cytologic








61

abnormalities were not related to hematologic changes or low

serum folate/vitamin B12 concentrations; however, after three

weeks of folic acid supplementation (10 mg/d), the abnormal

cytologic findings were reversed or improved. Later,

Butterworth et al. (1982) reported that daily oral folic acid

supplementation (10 mg/d) was associated with improvement in

the cytologic manifestations of dysplasia compared with

placebo-treated controls. The supplemented group also had

less severe biopsy readings after three months of

supplementation. On the basis of these findings, it was

suggested that folate deficiency either plays an integral role

in the dysplastic process or is occasionally misdiagnosed as

cervical dysplasia (Butterworth et al., 1982).

In a more recent study, Butterworth et al. (1992b)

examined the effect of high-dose oral supplements of folic

acid (10 mg/d for six months) on the course of cervical

dysplasia. Supplementation appeared to have no significant

effect on the course of established cases of dysplasia, with

a high rate of apparent regression occurring in both the

placebo-treated and the folic acid-supplemented groups. These

researchers attributed the difference between the findings of

this study and their earlier report (Butterworth et al., 1982)

to the use of a more adequate sample size, exclusion of

patients with atypia less than dysplasia and a longer period

of observation. Although they concluded that folic acid

supplementation does not alter the course of established








62

disease, they did not exclude the possibility that folate

deficiency played a role in carcinogenesis because they found

a higher prevalence of dysplasia associated with human

papillomavirus 16 (HPV-16) infection (an oncogenic strain

thought to cause cervical dysplasia) among women in the lower

two tertiles of red blood cell folate than in the highest

tertile. Based on this information, they concluded that

folate deficiency may act as a co-carcinogen during the

initiation of cervical dysplasia.

In a separate case-control study conducted by the same

research group (Butterworth et al., 1992a), infection with

HPV-16 was the strongest risk factor for cervical dysplasia,

and there was a statistically significant interaction between

low erythrocyte folate concentrations and the HPV-16 virus.

The conclusion reached by these investigators was that the

carcinogenic effect of HPV-16 infection is enhanced in women

with low concentrations of erythrocyte folate. Although the

mechanism of interaction between folate and HPV-16 was not

investigated in this study, the researchers suggested that a

folate deficiency might increase the possibility of

incorporating the viral genome into human DNA resulting in

transformation of the epithelial cells.

The potential role of folate deficiency in carcinogenesis

may not be limited to the cervix since premalignant lesions

occurring in other organs exhibit features similar to those

seen in cervical dysplasia (Heimburger et al., 1987). Studies








63

examining the relationship between folate status and bronchial

metaplasia suggest that folate deficiency may influence the

susceptibility of the bronchial mucosa to neoplastic

transformation. For example, Heimburger et al. (1987) found

that the serum and erythrocyte folate concentrations of men

who smoked an average of 25 cigarettes per day were

significantly lower than nonsmokers, with the lowest values

occurring in smokers with bronchial metaplasia. Serum and

erythrocyte folate concentrations were also significantly

lower in smokers with metaplasia compared to smokers without

metaplasia. In a subsequent double-blind intervention trial

(Heimburger et al., 1988), smokers with bronchial squamous

metaplasia were stratified according to smoking level and

randomly assigned to treatment with a placebo or 10 mg of

folic acid and 500 Ag of hydroxycobalamin for four months.

Reduction of atypia, as determined by direct cytological

comparison, was significantly greater in the supplemented

group. Unfortunately, it is impossible to determine if folic

acid alone was responsible for the favorable effect since

hydroxycobalamin was administered simultaneously. Never-

theless, these studies are provocative and provide fertile

ground for continued investigation in this area.

Compared with the general population, patients with

chronic ulcerative colitis are at greater risk for developing

cancer of the colon. It has been hypothesized that folic acid

supplementation may protect against the development of








64

dysplasia (a premalignant pathologic finding) and cancer in

ulcerative colitis. To test this hypothesis, Lashner et al.

(1989) conducted a case control study to examine the effect of

folic acid supplementation on the incidence of dysplasia or

cancer in 99 subjects with chronic ulcerative colitis. A 62%

lower incidence of neoplasia was associated with folic acid

supplementation compared to subjects not receiving supple-

mentation. Although this outcome was not statistically

significant, it did not change when adjustments were made for

known confounders, suggesting that this finding was not due

entirely to bias or confounding and that inadequate sample

size may have contributed to the lack of significance (Lashner

et al., 1989).

Folate deficiency may also play a role in the development

of esophageal cancer. Jaskiewicz et al. (1988) have examined

cytological specimens obtained by brush biopsy of the

esophagus from subjects living in an area of Africa where

squamous cell carcinoma of the esophagus is the most common

form of cancer. Biopsy results revealed morphologic features

similar to those seen with folate deficiency and dysplasia.

Assessment of folate status in this same population revealed

significantly lower erythrocyte folate concentrations in

subjects with dysplastic and cancer cells compared with

controls (van Helden et al., 1987). Additionally, the

erythrocyte folate concentrations of subjects living in high-

incidence districts was significantly lower than those in the








65

intermediate and low incidence districts. However, these

results must be interpreted cautiously since multiple nutrient

deficiencies, particularly in the high-incidence area, were

noted.

Altered immune function. Although the effect of

compromised folate status on immunocompetence has not been

widely researched, studies using folate-deficient animals and

observations of patients with megaloblastic anemia due to

folate deficiency suggest that alterations occur in both

humoral and cell-mediated immunity. This is not surprising,

since folate is essential for the synthesis of DNA, and a

deficiency of this nutrient would hinder the ability of the

sensitized cells to proliferate rapidly. Examples of the

effects of compromised folate status on immune function as

determined from human studies include delayed cutaneous

hypersensitivity and depressed peripheral lymphocyte response

to phytohemagglutinin. Similar changes in immune function

have been shown using animal models. In addition to these

changes, reductions in white blood cells, leukocytes,

granulocytes, antibody forming cells and T-cells have been

reported in various animal models subjected to an isolated

folate deficiency (Nauss and Newberne, 1981).


Methods Used to Assess Folate Status and Metabolism in Humans


Microbiological assay. The microbiological assay is

considered the most useful method for quantifying folate








66
levels in biological samples (Tamura, 1990). This method uses

the growth response of folate-sensitive microorganisms to

determine folate content. The assay is performed by comparing

the growth of a test organism added to folate-free media

containing an aliquot of the sample, to the growth of the same

organism added to folate-free media that has been enriched

with known concentrations of folic acid. Growth is measured

as turbidity following incubation under controlled conditions.

The microorganism used most often for this assay is

Lactobacillus case, although Streptococcus faecium or

Pediococcus cerevisiae may also be used.

The three species of bacteria that may be used for this

assay do not respond equally to the various forms of folate.

For example, S. faecium does not respond to N5-methyl-

tetrahydrofolate, nor to folate derivatives with more than two

molecules of glutamate. Pediococcus cerevisiae is even more

selective. It does not respond to N5-methyl-tetrahydrofolate,

and of the remaining folate derivatives, it can only use those

occurring as reduced monoglutamyl tetrahydrofolates (Tamura,

1990). Since neither S. faecium nor P. cerevisiae respond to

N5-methyl-tetrahydrofolate, they are not suitable test

organisms for determination of serum or erythrocyte folate

concentration. Lactobacillus case, however, responds to N5-

methyl-tetrahydrofolate, as well as other folate derivatives,

which makes it the best choice when analyzing biological

samples for total folate.








67

The fact that L. case, S. faecium, and P. cerevisiae

respond differently to folates with varying chain lengths,

one-carbon substituent groups and oxidation states offers

researchers the opportunity to estimate the amount of folate

derivatives present in a particular sample. This can be

useful when the quantity of sample available for analysis is

insufficient to perform column chromatography. For example,

if it is important to estimate the amount of folate

derivatives other than N5-methyl-tetrahydrofolate, L. case

can be used in combination with S. faecium. Similarly, P.

cerevisiae can be used in conjunction with L. case to

estimate folate derivatives other than Ns-methyl-

tetrahydrofolate and oxidized pteroylglutamic acid. This

deductive approach can also be used to determine whether

samples contain folate polyglutamates. If the activity of S.

faecium or P. cerevisiae increases after folate hydrolase

treatment, the sample contains folate polyglutamates with more

than two molecules of glutamate. Likewise, if L. casei

activity increases after enzyme treatment, the sample contains

polyglutamates with more than three molecules of glutamate

(Tamura, 1990).

Biological samples typically analyzed for folate content

include serum, whole blood, urine and tissues. Cellular

folates, such as those found in tissues, are usually in the

polyglutamate form and require cleavage to the monoglutamate

form before folate concentration using the microbiological








68

assay can be determined. Partially purified folate hydrolase

preparations from chicken pancreas, hog kidney and rat or

human plasma have been used for this purpose. Folate in the

erythrocytes is also in the polyglutamate form; however, the

presence of natural folate hydrolase in the blood precludes

the need for treating hemolyzed whole blood samples with an

exogenous folate hydrolase. Urine and serum samples do not

require prior treatment with folate hydrolase because the

folate present in these samples is in the monoglutamyl form.

Although the basic concept of the microbiological assay

has not changed over the years, several improvements have been

made. Examples of these improvements have been reviewed by

Tamura (1990) and include the addition of antioxidants (e.g.

ascorbic acid) to samples to protect labile reduced forms of

folate from oxidation and the use of a cryoprotected organism

to maintain constant growth-response curves. Recently, this

assay has been adapted to take advantage of the availability

of the 96-well microtiter plate reader (Horne and Patterson,

1988; Newman and Tsai, 1986; O'Broin and Kelleher, 1992).

Although the procedure followed with this adaptation is

similar in concept to the standard procedure, the microtiter

plate reader improves the efficiency of absorbance readings.

Rapid calculation of the results can be achieved by

interfacing the microtiter plate reader with a computer (Horne

and Patterson, 1988). These modifications can save a








69

tremendous amount of time and money without sacrificing

accuracy (Newman and Tsai, 1986; O'Broin and Kelleher, 1992).

Radiometric binding assay. The radiometric binding assay

is a competitive protein binding assay in which radio-labeled

folate and unbound folate in the sample compete for the

binding sites on folate binding proteins. This method is

usually performed by clinical laboratories using commercially

available radioassay kits (Brody, 1991; Tamura, 1990).

Compared with the microbiological assay, this competitive

binding assay is simpler to perform and is not affected by

bacterial contamination or the presence of antibiotics.

Despite these potential advantages, the usefulness of the

radiometric binding assay is limited because the proteins used

to bind folates do not have an equal affinity for all forms of

folate. Binding affinity seems to be influenced by the state

of oxidation, the one-carbon substituent group and glutamyl

chain-length. For this reason, the suitability of the

radioassay method for quantifying complex mixtures of

naturally occurring folates has been questioned. This method

may be acceptable for determining serum folate concentration,

however, because N5-methyl-tetrahydrofolate is the predominant

form of folate in serum, and this form of folate is tightly

bound by folate binding proteins. Conversely, if the

population being assessed is receiving large doses of folates

other than Ns-methyl-tetrahydrofolate, the radioassay may be

inappropriate (Brody, 1991; Tamura, 1990).








70
Folate bioavailability studies. Until recently, most of

the information known about the metabolic requirements and

bioavailability of folates in humans was derived from animal

bioassay studies, or from a limited number of human studies

which used plasma or urinary folate concentration in folate-

saturated human subjects as the response criteria. While

these studies have made an important contribution to our

understanding of folate metabolism, they have several

limitations which restrict their scope and applicability. A

potential limitation of animal bioassay studies is the use of

animal models that are not entirely appropriate for studying

human metabolism. For example, rats are not appropriate

models to use for folate polyglutamate absorption studies

because they exhibit little or no brush border folate

conjugase activity (Wang et al., 1985). Limitations of

bioassay studies conducted in human subjects include their

inability to provide information about in vivo metabolism and

the need to consume large quantities of tested foods or

purified folates, the later of which could change the rate of

digestion and absorption in a way that is different from that

which usually occurs (Gregory and Toth, 1990).

The development of new tools and techniques combined with

previous accomplishments has facilitated research in the area

of folate requirements and bioavailability. The synthesis of

various forms of folate, including folate polyglutamates

(Krumdieck and Baugh, 1969; Godwin et al., 1972), and the








71

ability to radio-label folate derivatives with tritium at

different locations in the molecule (Godwin et al., 1972),

provided valuable tools for initial studies of folate

metabolism in animals. Although radio-labeled folates have

been used in human studies, concern about their safety

precluded their use for long-term supplementation studies and

restricted the amount of labeled folate that could be

administered to no more than a tracer dose. The development

of stable-isotopically (deuterium) labeled folates (Gregory,

1990; Gregory and Toth, 1988a; 1988b) has circumvented these

problems, while providing the opportunity to trace the fate of

folate during normal metabolism and to compare this with the

fate of folate under altered conditions or physiological

states. Additionally, the availability of stable isotopes of

folate affords the luxury of evaluating several forms of

folate simultaneously and allows the researcher to determine

the kinetics of absorption and turnover of the administered

compound (Gregory and Toth, 1990). Other developments that

have facilitated research in this area include the development

of improved methods for separating various folate

monoglutamates using high-performance liquid chromatography

(HPLC) (Kashani and Cooper, 1985; Wilson and Home, 1984); the

development of affinity chromatography columns that allow for

easier separation of folates from the sample (Selhub et al.,

1980), which ultimately results in cleaner chromatographic

separation using HPLC (Selhub, 1989); and the development of








72

a mass spectrometric method to quantify the ratio of unlabeled

versus labeled folates (Toth and Gregory, 1988).

The first published studies of an in vivo application of

stable-isotopically labeled folates in human subjects were

performed by Gregory and coworkers (Gregory and Toth, 1988a;

Gregory et al., 1990). The initial report (Gregory and Toth,

1988a) described the results of a preliminary investigation of

gas chromatography/mass spectral (GCMS) analysis of urinary

folates. The second study (Gregory et al., 1990) evaluated

the adequacy of a saturation regimen of 2 mg/d of folic acid

and the effectiveness of simultaneous administration of two

forms of deuterium-labeled folate. Subjects participating in

this study received 2 mg/d unlabeled folic acid for one week

to enhance urinary excretion of absorbed folates. On the

morning prior to administration of the deuterium-labeled

folates, the subjects collected a 24 hour urine sample. The

next morning, after an overnight fast, subjects consumed apple

juice containing two different deuterium-labeled folates. The

subjects collected their urine for 48 hours following the

treatment. A constant diet was consumed beginning at the time

of the pre-dose urine collection through the end of the post-

dose urine collection. Dietary and total urinary folate

concentration was determined by microbiological assay using L.

casei. Urine samples were also prepared for mass spectral

analysis of labeled folates, after which the molar ratio of

each species of folate in the sample was calculated. The








73

results of these studies confirmed that stable-isotopically

labeled folates are well-suited for in vivo studies and that

the protocol outlined above is suitable for studying many

aspects of folate bioavailability and in vivo kinetics

(Gregory et al., 1990). Recently, Von der Porten et al.

(1992) used this methodology to study in vivo folate kinetics

in human subjects supplemented with deuterium-labeled folic

acid for four weeks. By using deuterium-labeled folate, these

researchers were able to determine rate constants and isotope

enrichment, which subsequently enabled them to assess tissue

uptake and equilibration, in vivo turnover rates and body pool

sizes of folate. It is expected that further application of

this technique will greatly enhance our knowledge of folate

metabolism and improve our ability to make appropriate

nutritional recommendations.


Zinc


Chemistry


Zinc has an atomic weight of 65.37. It is a first series

transition element with the electronic configuration [Argon]

4s2 3d10. This configuration confers properties to zinc that

distinguish it from other transition metals. For example,

zinc is diamagnetic rather than paramagnetic, and although it

can exist in several valence states, it is resistant to

oxidation and is found almost universally as the divalent ion

(Zn2+). Another property of zinc that is relevant to its








74

specific biological functions is its ability to form stable

complexes with side chains of proteins. The high charge

density of Zn2+ allows this metal to function as a Lewis acid

to withdraw electrons from electron-rich functional groups of

ligands resulting in the formation of noncovalently bound

coordination complexes. These features make zinc ideally

suited for its involvement in enzyme function and structure

(Solomons, 1988; Williams, 1989).


Absorption


The process of zinc absorption includes the uptake of

zinc by the intestinal mucosal cells, movement of zinc through

the mucosal cells and transfer into the portal circulation.

Paracellular movement of zinc to the portal circulation may

also occur. These aspects of zinc absorption are discussed in

the following paragraphs.

Although the study of zinc absorption has been given much

attention, the precise mechanism, location and control of this

process have not been fully delineated (Cousins and Hempe,

1990). Differences in experimental conditions may account for

the conflicting results of in vivo and in vitro experiments

designed to study zinc absorption, since a multitude of host

and environmental factors have been shown to influence this

process (Solomons and Cousins, 1984).

Zinc is absorbed throughout the small intestine, but the

segment with the highest capacity to absorb zinc has not yet








75

been determined (Cousins and Hempe, 1990). Rat studies used

to identify the major absorptive site have produced variable

results, with some studies (Davies, 1980; Methfessel and

Spencer, 1973; Van Campen and Mitchell, 1965) suggesting that

the greatest amount of zinc uptake occurs in the duodenum, and

others (Antonson et al., 1979; Emes and Arthur, 1975)

suggesting that greater zinc uptake occurs in the distal

portions of the intestine. Although Sahagian et al. (1966)

found the concentration of zinc from normal rat intestine to

be fairly uniform throughout each region, zinc uptake by

strips of rat intestine was greater in the duodenal and ileal

segments compared to the jejunal segment. These findings are

in contrast to those of an in vivo intestinal perfusion study

(Matseshe et al., 1980) conducted in humans. In this study,

Matseshe et al. found that more zinc left the distal duodenum

than what was contained in a test meal, suggesting that

endogenous secretions contributed to the total intraluminal

zinc concentration. For this reason, net duodenal zinc

disappearance, if any, could not be determined. Once the

intraluminal contents passed into the jejunum, zinc

disappeared gradually but incompletely. Ileal zinc absorption

was not determined in this study. In fact, all portions of

the small intestine may be functionally important in terms of

zinc absorption, with the duodenum having first access to

dietary and endogenous zinc, and subsequent sections having

the benefit of the action of digestive processes that may








76
increase the accessibility of this nutrient (Cousins and

Hempe, 1990; Lbnnerdal, 1989a).

The first phase of the absorptive process involves the

movement of zinc from the intestinal lumen into the mucosal

cell. Evidence that zinc absorption occurs rapidly and

involves a saturable, carrier-mediated component, as well as

nonsaturable diffusion has come from several rat studies.

Davies (1980) exposed ligated duodenal loops from rats to

different concentrations of zinc and found that these segments

exhibited saturation kinetics at low luminal zinc

concentrations. At higher luminal zinc concentrations, zinc

uptake was linear suggesting that zinc absorption also

occurred by diffusion. Menard and Cousins (1983) reported

similar results using isolated brush border membrane vesicles

from rats. Further evidence for a mediated component of zinc

absorption was derived from experiments with rats previously

fed a zinc-adequate or a zinc-deficient diet (Steel and

Cousins, 1985). Zinc absorption, as determined from zinc

accumulation in the portal perfusate, revealed that absorption

was saturable in both groups, and that zinc-deficient rats had

a more rapid rate of zinc absorption at all luminal zinc

concentrations. Zinc absorption appeared to involve both

mediated and nonmediated components, with most of the

absorption occurring by means of the mediated component in the

zinc-deficient group. These results suggest that the

saturable process is stimulated by zinc depletion, whereas the








77

nonsaturable process is unaffected by zinc deficiency and

proceeds in proportion to the intraluminal zinc concentration.

A more recent study conducted by Hoadley et al. (1987) is in

agreement with the findings of previous studies suggesting

that zinc absorption involves two kinetic processes.

Although there is general agreement that zinc uptake by

the intestinal mucosal cell involves a saturable, carrier-

mediated component, as well as nonsaturable diffusion, it is

not known if these processes represent components of a single

transport step occurring at the brush border membrane, or if

movement from the lumen to the portal circulation occurs by

two independent routes: transcellular and paracellular.

(Cousins and Hempe, 1990). Transcellular movement may involve

a protein that has recently been identified (Hempe and

Cousins, 1991), purified and partially characterized (Khoo and

Cousins, 1993). This protein, known as cysteine-rich

intestinal protein is a low molecular weight, zinc-binding,

cytosolic protein isolated from intestinal mucosal cells.

Cysteine-rich intestinal protein has two possible zinc binding

sites, and it is hypothesized that this protein serves as an

intracellular zinc carrier. This protein appears to bind more

zinc when intestinal metallothionein is not induced,

suggesting that intestinal metallothionein may interact with

cysteine-rich intestinal protein to regulate zinc absorption

and transport (Khoo and Cousins, 1993).








78
Movement of zinc from the intestinal lumen into the

mucosal cell may be affected by the intraluminal environment.

Zinc liberated from food matrices during the digestive process

may remain as the free ion or may form coordination complexes

with endogenous (pancreatic, biliary or mucosal) or exogenous

(dietary) intraluminal ligands (Solomons and Cousins, 1984).

Studies investigating the effect of intraluminal binding

ligands on zinc uptake have suggested that ligand binding may

affect zinc absorption by altering intestinal membrane

permeability and/or zinc gradients across intestinal cell

membranes (Cousins, 1985; Solomons and Cousins, 1984).

However, while there is little question that zinc-binding

ligands affect zinc absorption, it is doubtful that this

process is a prerequisite for movement of zinc across the

membrane surface (Cousins and Hempe, 1990).

Once zinc is inside the intestinal mucosal cell, it binds

to a variety of high molecular weight ligands, as well as to

intestinal metallothionein, a low molecular weight, cyteine-

rich metalloprotein. The amount of zinc bound to intestinal

metallothionein varies according to zinc status and intake.

Zinc binding to intestinal metallothionein restricts the

movement of zinc from the cell, thereby contributing to the

regulation of zinc absorption. Intestinal metallothionein

synthesis is induced at the transcriptional level by high

dietary zinc (Blalock et al., 1988; Menard et al., 1981). When

the rate of intestinal metallothionein synthesis is high, net








79
zinc release to the portal circulation is curtailed (Menard et

al., 1981). In zinc deficiency, intestinal metallothionein

concentration is low and more zinc is released into the portal

circulation (Hoadley, et al., 1988). Thus, it appears that

the induction of metallothionein synthesis in intestinal

mucosal cells could provide for the efficient regulation of

zinc transfer to the vascular compartment.

Based on the results of rat studies (Davies, 1980; Smith

and Cousins, 1980), transfer of zinc from the mucosal cell to

the portal circulation occurs more slowly than uptake and

accumulation within the cell. According to Davies (1980),

transfer of zinc into the portal circulation may occur in two

stages: 1) rapid transfer occurring over the first 30 minutes;

and 2) slower transfer occurring from 30 minutes to six hours

after receiving the dose, which may represent the release of

intracellularly bound zinc from zinc-binding proteins.

Consequently, movement of zinc from the intestinal mucosal

cell into the vascular compartment may be the rate-limiting

step in zinc absorption (Smith and Cousins, 1980). At low

luminal zinc concentrations, much of the zinc available is

released into the portal circulation; however, as the luminal

zinc concentration increases, less of this nutrient is

transported to the vascular system (Hambidge et al., 1986;

Smith and Cousins, 1980). An exception to this phenomenon

occurs when excessive loads of luminal zinc are available. In








80

this case, the ability to control the release of zinc into the

circulation is diminished (Smith and Cousins, 1980).


Transport. Distribution and Metabolism


Although it had been proposed that transferring was

responsible for portal zinc transport (Evans and Winter,

1975), several lines of investigation have shown that albumin

is the principal portal transport protein for zinc (Smith et

al., 1979). Smith et al. (1979) demonstrated the importance

of albumin in portal transport by comparing the percent of

65Zn absorbed when the vascular perfusate composition was

varied. The absence of albumin from the perfusate resulted in

negligible zinc transfer to the portal circulation, whereas

the isosmotic replacement of all plasma proteins with albumin

resulted in a twofold increase in zinc transfer over that of

the control perfusate. These data intimate that the extent of

zinc absorption may be affected by the concentration of

albumin in the blood (Cousins, 1985). Consequently, zinc

absorption may be impaired in disease states associated with

the development of hypoalbuminemia.

Zinc entering the portal circulation is rapidly

transported to the liver. The liver is the primary organ

involved in zinc metabolism, and a large portion of the zinc

in portal blood is exchanged with the liver (Cousins and

Hempe, 1990). In vitro experiments with rat liver parenchymal

cells demonstrated that zinc uptake was temperature and








81

energy-dependent, followed saturation kinetics and occurred in

two phases (Failla and Cousins, 1978a). The first phase was

characterized by rapid, saturable uptake. This was followed

by a slower phase comprised of both saturable and linear

components. Maximal uptake occurred at the normal plasma zinc

concentration (Cousins, 1989; Failla and Cousins, 1978a).

Zinc accumulation by these cells increased when physiological

concentrations of certain adrenal corticosteroids were added

suggesting that these hormones may perform an essential role

in the regulation of hepatic zinc metabolism. As a corollary,

it is possible that zinc has a role in glucocorticoid-mediated

alterations of hepatic metabolic processes (Failla and

Cousins, 1978b).

Distribution of zinc to the extrahepatic tissues occurs

mainly via the plasma. The plasma contains approximately 10

to 20% of the zinc in whole blood, whereas the erythron and

leukocytes contain the major portion of zinc in the blood.

Most of the plasma zinc is bound to albumin, although alpha-2-

macroglobulin, transferring, histidine and cysteine may also

transport small amounts of this metal. Zinc bound to albumin

is considered to be loosely bound and represents the

metabolically active, exchangeable zinc pool in the blood.

This pool is responsive to acute and chronic changes related

to stress, infection and dietary zinc. In contrast to plasma

zinc, the zinc in erythrocytes is mostly associated with

carbonic anhydrase, although small amounts may be associated








82

with superoxide dismutase and metallothionein (Cousins, 1989;

Cousins and Hempe, 1990; DiSilvestro and Cousins, 1983).

The rate of zinc incorporation into extrahepatic tissues

varies, as does the rate of zinc turnover in these tissues.

Zinc accumulation and turnover occurs rapidly in the kidney,

pancreas and spleen (Hambidge, et al., 1986). The rate of

zinc uptake by skeletal muscle and the central nervous system

is relatively slow, and the zinc in these tissues remains

tightly bound for long periods of time (Hambidge et al.,

1986). The zinc incorporated into hair is not available for

exchange either. Tissue-specific redistribution of body zinc

can occur during periods of zinc deprivation, stress and

infection/inflammation (Cousins and Leinart, 1988; Dunn and

Cousins, 1989; Giugliano and Millward, 1984; Huber and

Cousins, 1988; Jackson et al., 1982). For example, the zinc

concentration of muscle in zinc-deficient rats is protected,

while the concentration in bone, liver and plasma declines

(Giugliano and Millward, 1984; Jackson et al., 1982). This

redistribution may occur as a result of tissue-specific

induction of metallothionein.

In general, the largest concentration of intracellular

zinc is found in the cytosol, although smaller amounts are

present in the nuclear, microsomal and mitochondrial fractions

of cells (Hambidge et al., 1986). Zinc is also present in the

membrane and may enhance membrane stability (Bettger and

O'Dell, 1981). Within the cytosol, zinc is primarily bound to








83

large molecular weight proteins. These proteins may be zinc

metalloenzymes. In contrast, the amount of zinc bound to

metallothionein is relatively low under normal dietary

conditions; however, when dietary zinc is increased, the

metallothionein gene is induced and metallothionein synthesis

is elevated (Cousins and Lee-Ambrose, 1992; Cousins and

Leinart, 1988; Huber and Cousins, 1988). Consequently,

increased zinc binding to metallothionein in the liver,

pancreas, kidney and muscle occurs. Zinc may regulate the

expression of the metallothionein gene through a specific

nuclear metal-binding protein that also binds to unique DNA

sequences in the promoter region (Cousins and Hempe, 1990).

Synthesis of metallothionein, particularly in the liver, may

also occur in response to hormones, as well as physiological

stimuli such as stress, acute infection and shock (Cousins and

Hempe, 1990; Hambidge et al., 1986).


Excretion


The major route of zinc excretion is via the feces. Zinc

in the feces is derived from unabsorbed dietary zinc, as well

as endogenously secreted zinc. Sources of endogenous zinc

include pancreatic, biliary and mucosal secretions, as well as

zinc present in desquamated mucosal cells. Usually,

endogenously secreted zinc is efficiently reabsorbed, but

intraluminal factors such as the presence of phytic acid may








84
decrease the efficiency of absorption (Cousins and Hempe,

1990). Additionally, dietary intake of zinc may affect

reabsorption, with more zinc of endogenous origin being

excreted in the feces at higher levels of zinc intake (Jackson

et al., 1984).

Urinary zinc losses account for a fairly small amount of

the zinc excreted under normal physiologic conditions. The

usual range of zinc excreted in the urine of healthy adults is

about 300 to 600 gg/d (Gibson, 1990). Urinary zinc excretion

has been shown to decrease in human subjects consuming a zinc-

deficient experimental diet (0.28 mg zinc/d) (Baer and King,

1984). Injury, burns, infection, acute starvation and

pathologic conditions resulting in excessive muscle catabolism

have been associated with clinically significant increases in

urinary zinc excretion. Hyperzincuria is known to occur in

conjunction with proteinuria due to kidney dysfunction and in

patients with sickle cell disease and cirrhosis of the liver

(Cousins and Hempe, 1990; Gibson, 1990).

Dermal losses due to sweating and to the sloughing of

epithelial tissue also account for some of the zinc lost from

the body. As reported in a review by Hambidge et al. (1986),

the total amount of zinc lost in sweat from adults living in

a temperate North American climate has been estimated to be

0.4 to 2.8 mg/d. Dermal zinc losses have been found to be

influenced by dietary zinc intake, with reduced losses

occurring when zinc intake is marginal, and losses increasing




Full Text
ZINC STATUS RESPONSE TO FOLIC ACID SUPPLEMENTATION
AND THE EFFECT OF LEVEL OF ZINC INTAKE ON
FOLATE UTILIZATION IN HUMAN SUBJECTS
By
GAIL P. ABBOTT KAUWELL
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1993

Copyright 1993
by
Gail P. Abbott Kauwell

I dedicate this dissertation to all of the people who
were instrumental in helping me achieve this goal and to my
steadfast, loyal and loving companions, Chelsea and Curly.

ACKNOWLEDGMENTS
It is with great difficulty that I write this section of
my dissertation because it is impossible to properly
acknowledge everyone who deserves to be recognized and
thanked. Your support has come in many forms: encouragement,
ideas, storage space, equipment, supplies, technical
assistance, helping hands, friendly smiles and more. I do
remember and sincerely appreciate all the ways that you have
supported me through the trials and tribulations of completing
this research project and dissertation.
Although I do not wish to slight anyone's contributions
by not including their name in this acknowledgment, I would be
remiss if I did not take this opportunity to highlight some of
the people to whom I am most thankful. I am most grateful to
Dr. Lynn Bailey, the chairman of my supervisory committee,
whose encouragement, support, guidance and friendship has been
limitless. I also appreciate the guidance and support
provided by my committee members, Dr. Robert Cousins, Dr.
Jesse Gregory, Dr. Claudia Probart and Dr. Rachel Shireman.
In addition to my committee members, I want to recognize and
thank Dr. Susan Percival, who provided me with the opportunity
to work in her lab, and Peter Johnson for helping me smile
along the way. The subjects who participated in this study
IV

also deserve to be recognized for the dedication, interest and
enthusiasm they displayed throughout the duration of this
research project. Last, but certainly not least, I am
thankful for the support, encouragement and thoughtful advice
that has always been graciously provided to me by Dr. Richard
Gutekunst, Dean of the College of Health Related Professions.
v

TABLE OF CONTENTS
page
ACKNOWLEDGMENTS iv
LIST OF TABLES ix
LIST OF FIGURES X
LIST OF ABBREVIATIONS xii
ABSTRACT XV
CHAPTERS
1 INTRODUCTION 1
2 REVIEW OF THE LITERATURE 5
Folate 5
Chemistry 5
Metabolism 7
Biochemical Functions 17
Recommended Dietary Allowances for
Folate 21
Effects of High Doses of Folic Acid . . 23
Sources/Distribution and Stability of
Folate in Foods 2 5
Assessment of Folate Status 27
Folate Status in Special Population
Groups 36
Effects of Environmental Factors on
Folate Status 45
Consequences of Compromised Folate
Status 52
Methods Used to Assess Folate Status and
Metabolism in Humans 65
Zinc 73
Chemistry 73
Absorption 74
Transport, Distribution and Metabolism . 80
Excretion 83
Zinc Homeostasis 85
Biochemical Functions 85
vi

Recommended Dietary Allowances for Zinc 88
Effects of High Doses of Zinc 92
Food Sources of Zinc, Usual Intakes and
Bioavailability 94
Factors Affecting Zinc Status 99
Assessment of Zinc Status 101
Metallothionein 110
Zinc Deficiency 116
Folate-Zinc Interactions 118
Effect of Impaired Zinc Status on Folate
Absorption and Metabolism .... 118
Effect of Supplemental Folic Acid on Zinc
Status 122
3 RATIONALE FOR RESEARCH PROTOCOL 140
4 SUBJECTS, EXPERIMENTAL DESIGN, MATERIALS
AND METHODS 145
Subjects 145
Recruitment and Selection 145
Description of Subjects 146
Experimental Design 147
Materials and Methods 151
Description of Diet and Supplements . 151
Procedures Used to Foster, Monitor and
Assess Compliance 156
Procedure Used to Assess Adeguacy of
Blinding 157
Procedures Used to Prevent Zinc
Contamination 157
Urine Collection and Processing
Procedures 160
Blood Collection and Processing
Procedures 160
Preparation of Diet Composites .... 163
Determination of Dietary Zinc Content 164
Biochemical Analyses 167
Plasma and Urine Zinc Concentrations . 167
Erythrocyte Lysate Zinc Concentration 168
Protein Determination 169
Erythrocyte Metallothionein
Concentration 170
Determination of Serum Ferritin
Concentration 175
Determination of Serum, Whole Blood and
Urinary Folate Concentrations . . 176
Determination of Urinary Deuterium-
Labeled Folate 180
Statistical Analysis 184
Vll

5 RESULTS 186
Effect of Supplemental Folic Acid on
Zinc Status 189
Plasma Zinc 190
Erythrocyte Zinc 193
Serum Alkaline Phosphatase 195
Erythrocyte Metallothionein 198
Serum Ferritin 201
Urinary Zinc 203
Effect of Level of Zinc Intake on Folate
Utilization 206
Serum Folate 2 07
Erythrocyte Folate 207
Urinary Folate 211
6 DISCUSSION 219
Effect of Supplemental Folic Acid on
Zinc Status 219
Effect of Level of Zinc Intake on Folate
Utilization 227
7 SUMMARY AND CONCLUSIONS 231
APPENDICES
A SUBJECT SELECTION SCREENING TOOL 235
B ZINC RESTRICTED METABOLIC DIET 246
C CONTRACT 251
REFERENCE LIST 253
BIOGRAPHICAL SKETCH 286
viii

LIST OF TABLES
Table page
2-1 Comparison of 1980 and 1989 Recommended Dietary
Allowances (RDA) for folate 21
2-2 Physiologic functions of zinc 88
4-1 Average nutrient content of three-day diet with and
without supplements 152
4-2 Composition of protein shakes 153
5-1 Mean weights (± SD) of subjects on zinc-restricted
and zinc-adequate diets 187
5-2 Mean (± SD) usual calorie, protein, folate and zinc
intakes of subjects on zinc-restricted or zinc-
adequate diets 187
5-3 Molar ratios of zinc to folic acid for each
treatment combination 189
5-4 Overall mean (± SD) values of response variables
used to assess the effect of supplemental folic
acid on zinc status in subjects consuming zinc-
restricted or zinc-adequate diets 191
5-5 Overall mean (± SD) values of response variables
used to assess the response to folic acid
supplementation in subjects consuming zinc-
restricted or zinc-adequate diets 209
5-6 Overall mean (± SD) deuterium-labeled folate
excreted expressed as a percentage of total folate
intake, total urinary folate and oral dose . . . 215
IX

LIST OF FIGURES
Figure page
2-1 Structure of folic acid and folic acid
derivatives 6
2-2 Folate-mediated one-carbon metabolism 8
4-1 Experimental design 148
4-2 Schematic of enzyme-linked immunosorbent assay used
for determination of erythrocyte metallothionein
concentrations 173
5-1 Effect of supplemental folic acid on plasma zinc in
subjects consuming zinc-restricted or zinc-adequate
diets 192
5-2 Effect of supplemental folic acid on erythrocyte
zinc in subjects consuming zinc-restricted or zinc-
adequate diets 194
5-3 Effect of supplemental folic acid on serum alkaline
phosphatase in subjects consuming zinc-restricted
or zinc-adequate diets 197
5-4 Effect of supplemental folic acid on erythrocyte
metallothionein in subjects consuming zinc-
restricted or zinc-adequate diets 200
5-5 Effect of supplemental folic acid on serum ferritin
in subjects consuming zinc-restricted or zinc-
adequate diets 2 02
5-6 Effect of supplemental folic acid on urinary zinc
in subjects consuming zinc-restricted or zinc-
adequate diets 2 05
5-7 Serum folate response to folic acid supplementation
in subjects consuming zinc-restricted or zinc-
adequate diets 2 08
x

5-8
Erythrocyte folate response to folic acid
supplementation in subjects consuming zinc-
restricted or zinc-adequate diets 210
5-9 Urinary folate response to folic acid
supplementation in subjects consuming zinc-
restricted or zinc-adequate diets 212
5-10 Urinary deuterium-labeled folate response to folic
acid supplementation in subjects consuming zinc-
restricted or zinc-adequate diets 214
5-11 Percent of oral labeled folic acid plus dietary
folate excreted as deuterium-labeled folate in
subjects consuming zinc-restricted or zinc-adequate
diets 216
5-12 Percent of total urinary folate excreted as
deuterium-labeled folate in subjects consuming
zinc-restricted or zinc-adequate diets 217
5-13 Percent of oral deuterium-labeled folic acid
excreted as deuterium-labeled folate in subjects
consuming zinc-restricted or zinc-adequate diets 218
xi

LIST OF ABBREVIATIONS
Abbreviation
Meanincr
AAS
atomic absorption spectrophotometry
BSA
bovine serum albumin
C
Centigrade
14C
carbon 14
cm
centimeter
[14C]-PteGlu7
carbon 14-labeled
pteroylglutamylhexaglutamate
d
day
DPBS
Dulbecco's phosphate-buffered saline
d2fa
deuterium-labeled folic acid
dL
deciliter
DNA
deoxyribonucleic acid
EDTA
ethylenediamine tetraacetic acid
ELISA
enzyme-linked immunosorbent assay
F
Fahrenheit
FA
folic acid
FBP
folate-binding protein
FIGLU
formimino-glutamic acid
FDA
Food and Drug Administration
g
gram
g
gravity
xii

GCMS
gas chromatography-mass spectrometry
h
hour
HC1
hydrochloric acid
HPLC
high-performance liquid chromatography
[ 3H]PteGlu
tritiated pteroylmonoglutamic acid
HPV-16
human papillomavirus 16
IU
International Units
kcal
kilocalories
kg
kilogram
L
liter
M
molar
mg
milligram
mL
milliliter
mol
mole
mmol
millimole
MRC
Medical Research Council
MT
metallothionein
N
normal
NaCl
sodium chloride
NaOH
sodium hydroxide
NaN3
sodium azide
ng
nanogram
nm
nanometer
oz
ounce
P
probability
pABG
para-aminobenzoyl glutamate
xiii

PBS
phosphate-buffered saline
ppm
parts per million
RDA
Recommended Dietary Allowances
RNA
ribonucleic acid
rpm
revolutions per minute
SD
standard deviation
THF
tetrahydrofoíate
HCi
microcurie
m g
microgram
flh
microliter
IIM
micromolar
limol
micromole
v/v
volume/volume
y
year
Zn
zinc
XIV

Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
ZINC STATUS RESPONSE TO FOLIC ACID SUPPLEMENTATION
AND THE EFFECT OF LEVEL OF ZINC INTAKE ON
FOLATE UTILIZATION IN HUMAN SUBJECTS
By
Gail P. Abbott Kauwell
August 1993
Chairperson: Dr. Lynn B. Bailey
Major Department: Food Science and Human Nutrition
Changes in zinc status in response to folic acid
supplementation and the effect of level of zinc intake on
folate utilization were evaluated in human subjects. Twelve
healthy men were randomly assigned to consume a zinc-
restricted diet (3.5 mg zinc/d) or an identical diet
supplemented with 11 mg of zinc as zinc sulfate (14.5 mg
zinc/d total) for 25 days. Half of the subjects in each group
received 800 nq/d deuterium-labeled folic acid. The remaining
subjects received a placebo. After an 80-day washout period,
the study was repeated with the folic acid/placebo treatments
reversed. Fasting blood samples and 24-hour urine collections
were obtained at corresponding time points during both study
periods.
Plasma zinc, erythrocyte zinc, urinary zinc, serum
alkaline phosphatase and erythrocyte metallothionein
xv

concentrations were measured to determine zinc status. No
differences in mean values within or between the zinc diet
groups were detected (P>0.05) for these response variables.
A significant (P<0.001) time by supplement by diet interaction
was detected for erythrocyte metallothionein; however,
significant differences in the change in erythrocyte
metallothionein concentrations due to the supplemental effect
were not detected (P>0.05) within the zinc diet groups. No
significant differences due to the effect of the supplement
were detected (P>0.05) within or between the zinc diet groups
for the remaining response variables. These data suggest that
short-term supplementation with 800 /¿g folic acid/d does not
adversely affect zinc status in healthy men.
To determine the effect of zinc intake on folate
utilization, total urinary, serum and erythrocyte folate
concentrations, and urinary excretion of deuterium-labeled and
unlabeled folate were determined. Differences in mean serum,
erythrocyte, total urinary or urinary deuterium-labeled folate
concentrations between subjects fed 3.5 or 14.5 mg zinc/d were
not detected (P>0.05). Similarly, significant differences in
the percent of total folate ingested or the percent of total
urinary folate excreted as deuterium-labeled folate were not
detected (P>0.05) between the groups. These data suggest that
utilization of supplemental folic acid in healthy men is not
influenced by the level of zinc intake.
xvi

CHAPTER 1
INTRODUCTION
The crucial roles of folate and zinc in protein and
nucleic acid metabolism and genetic expression have
contributed to the intense research interest centered around
these two nutrients. Changes in intake, bioavailability
and/or metabolism of either or both of these nutrients can
have deleterious effects in humans and animals. Understanding
the specific mechanisms of action and relationships between
zinc and folate is essential to improving our ability to make
efficacious nutritional recommendations and policies that will
enhance the health and well-being of the human race.
Zinc is an integral constituent of at least 60 different
enzymes (Cousins and Hempe, 1990) including deoxyribonucleic
acid (DNA) transferase, ribonucleic acid (RNA) polymerases,
alkaline phosphatase, gamma aminolevulinic acid dehydratase,
carbonic anhydrase, carboxypeptidase, alcohol dehydrogenase
and glutamic, lactic and malic acid dehydrogenases (Vallee and
Galdes, 1984). Of particular interest with regard to studies
investigating the relationship between zinc and folate
nutriture was the discovery (Silink et al., 1975) that bovine
hepatic folate conjugase is a zinc metalloenzyme. The fact
that zinc is a component of so many metalloenzymes explains
1

2
the multiplicity of physiologic functions attributed to this
nutrient. Examples of these functions include development and
maintenance of the body's immune system, prevention of lipid
peroxidation, metabolism of energy containing nutrients,
hormonal interactions, bone formation and the replication and
differentiation of cells (Cousins, 1985; Cunnane, 1988;
Hambidge et al., 1986).
"Folate" is the generic term used in reference to the
many different naturally occurring forms of pteroyl-
monoglutamic acid and pteroylpolyglutamic acid. These
compounds have nutritional properties and chemical structures
similar to their parent compound, folic acid. Folate is a
coenzyme for many one carbon reactions, and like zinc, is
required for cell replication. Specifically, folate coenzymes
are essential for the synthesis of the pyrimidine,
thymidylate, which is required for DNA synthesis. Other
metabolic processes requiring folate coenzymes include the
interconversion of serine and glycine, methionine synthesis,
histidine degradation, methylation of biogenic amines,
generation of formate and purine biosynthesis (Brody, 1991).
Recent studies suggest that supplemental folic acid may be
important in reducing the incidence of neural tube defects
(Bower and Stanley, 1989; Czeizel and Dudás, 1992; Medical
Research Council Vitamin Study Research Group, 1991; Milunsky
et al., 1989; Mulinare et al., 1988; Smithells et al., 1981);
modulating cancer risk in a variety of tissues including the

3
lung (Heimburger et al., 1987; 1988), cervix (Butterworth et
al., 1982; 1992a; 1992b), esophagus (Jaskiewicz et al., 1988)
and colon (Lashner et al., 1989); and reducing elevated
homocysteine levels, an emerging independent risk factor for
coronary heart disease (Kang et al., 1986; 1987).
Despite the potential benefits associated with folic acid
supplementation, and the fact that it is widely believed to be
nontoxic to humans under normal circumstances (DiPalma and
Ritchie, 1977), concern has been expressed regarding the
relative safety of ingesting additional amounts of this
nutrient. The major safety issues as outlined by Butterworth
and Tamura (1989) focus on: the potential harm to users of
anticonvulsant medications; interference with the diagnosis of
vitamin B12 deficiency; the possibility of other unexpected
adverse health effects; and interference with zinc absorption
or metabolism. For the most part, all but the latter safety
issue have been adequately addressed.
The question of whether supplemental folic acid disturbs
zinc absorption or metabolism, and thus zinc status, has been
examined by numerous researchers (Butterworth et al., 1988;
Fuller et al., 1987; Ghishan et al., 1986; Keating et al.,
1987; Krebs et al., 1988; Milne, 1989; Milne et al., 1984;
Mukherjee et al., 1984; Simmer et al., 1987; Tamura et al.,
1992; Wilson et al., 1983). The results from these studies
have been equivocal. Of these studies, dietary intake was
controlled in only one research design (Milne et al., 1984);

4
consequently, the results and conclusions of these studies may
vary depending on the adequacy and comparability of intake
among subjects. Discrepant results may also be attributed to
the diversity of research designs and protocols used, as well
as the lack of a satisfactory index of zinc nutriture.
The present study was designed to overcome some of the
limitations of previous investigations by evaluating the
response of male human subjects to 0 /¿g/d and 800 /¿g/d of
stable-isotopically labeled (deuterium) folic acid under
controlled dietary conditions. The objective was to determine
if supplemental folic acid affected zinc status in subjects
consuming zinc-adequate (i.e. 14.5 mg/d) or zinc-restricted
(i.e. 3.5 mg/d) diets. Additionally, the use of deuterium-
labeled folic acid provided the opportunity to study folate
utilization under conditions of marginal and adequate zinc
intakes. This is the first time that folate utilization using
a stable isotope has been studied under controlled dietary
conditions. Another significant aspect of this study was the
determination of erythrocyte metallothionein concentrations in
addition to traditional measures of zinc status. Unlike other
indices of zinc status, the concentration of erythrocyte
metallothionein has been shown (Grider et al., 1990) to
respond quickly to acute dietary zinc deficiency and to
supplementation and may therefore be a more reliable and
sensitive indicator of zinc status.

CHAPTER 2
REVIEW OF THE LITERATURE
Folate
Chemistry
Folic acid, or pteroylglutamic acid (2-amino-4-hydroxy-6-
methyleneaminobenzoyl-L-glutamic acid pteridine), consists of
three distinct subunits: a pteridine moiety, para-aminobenzoic
acid and glutamic acid (Figure 2-1). The pteridine moiety is
linked by a methylene bridge to para-aminobenzoic acid, which
is then joined by peptide linkage to glutamic acid. Although
mammals can synthesize all the components of this vitamin,
they are not capable of de novo biosynthesis because they lack
the enzyme needed for coupling the pteridine molecule to para-
aminobenzoic acid (Cooper, 1984) . De novo synthesis of
folates does occur in plants and bacteria.
Folic acid is yellow and has a molecular weight of 441.4.
It is only slightly soluble in water in the acid form, but is
guite soluble in the salt form (Brody, 1991) . Folic acid
occurs only rarely in nature, although it is the form most
commonly found in vitamin supplements and is the parent
compound of the naturally occurring folate vitamin forms.
5

CTi
Figure 2-1. Structure of folic acid and folic acid derivatives. a) Folic acid;
b) Reduced folate pentaglutamate and possible one-carbon moieties.

7
Compounds having nutritional properties and chemical
structures similar to those of folic acid have been assigned
the generic descriptor "folate." Natural folates occur in the
reduced 7,8-dihydro- and 5,6,7,8-tetrahydro- forms. Folates
may also contain one-carbon substituent groups (Figure 2-1).
These include: 5-methyl-, 10-formyl-, 5-formyl-, 5,10-
methenyl-, 5,10-methylene-, and 5-formimino-tetrahydrofoíate
(Brody, 1991). Most of these naturally occurring folates can
be degraded by heat, oxidation and/or ultraviolet light,
although some tetrahydrofolate derivatives are more stable
than others. For example, N5-methyl-tetrahydrofoíate is
relatively heat stable, but is destroyed by acid, as is N5'10-
methylene- tetrahydrofolate (Krumdieck, 1990; O'Brion et al.,
1975). For the most part, dietary folates occur in the form
of pteroylpolyglutamates (Halsted, 1979) containing three to
seven glutamic acid residues (Figure 2-1). Although the
principal pteroylpolyglutamate in food is N5-methyl-
tetrahydrofoíate, over 150 different forms of folate have been
reported to exist (Sauberlich, 1987).
Metabolism
Absorption. The first stage of intestinal folate
absorption involves the hydrolysis of pteroylpolyglutamates to
pteroylmonoglutatmes (Butterworth et al., 1969). Hydrolysis
is performed by the gamma glutamylcarboxypeptidases, commonly
grouped together and referred to as "folate conjugase".

COOH
chch2ch2cooh
—NH
(a)
Glu-y-Glu-y-Glu-y-Glu-y-Glu
Derivatives
R
(b)
5 - Formyl
10 - Formyl
5 - Formimino
10 - Methenyl
5,10 - Methylene
5 - Methyl
-CHO
-CHO
- CH = NH
= CH -
-CH-
-ch3
Figure 2-2. Folate-mediated one-carbon metabolism (Brody, 1991).
oo

9
Folate conjugase successively cleaves the gamma glutamyl
peptide bonds of pteroylpolyglutamates to the monoglutamate
form. Reisenauer and Halsted (1987) have estimated that the
activity of human jejunal brush border folate conjugase is
sufficient enough to preclude this step from limiting the rate
of absorption. However, research showing that the
bioavailability of folate polyglutamates is considerably less
than that of folate monoglutamates (Gregory et al., 1991)
suggests that hydrolysis of dietary polyglutamyl folates is a
rate-limiting step in absorption.
Two separate folate conjugases have been identified in
the human jejunum (Reisenauer et al., 1977). One of these is
soluble and located intracellularly, while the other is
membrane-bound and concentrated in the brush border. These
folate conjugases differ with respect to molecular weight, pH
optima and inhibition characteristics.
Brush border folate conjugase is a zinc-dependent
exopeptidase that sequentially cleaves polyglutamates to
monoglutamates and has a pH optimum near neutrality (Chandler
et al., 1986; Reisenauer et al., 1977). Chronic alcohol
consumption (Naughton et al., 1989; Reisenauer et al., 1989),
zinc deficiency (Tamura et al., 1978), mucosal damage
(Halsted, et al., 1986) or exposure to naturally occurring
inhibitors in food (Bhandari and Gregory, 1990) may exert a
negative effect on brush border folate conjugase activity and
subsequent folate absorption. Interestingly, brush border

10
folate conjugase activity does not appear to be affected by
the aging process (Bailey et al., 1984a). In contrast to
brush border folate conjugase, intracellular folate conjugase
is found in the lysosomes of intestinal cells and functions as
an endopeptidase with an acidic pH optimum (Wang et al.,
1986). The role of intracellular folate conjugase is unknown;
however, Wang et al. (1986) have proposed that its role in
cellular folate metabolism is unrelated to the digestion of
dietary folates.
Transport of folate across the brush border membrane is
the second stage of folate absorption. This is a complex
process that has not been entirely elucidated. Transport
occurs mainly in the jejunum and is believed to involve a
carrier system that is saturable, pH dependent (i.e. pH
optimum of 6.0), energy dependent and sodium dependent (Rose
et al., 1978; Said et al., 1987; Selhub et al., 1983). This
carrier-mediated system is thought to include a folate-binding
protein located in the jejunal brush border membrane. This
folate-binding protein is either the transport protein or is
an important component of the intestinal transport system
(Reisenauer, 1980). Data from competitive inhibition studies
(Said, et al., 1987; Selhub et al., 1984) support the
conclusion that the transport system may be the same for all
monoglutamate forms of folate. The carrier-mediated system
becomes saturated at luminal concentrations of 10-20 ¿¿M
(Selhub et al., 1984). In addition to carrier-mediated

11
transport, nonsaturable absorption involving passive diffusion
may also occur (Selhub et al., 1983). Absorption of folate by
this process occurs linearly at much higher folate
concentrations.
Decreased hydrolysis of folate polyglutamates and/or
interference with the transport of folate mononglutamates
across the intestinal brush border membrane can inhibit folate
absorption. Studies concerning the impact of dietary
components (i.e. conjugase inhibitors, dietary fiber, etc.);
dietary composition; the actual forms of folate ingested;
nutritional status; alcohol consumption; and the effect of
nutrient interactions on the extent of folate absorption have
been reviewed by Bailey (1988). The conclusions drawn from
these studies are often conflicting, confirming the need for
further research employing more consistent and appropriate
experimental designs and methodologies. A case in point is
the nutrient interaction that is hypothesized to occur between
zinc and folic acid.
Nondietary factors that may interfere with folate
absorption include altered gastrointestinal function and the
use of medications. Damage to the intestinal epithelial
cells, such as that which occurs in Crohn's disease (Hoffbrand
et al., 1968), celiac disease (Halsted et al., 1977; 1978;
Hoffbrand et al., 1970) and tropical sprue (Corcino et al.,
1976; Halsted, 1980b), or changes in the intraluminal
environment due to achlorhydria (Russell et al., 1986), may

12
adversely affect folate absorption. Medications that may
interfere with folate absorption include sulfasalazine
(Franklin and Rosenberg, 1973) and diphenylhydantoin (Gerson
et al., 1972; Hoffbrand and Necheles, 1968; Rosenberg et al.,
1968). Antacids and histamine receptor antagonists may also
adversely affect folate absorption by increasing the
intestinal pH to levels that exceed the optimum for folate
conjugase activity and carrier-mediated transport (Russell et
al., 1979). Folate analogs such as methotrexate, trimethoprim
and pyrimethamine, which are used primarily as dihydrofolate
reductase inhibitors, also suppress intestinal folate
absorption (Selhub et al., 1983).
Transport. Upon entry into the intestinal mucosal cells,
reduced dietary folates are converted, for the most part, to
N5-methyl-tetrahyrofoíate monoglutamate (Cooper, 1984; Pratt
and Cooper, 1971) . This is the predominant form in the portal
circulation under normal physiologic conditions. Reduction
and methylation of folic acid can also occur, although much of
this form of the vitamin appears in the portal blood unchanged
(Cooper, 1984) .
Serum folate binding proteins are involved with the
transport and distribution of folates to the liver and other
tissues. Two types of serum folate-binding proteins have been
identified (Wagner, 1985), one is specific with a high-
affinity binding capacity; the other, which is thought to be
albumin (Solimán and Olesen, 1976), is nonspecific, with a

13
low-affinity binding capacity. Approximately two-thirds of
serum folate is protein-bound, with the majority bound to
albumin.
A small amount (10 to 20%) of absorbed folates in reduced
substituted form is taken up by the liver on the first pass,
while the majority is distributed to other tissues (Steinberg,
1984). Since the liver is capable of secreting N5-methyl-
tetrahydrofoíate monoglutamate into the bile, the potential
exists for continual enterohepatic circulation of folate
(Steinberg, 1984). This idea is not supported, however, by
the results of an isotopic labeling study (Krumdieck et al.,
1978) that examined the routes of folate excretion following
administration of [14C]folic acid to a human subject. The
results of this study suggested that a significant portion of
biliary folate was not reabsorbed.
Contrary to the fate of reduced substituted forms of
folate entering the portal hepatic vein, folic acid entering
this vessel is taken up almost exclusively by the liver. Some
of this folic acid is used for polyglutamate synthesis, and
the rest is converted to N5-methyl-tetrahydrofoíate and
secreted into the bile (Lavoie and Cooper, 1974) . When a
large oral dose of folic acid is administered, most of it is
recovered in the urine in its original form (Brody, 1991) .
Tissue deposition and storage. Transport of folate
across cellular membranes is an energy reguiring process,
where anion gradients may serve as the energy source

14
(Yang et al., 1984). The N5-methyl-tetrahydrofoíate
transported across cell membranes must be converted to the
polyglutamate form to assume its role as a functional
coenzyme. Polyglutamates cannot cross biological membranes,
so polyglutamation also serves to trap folates inside cells at
concentrations that are one to two orders of magnitude greater
than those of the extracellular fluid. Conversion to the
polyglutamate form reguires methionine synthetase, a vitamin
B12-dependent enzyme, and pteroylpolyglutamate synthetase.
Methionine synthetase is responsible for removing the methyl
group, and pteroylpolyglutamate synthetase is responsible for
the addition of glutamyl residues. The resulting
tetrahydrofoíate polyglutamates usually contain between four
to seven glutamyl residues.
The total body folate content is in the range of 5 to 10
mg. The liver is considered to be the primary storage organ
containing about 50% of the total body folate (Herbert and
Colman, 1988). A variety of folate derivatives with various
chain lengths is present in the liver and other tissues, with
one particular species usually dominating (Shane, 1990).
Alterations in the distribution of folate polyglutamates have
been observed under certain physiological and nutritional
conditions. For example, longer chain length folates tend to
accumulate under circumstances whereby cellular folate
concentrations are diminished (i.e. folate deficiency;
methionine deficiency; etc.) (Cook et al., 1987). The

15
significance of this particular change has not been
determined, although it has been reasoned that extending the
chain length does not affect the short-term regulation of one-
carbon metabolism since this change occurs very slowly (Shane,
1990).
Utilization of endogenous folate pools may provide a
mechanism for maintaining normal folate supplies to critical
tissues during acute or chronic folate deprivation. A small
pool of intracellular folate monoglutamates is thought to be
available for this purpose. As deprivation continues, the
liver and other "storage tissues" (i.e. the kidney) may
generate folate monoglutamates through the action of
folylpolyglutamyl hydrolase on folate polyglutamates.
Additionally, the amount of folate presented to the liver is
thought to decrease, thereby reducing hepatic monoglutamate
uptake and new polyglutamate synthesis. Thus, over time,
hepatic folate stores decline.
Folate derived from dying cells may be another potential
source of this nutrient during periods of prolonged
deprivation (Steinberg, 1984). For example, erythrocytes
could make an important contribution to folate homeostasis
because of their high folate content and limited life span. A
study (Hillman et al., 1982) of heat-treated, senescent
erythrocytes has shown that labeled folate can be recovered in
the liver and bile. Therefore, folate salvaged from expired

16
erythrocytes may be redistributed through the enterohepatic
circulation.
Catabolism and excretion. Urinary excretion of intact
folates in well-nourished humans consuming a nutritionally
adequate diet is approximately 5-40 ¿xg per day (Herbert,
1987). The folate compounds identified in the urine include
N5-methyl-tetrahydrofolate, N10-formyl-tetrahydrofolate, N5,10-
methenyl-tetrahydrofoíate and N5-formyl-tetrahyrofoíate
(Chanarin, 1979) . Excretion of intact folates is limited due
to renal tubular reabsorption of this nutrient and the degree
of catabolism that occurs in vivo. Products of folate
catabolism occurring in the urine include pteridines, para-
acetamidobenzoyl glutamate and para-acetamidobenzoate (Anon.,
Nutrition Reviews, 1990; Chanarin, 1979). The latter two
catabolites appear to be the major excretory products,
suggesting that the principal route of catabolism occurs by
cleavage of the C9-N10 bond. The cleavage mechanism has been
the subject of much controversy. Current in vitro studies
suggest the existence of more than one mechanism for folate
cleavage (Anon., Nutrition Reviews, 1990).
Folate can also be excreted in the feces. The amount
excreted in the feces has been reported, in some cases, to be
higher than the estimated dietary intake, presumably because
of folate synthesis by colonic bacteria (Brody, 1991).
Consequently, fecal folate excretion is not considered a
reliable index of folate metabolism.

17
Biochemical Functions
Reduction of the pteridine ring to the tetrahydro-form,
elongation of the glutamyl side chain and acquisition of one-
carbon units at the N5 and/or N10 positions of the pteridine
ring system must occur in order to produce the intracellular,
metabolically active forms of folate. Pteridine ring
reduction is accomplished by the cytosolic enzyme, 7,8-
dihydrofoíate reductase. Folic acid and dihydrofolic acid can
serve as substrates for this enzyme. Reduction to the
tetrahydrofoíate form, and demethylation, in the case of N5-
methyl-tetrahydrofoíate, must occur before elongation of the
side-chain or acquisition of one-carbon substituent groups can
proceed. Side-chain elongation is achieved by the action of
folate polyglutamate synthetase. This is an adenosine
triphosphate (ATP)-dependent enzyme which joins glutamyl
residues to the vitamin by peptide bonds in an oligo-gamma-
glutamyl linkage. Once these steps have been accomplished,
one-carbon units at the oxidation level of formate,
formaldehyde or methanol can be added. The major contributor
of one-carbon groups is serine, although formimino-glutamate
(FIGLU), formylglutamate and formate may also serve as the
single-carbon source. The resulting tetrahyrofoíate
derivatives (N5-methyl-, N5-formyl-, N10-formyl-, N5,10-
methylene-, N5,10-methenyl- and N5-f ormimino-) are used as
coenzymes and serve as donors and acceptors of one-carbon
units in a host of reactions involved in amino acid and

18
nucleotide metabolism. Examples of specific biochemical
functions of folate include: purine and pyrimidine
biosynthesis; the generation and utilization of formate; and
amino acid interconversions including the catabolism of
histidine to glutamic acid, the interconversion of serine and
glycine and the conversion of homocysteine to methionine.
These reactions are collectively referred to as one-carbon
metabolism (Brody, 1991; Krumdieck, 1990).
The cyclical nature of intracellular folate metabolism is
exemplified by tracing the potential fates of tetrahydrofoíate
polyglutamates (see Figure 2-2). In mammalian tissues, it
appears that the major cycle of one-carbon metabolism involves
the conversion of tetrahydrofolate polyglutamate to N5,10-
methylene-tetrahydrofolate by serine hydroxymethyl-
transferase. This one-carbon derivative is a key intermediate
that can be: oxidized to N5'10-methenyl-tetrahydrofoíate (by
N5,10-methylene-tetrahydrofoíate dehydrogenase) for use in de
novo synthesis of purines; used for the synthesis of
thymidylate (by thymidylate synthetase); or reduced to N5-
methyl-tetrahydrofoíate (by N5'10-methylene-tetrahydrofoíate
reductase) for use in the biosynthesis of methionine. The
latter two reactions are irreversible reactions that compete
for the N5'10-methylene-tetrahydrofoíate intermediate.
Regardless of the metabolic path into which the N5'10-
methylene-tetrahydrofoíate is directed, its eventual fate
(under normal physiologic conditions) is the loss of its one-

19
carbon substituent group, and its return to either dihydro- or
tetrahyro-folate polyglutamate (Brody, 1991; Krumdieck, 1990).
Regulation of one-carbon transfer reactions is highly
complex. Changes in the concentration of substrates, products
and cofactors serve as a means for guickly activating or
inhibiting certain folate-reguiring reactions. An example of
this complex regulatory process is one that has been given
much attention and involves the fate of N5'10-methylene-
tetrahydrofoíate. As noted above and depicted in Figure 2-2,
this intermediate can be used to synthesize several different
products. Methylene-tetrahydrofoíate reductase, the enzyme
catalyzing the conversion of N5'10-methylene-tetrahydrofoíate
to N5-methyl-tetrahydrofoíate, is very highly regulated. When
insufficient amounts of methionine are available, inhibition
of the reductase enzyme is relaxed allowing for increased
production of methionine from homocysteine. The methionine
produced by this reaction is converted to S-adenosyl
methionine, which then serves as a methyl donor to form
various methylated products. However, when S-adenosyl
methionine accumulates, methylene- tetrahydrofoíate reductase
activity is curtailed through feedback inhibition. Methylene-
tetrahydrof oíate reductase is also inhibited by the
accumulation of dihydrofolate polyglutamates, a situation that
occurs when thymidylate synthesis increases. Inhibition of
this enzyme under these circumstances allows for continued

20
commitment of folate to nucleotide biosynthesis (Brody, 1991;
Krumdieck, 1990).
In addition to the regulatory influences posed by changes
in the concentration of substrates, products and cofactors,
evidence for a second type of regulation has been reviewed by
Krumdieck (1990). This newly proposed regulatory process is
thought to be a slow-response mechanism based on covalent
modification of the polyglutamyl chain length. The idea that
a second form of regulation existed was generated from in vivo
studies demonstrating that the glutamyl chain length of
cellular folates varies in response to physiological or
pathological stimuli (i.e. developmental age, tissue
regeneration, infection, starvation, alcohol ingestion and
methionine-choline deficiency) that alter the steady-state
eguilibrium of one-carbon metabolism. Additionally, the
finding that chain-length distribution of folate
polyglutamates differs from organ to organ in the same species
adds credibility to this concept because it is unlikely that
the requirements for one-carbon transfer reactions are the
same for all organs. Since changes in polyglutamate chain
length are slow to develop and only respond to persistent
stimuli, it is thought that the purpose of this regulatory
mechanism is to correct prolonged deviations rather than brief
fluctuations that may occur in an otherwise steady-state
(Krumdieck, 1990).

21
Table 2-1. Comparison of 1980 and 1989 Recommended Dietary
Allowances (RDA) for folate.
Category/age
1980 RDA*
Mg/d
1989 RDA**
Mg/d
Infants
0.0-0.5
30
25
0.5-1.0
45
35
Children
1-3
100
50
4-6
200
75
7-10
300
100
Men
11-14
400
150
15-18
400
200
19-24
400
200
25-50
400
200
51+
400
200
Women
11-14
400
150
15-18
400
180
19-24
400
180
25-50
400
180
51+
400
180
Pregnant women
800
400
Lactating women
First 6 months
500
280
Second 6 months
500
260
*Data from Food and
**Data from Food and
Nutrition Board, 1980.
Nutrition Board, 1989.
Recommended Dietary Allowances for Folate
The 1989 Recommended Dietary Allowances (RDA) for folate
(Food and Nutrition Board, 1989a) represent a significant

22
reduction from the previously recommended amounts (Food and
Nutrition Board, 1980) for all age, sex and special population
categories (see Table 2-1). The rationale for the reduction
of the RDA for folate was based on two types of data: the
quantity of folate required to invoke established physiologic
responses or replace daily losses, after adjusting for
bioavailability and individual variability; and estimates of
dietary folate consumption related to the prevalence of
deficiency in population groups (Bailey, 1990a; Bailey, 1992).
Although some researchers (Herbert, 1987; Reisenauer and
Halsted, 1987) support the view that the existing data are
sufficient to warrant a reduction in the recommended level of
folate intake, others (Bailey, 1992; Sauberlich et al., 1987)
cast doubt on the appropriateness of the new RDA. In a
critique of the research used to establish the 1989 RDA,
Bailey (1992) submits that this new RDA may be insufficient to
provide an adequate margin of safety for specific populations.
The rationale for her conclusion is based on the following
facts: 1) in some cases, the population surveys used to make
conclusions about folate requirements were not specifically
designed to assess folate status; 2) the studies used to
support the 1989 RDA were not always comparable in terms of
the forms of folate used (i.e. synthetic folic acid versus
dietary folate); 3) the correction factor used to account for
folate bioavailability is only an estimate based on limited
data from studies employing different experimental

23
methodologies; and 4) the food composition tables for folate
are incomplete and may not accurately reflect the actual
amount of folate ingested or available. This final problem is
due to the fact that estimates of the folate content of foods
vary due to the method of analysis; the type of food consumed;
the method of food processing, preparation, storage and
handling; and the effect of nutrient interactions.
Effects of High Doses of Folic Acid
Folate is considered nontoxic in small doses as well as
in doses that exceed the RDA several hundredfold (Brody, 1991;
Butterworth and Tamura, 1989; DiPalma and Ritchie, 1977;
Herbert and Colman, 1988). The water soluble nature of
folate, and the apparent reguirement for attachment to
saturable folate-binding proteins as a condition for storage,
probably account for the relative nontoxicity of this vitamin.
These features provide a mechanism for rapid excretion of
folate when the serum- and tissue-binding capacity are
exceeded (Herbert and Colman, 1988).
Adverse effects of supplemental folic acid were not noted
in adult humans receiving 400 mg/d for 5 months, or after 10
mg/d for 5 years (Brody, 1991). Similarly, no adverse effects
were reported in a group of well-nourished women ingesting an
oral folic acid supplement (10 mg/d) for 4 months (Butterworth
et al., 1982). Contrary to these reports, insomnia and
irritability were noted in subjects consuming folic acid in

24
the amount of 15 mg/d (Hunter et al., 1970). This effect has
not been confirmed in subsequent studies (Alhadeff et al.,
1984) .
Despite the apparent relative safety of supplemental
doses of folic acid in normal adult subjects, large doses of
this nutrient (5 mg/d) (Brody, 1991) can have deleterious
effects when administered to individuals with undiagnosed
and/or untreated pernicious anemia. Administration of folic
acid supplements obscures the diagnosis of pernicious anemia
by correcting the macrocytic anemia associated with this
condition, but fails to alleviate the concurrent neurologic
lesions. Conseguently, neurologic damage progresses
unchecked. Assessment of vitamin B12 status prior to the
initiation of folic acid supplementation can prevent this
potentially harmful outcome.
Large doses of folic acid (100 or more times the RDA) may
also be harmful to individuals with epilepsy who are receiving
continuous phenytoin therapy. Supplementation at this level
may precipitate convulsions (Herbert, 1987). Lower doses of
folic acid (0.1 to 1 mg/d) have not been shown to impair
seizure control (Roe, 1989).
It has been suggested that high intakes of folic acid may
interfere with zinc absorption and/or metabolism (Ghishan et
al., 1986; Milne, 1989; Milne et al., 1984; Mukherjee et al.,
1984; Simmer, et al., 1987; Wilson et al., 1983). This topic

25
will be addressed in more detail in a subsequent section of
this dissertation.
In contrast to some of the potentially harmful effects of
supplemental folic acid, there is evidence to suggest that
additional amounts of this vitamin may be beneficial in
modulating cancer risk in a variety of tissues including
cervical dysplasia (Butterworth et al., 1982; Butterworth et
al., 1992a; Butterworth et al., 1992b), bronchial metaplasia
(Heimburger et al., 1987; Heimburger et al., 1988), and
neoplasms associated with the esophagus (Jaskiewicz et al.,
1988) and colon (Lashner, et al., 1989). Supplemental folic
acid has also been associated with a reduction in the risk of
occurrence/recurrence of neural tube defects (Bower and
Stanley, 1989; Czeizel and Dudás, 1992; Medical Research
Council Vitamin Study Research Group, 1991; Milunsky et al.,
1989; Mulinare et al., 1988; Smithells et al., 1981).
Sources/Distribution and Stability of Folate in Foods
Although there is a need for more complete data
concerning the folate content and bioavailability of foods, a
considerable amount of information concerning food sources of
folate and the forms of the vitamin present in these foods has
been generated. The most concentrated sources of folate
include liver, citrus fruits, raw broccoli and dark green
leafy vegetables such as raw spinach. Cooked greens,
including spinach, turnip and mustard greens also contain

26
folate, but in lower amounts. The reduction in folate content
due to cooking occurs primarily due to leaching of the vitamin
into the cooking water, although thermal and oxidative
destruction may also occur. Legumes are another highly
concentrated source of folate; however, it must be recognized
that they contain heat-activated conjugase inhibitors which
may decrease the availability of folate polyglutamates. This
is true for other folate containing foods such as cooked
cabbage. Other good sources of folate include fortified
breakfast cereals. Most fortified cereals supply at least 25%
of the United States Recommended Daily Allowance for this
nutrient. In addition to the more concentrated sources of
dietary folate, it is important to consider the potential
contribution that foods containing modest amounts of folate
can have on total intake if these foods are consumed
frequently and/or in large quantities. Examples of these
include ground beef and whole-grain breads and cereals, as
well as tea (Bailey, 1990a; Bailey, 1992).
Very little folic acid is naturally present in food,
although this is the form used in food fortification because
of its exceptional stability characteristics (Gregory, 1989).
Folates in food occur almost exclusively in reduced
polyglutamate forms, with the predominant forms being N5-
methyl-tetrahydrofolate, N10-formyl-tetrahydrofoíate and
unsubstituted tetrahydrofoíate polyglutamates of varying
chain-lengths (Gregory, 1989). Reduced folates, with the

27
exception of N5-formyl- tetrahydrofoíate, are potentially more
labile and subject to oxidation under aerobic conditions,
especially in the presence of heat, light and/or metal ions
(Cooper et al., 1978; Gregory, 1989). The actual stability of
folate in foods has been difficult to determine, with reported
losses of folate activity being highly variable. This
variability may be due to differences in oxygen exposure
during cooking, the amount of cooking water present and/or
intrinsic differences in ascorbic acid content of different
foods (Gregory, 1989). When partial or full oxidation of
tetrahydrofoíate derivatives occurs, they may be further
catabolized to yield compounds that are physiologically
inactive with respect to human nutrition. If these compounds
are capable of supporting growth responses in microorganisms
used to measure the folate content of foods, overestimation of
the biologically useful folate content of foods could occur.
Assessment of Folate Status
The nutritional status of an individual indicates the
degree to which physiological needs for nutrients are being
met. Nutritional status is often evaluated using dietary
history and intake data, and biochemical and clinical
parameters.
Dietary intake. Estimation of the dietary folate content
of foods and the evaluation of folate intake by individuals
and population groups is complicated by many factors.

28
Researchers and practitioners need to recognize the
limitations of the available folate databases and the problems
inherent in collecting dietary intake and food freguency data
when using this information to evaluate nutritional status.
Examples of problems associated with folate food composition
data include: the use of different analytical technigues to
determine the folate content of foods; missing information for
foods that have not been analyzed; failure to incorporate
information on the bioavailability of folate from various
foods; and the effect of different methods of food
preparation, storage and handling (Bailey, 1990a; Bailey,
1992; Gregory, 1989).
The accurate recording and evaluation of individual
dietary intake and food freguency data is also problematic.
For example, it is often difficult for individuals to
correctly remember the types and/or amounts of foods they have
consumed. Food selection may also be affected as a result of
being asked to record food intake. Factors such as age, mood,
intelligence, attention span, freguency of exposure to the
process and perceived importance of the information can also
affect the ability to recall and/or record food intake
information (Blake et al., 1989; Karvetti and Knuts, 1985;
Lissner et al., 1989).
Biochemical and clinical measures of folate status. The
progressive changes in biochemical and clinical parameters
occurring during folate depletion were determined and

29
described by Herbert (1962) in a depletion study in which he
consumed a folate-deficient diet for four months. As a result
of this experiment, Herbert categorized folate depletion into
four stages: early negative folate balance; nutrient
depletion; biochemical nutrient deficiency; and clinical
nutrient deficiency.
During the initial stage of folate depletion, serum
folate values become depressed. Serum folate concentrations
are very responsive to recent dietary intake, with levels
becoming low after consuming a folate deficient diet for only
two to three weeks. A value of less than 3 ng/mL is
indicative of negative folate balance. Values from 3 to 6
ng/mL represent marginally negative folate balance, with
normal serum folate values ranging from 6 to 25 ng/mL. The
sensitivity of serum folate to recency of dietary folate
intake make it a poor indicator of the degree of folate
deficiency. To determine the severity of folate depletion,
parameters indicative of body stores and changes in metabolic
function need to be measured concurrently (Herbert, 1987;
1990).
The second stage of folate depletion is characterized by
a decline in body folate stores. The largest amount of folate
is stored in the liver. When folate intake is deficient,
normal liver folate stores can be maintained for approximately
four months. Coincidentally, the average life span of normal
erythrocytes is four months as well. Since the erythrocyte

30
folate concentration is actually a measure of folate status at
the time the erythrocyte was synthesized, erythrocyte folate
concentrations usually parallel liver folate stores. This
relationship, combined with the greater accessibility of
erythrocytes, has resulted in the routine use of erythrocyte
folate concentration as a means for determining tissue stores.
Thus, stage two is identified by measuring erythrocyte folate
concentrations, which decrease to less than 140 ng/mL when
tissue stores are low. Erythrocyte folate values between 140
to 160 ng/mL suggest marginal depletion, and concentrations
above 160 ng/mL indicate normal folate status (Herbert, 1987;
1990).
Severe depletion of folate stores, characterized by
impaired folate-dependent metabolism, represents the third
stage of depletion. Folate coenzymes are required for many
metabolic functions including the synthesis of thymidylate.
A severe folate deficiency retards the synthesis of this
nucleotide, and thus interferes with DNA synthesis.
Subsequently, deranged DNA synthesis results in morphologic
changes in erythrocytes and neutrophils. Neutrophils become
hypersegmented because more constriction bands are formed,
constricting the DNA into more lobes. Hypersegmentation of
neutrophils is identifiable early in the course of impaired
metabolism because of the short half-life of these cells. The
criterion used to define hypersegmentation of neutrophils is
a lobe average equal to or greater than 3.5 lobes per cell

31
(Herbert, 1987; 1990). This hematologic alteration is thought
to be a sensitive screening tool although it is an unreliable
indicator of folate status during pregnancy (Herbert et al.,
1975) and in a small percentage (1%) of otherwise normal
adults with congenital polymorphonuclear leukocyte
segmentation (Herbert, 1964) .
The morphologic changes manifested in erythrocytes during
the third stage of depletion include an increase in size and
conversion to an oval shape. At this stage, morphologic
damage is confined to the youngest erythrocytes, which are not
yet in the majority because of their longer life span, so
macroovalocytosis and an increase in the mean corpuscular
hemoglobin concentration are not evident. Other changes that
occur during stage three of depletion include a further
decline in liver and erythrocyte folate concentration
(Herbert, 1990).
The final depletion stage is clinically manifested as
normochromic, macrocytic anemia. At this point, the majority
of the erythrocytes are larger than normal resulting in an
increase in the mean corpuscular volume. The hemoglobin level
also declines due to decreased erythropoiesis. Further
decreases in liver and erythrocyte folate concentrations may
also be noted (Herbert, 1990).
The type of anemia caused by a folate deficiency is
clinically indistinguishable from that caused by a deficiency
of vitamin B12. Anemia due to vitamin B12 deficiency is

32
thought to be caused by a secondary deficiency of folate. It
has been hypothesized that this secondary deficiency develops
as a result of trapping folate in the N5-methyl-
tetrahydrofoíate form.
The predominant form of folate in the serum, liver, and
most likely, other body storage depots, is N5-methyl-
tetrahydrofoíate. Another source of this form of folate is
that which is synthesized from N5,10-methylene-tetrahydrofoíate
when control of N5,10-methylene-tetrahydrofoíate reductase is
relaxed. Conversion of N5-methyl-tetrahydrofoíate to
tetrahydrofoíate is catalyzed by the vitamin B12-dependent
enzyme methionine synthetase. When vitamin B12 is present in
adequate amounts, the methyl group from N5-methyl-
tetrahydrofoíate is removed resulting in the regeneration of
tetrahydrofoíate. This reaction is important for two reasons:
1) there is no other mechanism for regenerating
tetrahydrofoíate in human cells; and 2) tetrahydrofoíate is a
precursor to many other folate coenzymes, including N5,10-
methylene-tetrahydrofoíate, which is ultimately used for DNA
synthesis. According to the "methyl-folate trap" hypothesis
(Herbert and Zalusky, 1962), folate gets "trapped" in the N5-
methyl-tetrahydrofolate form when vitamin B12 is deficient.
This form of folate is not usable for any other folate-
requiring reactions, including the production of thymidylate.
Inadequate production of thymidylate interferes with the
synthesis of DNA and eventually causes the development of a

33
macrocytic anemia that is hematologically identical to that
caused by folate deficiency (Herbert, 1990).
The need to properly identify the underlying cause of
macrocytic anemia prior to initiating treatment is essential
in order to avoid deleterious consequences. For this reason,
concurrent assessment of vitamin B12 and folate status is
recommended. As an alternative, the deoxyuridine suppression
test can be used to distinguish between macrocytic anemia
caused by a deficiency of folate, vitamin B12 or both of these
nutrients. As previously discussed, in the absence of folate
and/or B12 deficiency, deoxyuridine is converted to thymidine
by thymidine synthetase. Thymidine is subsequently
incorporated into DNA. The deoxyuridine suppression test is
an in vitro test that measures the activity of thymidine
synthetase by comparing the amount of unlabeled versus labeled
thymidine incorporated into DNA, when labeled thymidine is
added to bone marrow cells or phytohemagglutinin-stimulated
lymphocytes. Labeled thymidine incorporation is suppressed
when folate and vitamin B12 are present in adequate amounts.
Conversely, when a deficiency of folate or vitamin B12 is
present, the conversion of deoxyuridine to thymidine is
reduced and more labeled thymidine is incorporated into DNA.
When suppression of labeled thymidine is low, the underlying
cause can be determined by adding N5-methyl-tetrahydrofoíate
to the medium. If folate is deficient, the suppression rate
will be increased when this coenzyme form is added. If the

34
cause is due to a vitamin B12 deficiency, the addition of N5-
methyl-tetrahydrofoíate will have no effect (Brody, 1991;
Herbert, 1990).
In the past, problems associated with sample collection
and processing techniques have limited the application of the
deoxyuridine suppression test to laboratory research settings.
The development of a method using a whole blood (O.lmL)
lymphocyte culture instead of cultures of separated
lymphocytes has made this test more suitable for use in
clinical laboratories, and perhaps, survey studies (Das et
al., 1980); however, as a measure of folate status, it is no
more informative than erythrocyte folate concentration (Tamura
et al., 1990).
The histidine load test is another method that can be
used to assess folate status. Formimino-glutamate, a product
of histidine catabolism, is further catabolized to glutamate,
ammonia and carbon dioxide by tetrahydrofoíate formimino-
transferase, a folate-requiring enzyme. When folate is
deficient, urinary excretion of FIGLU is elevated, with
excretion being particularly high after an oral dose of L-
histidine. Despite the sensitivity of this test to folate
deficiency, it is not specific for folate deficiency since a
deficiency of vitamin B12 will also cause an increase in FIGLU
excretion. Additionally, FIGLU excretion is affected by other
diseases and physiological conditions, so its application is
usually limited to scientific studies (Brody, 1991).

35
Measurement of plasma homocysteine has recently been
proposed as a new method for assessing folate status. Under
normal conditions, approximately 50% of the available
homocysteine is converted to methionine by the folate-vitamin
B12-requiring remethylation reaction catalyzed by methionine
synthetase (see Figure 2-2). Inhibition of this reaction due
to vitamin B12 deficiency or inborn errors of folate or
vitamin B12 metabolism, results in an accumulation of
homocysteine in the blood (Krumdieck, 1990). Kang et al.
(1987) and Stabler et al. (1988) investigated the potential
association of folate deficiency with homocysteinemia and
found a negative correlation between serum folate
concentrations and protein-bound homocysteine. Stabler et al.
(1988), reported elevated serum homocysteine concentrations in
18 of 19 folate-deficient patients. Compromised folate status
was attributed to nutritional inadequacy in 17 of these
subjects. However, hyperhomocysteinemia was also found to
occur in patients with a deficiency of vitamin B12 (Stabler et
al., 1988), so the total serum homocysteine concentration must
be used in combination with other parameters in order to
distinguish folate deficiency from a deficiency of vitamin
B12. Determination of serum methylmalonic acid concentration,
which is normal in patients with folate deficiency, has been
recommended for this purpose (Stabler et al., 1988).

36
Folate Status in Special Population Groups
Folate status is affected by physiological changes
occurring at different stages of the life cycle. The
potential impact of growth, development and maturation on
folate requirements, metabolism, and subsequently, status, in
selected population groups, is summarized below.
Infants. The rapid rate of growth that occurs during the
first year of life influences folate requirements. Folate
requirements are also affected by the developmental immaturity
of the infant. For example, low secretion of pancreatic
proteases and gastric and biliary secretions during the first
months of life may significantly affect the bioavailability of
food folate and thereby influence folate requirements and
status (Picciano, 1990).
It has been reported that blood folate values of infants
at birth are higher than values for pregnant and lactating
mothers (Ek and Magnus, 1979) or normal adults (Smith et al.,
1985; Vanier and Tyas, 1966), and that these values decline
when solid foods are introduced (Smith et al., 1985). A study
(Smith et al., 1985) designed to assess the folate status of
infants supports the concept that milk alone (human or
proprietary formulas) is an important dietary source of folate
during the first year of life, and inclusion of this food can
provide sufficient folate to maintain blood folate
concentrations within an acceptable range.

37
Preterm, low-birth-weight infants have greater folate
requirements than term infants (Rodriguez, 1978). Depressed
serum and erythrocyte folate concentrations and megaloblastic
anemia have been observed in this population subgroup (Gray
and Butler, 1965; Roberts et al., 1969; Strelling et al.,
1966). The efficacy of supplemental folate given to low-
birth-weight infants was demonstrated by Burland et al.
(1971) . After comparing the serum and erythrocyte folate
values of supplemented infants to those of unsupplemented
infants during the first nine months of life, these
researchers concluded that folate supplementation should be
provided to all low-birth-weight infants, but that more
research was needed to determine the optimal route, dose and
duration of therapy. Dallman (1974) has stated that folate
supplementation at the level of 50 Mg/d for well infants
weighing less than 2,000 g at birth is warranted. The
Subcommittee on Pediatric Parenteral Nutrient Requirements
(Greene et al., 1988) of the American Society for Clinical
Nutrition has recommended 56 nq/kq/d of folic acid for
preterm infants receiving parenteral nutrition.
Adolescents. The rapid growth experienced by adolescents
is characterized by increases in lean body mass, skeletal
tissue and blood volume. Folate requirements are elevated
during this accelerated growth period because of the role
folate plays in cell division. Superimposed on the extra
demands for folate due to growth, are the effects of other

38
factors that may adversely affect folate status. These
include poor diet, alcohol and drug use, smoking and the
possibility of pregnancy in the sexually mature female.
Compromised folate status is prevalent among adolescents,
particularly those living in low-income households. Bailey et
al. (1982a) reported that 45% of rural black and white
adolescent males and females living in low-income households
had erythrocyte folate concentrations below 140 ng/mL. Serum
folate concentrations were less than 6 ng/mL in 56% of these
subjects. A similar trend was found in adolescents from urban
low-income households, where 42% of the subjects had
erythrocyte folate concentrations less than 140 ng/mL, and 45%
had serum folate concentrations below 6 ng/mL (Bailey et al.,
1982b). When assessed as a function of sexual maturation,
these researchers (Bailey et al., 1982 a; 1982 b) found that
serum folate concentrations declined as sexual maturation
progressed. A similar relationship was reported by Daniel et
al. (1975). Based on erythrocyte folate concentration, Tsui
and Nordstrom (1990) found the prevalence of folate deficiency
to be 13% among males and 40% among females. In the same
study, analysis of seven day food records revealed that for
all race, sex and age groups, subjects who were folate
deficient had significantly lower folate intakes than those
with normal folate concentrations. The type of dietary
pattern that contributed to inadeguate folate consumption in
this study was not addressed, but a study by Bailey et al.

39
(1984b) suggests that poor folate status among adolescents may
be due in part to their limited consumption of vegetables and
fruit.
Pregnancy. The RDA for folate is doubled during
pregnancy. This large increase in the recommended intake
reflects the fact that cell division and multiplication is
occurring rapidly. Increased dietary folate, along with
increased amounts of other nutrients, are needed to support
the physiological and compositional changes occurring at this
time. Increased maternal erythropoiesis, uterine and mammary
tissue expansion, placental and fetal growth and greater
urinary folate losses all contribute to the increased demand
for folate during pregnancy (National Academy of Sciences,
1990). During the final weeks of the normal gestational
period, the rate of active transport of folate across the
placenta is elevated. This places further demands on maternal
folate stores and may explain why serum and erythrocyte folate
values are usually several-fold higher in neonates than in
pregnant and lactating mothers (Bailey, 1990b).
As with other population subgroups, estimates of dietary
folate intake during pregnancy by women residing in the United
States are limited. A recent study (Huber et al., 1988) of
566 pregnant women who were primarily white, middle class and
at least 20 years old, found that only 8.5% of the women
derived their folate intake entirely from the diet. The mean
folate intake of this group was 257 Mg/d. The remaining women

40
(91.5%) consumed folic acid supplements and had a mean intake
of 1087 /¿g/d. The women who did not take folic acid
supplements had significantly lower serum and erythrocyte
folate concentrations compared to those who used supplements.
Data from the 1985 Continuing Survey of Food Intake (United
States Department of Agriculture, 1987) indicated that the
mean folate intake by women (nonpregnant) between the ages of
19 and 34 (all income levels) was 217 M9/d. These data
suggest that unless women are motivated to make dietary
changes, or are instructed or knowledgeable enough to
recognize the need to take a folic acid supplement during
pregnancy, the recommended allowance for folate may not be
met. This is particularly disconcerting in light of the
protective effect of folate against neural tube defects
(Anon., Morbidity and Mortality Weekly, 1992).
A high prevalence of folate deficiency has been suggested
by population studies of pregnant women in whom blood folate
concentrations were measured. Herbert et al. (1975) studied
110 low-income, predominantly black or Puerto Rican women
living in New York City. These researchers reported that 20%
of the subjects had serum folate values below 3 ng/mL, and 16%
of the subjects had erythrocyte folate values below 150 ng/mL.
Bailey et al. (1980) analyzed blood samples from low-income
women in Florida and found that 29% had erythrocyte folate
values below 140 ng/mL. Bailey did not attribute the low
erythrocyte folate concentrations found in these studies to

41
hemodilution because erythrocyte folate concentrations are
indicative of the folate available to precursor red cells in
the bone marrow at the time the currently circulating cells
were developed (Bailey, 1990b).
The value of folic acid supplementation in the prevention
of folate deficiency during pregnancy was demonstrated in a
study of African women (Colman et al., 1975). Pregnant women
receiving folic acid supplemented cereals experienced
significant increases in serum and erythrocyte folate
concentrations, whereas the erythrocyte concentrations in
unsupplemented women decreased by an average of 42 ng/mL
during the last month of gestation (33 days). Despite a
hemoglobin concentration of 11 g/dL or more at the start of
the study, the supplemented women experienced an increase in
hemoglobin concentration suggesting that hematopoiesis was
limited by folate deficiency. These researchers suggested
that this finding gives further credence to the need for folic
acid supplementation in this population.
Not all researchers agree that low serum folate
concentrations during pregnancy are associated with maternal
complications or congenital malformations of the fetus (Hall
et al., 1976). For example, Hall et al. (1976) examined over
2700 women at four time points and found progressive
reductions in serum folate values at each stage. Maternal
complications and fetal outcome were not reported for any of
these pregnancies. Women from low socioeconomic groups,

42
smokers, multigravidae and women with twin pregnancies had
greater declines in serum folate. With the exception of the
smokers, however, significant reductions in mean serum folate
concentrations were not detected. It was concluded that this
decline was due to plasma volume expansion and did not warrant
routine folate supplementation.
Lactation. The effect of lactation on maternal and
infant status and milk folate content in unsupplemented women
has been described by several researchers. Smith et al.
(1983) found that blood folate concentrations were lower in
well-nourished, unsupplemented lactating women compared to
supplemented lactating women and normal nonlactating controls.
While the erythrocyte folate concentration of the
unsupplemented women declined, the folate content of their
milk was comparable to that of the supplemented women. There
was no difference in the blood folate concentrations of
infants of supplemented or unsupplemented mothers. Similar
results were found in a study described by Metz (1970). In
this study, lactating women fed a controlled low-folate diet
experienced rapid reductions in serum folate concentrations,
while the folate content of their milk remained constant.
When folate status during pregnancy and lactation is
severe enough to cause megaloblastic anemia, oral
administration of supplemental folic acid to lactating mothers
has been shown to improve their milk folate content (Cooperman
et al., 1982). Milk folate concentrations have also been

43
increased in lactating women of low socioeconomic status when
folic acid supplements were administered (Sneed et al. , 1981).
However, serum folate concentration appears to increase, while
milk folate content remains constant when maternal blood
folate levels are already within an acceptable range (Tamura
et al., 1980). These findings suggest that a regulatory
mechanism controls the level of milk folate secretion (Tamura
et al., 1980) with the concentration of folate in breast milk
being maintained at the expense of maternal reserves and
status (Smith et al., 1983). Folic acid supplementation
during pregnancy and lactation may help to protect the folate
status of lactating women (Smith et al., 1983).
Elderly. While some researchers (Baker et al., 1978)
have suggested that age related changes adversely affect
folate absorption, other investigators (Bailey et al., 1984a)
have concluded that folate absorption is not affected by the
aging process. Although alterations in other aspects of
folate metabolism may exist, as intimated by data showing
depressed erythrocyte folate uptake in elderly subjects
(Ettinger and Colman, 1985), assessment studies of population
subgroups of the elderly suggest that socioeconomic and
environmental factors are probably the most important
contributors to the development of compromised folate status.
The potential effect of socioeconomic level on folate
status in the elderly population is apparent when comparing
assessment studies conducted in population subgroups with

44
different income levels. The majority of high-income elderly
subjects participating in studies conducted in New Mexico
(Garry et al., 1984) and Florida (Wagner et al., 1981) had
normal serum and erythrocyte folate concentrations. Only 3%
and 6% of the participants in the New Mexico and Florida
studies, respectively, had erythrocyte folate values below 140
ng/mL. Conversely, Bailey et al. (1979) found that 60% of
elderly Floridians from very poor socioeconomic backgrounds
had erythrocyte folate concentrations below 140 ng/mL, as well
as evidence of macrocytic anemia.
Environmental factors that may place the elderly at
higher risk for the development of compromised folate status
have been reviewed by Bailey (1990b) and Sauberlich (1990).
These factors include: institutionalization; chronic use of
prescription and/or nonprescription medications; the presence
of disease; and the consumption of alcohol.
Although not specifically addressed for each stage of the
life cycle covered above, it is important to consider the
impact that cultural food habits and customs, food
preferences, educational level, state of mental and physical
health, socioeconomic factors and food availability have on
food selection and nutrient intake. While physiological
changes occurring throughout the life cycle can have a
profound affect on nutrient needs and metabolism, Sauberlich
(1990) has concluded that the most common cause of compromised
folate status is inadequate dietary intake.

45
Effects of Environmental Factors on Folate Status
Prescription and nonprescription medications. Certain
prescription and nonprescription medications interfere with
folate absorption and/or metabolism, and depending on the dose
and duration of use, may result in compromised folate status.
Examples of these drugs include folate antagonists,
anticonvulsants, histamine receptor blockers, antacids, anti¬
inflammatory agents, aspirin and possibly oral contraceptive
agents.
Folate antagonists are used in cancer chemotherapy or to
treat infections such as malaria. Methotrexate is an example
of a folate antagonist. This drug interferes with folate
metabolism by inhibiting dihydrofolate reductase, resulting in
a functional folate deficiency and anemia. Methotrexate can
also cause reversible mucosal ulceration (Roe, 1989). The
resulting damage to enterocytes can impair the absorption of
folate and other nutrients.
Rosenberg et al. (1982) have noted that the frequent use
of antacids and histamine receptor blockers among the elderly
poses a theoretical risk to this population in terms of their
folate status. Histamine receptor blockers such as
cimetidine, and antacids like sodium bicarbonate, may impair
folate status by increasing the intestinal pH to levels that
exceed the optimum for folate conjugase activity and carrier-
mediated transport and/or passive diffusion (Mackenzie and
Russell, 1976; Russell et al., 1979). However, folate

46
bioavailability was not reduced by chronic bicarbonate
administration in a rat bioassay (Hoppner and Lampi, 1988) .
Furthermore, evidence of folate deficiency due to use of these
medications is lacking (Rosenberg et al., 1982).
Sulfasalazine is an anti-inflammatory agent frequently
used in the treatment of inflammatory bowel disease. Franklin
and Rosenberg (1973) have demonstrated that this drug
interferes with folate absorption. It appears that
sulfasalazine interferes with the absorptive process through
competitive inhibition (Strum, 1981), and by inhibition of
jejunal brush border folate conjugase activity (Reisenauer and
Halsted, 1981) . Sulfasalazine also has the potential to
disturb folate metabolism since it can inhibit several hepatic
folate-dependent enzymes (Selhub et al., 1978). Patients
taking daily therapeutic doses of sulfasalazine are at risk of
developing folate deficiency and should be provided with
therapeutic doses of folic acid in order to prevent a
deficiency of this nutrient.
The interaction between folate and diphenylhydantoin, an
anti-convulsant medication used for treatment of epilepsy, may
actually be a two-way interaction (Reynolds, 1973; Rivey et
al., 1984) . Chronic use of this drug has been associated with
folate deficiency and anemia, although the progression of the
deficiency to megaloblastic anemia is rare (Gerson et al.,
1972; Hoffbrand and Necheles, 1968; Reynolds, 1973; Rivey et
al., 1984; Rosenberg et al., 1968) and administration of

47
phenytoin has not been shown to affect the kinetics of folate
excretion (Krumdieck et al., 1978). Conversely, long-term,
high-dose folic acid supplementation of folate-deficient
patients taking diphenylhydantoin may result in lowered serum
concentrations of this drug in selected patients, with the
potential for loss of control of the seizure disorder
(Reynolds, 1973; Rivey et al., 1984).
The mechanism responsible for the development of
compromised folate status in patients receiving
diphenylhydantoin is unclear, but several hypotheses have been
espoused. These hypotheses have been reviewed by Rivey et al.
(1984). They include: drug inhibition of brush border folate
conjugase (Hoffbrand and Necheles, 1968; Rosenberg et al.,
1968); elevation of the intraluminal pH resulting in
malabsorption of dietary folate (Benn et al., 1971); drug-
induced impairment of folate transport into tissues (Krumdieck
et al., 1978); and induction of folate-requiring metabolic
processes in the liver (Maxwell et al., 1972). Studies
employed to prove and/or disprove each of these hypotheses
have provided seemingly contradictory results. Rivey et al.
(1984) have suggested that diphenylhydantoin may affect folate
homeostasis by multiple mechanisms.
Alter et al. (1971) and Lawrence et al. (1984) have
reported that aspirin in therapeutic doses can reduce serum
folate concentrations. After aspirin is discontinued, serum
folate values rapidly increase. Individuals with rheumatoid

48
arthritis, as well as those taking therapeutic doses of
aspirin for the prevention of heart attacks, may be
particularly vulnerable to the effects of chronic aspirin
ingestion. Elderly individuals are more likely to use
aspirin for these purposes, which may make them more
susceptible to folate depletion depending on the amount and
frequency of use.
Several investigators have noted an association between
the use of oral contraceptive agents and low serum and
erythrocyte folate values (Butterworth et al., 1982;
Pietarinen et al., 1977; Smith et al., 1975). Other
researchers have found no difference in folate status of oral
contraceptive users versus nonusers (Paine et al., 1975; Ross
et al., 1976; Whitehead et al., 1973). Results of studies
comparing blood folate concentrations of women using oral
contraceptive agents within six months of conception to those
of nonusers have also been equivocal (Bailey, 1980; Martinez
and Roe, 1977). Factors such as the specific formulation of
the pill (i.e. level of estrogen), duration of pill use and/or
dietary intake may affect the body's response to oral
contraceptive agents. Based on observations that
megaloblastic changes in the cervical epithelium of women
taking oral contraceptive agents were reversed with oral folic
acid supplements (Whitehead et al., 1973), some researchers
have speculated that oral contraceptive use may lead to a

49
localized folate deficiency in the cervix (Butterworth et al.,
1982) .
Smoking. The effects of smoking in nonpregnant women
were investigated by Wittier et al. (1982). The mean serum
and erythrocyte folate concentrations of the smokers who
participated in this study were lower than those of the
nonsmokers. No differences between smokers were detected,
however, with regard to the number of cigarettes smoked per
day.
Heimburger et al. (1987; 1988) compared the folate status
of male smokers to that of a control group of nonsmokers and
found significantly lower serum and erythrocyte folate
concentrations in the smokers. Smokers with metaplasia had
lower blood folate concentrations than did smokers without
metaplasia, and folate concentrations appeared to decrease
with increasing severity of metaplasia. These studies suggest
that smoking may adversely affect folate status.
Alcohol. Compromised folate status is common in chronic
alcoholics and is probably caused by a combination of factors
including: poor dietary habits; intestinal malabsorption;
decreased hepatic uptake; and increased urinary folate
excretion (Halsted, 1980). The effects of binge drinking on
folate absorption have been illustrated by studies using
orally administered radio-labeled folic acid. Evidence of
folic acid malabsorption was provided by decreased plasma
concentrations of radioactivity after oral doses of tritiated

50
pteroylmonoglutamic acid ([3H]PteGlu) (Halsted et al., 1967)
and by reduced luminal disappearance of [3H]PteGlu during
jejunal perfusion (Halsted et al., 1971). The underlying
defect responsible for the limited jejunal uptake of folic
acid was thought to be due to inadeguate intake of dietary
folate during chronic alcohol ingestion (Halsted et al.,
1971). As discussed by Halsted (1990), a subsequent human
study that examined the effects of a folate deficient diet on
the absorption of [3H]PteGlu (Halsted et al., 1973), and an
absorption study conducted with monkeys fed diets containing
50% of their calories as ethanol (Romero et al., 1981),
prompted researchers to hypothesize that folate malabsorption
in chronic alcoholism results from the combined effects of
folate deficiency and ethanol exposure.
Recent research has attempted to delineate the
pathogenesis of folate malabsorption occurring in alcoholics.
To separate the effects of poor diet from those of alcohol
exposure, studies using miniature pigs exposed to alcohol for
a short period of time and in the absence of folate deficiency
have been performed (Naughton et al., 1989; Reisenauer et al.,
1989) . These studies suggest that inhibition of brush border
folate conjugase, resulting in decreased hydrolysis of folate
polyglutamates, may be the earliest functional lesion
contributing to folate malabsorption and deficiency in
alcoholism (Halsted, 1990).

51
Disease. Folate deficiency has been associated with a
variety of diseases including certain hemolytic diseases
(Brody, 1991) , cancer of the head and neck (Brody, 1991),
inborn errors of metabolism (Brody, 1991) and diseases of the
intestinal mucosa (Corcino et al., 1976; Halsted et al., 1977;
1978; Hoffbrand et al., 1968; 1970). The proposed underlying
etiology of folate deficiency in these diseases is defective
absorption and/or altered folate metabolism. These defects
lead to an increased folate requirement. For example, the
increased damage to red blood cells occurring in hemolytic
diseases results in increased cell division in the bone
marrow. This increase in cell division is thought to elevate
the requirement for folate (Brody, 1991). Altered metabolism
may also be responsible for the folate deficiency that
develops in patients with certain types of cancer,
particularly cancer of the head and neck (Brody, 1991). The
effects of altered folate metabolism in these individuals is
independent of the effects of anticancer drugs such as
methotrexate which can also adversely affect folate status.
Patients with inborn errors of folate metabolism may have
lower levels of folate-dependent enzymes, as well as defective
folate absorption, thereby increasing their folate requirement
(Brody, 1991).
Malabsorption is thought to be the major defect
contributing to compromised folate status associated with
untreated gastrointestinal diseases such as celiac sprue and

52
tropical sprue. Luminal disappearance of [3H]PteGlu and
pteroyl [ 14C]-glutamylhexaglutamate ([ 14C]-PteGlu7) inpatients
with celiac sprue (Halsted et al., 1977; 1978) and tropical
sprue (Corcino et al., 1976) is significantly less than
luminal disappearance in normal subjects. When appropriate
medical and nutritional management of these diseases is
instituted, luminal disappearance increases significantly
(Corcino et al., 1976; Halsted et al., 1977; 1978). Further
evidence corroborating the idea that malabsorption adversely
affects folate status, at least in terms of celiac sprue,
comes from two studies. A significant decrease in the
hydrolysis of perfused [ 14C]-PteGlu7 in patients with celiac
sprue was noted in one study (Halsted et al., 1977). The
other experiment showed that brush border folate conjugase
activity in jejunal biopsy specimens taken from patients with
celiac sprue was significantly lower than the activity of this
enzyme in specimens from normal subjects (Halsted et al.,
1986).
Consequences of Compromised Folate Status
Megaloblastic anemia is probably the most commonly
recognized clinical problem associated with folate deficiency,
but research is beginning to identify many new potential
conseguences of compromised folate status. Negative outcomes
more recently associated with folate deficiency include neural
tube defects, compromised infant birth weight, increased

53
potential for neoplastic changes and altered immune function.
A brief review of the evidence linking folate deficiency to
these problems is presented in this section.
Neural tube defects. Neural tube defects, which include
spina bifida, anencephaly and encephalocele, are among the
most common severe congenital malformations. The idea that
folate deficiency contributed to the causation of fetal
malformations was introduced almost thirty years ago by
Hibbard (1964) and Hibbard and Smithells (1965). Smithells
et al. (1980; 1981) conducted the first intervention trial
which suggested that supplementation with folic acid (0.36
mg/d) or other vitamins near the time of conception might
reduce the risk of recurrence of birth defects categorized as
neural tube defects. The results of this study could not be
taken as definitive proof of a protective effect for folate,
however, due to the co-administration of folic acid with other
vitamins, the lack of randomization of subjects to the control
and treatment groups and the absence of a double-blind design.
Another early intervention trial was conducted by
Laurence et al. (1981). This was a small study that employed
a controlled, randomized, double-blind design to test the
effects of folic acid supplementation (4 mg/d) alone. The
data provided inconclusive results when analyzed using the
original randomization scheme; however, when the data for
women who did not take their supplements were transferred to
the control group, the supplemented subjects had a

54
significantly lower recurrence rate. Preliminary results of
a study using a similar design and a small number of subjects,
found a reduction in the recurrence rate after a
supplementation program had been initiated, compared to the
recurrence rate before a supplementation program had been
established (Holmes-Siedle et al. 1982).
These early studies supported the concept of a protective
role for folic acid, but their flawed designs precluded the
ability to make any definitive conclusions and
recommendations. The results of a recent study (Medical
Research Council (MRC) Vitamin Study Research Group, 1991),
however, have established that supplementation with folic acid
around the time of conception can decrease the risk of neural
tube defects in women who have previously given birth to an
affected infant. This study was a large, multi-center,
double-blind intervention trial that randomized participants
to one of four groups: 1) folic acid alone; 2) folic acid plus
other vitamins; 3) other vitamins alone; and 4) no
supplements. This design allowed the researchers to determine
if vitamins other than folic acid conferred a protective
effect. The results demonstrated that folic acid, rather than
the combination of other vitamins, was responsible for the
improved outcome.
While the MRC Vitamin Study Research Group (1991)
findings are positive with respect to folate's role in
reducing the risk of recurrent neural tube defects, they do

55
not address the association of folate intake with the
occurrence of neural tube defects. In the United States, most
(approximately 95%) of the neural tube defect-affected infants
and fetuses occur in pregnancies of women who have not
previously given birth to an infant with a neural tube defect.
Thus, in order to reduce the overall prevalence of neural tube
defects, folic acid supplementation must exert a protective
effect on the developing embryos of women who have no history
of a neural tube defect-affected pregnancy. The MRC study
also leaves open to question the minimum dose and form of
folate needed to confer a protective effect, since subjects
were given daily supplements containing 4 mg of folic acid.
Over the last five years, several observational studies
have been conducted to evaluate the impact of periconceptional
folic acid/vitamin supplementation on the occurrence of neural
tube defects. Three of these studies were case-control
studies (Bower and Stanley, 1989; Mills et al., 1989; Mulinare
et al., 1988) and one was a prospective cohort study (Milunsky
et al., 1989). All of these studies involved women who had no
history of a neural tube defect-affected infant/fetus. The
occurrence of neural tube defects in women who reported taking
multivitamins containing folic acid (approximately 0.4-0.8 mg
folic acid per day) for at least one month prior to conception
through the first trimester of pregnancy were compared to
women who did not take supplements. Dietary intake of folate
was also considered in some studies. A protective effect was

56
associated with multivitamin supplement use and/or higher
levels of dietary folate intake in three (Bower and Stanley,
1989; Milunsky et al., 1989; Mulinare et al., 1988) of the
four studies.
Two new studies have provided additional support in favor
of a protective effect of folate. One study was a randomized
controlled trial conducted in Hungary (Czeizel and Dudás,
1992). Subjects were 18-35 years old, were not pregnant at
the time of recruitment and had no history of infertility or
fetal death. The volunteers were randomized to receive either
a placebo or a multivitamin supplement (including 0.8 mg folic
acid) . There were no cases of neural tube defects in the 2104
participants receiving the multivitamin, whereas, the placebo
group had six occurrences and 2046 unaffected pregnancies.
Although the criteria for inclusion did not specifically
exclude women with a history of a neural tube defect-affected
pregnancy, it is likely that most women falling into this
category would have already been recruited for the MRC study
and would not have been available to participate in the study
just described. Under this assumption, these results have
been interpreted as providing evidence that folic acid can
reduce the risk of occurrence of neural tube defects; and that
the quantity of folic acid needed to produce such an effect is
much less than the 4 mg daily dose used in the MRC trial.
The results of another study examining the rate of
occurrence of neural tube defects in folate supplemented and

57
unsupplemented women has recently been published (Werler et
al., 1993). This large case-control study was conducted in
Boston, Philadelphia and Toronto. The control group consisted
of 2615 infants with birth defects other than neural tube
defects and oral clefts. This group was compared to 443 cases
with neural tube defects. The prevalence of use of folic
acid-containing multivitamins during the periconceptional
period was compared between mothers of cases and controls.
Daily folic acid supplementation was found to reduce the risk
of occurrent neural tube defects by 60%. Since the most
commonly used dose of folic acid was 0.4 mg, these data were
considered to be consistent with the hypothesis that 0.4 mg
supplemental folic acid per day is sufficient to decrease the
risk of neural tube defects among pregnancies of women in the
general population.
In the wake of the findings of the MRC trial (1991) and
the studies by Czeizel and Dudás (1992) and Werler et al.
(1993) the data from observational studies have been
reevaluated. Taken together, the results have been
interpreted as providing support for the hypothesis that folic
acid will decrease the risk of occurrence, as well as the
recurrence, of neural tube defects. Accordingly, the United
States Public Health Service has recently recommended that:
All women of childbearing age in the United States who
are capable of becoming pregnant should consume 0.4 mg of
folic acid per day for the purpose of reducing their risk
of having a pregnancy affected with spina bifida or other
NTDs. Because the effects of higher intakes are not well
known but include complicating the diagnosis of vitamin

58
B12 deficiency, care should be taken to keep total folate
consumption at <1 mg per day, except under the
supervision of a physician. Women who have had a prior
NTD-affected pregnancy are at high risk of having a
subsequent affected pregnancy. When these women are
planning to become pregnant, they should consult their
physicians for advice. (Anon., Morbidity and Mortality
Weekly, 1992, p. 1)
Recognition of the importance of folate in reducing the
risk of bearing a child with a neural tube defect is certain
to stimulate research focused on understanding the mechanism
whereby folate exerts its protective effect. Particular
attention is likely to be directed at identifying the
existence of underlying defects in folate metabolism.
Compromised birth weight. Folate deficiency occurring
during pregnancy has been associated with low infant birth
weight. Several researchers have reported increased infant
birth weights when folic acid supplements were given to
folate-deficient pregnant women (Baumslag et al., 1970;
Iyengar and Rajalakshmi et al., 1975; and Rolschau et al.,
1979) . Placental weights were measured in two of these
studies (Iyengar and Rajalakshmi et al., 1975; Rolschau et
al., 1979), and a positive association with birth weight was
noted. These findings suggest that folic acid supplementation
improves fetal outcome by improving nutrition via increased
placental size (Bailey, 1990b). Additional evidence
suggesting a positive relationship between folic acid
supplementation and infant birth weight has recently been
published by Goldenberg et al. (1992). These researchers
found that maternal serum folate concentrations, occurring

59
within a range suggestive of compliance with the
supplementation regimen, were associated with higher infant
birth weight and a decreased rate of fetal growth retardation.
Neoplastic changes. The potential relationship between
folate and cancer was identified as early as 1944 when several
researchers noted that large doses of folic acid directly
interfered with the growth of certain tumors. Subsequently,
it was found that administration of folate antagonists
produced beneficial effects in patients with certain forms of
cancer. This discovery, although it revolutionized the
treatment of childhood leukemia and other forms of cancer,
probably contributed to the paucity of further research
regarding the potential anticancer effect of folate
(Butterworth, 1991). Renewed interest in the anticancer
potential of folate has been sparked by recent findings
suggesting that inadequate dietary intake of folate and/or
compromised folate status may be associated with an increased
risk or prevalence of certain forms of cancer; and that
optimal folate intake may provide a protective effect.
The initiation of carcinogenic activity is thought to
occur due to altered regulation of genetic expression of both
endogenous and exogenous oncogenes. Although the role of
folate deficiency in carcinogenesis has not been completely
elucidated, the co-carcinogenic effect of a deficiency of this
nutrient is thought to be related to folate's role in the
maintenance of chromosome structure and function. It has been

60
well established that folate is important in the regulation of
methyl groups used for DNA methylation. A deficiency of
methyl groups due to folate deficiency results in
undermethylated DNA and substitution of uridylate for
thymidylate. Subseguently, this interferes with histone
binding and results in the transcription of genetic seguences
that would ordinarily be suppressed. Additionally, improper
histone binding results in increased exposure of DNA to attack
by endogenous nucleases, thereby raising the risk of
chromosome breaks and incorporation of viral genomes.
Chromosome breaks associated with folate deficiency occur at
specific heritable fragile sites and many folate-sensitive
breaks occur at positions known to be associated with
translocations seen in cancer. It is thought that these
translocations disturb regulatory patterns of contiguous
segments of genetic information (Butterworth, 1991; Eto and
Krumdieck, 1986).
Compromised folate status has been associated with
increased risk of dysplasia or cancer of the cervix, colon,
bronchus and esophagus. The research documenting these
associations is briefly reviewed in the following paragraphs.
The concept of "localized folate deficiency" was
introduced as a result of a study conducted by Whitehead et
al. (1973). These researchers reported the occurrence of
megaloblastic features in cervical epithelial cells from women
taking oral contraceptive agents. These cytologic

61
abnormalities were not related to hematologic changes or low
serum folate/vitamin B12 concentrations; however, after three
weeks of folic acid supplementation (10 mg/d), the abnormal
cytologic findings were reversed or improved. Later,
Butterworth et al. (1982) reported that daily oral folic acid
supplementation (10 mg/d) was associated with improvement in
the cytologic manifestations of dysplasia compared with
placebo-treated controls. The supplemented group also had
less severe biopsy readings after three months of
supplementation. On the basis of these findings, it was
suggested that folate deficiency either plays an integral role
in the dysplastic process or is occasionally misdiagnosed as
cervical dysplasia (Butterworth et al., 1982).
In a more recent study, Butterworth et al. (1992b)
examined the effect of high-dose oral supplements of folic
acid (10 mg/d for six months) on the course of cervical
dysplasia. Supplementation appeared to have no significant
effect on the course of established cases of dysplasia, with
a high rate of apparent regression occurring in both the
placebo-treated and the folic acid-supplemented groups. These
researchers attributed the difference between the findings of
this study and their earlier report (Butterworth et al., 1982)
to the use of a more adequate sample size, exclusion of
patients with atypia less than dysplasia and a longer period
of observation. Although they concluded that folic acid
supplementation does not alter the course of established

62
disease, they did not exclude the possibility that folate
deficiency played a role in carcinogenesis because they found
a higher prevalence of dysplasia associated with human
papillomavirus 16 (HPV-16) infection (an oncogenic strain
thought to cause cervical dysplasia) among women in the lower
two tertiles of red blood cell folate than in the highest
tertile. Based on this information, they concluded that
folate deficiency may act as a co-carcinogen during the
initiation of cervical dysplasia.
In a separate case-control study conducted by the same
research group (Butterworth et al., 1992a), infection with
HPV-16 was the strongest risk factor for cervical dysplasia,
and there was a statistically significant interaction between
low erythrocyte folate concentrations and the HPV-16 virus.
The conclusion reached by these investigators was that the
carcinogenic effect of HPV-16 infection is enhanced in women
with low concentrations of erythrocyte folate. Although the
mechanism of interaction between folate and HPV-16 was not
investigated in this study, the researchers suggested that a
folate deficiency might increase the possibility of
incorporating the viral genome into human DNA resulting in
transformation of the epithelial cells.
The potential role of folate deficiency in carcinogenesis
may not be limited to the cervix since premalignant lesions
occurring in other organs exhibit features similar to those
seen in cervical dysplasia (Heimburger et al., 1987). Studies

63
examining the relationship between folate status and bronchial
metaplasia suggest that folate deficiency may influence the
susceptibility of the bronchial mucosa to neoplastic
transformation. For example, Heimburger et al. (1987) found
that the serum and erythrocyte folate concentrations of men
who smoked an average of 25 cigarettes per day were
significantly lower than nonsmokers, with the lowest values
occurring in smokers with bronchial metaplasia. Serum and
erythrocyte folate concentrations were also significantly
lower in smokers with metaplasia compared to smokers without
metaplasia. In a subsequent double-blind intervention trial
(Heimburger et al., 1988), smokers with bronchial squamous
metaplasia were stratified according to smoking level and
randomly assigned to treatment with a placebo or 10 mg of
folic acid and 500 /xg of hydroxycobalamin for four months.
Reduction of atypia, as determined by direct cytological
comparison, was significantly greater in the supplemented
group. Unfortunately, it is impossible to determine if folic
acid alone was responsible for the favorable effect since
hydroxycobalamin was administered simultaneously. Never¬
theless, these studies are provocative and provide fertile
ground for continued investigation in this area.
Compared with the general population, patients with
chronic ulcerative colitis are at greater risk for developing
cancer of the colon. It has been hypothesized that folic acid
supplementation may protect against the development of

64
dysplasia (a premalignant pathologic finding) and cancer in
ulcerative colitis. To test this hypothesis, Lashner et al.
(1989) conducted a case control study to examine the effect of
folic acid supplementation on the incidence of dysplasia or
cancer in 99 subjects with chronic ulcerative colitis. A 62%
lower incidence of neoplasia was associated with folic acid
supplementation compared to subjects not receiving supple¬
mentation. Although this outcome was not statistically
significant, it did not change when adjustments were made for
known confounders, suggesting that this finding was not due
entirely to bias or confounding and that inadequate sample
size may have contributed to the lack of significance (Lashner
et al., 1989).
Folate deficiency may also play a role in the development
of esophageal cancer. Jaskiewicz et al. (1988) have examined
cytological specimens obtained by brush biopsy of the
esophagus from subjects living in an area of Africa where
squamous cell carcinoma of the esophagus is the most common
form of cancer. Biopsy results revealed morphologic features
similar to those seen with folate deficiency and dysplasia.
Assessment of folate status in this same population revealed
significantly lower erythrocyte folate concentrations in
subjects with dysplastic and cancer cells compared with
controls (van Helden et al., 1987). Additionally, the
erythrocyte folate concentrations of subjects living in high-
incidence districts was significantly lower than those in the

65
intermediate and low incidence districts. However, these
results must be interpreted cautiously since multiple nutrient
deficiencies, particularly in the high-incidence area, were
noted.
Altered immune function. Although the effect of
compromised folate status on immunocompetence has not been
widely researched, studies using folate-deficient animals and
observations of patients with megaloblastic anemia due to
folate deficiency suggest that alterations occur in both
humoral and cell-mediated immunity. This is not surprising,
since folate is essential for the synthesis of DNA, and a
deficiency of this nutrient would hinder the ability of the
sensitized cells to proliferate rapidly. Examples of the
effects of compromised folate status on immune function as
determined from human studies include delayed cutaneous
hypersensitivity and depressed peripheral lymphocyte response
to phytohemagglutinin. Similar changes in immune function
have been shown using animal models. In addition to these
changes, reductions in white blood cells, leukocytes,
granulocytes, antibody forming cells and T-cells have been
reported in various animal models subjected to an isolated
folate deficiency (Nauss and Newberne, 1981).
Methods Used to Assess Folate Status and Metabolism in Humans
Microbiological assay. The microbiological assay is
considered the most useful method for guantifying folate

66
levels in biological samples (Tamura, 1990). This method uses
the growth response of folate-sensitive microorganisms to
determine folate content. The assay is performed by comparing
the growth of a test organism added to folate-free media
containing an aliquot of the sample, to the growth of the same
organism added to folate-free media that has been enriched
with known concentrations of folic acid. Growth is measured
as turbidity following incubation under controlled conditions.
The microorganism used most often for this assay is
Lactobacillus casei, although Streptococcus faecium or
Pediococcus cerevisiae may also be used.
The three species of bacteria that may be used for this
assay do not respond equally to the various forms of folate.
For example, S. faecium does not respond to N5-methyl-
tetrahydrofoíate, nor to folate derivatives with more than two
molecules of glutamate. Pediococcus cerevisiae is even more
selective. It does not respond to N5-methyl-tetrahydrofoíate,
and of the remaining folate derivatives, it can only use those
occurring as reduced monoglutamyl tetrahydrofdates (Tamura,
1990). Since neither S. faecium nor P. cerevisiae respond to
N5-methyl-tetrahydrofoíate, they are not suitable test
organisms for determination of serum or erythrocyte folate
concentration. Lactobacillus casei, however, responds to N5-
methyl-tetrahydrofoíate, as well as other folate derivatives,
which makes it the best choice when analyzing biological
samples for total folate.

67
The fact that L. casei, S. faecium, and P. cerevisiae
respond differently to folates with varying chain lengths,
one-carbon substituent groups and oxidation states offers
researchers the opportunity to estimate the amount of folate
derivatives present in a particular sample. This can be
useful when the quantity of sample available for analysis is
insufficient to perform column chromatography. For example,
if it is important to estimate the amount of folate
derivatives other than N5-methyl-tetrahydrofolate, L. casei
can be used in combination with S. faecium. Similarly, P.
cerevisiae can be used in conjunction with L. casei to
estimate folate derivatives other than N5-methyl-
tetrahydrofoíate and oxidized pteroylglutamic acid. This
deductive approach can also be used to determine whether
samples contain folate polyglutamates. If the activity of S.
faecium or P. cerevisiae increases after folate hydrolase
treatment, the sample contains folate polyglutamates with more
than two molecules of glutamate. Likewise, if L. casei
activity increases after enzyme treatment, the sample contains
polyglutamates with more than three molecules of glutamate
(Tamura, 1990).
Biological samples typically analyzed for folate content
include serum, whole blood, urine and tissues. Cellular
folates, such as those found in tissues, are usually in the
polyglutamate form and require cleavage to the monoglutamate
form before folate concentration using the microbiological

68
assay can be determined. Partially purified folate hydrolase
preparations from chicken pancreas, hog kidney and rat or
human plasma have been used for this purpose. Folate in the
erythrocytes is also in the polyglutamate form; however, the
presence of natural folate hydrolase in the blood precludes
the need for treating hemolyzed whole blood samples with an
exogenous folate hydrolase. Urine and serum samples do not
require prior treatment with folate hydrolase because the
folate present in these samples is in the monoglutamyl form.
Although the basic concept of the microbiological assay
has not changed over the years, several improvements have been
made. Examples of these improvements have been reviewed by
Tamura (1990) and include the addition of antioxidants (e.g.
ascorbic acid) to samples to protect labile reduced forms of
folate from oxidation and the use of a cryoprotected organism
to maintain constant growth-response curves. Recently, this
assay has been adapted to take advantage of the availability
of the 96-well microtiter plate reader (Horne and Patterson,
1988; Newman and Tsai, 1986; O'Broin and Kelleher, 1992).
Although the procedure followed with this adaptation is
similar in concept to the standard procedure, the microtiter
plate reader improves the efficiency of absorbance readings.
Rapid calculation of the results can be achieved by
interfacing the microtiter plate reader with a computer (Horne
and Patterson, 1988). These modifications can save a

69
tremendous amount of time and money without sacrificing
accuracy (Newman and Tsai, 1986; O'Broin and Kelleher, 1992).
Radiometric binding assay. The radiometric binding assay
is a competitive protein binding assay in which radio-labeled
folate and unbound folate in the sample compete for the
binding sites on folate binding proteins. This method is
usually performed by clinical laboratories using commercially
available radioassay kits (Brody, 1991; Tamura, 1990).
Compared with the microbiological assay, this competitive
binding assay is simpler to perform and is not affected by
bacterial contamination or the presence of antibiotics.
Despite these potential advantages, the usefulness of the
radiometric binding assay is limited because the proteins used
to bind folates do not have an equal affinity for all forms of
folate. Binding affinity seems to be influenced by the state
of oxidation, the one-carbon substituent group and glutamyl
chain-length. For this reason, the suitability of the
radioassay method for quantifying complex mixtures of
naturally occurring folates has been questioned. This method
may be acceptable for determining serum folate concentration,
however, because N5-methyl-tetrahydrofoíate is the predominant
form of folate in serum, and this form of folate is tightly
bound by folate binding proteins. Conversely, if the
population being assessed is receiving large doses of folates
other than N5-methyl-tetrahydrofoíate, the radioassay may be
inappropriate (Brody, 1991; Tamura, 1990).

70
Folate bioavailabilitv studies. Until recently, most of
the information known about the metabolic requirements and
bioavailability of folates in humans was derived from animal
bioassay studies, or from a limited number of human studies
which used plasma or urinary folate concentration in folate-
saturated human subjects as the response criteria. While
these studies have made an important contribution to our
understanding of folate metabolism, they have several
limitations which restrict their scope and applicability. A
potential limitation of animal bioassay studies is the use of
animal models that are not entirely appropriate for studying
human metabolism. For example, rats are not appropriate
models to use for folate polyglutamate absorption studies
because they exhibit little or no brush border folate
conjugase activity (Wang et al., 1985). Limitations of
bioassay studies conducted in human subjects include their
inability to provide information about in vivo metabolism and
the need to consume large quantities of tested foods or
purified folates, the later of which could change the rate of
digestion and absorption in a way that is different from that
which usually occurs (Gregory and Toth, 1990) .
The development of new tools and techniques combined with
previous accomplishments has facilitated research in the area
of folate requirements and bioavailability. The synthesis of
various forms of folate, including folate polyglutamates
(Krumdieck and Baugh, 1969; Godwin et al., 1972), and the

71
ability to radio-label folate derivatives with tritium at
different locations in the molecule (Godwin et al., 1972),
provided valuable tools for initial studies of folate
metabolism in animals. Although radio-labeled folates have
been used in human studies, concern about their safety
precluded their use for long-term supplementation studies and
restricted the amount of labeled folate that could be
administered to no more than a tracer dose. The development
of stable-isotopically (deuterium) labeled folates (Gregory,
1990; Gregory and Toth, 1988a; 1988b) has circumvented these
problems, while providing the opportunity to trace the fate of
folate during normal metabolism and to compare this with the
fate of folate under altered conditions or physiological
states. Additionally, the availability of stable isotopes of
folate affords the luxury of evaluating several forms of
folate simultaneously and allows the researcher to determine
the kinetics of absorption and turnover of the administered
compound (Gregory and Toth, 1990). Other developments that
have facilitated research in this area include the development
of improved methods for separating various folate
monoglutamates using high-performance liquid chromatography
(HPLC) (Kashani and Cooper, 1985; Wilson and Horne, 1984) ; the
development of affinity chromatography columns that allow for
easier separation of folates from the sample (Selhub et al.,
1980), which ultimately results in cleaner chromatographic
separation using HPLC (Selhub, 1989); and the development of

72
a mass spectrometric method to quantify the ratio of unlabeled
versus labeled folates (Toth and Gregory, 1988) .
The first published studies of an in vivo application of
stable-isotopically labeled folates in human subjects were
performed by Gregory and coworkers (Gregory and Toth, 1988a;
Gregory et al., 1990). The initial report (Gregory and Toth,
1988a) described the results of a preliminary investigation of
gas chromatography/mass spectral (GCMS) analysis of urinary
folates. The second study (Gregory et al., 1990) evaluated
the adequacy of a saturation regimen of 2 mg/d of folic acid
and the effectiveness of simultaneous administration of two
forms of deuterium-labeled folate. Subjects participating in
this study received 2 mg/d unlabeled folic acid for one week
to enhance urinary excretion of absorbed folates. On the
morning prior to administration of the deuterium-labeled
folates, the subjects collected a 24 hour urine sample. The
next morning, after an overnight fast, subjects consumed apple
juice containing two different deuterium-labeled folates. The
subjects collected their urine for 48 hours following the
treatment. A constant diet was consumed beginning at the time
of the pre-dose urine collection through the end of the post¬
dose urine collection. Dietary and total urinary folate
concentration was determined by microbiological assay using L.
casei. Urine samples were also prepared for mass spectral
analysis of labeled folates, after which the molar ratio of
each species of folate in the sample was calculated. The

73
results of these studies confirmed that stable-isotopically
labeled folates are well-suited for in vivo studies and that
the protocol outlined above is suitable for studying many
aspects of folate bioavailability and in vivo kinetics
(Gregory et al., 1990). Recently, Von der Porten et al.
(1992) used this methodology to study in vivo folate kinetics
in human subjects supplemented with deuterium-labeled folic
acid for four weeks. By using deuterium-labeled folate, these
researchers were able to determine rate constants and isotope
enrichment, which subsequently enabled them to assess tissue
uptake and equilibration, in vivo turnover rates and body pool
sizes of folate. It is expected that further application of
this technique will greatly enhance our knowledge of folate
metabolism and improve our ability to make appropriate
nutritional recommendations.
Zinc
Chemistry
Zinc has an atomic weight of 65.37. It is a first series
transition element with the electronic configuration [Argon]
4s2 3d10. This configuration confers properties to zinc that
distinguish it from other transition metals. For example,
zinc is diamagnetic rather than paramagnetic, and although it
can exist in several valence states, it is resistant to
oxidation and is found almost universally as the divalent ion
(Zn2+) . Another property of zinc that is relevant to its

74
specific biological functions is its ability to form stable
complexes with side chains of proteins. The high charge
density of Zn2+ allows this metal to function as a Lewis acid
to withdraw electrons from electron-rich functional groups of
ligands resulting in the formation of noncovalently bound
coordination complexes. These features make zinc ideally
suited for its involvement in enzyme function and structure
(Solomons, 1988; Williams, 1989).
Absorption
The process of zinc absorption includes the uptake of
zinc by the intestinal mucosal cells, movement of zinc through
the mucosal cells and transfer into the portal circulation.
Paracellular movement of zinc to the portal circulation may
also occur. These aspects of zinc absorption are discussed in
the following paragraphs.
Although the study of zinc absorption has been given much
attention, the precise mechanism, location and control of this
process have not been fully delineated (Cousins and Hempe,
1990). Differences in experimental conditions may account for
the conflicting results of in vivo and in vitro experiments
designed to study zinc absorption, since a multitude of host
and environmental factors have been shown to influence this
process (Solomons and Cousins, 1984).
Zinc is absorbed throughout the small intestine, but the
segment with the highest capacity to absorb zinc has not yet

75
been determined (Cousins and Hempe, 1990). Rat studies used
to identify the major absorptive site have produced variable
results, with some studies (Davies, 1980; Methfessel and
Spencer, 1973; Van Campen and Mitchell, 1965) suggesting that
the greatest amount of zinc uptake occurs in the duodenum, and
others (Antonson et al., 1979; Ernes and Arthur, 1975)
suggesting that greater zinc uptake occurs in the distal
portions of the intestine. Although Sahagian et al. (1966)
found the concentration of zinc from normal rat intestine to
be fairly uniform throughout each region, zinc uptake by
strips of rat intestine was greater in the duodenal and ileal
segments compared to the jejunal segment. These findings are
in contrast to those of an in vivo intestinal perfusion study
(Matseshe et al., 1980) conducted in humans. In this study,
Matseshe et al. found that more zinc left the distal duodenum
than what was contained in a test meal, suggesting that
endogenous secretions contributed to the total intraluminal
zinc concentration. For this reason, net duodenal zinc
disappearance, if any, could not be determined. Once the
intraluminal contents passed into the jejunum, zinc
disappeared gradually but incompletely. Ileal zinc absorption
was not determined in this study. In fact, all portions of
the small intestine may be functionally important in terms of
zinc absorption, with the duodenum having first access to
dietary and endogenous zinc, and subseguent sections having
the benefit of the action of digestive processes that may

76
increase the accessibility of this nutrient (Cousins and
Hempe, 1990; Lonnerdal, 1989a).
The first phase of the absorptive process involves the
movement of zinc from the intestinal lumen into the mucosal
cell. Evidence that zinc absorption occurs rapidly and
involves a saturable, carrier-mediated component, as well as
nonsaturable diffusion has come from several rat studies.
Davies (1980) exposed ligated duodenal loops from rats to
different concentrations of zinc and found that these segments
exhibited saturation kinetics at low luminal zinc
concentrations. At higher luminal zinc concentrations, zinc
uptake was linear suggesting that zinc absorption also
occurred by diffusion. Menard and Cousins (1983) reported
similar results using isolated brush border membrane vesicles
from rats. Further evidence for a mediated component of zinc
absorption was derived from experiments with rats previously
fed a zinc-adequate or a zinc-deficient diet (Steel and
Cousins, 1985). Zinc absorption, as determined from zinc
accumulation in the portal perfusate, revealed that absorption
was saturable in both groups, and that zinc-deficient rats had
a more rapid rate of zinc absorption at all luminal zinc
concentrations. Zinc absorption appeared to involve both
mediated and nonmediated components, with most of the
absorption occurring by means of the mediated component in the
zinc-deficient group. These results suggest that the
saturable process is stimulated by zinc depletion, whereas the

77
nonsaturable process is unaffected by zinc deficiency and
proceeds in proportion to the intraluminal zinc concentration.
A more recent study conducted by Hoadley et al. (1987) is in
agreement with the findings of previous studies suggesting
that zinc absorption involves two kinetic processes.
Although there is general agreement that zinc uptake by
the intestinal mucosal cell involves a saturable, carrier-
mediated component, as well as nonsaturable diffusion, it is
not known if these processes represent components of a single
transport step occurring at the brush border membrane, or if
movement from the lumen to the portal circulation occurs by
two independent routes: transcellular and paracellular.
(Cousins and Hempe, 1990). Transcellular movement may involve
a protein that has recently been identified (Hempe and
Cousins, 1991), purified and partially characterized (Khoo and
Cousins, 1993). This protein, known as cysteine-rich
intestinal protein is a low molecular weight, zinc-binding,
cytosolic protein isolated from intestinal mucosal cells.
Cysteine-rich intestinal protein has two possible zinc binding
sites, and it is hypothesized that this protein serves as an
intracellular zinc carrier. This protein appears to bind more
zinc when intestinal metallothionein is not induced,
suggesting that intestinal metallothionein may interact with
cysteine-rich intestinal protein to regulate zinc absorption
and transport (Khoo and Cousins, 1993).

78
Movement of zinc from the intestinal lumen into the
mucosal cell may be affected by the intraluminal environment.
Zinc liberated from food matrices during the digestive process
may remain as the free ion or may form coordination complexes
with endogenous (pancreatic, biliary or mucosal) or exogenous
(dietary) intraluminal ligands (Solomons and Cousins, 1984).
Studies investigating the effect of intraluminal binding
ligands on zinc uptake have suggested that ligand binding may
affect zinc absorption by altering intestinal membrane
permeability and/or zinc gradients across intestinal cell
membranes (Cousins, 1985; Solomons and Cousins, 1984).
However, while there is little question that zinc-binding
ligands affect zinc absorption, it is doubtful that this
process is a prerequisite for movement of zinc across the
membrane surface (Cousins and Hempe, 1990).
Once zinc is inside the intestinal mucosal cell, it binds
to a variety of high molecular weight ligands, as well as to
intestinal metallothionein, a low molecular weight, cyteine-
rich metalloprotein. The amount of zinc bound to intestinal
metallothionein varies according to zinc status and intake.
Zinc binding to intestinal metallothionein restricts the
movement of zinc from the cell, thereby contributing to the
regulation of zinc absorption. Intestinal metallothionein
synthesis is induced at the transcriptional level by high
dietary zinc (Blalock et al., 1988; Menard et al., 1981). When
the rate of intestinal metallothionein synthesis is high, net

79
zinc release to the portal circulation is curtailed (Menard et
al., 1981). In zinc deficiency, intestinal metallothionein
concentration is low and more zinc is released into the portal
circulation (Hoadley, et al., 1988). Thus, it appears that
the induction of metallothionein synthesis in intestinal
mucosal cells could provide for the efficient regulation of
zinc transfer to the vascular compartment.
Based on the results of rat studies (Davies, 1980; Smith
and Cousins, 1980) , transfer of zinc from the mucosal cell to
the portal circulation occurs more slowly than uptake and
accumulation within the cell. According to Davies (1980),
transfer of zinc into the portal circulation may occur in two
stages: 1) rapid transfer occurring over the first 30 minutes;
and 2) slower transfer occurring from 30 minutes to six hours
after receiving the dose, which may represent the release of
intracellularly bound zinc from zinc-binding proteins.
Consequently, movement of zinc from the intestinal mucosal
cell into the vascular compartment may be the rate-limiting
step in zinc absorption (Smith and Cousins, 1980). At low
luminal zinc concentrations, much of the zinc available is
released into the portal circulation; however, as the luminal
zinc concentration increases, less of this nutrient is
transported to the vascular system (Hambidge et al., 1986;
Smith and Cousins, 1980). An exception to this phenomenon
occurs when excessive loads of luminal zinc are available. In

80
this case, the ability to control the release of zinc into the
circulation is diminished (Smith and Cousins, 1980) .
Transport. Distribution and Metabolism
Although it had been proposed that transferrin was
responsible for portal zinc transport (Evans and Winter,
1975), several lines of investigation have shown that albumin
is the principal portal transport protein for zinc (Smith et
al., 1979). Smith et al. (1979) demonstrated the importance
of albumin in portal transport by comparing the percent of
65Zn absorbed when the vascular perfusate composition was
varied. The absence of albumin from the perfusate resulted in
negligible zinc transfer to the portal circulation, whereas
the isosmotic replacement of all plasma proteins with albumin
resulted in a twofold increase in zinc transfer over that of
the control perfusate. These data intimate that the extent of
zinc absorption may be affected by the concentration of
albumin in the blood (Cousins, 1985). Consequently, zinc
absorption may be impaired in disease states associated with
the development of hypoalbuminemia.
Zinc entering the portal circulation is rapidly
transported to the liver. The liver is the primary organ
involved in zinc metabolism, and a large portion of the zinc
in portal blood is exchanged with the liver (Cousins and
Hempe, 1990). In vitro experiments with rat liver parenchymal
cells demonstrated that zinc uptake was temperature and

81
energy-dependent, followed saturation kinetics and occurred in
two phases (Failla and Cousins, 1978a). The first phase was
characterized by rapid, saturable uptake. This was followed
by a slower phase comprised of both saturable and linear
components. Maximal uptake occurred at the normal plasma zinc
concentration (Cousins, 1989; Failla and Cousins, 1978a).
Zinc accumulation by these cells increased when physiological
concentrations of certain adrenal corticosteroids were added
suggesting that these hormones may perform an essential role
in the regulation of hepatic zinc metabolism. As a corollary,
it is possible that zinc has a role in glucocorticoid-mediated
alterations of hepatic metabolic processes (Failla and
Cousins, 1978b).
Distribution of zinc to the extrahepatic tissues occurs
mainly via the plasma. The plasma contains approximately 10
to 20% of the zinc in whole blood, whereas the erythron and
leukocytes contain the major portion of zinc in the blood.
Most of the plasma zinc is bound to albumin, although alpha-2-
macroglobulin, transferrin, histidine and cysteine may also
transport small amounts of this metal. Zinc bound to albumin
is considered to be loosely bound and represents the
metabolically active, exchangeable zinc pool in the blood.
This pool is responsive to acute and chronic changes related
to stress, infection and dietary zinc. In contrast to plasma
zinc, the zinc in erythrocytes is mostly associated with
carbonic anhydrase, although small amounts may be associated

82
with superoxide dismutase and metallothionein (Cousins, 1989;
Cousins and Hempe, 1990; DiSilvestro and Cousins, 1983) .
The rate of zinc incorporation into extrahepatic tissues
varies, as does the rate of zinc turnover in these tissues.
Zinc accumulation and turnover occurs rapidly in the kidney,
pancreas and spleen (Hambidge, et al., 1986). The rate of
zinc uptake by skeletal muscle and the central nervous system
is relatively slow, and the zinc in these tissues remains
tightly bound for long periods of time (Hambidge et al.,
1986). The zinc incorporated into hair is not available for
exchange either. Tissue-specific redistribution of body zinc
can occur during periods of zinc deprivation, stress and
infection/inflammation (Cousins and Leinart, 1988; Dunn and
Cousins, 1989; Giugliano and Millward, 1984; Huber and
Cousins, 1988; Jackson et al., 1982). For example, the zinc
concentration of muscle in zinc-deficient rats is protected,
while the concentration in bone, liver and plasma declines
(Giugliano and Millward, 1984; Jackson et al., 1982). This
redistribution may occur as a result of tissue-specific
induction of metallothionein.
In general, the largest concentration of intracellular
zinc is found in the cytosol, although smaller amounts are
present in the nuclear, microsomal and mitochondrial fractions
of cells (Hambidge et al., 1986) . Zinc is also present in the
membrane and may enhance membrane stability (Bettger and
O'Dell, 1981). Within the cytosol, zinc is primarily bound to

83
large molecular weight proteins. These proteins may be zinc
metalloenzymes. In contrast, the amount of zinc bound to
metallothionein is relatively low under normal dietary
conditions; however, when dietary zinc is increased, the
metallothionein gene is induced and metallothionein synthesis
is elevated (Cousins and Lee-Ambrose, 1992; Cousins and
Leinart, 1988; Huber and Cousins, 1988). Consequently,
increased zinc binding to metallothionein in the liver,
pancreas, kidney and muscle occurs. Zinc may regulate the
expression of the metallothionein gene through a specific
nuclear metal-binding protein that also binds to unique DNA
sequences in the promoter region (Cousins and Hempe, 1990).
Synthesis of metallothionein, particularly in the liver, may
also occur in response to hormones, as well as physiological
stimuli such as stress, acute infection and shock (Cousins and
Hempe, 1990; Hambidge et al., 1986).
Excretion
The major route of zinc excretion is via the feces. Zinc
in the feces is derived from unabsorbed dietary zinc, as well
as endogenously secreted zinc. Sources of endogenous zinc
include pancreatic, biliary and mucosal secretions, as well as
zinc present in desquamated mucosal cells. Usually,
endogenously secreted zinc is efficiently reabsorbed, but
intraluminal factors such as the presence of phytic acid may

84
decrease the efficiency of absorption (Cousins and Hempe,
1990). Additionally, dietary intake of zinc may affect
reabsorption, with more zinc of endogenous origin being
excreted in the feces at higher levels of zinc intake (Jackson
et al., 1984) .
Urinary zinc losses account for a fairly small amount of
the zinc excreted under normal physiologic conditions. The
usual range of zinc excreted in the urine of healthy adults is
about 300 to 600 [iq/d (Gibson, 1990). Urinary zinc excretion
has been shown to decrease in human subjects consuming a zinc-
deficient experimental diet (0.28 mg zinc/d) (Baer and King,
1984). Injury, burns, infection, acute starvation and
pathologic conditions resulting in excessive muscle catabolism
have been associated with clinically significant increases in
urinary zinc excretion. Hyperzincuria is known to occur in
conjunction with proteinuria due to kidney dysfunction and in
patients with sickle cell disease and cirrhosis of the liver
(Cousins and Hempe, 1990; Gibson, 1990).
Dermal losses due to sweating and to the sloughing of
epithelial tissue also account for some of the zinc lost from
the body. As reported in a review by Hambidge et al. (1986),
the total amount of zinc lost in sweat from adults living in
a temperate North American climate has been estimated to be
0.4 to 2.8 mg/d. Dermal zinc losses have been found to be
influenced by dietary zinc intake, with reduced losses
occurring when zinc intake is marginal, and losses increasing

85
following zinc repletion (Milne et al., 1984). This apparent
conservation of zinc during a period of inadeguate zinc intake
may represent a homeostatic mechanism (Hambidge et al., 1986).
Menstrual and seminal excretions represent additional
routes by which zinc losses may occur in females and males,
respectively. The average amount of zinc lost with each
menstrual cycle represents a loss of approximately 15 nq/d
over the course of a month (Hambidge et al., 1986). Seminal
zinc losses are estimated to be approximately 0.6 mg/ejaculum
(Baer and King, 1984).
Zinc Homeostasis
Whole body zinc homeostasis is achieved primarily by
changes in absorption and excretion in response to changes in
dietary zinc intake. The results of an animal study in which
rats were fed increasing amounts of zinc (Coppen and Davies,
1987) suggest that zinc homeostasis at lower levels of zinc
intake is a function of increased efficiency of absorption and
retention, but when dietary zinc intake is excessive,
homeostasis is modulated solely by changes in zinc excretion.
Biochemical Functions
The unique chemistry of zinc and its biological abundance
make it ideally suited for a multitude of biochemical and
physiological functions. Of these functions, zinc is most
noted for its role as a component of over 60 different

86
metalloenzymes, including those involved in the metabolism of
proteins, carbohydrates, lipids and nucleoproteins. The
specific metalloenzyme roles of zinc have been characterized
as catalytic, structural and regulatory. The catalytic action
of zinc is probably due to its ability to bind directly with
the substrate and/or through a metal-bound molecule of water.
Examples of zinc metalloenzymes with catalytic roles include
carbonic anhydrase and carboxypeptidase. The structural role
of zinc is related to its ability to provide structural
integrity to zinc-reguiring enzymes, such as superoxide
dismutase, by stabilizing the guaternary structure of the
enzyme complex. Alteration of this structure due to the loss
of zinc results in disruption of enzyme activity. Apart from
its catalytic and structural roles, zinc may also serve a
regulatory function by inhibiting enzymatic function, as it
does in fructose-1,6-bisphosphatase (Hambidge et al., 1986).
There is evidence that in addition to the enzymatic
functions of zinc, this nutrient may have important
nonenzymatic biological functions. For example, zinc may play
a role in gene transcription and expression by promoting
structural changes in DNA-binding proteins that allow these
proteins to bind more strongly or specifically to DNA via
motifs called zinc fingers. Although the impact of zinc
deficiency on zinc fingers reguires further investigation, it
is possible that zinc deficiency could result in improper
folding or structural configuration of the DNA-binding

87
proteins, resulting in altered gene expression, cell
dysfunction and ultimately, overt clinical manifestations of
zinc deficiency (Cousins and Hempe, 1990).
Bettger and O'Dell (1981) have suggested that zinc may
function to stabilize cell membrane structure. These
researchers found that erythrocytes from zinc-deficient rats
contained significantly less zinc and exhibited increased
osmotic fragility compared to erythrocytes from control
animals. The fact that elevated lipid peroxidation has been
observed in tissues from zinc-deficient animals (Sullivan et
al., 1980) lends support to this idea and may provide an
explanation for the development of skin disorders and other
symptoms associated with zinc deficiency. Although a
mechanism for this proposed cytoprotective function has not
been identified, it is possible that stabilization may occur
through phospholipid or thiol group linkages with zinc
(Cousins and Hempe, 1990).
The numerous physiologic functions ascribed to zinc are
listed in Table 2-2. Most of these functions have been
identified based on the results of animal and/or human studies
in which a zinc-deficient state was experimentally induced, or
clinical observations of zinc-deficient humans. In many
cases, the underlying biochemical mechanism(s) responsible for
the physiological effect has not been determined.

88
Table 2-2. Physiologic functions of zinc.
Antioxidant role via metalloproteins
Cellular growth
Cellular replication
Fertility and reproduction
Hormone production, storage and secretion
Immune system development and maintenance
Protein, carbohydrate and lipid metabolism
Sexual maturation
Taste and appetite
Recommended Dietary Allowances for Zinc
Estimates of human zinc requirements have been made using
balance studies and by measuring endogenous zinc losses along
with fractional zinc absorption. Neither of these methods is
entirely satisfactory because the results can be influenced by
homeostatic mechanisms that control zinc absorption and
excretion. An alternative approach would be to determine the
amount of zinc that maintains normal physiological and
metabolic functions. This approach has been limited by the
lack of a sensitive and specific indicator(s) of zinc status
(King, 1986).
The 1989 Recommended Dietary Allowances (RDA) for zinc
were based on the results of balance studies and estimated
zinc losses in healthy adults. Balance studies have suggested

89
that at least 12 mg zinc/d is necessary to achieve
equilibrium. Estimates of zinc losses range from 2.2 to 2.8
mg/d in healthy young males consuming a mixed diet. Based on
these loss estimates, it has been assumed that an average of
approximately 2.5 mg/d absorbed zinc is needed to maintain
equilibrium. Using an absorption efficiency of 20% to allow
for poor zinc absorption associated with high fiber diets, the
resulting dietary requirement amounts to 12.5 mg/d, which
closely approximates the estimate determined from balance
studies. To allow for a margin of safety, the 1989 RDA for
zinc has been set at 15 mg/d for adolescent and adult men,
whereas the RDA for adolescent and adult females has been set
at 12 mg/d because of their smaller body size (Food and
Nutrition Board, 1989b) . The RDA for zinc for elderly
Americans is set at the same level as that for younger adults,
although the results of a stable isotope study (Turnland et
al., 1986) suggest the possibility that this population group
may have a higher requirement due to a reduced ability to
absorb zinc. Conversely, the reduction in zinc absorption may
reflect a lower requirement for absorbed zinc.
The increase in the requirement for zinc during pregnancy
may be a reflection of the important role zinc plays in
cellular replication and differentiation. Estimates of the
average amount of additional zinc needed during pregnancy
(i.e. approximately 100 mg zinc/normal pregnancy) have been
made by Sandstead (197 3) and by Swanson and King (1987) .

90
Based on these estimates and the lack of evidence for
increased absorption efficiency during pregnancy (Swanson et
al., 1983), a dietary intake of 15 mg/d is recommended during
pregnancy (Food and Nutrition Board, 1989b).
The average daily milk production and the average zinc
content of human milk during the first and second six months
after delivery were used to estimate the daily requirement
during lactation. This estimate was adjusted to account for
an absorption efficiency of 20% and a coefficient of variation
of 12.5%, resulting in a recommended allowance of 19 mg/d and
16 mg/d during the first and second six months of lactation,
respectively (Food and Nutrition Board, 1989b).
Full-term, breast-fed infants rarely show signs of zinc
deficiency, so it has been assumed that the zinc content of
human milk, along with the infant's liver stores, are adequate
to meet daily requirements during the first six months of life
(Food and Nutrition Board, 1989b). The bioavailability of
zinc from human milk is higher than that from infant formulas
(Blakeborough et al., 1986; Sandstrom et al., 1983). The high
concentration of zinc-binding ligands such as citric acid
and/or picolinic acid was once thought to be responsible for
the higher bioavailability (Eckhert et al., 1977). However,
the high lactalbumin content of human milk, as opposed to the
high casein content of cow's milk-based formulas, appears to
be the major determinant of zinc bioavailability (Cousins and
Smith 1980; Lonnerdal, 1989b; Roth and Kirchgessner, 1985;

91
Sandstróm et al., 1983). Although the zinc content of human
milk decreases as the infant ages, the contribution of zinc
from solid foods is thought to be sufficient to meet the
infant's needs during the second six months of life.
Compared to breast-fed infants, the estimated requirement
for formula-fed infants is higher because the bioavailability
of zinc from formula, particularly soy-based formulas, is
lower (Casey et al., 1981; Lonnerdal et al., 1984). The
current RDA for formula-fed infants was based on a study
(Walravens and Hambidge, 1976) demonstrating better growth of
male infants fed a formula supplemented with 4 mg zinc/L (i.e.
5.8 mg zinc/L) compared with infants consuming the same
formula without additional zinc (i.e. 1.8 mg zinc/L).
Assuming that formula consumption is 750 mL/d, plus two
standard deviations, the RDA for formula-fed infants was set
at 5 mg/d (Food and Nutrition Board, 1989).
Consideration has been given to the possibility that
iron-fortified infant formulas or infant foods may impair zinc
absorption and adversely affect zinc status (Craig et al.,
1984; Fairweather-Tait and Southon, 1989). However, the
American Academy of Pediatrics Committee on Nutrition (1989)
has rendered the opinion that the available evidence does not
support a detrimental effect of iron-fortified formulas on
infants' zinc status.
The recommended dietary allowance for zinc for
preadolescent children is 10 mg/d. This recommendation was

92
made based on a study of Spanish-American children (Walravens
et al., 1983) with zinc intakes of approximately 5 to 6 mg/d.
These children displayed signs of marginal zinc deficiency and
their height-for-age was below the tenth percentile. When
their diet was supplemented to a total intake of 10 mg/d,
their rate of linear growth improved. Evidence of a growth-
limiting effect due to marginal zinc deficiency, and a growth¬
enhancing effect due to zinc supplementation, has also been
demonstrated in Southern Ontario boys aged 5 to 7 (Gibson et
al., 1989).
Effects of High Doses of Zinc
Cases of acute and chronic zinc toxicity have been
reported in humans. Accounts of acute toxicity are relatively
rare and have been primarily associated with food poisoning
incidents (Fosmire, 1990). In these cases (Brown et al.,
1964), acidic foods or beverages were stored in galvanized
containers for long periods of time. Presumably, sufficient
zinc was leached from the coating of these containers to cause
toxic manifestations which included nausea, vomiting,
epigastric pain, abdominal cramps and diarrhea. Different
symptoms, including lethargy, light-headedness and slight
staggering of gait, were reported (Murphy, 1970) in a case
where 12 g elemental zinc was ingested over a period of 36 h.
Fosmire (1990) has suggested that the form of zinc salt

93
ingested may influence which manifestations of toxicity will
be observed.
Zinc toxicity resulting from moderately elevated intakes
of zinc (i.e. 100 to 300 mg/d) is more common than acute zinc
toxicity (Fosmire, 1990). Potential consequences of prolonged
intakes within this range include induction of copper
deficiency, impaired immune response and altered plasma
lipoprotein profiles.
Copper deficiency, manifested by low plasma copper,
anemia and neutropenia has been reported in patients with
sickle cell anemia (Prasad et al., 1978b) and patients with
nonresponsive celiac disease (Porter et al., 1977) treated
with 150 mg zinc/d for 23 or 13 months, respectively.
Impaired copper status has been reported with lower levels of
supplementation as well (i.e. 50 mg/d for 6 or 10 weeks)
(Fischer et al., 1984; Yadrick et al., 1989).
Evidence of impaired immune response in young men
consuming 300 mg zinc/d for 6 weeks was reported by Chandra,
1984; however, supplementation with 100 mg zinc/d for 3 months
in an elderly population did not result in impaired immune
status (Bogden et al., 1988). Differences in the ages of the
subjects and the supplementation regimens may account for the
discrepant results (Fosmire, 1990).
Altered lipoprotein profiles have been reported (Chandra,
1984) in subjects given 300 mg zinc/d for 6 weeks. This dose
of zinc resulted in a significant decrease in high-density

94
lipoprotein cholesterol and a significant increase in low-
density lipoprotein cholesterol levels. Reduced high-density
lipoprotein cholesterol levels have also been reported in
subjects consuming 160 mg zinc/d for 6 weeks (Hooper et al.,
1980).
Food Sources of Zinc, Usual Intakes and Bioavailabilitv
Major dietary sources of zinc include foods with a high
protein content such as oysters and other shellfish, meat,
liver and dairy products. The zinc content of meat varies
depending on the species and the specific tissue, with dark
red meat generally containing more zinc than white meat.
Whole grains, legumes and nuts are also good sources of this
nutrient, but the zinc in these foods is generally less
available for absorption and can be affected by growing
conditions and processing. Vegetables and fruits contain
small amounts of zinc. Sweets, fats and oils are relatively
low in zinc.
The zinc content of the United States food supply in 1985
was estimated to be 12.3 mg/person/d (Moser-Veillon, 1990).
Although this estimate does not meet the zinc RDA for adult
men, it should be recognized that the per-person data included
women and children for whom the zinc RDA is lower. This
estimate assumes an equal distribution of foods and nutrients
across the entire population and does not account for the
effect of food choices or zinc bioavailability.

95
Data from the Food and Drug Administration's (FDA) Total
Diet Study (Pennington and Young, 1991) indicate that American
infants generally meet their RDA for zinc. In the case of
young children, the findings of FDA's Total Diet Study and the
Nationwide Food Consumption Survey (United States Department
of Agriculture, 1987) , are not as encouraging, with many
children consuming diets with zinc contents below the RDA.
During adolescence and adulthood, gender differences in zinc
intakes become apparent, with males tending to have more
adequate intakes of this nutrient. For example, FDA's Total
Diet Study found that the diets of teenage boys, but not
girls, met the RDA for zinc. Similarly, the 1985 Nationwide
Food Consumption Survey (United States Department of
Agriculture, 1986) found that men between the ages of 19 to 50
years consumed an average of 94% of their RDA for zinc, while
FDA's Total Diet Study reported an average of 109% of the RDA
for men between the ages of 25 to 30 years. In contrast, the
1986 Nationwide Food Consumption Survey (United States
Department of Agriculture, 1987) found that women between the
ages of 2 0 to 49 years consumed about half of the RDA for
zinc. Low dietary zinc intakes among women have been
confirmed by other studies including FDA's Total Diet Study
and the National Health and Nutrition Examination Survey II.
The difference in total zinc intake between males and females
has been attributed to differences in energy consumption since

96
the zinc density of diets consumed by men and women were
similar (Moser-Veillon, 1990).
Since zinc intake is associated with caloric consumption,
it is not surprising that elderly people, who may restrict
their food intake due to illness, poverty and/or difficulty in
chewing, often have lower zinc intakes than younger adults.
The estimated zinc intake of elderly people is 7 to 10 mg/d
(Greger, 1989). Zinc bioavailability may also be reduced in
this group due to reduced meat consumption and increased
consumption of phytic acid containing foods such as cereals.
As discussed in a review by Cousins and Hempe (1990),
even though the zinc content of foods can be used to estimate
dietary intake of this nutrient, the bioavailability of zinc
from different foods and food combinations is highly variable.
For this reason, dietary intake may be a poor indicator of the
amount of zinc that is actually available for use by the body.
A more appropriate indicator of zinc supply would be the
amount of bioavailable zinc in the diet; however, this is
difficult to determine because estimates can be affected by
zinc status, nutrient interactions and the experimental
methods used to make these determinations (Cousins and Hempe,
1990).
Factors that may enhance or inhibit zinc availability and
absorption have been reviewed by Cousins and Hempe (1990).
Substances that may form insoluble complexes with zinc and
thus reduce the availability of this nutrient include: fiber,

97
phytic acid, oxalic acid, calcium, iron and high con¬
centrations of ethylenediamine tetraacetic acid (EDTA).
Dietary factors that may enhance zinc absorption, such as
amino acids (histidine, cysteine, lysine and glycine), citric
acid, picolinic acid and low concentrations of EDTA, are
thought to improve zinc absorption by forming digestible and
absorbable zinc chelates.
Rat studies have shown that phytic acid has an adverse
effect on zinc absorption when the phytic acid:zinc ratio
exceeds 12 to 15 (Morris and Ellis, 1980). Phytic acid alone,
however, does not appear to adversely effect zinc
bioavailability in humans at a phytic acid:zinc ratio of 10
(Ellis et al., 1987). Estimates (Ellis et al., 1982) of the
phytic acid:zinc ratio of omnivorous and vegetarian diets
typically consumed in the United States (i.e. a molar ratio of
3.3 for meat-based diets, 4.5 for lacto-ovo vegetarian diets
and 7.6 for soy-based diets) suggest that the phytic acid
content is not high enough to impair zinc bioavailability.
The effect of phytic acid is accentuated, however, in the
presence of high intraluminal calcium (Forbes and Erdman,
1983). Based on human experiments (Ellis et al., 1987)
applying the phytic acid x calcium:zinc ratio, phytic acid
does not appear to adversely effect zinc absorption from
omnivorous diets. It is possible, however, that zinc
bioavailability may be impaired in lacto-ovo vegetarians
consuming large guantities of calcium.

98
A competitive interaction between iron and zinc may also
exist; however, the results of human studies are
controversial. A possible explanation for the conflicting
results may be due to factors such as the form of iron, the
presence of a meal and/or the amounts of zinc and iron fed
(Storey and Greger, 1987). For example, when egual amounts of
ferrous iron and zinc (as sulfate) were ingested
simultaneously, zinc absorption was depressed; however, zinc
absorption was not affected when heme iron or a food source of
zinc were used (Solomons and Jacob, 1981). Additionally, when
human subjects consumed iron supplements with a meal, zinc
absorption was not impaired (Sandstrom et al., 1985). It is
likely that under most dietary conditions, an interaction
between iron and zinc is not sufficient to influence zinc
reguirements; however, zinc absorption may be adversely
affected in pregnant women taking iron supplements (Simmer et
al., 1987) .
Animal studies (Stuart et al., 1986) have shown that zinc
absorption increases when dietary protein increases, and that
zinc is more bioavailable from animal than plant protein
sources. The effect of protein on zinc absorption in humans,
however, is controversial. The inconsistent effect of protein
on zinc absorption in human studies may be due to other
factors associated with proteins such as phosphorus and phytic
acid content, as well as zinc status and previous intake. As
noted above, some amino acids have been associated with

99
improved zinc absorption because they form soluble complexes
with zinc (Solomons and Cousins, 1984). It is possible that
part of the positive effect of protein on zinc absorption
observed in animal studies is due to the effect of additional
amounts of these amino acids.
Factors Affecting Zinc Status
As noted above, the physiological stage of growth and
development affects zinc requirements, and the amount and
bioavailability of dietary zinc consumed, relative to zinc
need, has an impact on zinc status. Other factors that can
affect zinc status include diseases, alcohol consumption and
medication use. The potential effect of these factors on zinc
status is discussed below.
Diseases. Zinc intake, absorption, excretion and/or
requirements can be affected by the presence of certain
diseases (Cunnane, 1988), and many diseases are associated
with anorexia and/or decreased taste acuity. These problems
may reduce overall food intake, resulting in a reduction in
zinc intake. Impaired zinc absorption resulting from diseases
associated with intestinal mucosal damage, such as Crohn's
disease (McClain et al., 1980; Solomons et al., 1977), and
from diseases in which there is a lack of appropriate
absorption ligands, such as acrodermatitis enteropathica
(Aggett, 1989) and cystic fibrosis (Caillie-Bertrand et al.,
1982), can also adversely affect zinc status. Impaired zinc

100
status has also been reported in diseases associated with
excessive zinc losses such as sickle cell disease (Prasad et
al., 1976) and insulin-dependent diabetes mellitus (Canfield
et al., 1984). Increased zinc requirements are associated
with neoplastic diseases due to rapid tissue turnover
(Solomons, 1988).
Alcohol. The etiology of impaired zinc status observed
in some alcoholics is probably multifactorial, with inadequate
dietary intake, intestinal malabsorption, pancreatic
insufficiency, decreased albumin affinity and excessive
urinary excretion contributing to abnormal zinc metabolism
(Solomons, 1988) . Zinc status does not appear to be
compromised as a result of occasional alcohol use, or in all
individuals suffering from alcoholism, suggesting that the
effect on overall zinc homeostasis is dependent on the amount
and frequency of alcohol consumed and the extent of alcohol-
induced tissue damage (Cunanne, 1988).
Medications. Medications that may interfere with zinc
status include antineoplastic agents, aspirin and
penicillamine. Antineoplastic agents and aspirin may chelate
zinc, making it less available to the body. Penicillamine
interferes with zinc status by increasing urinary zinc
excretion (Cunanne, 1988).

101
Assessment of Zinc Status
Assessment of zinc status has been difficult because a
specific and sensitive indicator has not been available for
this purpose. In the absence of a single reliable measure of
zinc status, a variety of biochemical and functional indices
have been used. These indices include measurements of zinc in
tissues or fluids such as plasma/serum, erythrocytes,
leukocytes, neutrophils, urine and hair. Functional indices
such as changes in zinc metalloenzyme activity, dark
adaptation, taste acuity and macrophage chemotaxis have also
been used to assess zinc status. The limitations of these
indices are discussed in the following paragraphs.
Plasma/serum zinc concentration. Plasma or serum zinc
concentration is the most commonly used index of zinc status
(Cousins and Hempe, 1990; Solomons, 1979). Individuals with
fasting morning plasma/serum zinc values below 70 ^iq/dh
(< 10.71 /nmol/L) are considered to be at risk for zinc
deficiency (Gibson, 1990). Decreased plasma/serum zinc
concentrations have been reported in patients whose sole
source of nutrition consisted of total parenteral nutrition
solutions that did not contain zinc (Arakawa et al., 1976;
Fleming et al. , 1976). Experimentally-induced severe zinc
deficiency has also resulted in decreased plasma/serum
concentrations of this nutrient (Baer and King, 1984; Gordon
et al., 1982; Hess et al., 1977;
Prasad et al., 1978a).

102
Studies of pregnant women suggest that the normal pattern of
decline in plasma zinc throughout gestation may be useful as
an indicator of zinc utilization, and that deviations from
this pattern may indicate abnormal utilization, and thus,
impaired status (Swanson and King, 1987).
One of the problems associated with the use of
plasma/serum zinc concentration as an index of zinc status is
that this measure is subject to homeostatic control. As a
result, normal plasma/serum zinc concentrations may be
maintained despite inadequate zinc intake and/or absorption.
For example, in a study designed to evaluate zinc utilization
in young men fed adequate and low zinc intakes, Wada et al.
(1985) found that plasma zinc concentrations did not fall when
dietary zinc was decreased to 5.5 mg/d; however, fecal zinc
excretion was reduced. Similarly, zinc losses in sweat
decreased by 65%, but plasma zinc concentrations did not
decline during an 18 week study of subjects consuming 3.6 mg
zinc/d (Milne et al., 1983). Studies of infants and children
suggest that mild zinc deficiency is associated with growth
retardation in the absence of changes in plasma zinc
concentrations (Walravens and Hambidge, 1976; Walravens et
al., 1983; and Walravens et al., 1989). These findings
suggest that the initial response to mild zinc deficiency is
a reduction in growth rate or zinc excretion. As discussed by
King (1990), these adaptive responses appear to result in the
maintenance of plasma/serum zinc concentrations, and it is not

103
until the homeostatic capacity is exceeded that plasma/serum
zinc concentrations decline. The eventual reduction in
plasma/serum zinc represents a loss of zinc from bone and
liver and signals the impending development of metabolic and
clinical signs of zinc deficiency (King, 1990). Thus,
plasma/serum zinc concentrations is a marker of the size of
the exchangeable zinc pool (King, 1990).
Another problem associated with the use of plasma/serum
zinc concentration as an index of zinc status is that it is
altered by several nonnutritional factors. These factors
include: infection (Solomons et al., 1978; Wannemacher et al.,
1975), diurnal variations (Gordon et al., 1982), hormonal
state (Cousins, 1985) and short-term fasting (Henry and Elmes,
1975). Changes in circulating zinc associated with these
factors are not due to altered zinc status, but to metabolic
disturbances that result in zinc redistribution to other
tissues (Golden, 1989). For example, 65Zn uptake was
increased in the liver, bone marrow and thymus and reduced in
the bone, skin and intestine when interleukin-1 was
administered to rats (Cousins and Leinart, 1988). This
redistribution of tissue zinc was accompanied by a temporary
decline in circulating zinc. Consequently, plasma/serum zinc
concentration can not be used as a specific indicator of zinc
status unless the effects of other metabolic conditions can be
differentiated from the effects due to actual changes in zinc
status (Golden, 1989; King, 1990).

104
Oral zinc tolerance test. This test measures the
increase in plasma zinc from baseline (i.e. fasting) to four
hours after the ingestion of a pharmacological dose of zinc.
The results of the oral zinc tolerance test compare favorably
with those obtained by direct measurements of 65Zn absorption
(Valberg et al., 1985), suggesting that this test is a valid
measure of zinc absorption. However, this test does not
appear to be a reliable indicator of zinc status because it
can be affected by homeostatic mechanisms influencing zinc
absorption (Fickel et al., 1986). The results of this test
can also be influenced by factors other than absorption,
including variations in gastric emptying, peripheral uptake
and renal excretion (Valberg et al., 1985).
Erythrocyte zinc concentration. Studies in which
erythrocyte zinc concentration has been measured as an index
of zinc status have produced equivocal results. For example,
a significant decrease in the mean erythrocyte zinc
concentration of volunteers consuming 0.6 to 1.0 mg zinc/d for
78 days was reported by Buerk et al. (1973). Prasad et al.
(1978a) also noted significant decreases in erythrocyte zinc
in three of four subjects who consumed diets containing 2.7 or
3.5 mg zinc/d for two months. Conversely, Baer and King
(1984), in a zinc depletion study (0.28 mg zinc/d) lasting
four to nine weeks, and Rabbani et al. (1987), in a zinc
stabilization-depletion-repletion study lasting 56 weeks,
failed to show a change in mean erythrocyte zinc

105
concentrations. These apparently conflicting results might be
explained once more is learned about the metabolism of zinc in
erythrocytes, the dependence of erythrocyte zinc on
circulating levels of zinc and the influence of factors such
as the duration, severity and rapidity of onset of zinc
deficiency. The lack of standardization in terms of the form
of the erythrocyte sample used (i.e. washed cells, cell
lysates, cell membranes, density gradient-separated cells,
etc.) and the units employed to express erythrocyte zinc
concentrations (i.e. ;xg/g protein, ;xg/g hemoglobin, ^g/ml
erythrocyte lysate, etc.) may also account for divergent
results.
Leukocyte zinc and neutrophil zinc concentrations.
Several researchers have suggested that the zinc content of
leukocytes (Meadows et al., 1981; 1983; Prasad et al., 1978a)
and specific cellular types of leukocytes (i.e. neutrophils)
(Prasad and Cossack, 1982) may be more reliable indices of
zinc status than plasma/serum or erythrocyte zinc
concentrations. Despite these reports, there are several
methodological problems associated with the use of these
techniques, and the validity of using these measures as
indices of zinc status remains uncertain (Gibson, 1990).
Urinary zinc concentration. Low urinary zinc excretion
was first reported in dwarfs from the Middle East suffering
from severe zinc deficiency (Prasad et al., 1963a). Reduction
in urinary zinc excretion has also been reported in

106
experimentally induced zinc deficiency (Baer and King, 1984;
Hess et al., 1977). In fact, Baer and King (1984) found that
urinary zinc responded more rapidly to changes in zinc intake
than plasma zinc and suggested that the former might be useful
for evaluating zinc nutriture.
Urinary zinc excretion also appears to be sensitive to
marginal zinc intakes, although the length of time the
subjects are exposed to a reduced intake, the level to which
the intake is reduced and the calcium and phytate contents of
the diet may affect the overall response. For example,
urinary zinc losses were not reduced after nine days in
subjects fed a diet containing 5.5 mg zinc/d (Wada et al.,
1985), nor were they reduced after six weeks of consuming a
diet containing 7.2 mg zinc/d (Thomas et al., 1992). There
were, however, significant reductions in urinary zinc
excretion after consuming a diet containing 3.2 mg zinc/d for
six weeks (Thomas et al., 1992) and after adhering to a
feeding protocol that consisted of one week of 0.6 mg zinc/d
and two weeks of a diet containing 4 mg zinc/d plus additional
phytate and calcium (Ruz et al., 1991).
Despite the apparent sensitivity of urinary zinc
excretion to dietary zinc intake, the large variability in
zinc excretion in normal healthy subjects fed a zinc adeguate
diet suggests that urinary zinc excretion may be affected by
differences in tissue zinc status (King, 1986). This may
hamper the usefulness of urinary zinc excretion as an index of

107
zinc status. Furthermore, urinary zinc excretion can be
affected by other factors, with hyperzincuria and zinc
deficiency occurring concurrently in individuals with sickle
cell disease and cirrhosis of the liver. Hyperzincuria also
occurs in certain renal diseases and infections and after
injury, burns, acute starvation and treatment with
chlorothiazide (Prasad, 1983). Consequently, urinary zinc
excretion may only be useful as a measure of zinc status in
apparently healthy individuals (Gibson, 1990).
Activity of zinc-dependent enzymes. Measurement of the
activities of various zinc-dependent enzymes has been proposed
as a way to assess zinc status. Of these enzymes, measurement
of serum alkaline phosphatase activity is most common. Low
serum alkaline phosphatase activity has been reported in human
zinc deficiency due to acrodermatitis enteropathica (Weismann
and Hoyer, 1985) and in patients receiving unsupplemented
total parenteral nutrition (Kay et al., 1976; Weismann and
Hoyer, 1985). The activity of this enzyme has been shown to
increase when zinc supplements are administered to patients
with these conditions (Weismann and Hoyer, 1985). Similar
responses have been shown to occur in experimental zinc
deficiency followed by repletion (Baer et al., 1985; Prasad et
al., 1978a). Conversely, Hess et al. (1977) found no
consistent change in serum alkaline phosphatase activity
during an experimental zinc depletion study lasting five
weeks. No significant changes in serum alkaline phosphatase

108
activity were noted by Ruz et al. (1991) either, during
experimentally-induced mild zinc deficiency and repletion.
The controversial nature of these results, and the fact that
the activity of this enzyme can be affected by concurrent
liver/bone disease, suggest that use of serum alkaline
phosphatase activity as a determinant of zinc status should be
reserved for use in normal healthy subjects and should not be
employed as the sole criteria for diagnosing zinc deficiency.
Angiotensin-converting enzyme is a zinc metalloenzyme
that converts angiotensin I to angiotensin II. Activity of
this enzyme has been reported to be lower in zinc-deficient
rats than in zinc-supplemented rats and can be increased by in
vitro addition of zinc, with a greater percent increase in
activity occurring in zinc-deficient rats compared to controls
(Reeves and O'Dell, 1985). Low angiotensin-converting enzyme
activity has also been associated with low serum zinc
concentrations in human subjects who have lung cancer (Bakan
et al., 1988). Based on these findings, it has been suggested
that determination of angiotensin-converting enzyme activity
might be useful for assessing zinc status in humans; however,
studies in which experimental zinc deficiency has been induced
in human subjects (Milne et al., 1987; Ruz et al., 1991) do
not support this idea.
Zinc content of hair. As noted by Gibson (1990), the
zinc content of hair has been proposed as an index of chronic
suboptimal zinc status in children, providing the confounding

109
effect of severe protein-energy malnutrition is absent.
Support for this idea has come from studies of children with
marginal zinc deficiency, characterized by low growth
percentiles, who also had low hair zinc concentrations (Buzina
et al., 1980; Gibson et al., 1989; Hambidge et al., 1972;
Smit-Vanderkooy and Gibson, 1987; Xue-Cun et al., 1985).
However, zinc supplementation was not always effective in
improving hair zinc concentrations in these children, causing
concern about the appropriateness of using hair zinc
concentration as a measure of zinc status.
Studies comparing hair zinc concentrations to circulating
levels of zinc have also produced conflicting results. For
example, Klevay (1970) found a significant correlation between
hair zinc and serum zinc concentrations in a group of
Panamanian children. Conversely, a cross-sectional study
(McBean et al., 1971) of Iranian children, and a short-term
longitudinal zinc depletion/repletion study (Lane et al.,
1982), did not find a positive correlation between plasma zinc
and hair zinc concentrations. These discrepancies may be
explained in part by the fact that the zinc content of the
hair shaft reflects the guantity of zinc available to the hair
follicles over an earlier time interval. Conseguently,
positive correlations between hair zinc and plasma zinc
concentrations in children may only be evident in chronic zinc
deficiency (Gibson, 1990).

110
The usefulness of hair zinc concentration as an indicator
of suboptimal zinc status in adults is questionable. Mean
hair zinc concentration was not affected in response to
experimentally-induced acute zinc depletion (Baer and King,
1984; Ruz et al., 1991). Conversely, low hair zinc
concentrations have been reported in individuals who have
chronic diseases that are associated with impaired zinc status
such as sickle cell disease (Prasad et al., 1975; 1976) and
acrodermatitis enteropathica (Amador et al., 1975), but this
finding was not consistent among patients with Crohn's disease
(Solomons et al., 1977). Physiological state may also affect
the hair zinc concentration since some (Hambidge and
Droegemueller, 1974; Vir et al., 1981), but not all (Campbell-
Brown et al., 1985; Hambidge et al., 1983), researchers have
reported a decrease in hair zinc concentration during
gestation. Other confounding factors include environmental
contaminants, gender, age, season, hair color, hair
treatments, rate of hair growth and rate of zinc delivery to
the hair root (Hambidge, 1982).
Metallothionein
As suggested by the foregoing discussion, a single
reliable method for diagnosing zinc deficiency and assessing
zinc reserves has not been identified. Two new methods that
may be useful indicators of zinc status in humans are the
measurement of plasma and erythrocyte metallothionein

Ill
concentrations (Golden, 1989) . A brief review of the
structure, function and regulation of metallothionein,
followed by a discussion of the potential usefulness of
erythrocyte and plasma metallothionein concentrations for
assessment of zinc status, are presented in the succeeding
paragraphs.
Metallothionein is the term used to refer to a family of
low molecular weight, cytosolic proteins that are capable of
binding heavy metals such as cadmium, copper and zinc. These
proteins are single-chain polypeptides containing 60 to 61
amino acids, with cysteine as the predominate amino acid
residue. Other features of the primary structure include the
fact that the native protein contains no disulfide bonds,
histidine residues or aromatic amino acids. The molecular
weight of mammalian metallothionein, as determined from
seguence data, is about 6000 Daltons for the native protein,
although the actual molecular weight can range from 6500 to
7000 Daltons depending on the metal composition (Dunn et al.,
1987) .
The physiological significance of metallothionein has not
been clearly identified; however, proposed functions of this
protein include homeostatic and cytoprotective roles.
Potential cytoprotective roles include detoxification of heavy
metals, free radical scavenging and protection against
ultraviolet light and X-ray damage (Dunn et al., 1987). One
example of a homeostatic function is the ability of intestinal

112
metallothionein to block zinc absorption when zinc intake is
high (Cousins, 1985). Metallothionein may also regulate the
movement of zinc within cells and may participate in ligand-
exchange reactions by donating ions needed for activation of
metalloenzymes and/or zinc-requiring domains of DNA-binding
factors (i.e. zinc fingers) (Cousins and Hempe, 1990; Dunn et
al., 1987).
Although metallothionein isoforms have been isolated from
most vertebrate tissues, they are particularly abundant in the
liver, kidney and intestine. Very low concentrations of
metallothionein isoforms have been found in plasma, urine and
bile. The major isoforms of metallothionein, metallothionein-
1 and metallothionein-2, are found in most vertebrate tissues.
Expression of these isoforms is influenced by species, tissue
type, physiological state and exposure to metals (Dunn et al.,
1987). Although the predominant form of metallothionein is
metallothionein-2 (Dunn et al., 1987), a recent study by Huber
and Cousins (1993) showed that the primary gene expressed in
the bone marrow of rats is metallothionein-1. The predominate
form in human bone marrow/erythrocytes has not been
determined.
Metallothionein biosynthesis in the liver and intestine
is influenced by the zinc status of the animal, with low
concentrations in zinc-deficient animals and increased
quantities following zinc repletion (Richards and Cousins,
1976). Zinc has been shown to induce metallothionein

113
synthesis in rats by increasing the rate of metallothionein
gene transcription (Blalock et al., 1988). Evidence for
metallothionein induction by zinc in humans has also been
obtained (Grider et al., 1990). The proposed mechanism
involves binding of the metal to a nuclear regulatory factor
which subsequently binds to the DNA sequence of the metal
regulatory element (Cousins et al., 1988; Hamer, 1986).
Evidence supporting this hypothesis was recently reported by
Cousins and Lee-Ambrose (1992). These researchers
demonstrated that nuclear zinc uptake and metallothionein gene
expression are influenced proportionately by the level of
dietary zinc intake in rats, and that this newly acquired zinc
binds to nuclear zinc-binding factors, one of which appears to
bind to one of the known metal regulatory element sequences.
In addition to metallothionein induction by metal ions,
hepatic metallothionein synthesis is also influenced by
hormones (i.e. glucocorticoids, glucagon and epinephrine), as
well as other factors, such as cyclic adenyl monophosphate,
interferon, interleukin-1, food restriction and tissue injury
(Dunn et al., 1987). The exact mechanisms whereby each of
these factors affect metallothionein synthesis have not been
fully delineated, but there is significant tissue specificity.
The concentration of metallothionein in the plasma has
been shown (Mehra and Bremner, 1984) to correlate with dietary
zinc intake in neonatal rats and is a reflection of
changes in the concentration of hepatic metallothionein.

114
Sato et al. (1984) showed that plasma and hepatic
metallothionein concentrations were reduced to nondetectable
levels in zinc-deficient rats, but increased in adequately
nourished rats exposed to stress or infection. This is in
contrast to plasma zinc concentrations which are reduced in
response to both a decrease in the size of the exchangeable
zinc pool and in response to certain metabolic conditions
(i.e. stress, infection and/or hormones). Based on these
findings, it was proposed (Golden, 1989; King, 1990) that
plasma metallothionein concentrations could be used to
differentiate between low plasma zinc concentrations occurring
in response to a reduction in the size of the exchangeable
zinc pool, versus low concentrations occurring in response to
metabolic conditions. Accordingly, low plasma zinc and low
plasma metallothionein concentrations would suggest a
reduction in the size of the exchangeable zinc pool due to low
zinc intakes; whereas, elevated plasma metallothionein and low
plasma zinc concentrations would suggest that tissue zinc is
being redistributed in response to factors other than zinc
deficiency (Golden, 1989; King, 1990). A potential problem
associated with the use of this approach to diagnose zinc
deficiency is that plasma metallothionein concentrations in
zinc-deficient animals subjected to stress may be close to
those of normal animals (Sato et al., 1984), making it
difficult to differentiate between these conditions. A second
problem is that plasma metallothionein concentrations are

115
difficult to measure, especially in adult animals, because
concentrations are close to the detection limit of most
immunoassays (Bremner et al., 1987).
Recognition of the potential problems associated with the
use of plasma metallothionein concentrations to diagnose zinc
deficiency stimulated interest in determining if other body
fluids might be appropriate indicators of zinc status. Animal
studies (Bremner et al., 1987) showed that erythrocyte
metallothionein concentrations were very sensitive to changes
in dietary zinc but not copper or selenium supply, were not
affected by stress or infection and were at least ten-fold
higher than plasma metallothionein concentrations. These
findings resulted in the idea that the erythrocyte
metallothionein concentration might be a sensitive, reliable
and more convenient method for assessing zinc status.
A human metallothionein enzyme-linked immunosorbent assay
(ELISA) has been developed (Grider et al., 1989) and tested
(Grider et al., 1990; Thomas et al., 1992). Grider et al.
(1990) showed that erythrocyte metallothionein concentrations
were very responsive to changes in zinc intake in human
subjects. Erythrocyte metallothionein concentrations fell to
68% of the initial value after 6 days of consuming a zinc-
deficient diet (-0.5 mg/d), whereas fasting plasma zinc
concentrations were only reduced by 7%. Conversely,
supplementation with 50 mg zinc/d resulted in a seven-fold
increase in erythrocyte metallothionein concentrations in just

116
seven days. Thomas et al. (1992) confirmed the responsiveness
of erythrocyte metallothionein to zinc-deficient intakes in
human subjects and also found that comparisons of the change
in erythrocyte metallothionein concentrations in subjects fed
graded levels of zinc for six weeks could be used to
distinguish between low and adeguate levels of dietary zinc
intake. Thus, it appears that the concentration of
metallothionein in erythrocytes can be used as a sensitive and
reliable indicator of zinc status in humans under controlled
dietary conditions.
Zinc Deficiency
Zinc deficiency in humans was first documented in the
early 1960s (Prasad et al., 1961; 1963b) in Egyptian and
Iranian male adolescents who were consuming vegetable protein-
based diets. The symptoms observed in these young men
included retarded growth, delayed sexual development,
hypogonadism, rough skin, severe anemia and lethargy. Other
symptoms frequently associated with zinc deficiency include:
anorexia, alopecia, reduced taste acuity, emotional disorders
(e.g. depression; irritability), diarrhea, delayed wound
healing and impaired immune function (Aggett, 1989; Hambidge
et al., 1986; Solomons, 1988).
The development of zinc deficiency is thought to occur in
progressive stages arbitrarily referred to as mild and severe
(Hambidge, 1989) . The occurrence of severe zinc deficiency,

117
which is relatively uncommon, is usually associated with
diseases such as acrodermatitis enteropathica or malabsorption
syndromes (Aggett, 1989). Cases of severe zinc deficiency
have also been documented in patients receiving unsupplemented
total parenteral nutrition (Arakawa et al., 1976; Kay et al.,
1976). Although the clinical features of zinc deficiency are
relatively nonspecific, severe zinc deficiency is usually
associated with a complex of symptoms that signal the
possibility of a zinc-deficient state (Aggett, 1989; Hambidge
et al., 1986). These symptoms include dermatitis,
neuropsychiatric changes, diarrhea, alopecia, anorexia and
weight loss. Plasma zinc concentrations are usually depressed
as well. The response to increased zinc intake and/or
supplementation is usually rapid and is taken as confirmation
of the diagnosis (Hambidge et al., 1986). Untreated zinc
deficiency results in death (Prasad, 1991).
The lack of a sensitive and specific laboratory index of
zinc deficiency, as well as the lack of specific clinical
features, have hampered the ability to detect mild human zinc
deficiency (Hambidge, 1989). Despite these problems, there is
consensus that mild zinc deficiency of dietary origin or
resulting from altered zinc metabolism/requirements occurs in
humans. Population groups considered to be most at risk for
mild zinc deficiency include infants, children and pregnant
women, as well as individuals with diseases such as Crohn's
disease, cystic fibrosis, sickle cell disease and insulin-

118
dependent diabetes mellitus. Symptoms suggestive of mild zinc
deficiency include impaired immune function, neurosensory
changes and, in infants and children, a reduction in growth
rate and/or the quality of growth (Hambidge, 1989).
Folate-Zinc Interactions
There are two main concerns with regard to folic acid-
zinc interactions. The first concern is related to the role
of zinc in folate absorption and metabolism; the second is
related to the potential adverse effect of supplemental folic
acid on zinc absorption and zinc status. A review of the
research designed to investigate these relationships is
presented in the following sections of this dissertation.
Effect of Impaired Zinc Status on Folate Absorption and
Metabolism
One of the first reports suggesting a relationship
between impaired zinc status and folate absorption and/or
metabolism was prepared by Williams and Mills (1973). These
researchers observed a reduction in hepatic folate
concentration in rats fed zinc-deficient diets, although a
significant change in serum folate concentration was not
detected. This report, as well as a study showing that bovine
hepatic folate conjugase (Silink et al., 1975) was a zinc-
dependent enzyme, provided the basis for subsequent
experiments designed to investigate the effect of impaired
zinc status on folate absorption and metabolism.

119
Several researchers have investigated the effect of
dietary zinc deficiency on intestinal mucosal and/or
pancreatic folate conjugase activity and absorption of
pteroylpolyglutamates in rats. Canton et al. (1989) reported
significantly reduced folate conjugase activities in
pancreatic tissue and intestinal luminal wash in zinc-
deficient versus zinc-adequate rats. Zinc-deficient rats also
had significantly reduced plasma folate concentrations.
Significantly reduced pancreatic folate conjugase activities
and plasma folate concentrations were noted in subsequent
studies of similar design (Canton and Cremin, 1990; Hewedy et
al., 1991).
Recently, Tamura and Kaiser (1991) measured intestinal
mucosal folate conjugase activity, using intestinal mucosal
homogenates, and absorption of [14C]PteGlu7 and [3H]PteGlu in
rats fed a zinc-deficient diet. No significant differences
were found in intestinal mucosal folate conjugase activity or
absorption of [14C]PteGlu7 or [3H]PteGlu in rats fed the zinc-
deficient diet compared with zinc-supplemented control rats.
These researchers concluded that intestinal mucosal folate
conjugase is not zinc-dependent in rats and that zinc
deficiency does not impair intestinal absorption of
polyglutamates.
As noted by Tamura and Kaiser (1991), the discrepancy
between the results of their study and the former studies may
be attributed to the following: differences in the strain of

120
rats used (i.e. Wistar versus Sprague-Dawley); differences in
the methods used to assess folate absorption (i.e. measurement
of plasma folate concentrations for 3 h after the folate dose
versus measurement of 24 h urinary excretion and fecal loss of
radioactivity); the source of pteroylpolyglutamate (i.e.
polyglutamates derived from yeast, which may have contained
folate hydrolase inhibitors, versus synthetic [14C]PteGlu)
and; inclusion versus exclusion of saturating doses of folic
acid prior to the oral tests. Regardless of the underlying
explanation(s) for the discrepancies between these studies,
the significance of these results to humans is guestionable
since rats exhibit little or no brush border folate conjugase
activity (Wang et al., 1985), and the contribution of human
pancreatic folate conjugase to the hydrolysis and absorption
of pteroylpolyglutamates is uncertain (Bhandari et al., 1990;
Jagerstad et al., 1976).
Tamura et al. (1978) examined the effect of severe zinc
depletion on the absorption of folic acid in human subjects by
measuring serum folate concentrations after oral
administration of pteroylmonoglutamate and pteroyl¬
polyglutamate. They found that zinc deficiency adversely
affected absorption of pteroylpolyglutamate, but not
pteroylmonoglutamate. Enzyme activity was not directly
measured, and no attempt was made to determine whether there
was a genera], or intestinal decrease in protein and DNA
synthesis in zinc deficiency; however, based on the findings

121
of Silink et al. (1975), they hypothesized that zinc was
essential for maintaining normal activity of intestinal
conjugase in humans. The results of subsequent studies (Day
and Gregory, 1984; Gregory et al., 1987; Wang et al., 1985)
showing that human intestinal brush border folate conjugase is
indeed zinc-dependent support this hypothesis.
No other human studies designed to confirm the findings
of Tamura et al. (1978) or to study the effect of marginal
zinc intakes on folate absorption and utilization over time
have been conducted. Since marginal zinc intake/deficiency is
more prevalent than severe zinc deficiency (Hambidge, 1989),
research directed at studying the effect of marginal zinc
intakes on folate utilization would appear to be more relevant
than studies of individuals with severe zinc deficiency.
In addition to the potential adverse effect of zinc
deficiency on the hydrolysis and absorption of folate, it is
possible that a deficiency of this nutrient may indirectly
alter folate metabolism. Tamura et al. (1987) found that
hepatic methionine synthetase activity was significantly
higher in zinc-deficient rats compared with zinc-adequate
control rats (i.e. pair-fed and ad libitum), while hepatic
N5'10-methy lene-tetrahydr of oíate reductase activity was similar
in all groups. Other changes noted included decreased total
liver and plasma folate concentrations and significantly
greater oxidation of formate and histidine in zinc-deficient
rats, suggesting that the amount of available non-N5-methyl-

122
tetrahydrofoíate increases in zinc deficiency. Based on these
findings, Tamura et al. (1987) hypothesized that increased
methionine synthetase activity associated with zinc deficiency
in rats may regulate the tissue distribution of folate
coenzymes. Whether or not these changes occur in humans has
not been determined, but the concept that impaired zinc status
may indirectly alter folate metabolism is intriguing.
Effect of Supplemental Folic Acid on Zinc Status
A review of the research related to the question of the
effect of supplemental folic acid on zinc status is presented
below. Studies suggesting a negative effect are reviewed
first, followed by those suggesting no effect. The results of
a new study examining this issue are presented in Chapter 5.
The suggestion that consumption of modest amounts of
supplemental folic acid might have an antagonistic effect on
zinc status was first presented by Milne et al. (1984) . These
researchers examined the effect of supplemental folic acid in
eight men fed 7.5 mg zinc/d for 4 weeks, 3.5 mg zinc/d for 16
week and 33.5 mg zinc/d for 4 weeks. The folate content of
the diet was 150-180 ;xg/d. Supplemental folic acid (400 /ug
every other day) was provided to the same four subjects during
each diet period. Plasma zinc concentrations were not
significantly different between either of the folate treatment
groups at any level of zinc intake; however, the folic acid
supplemented group had significantly higher fecal zinc and

123
significantly lower urinary zinc losses when their zinc intake
was either 7.5 or 3.5 mg/d. No significant difference in
fecal zinc losses was observed when zinc intake was 33.5 mg/d.
Net zinc balance was not significantly different between the
treatment groups at any level of zinc intake. Despite the
lack of a significant effect on zinc balance, the authors
concluded that supplemental folic acid influences zinc
homeostasis and that the mechanism may involve the formation
of an insoluble chelate and impairment of absorption.
Interestingly, there were no changes between the treatment
groups in their excretion patterns of iron and copper;
minerals with which folic acid is known to form stable
complexes.
In an observational study of 450 pregnant women,
Mukherjee et al. (1984) noted a significant association
between the occurrence of pregnancy complications and the
combination of low maternal plasma zinc and high maternal
plasma folate concentrations. This combination of maternal
blood values was also associated with the occurrence of fetal
distress. These investigators speculated that, in addition to
iron, folic acid present in prenatal vitamin/mineral
supplements might inhibit the intestinal absorption of zinc
causing impaired zinc status and the development of
fetomaternal complications.
Using a series of in vivo and in vitro rat experiments,
Ghishan et al. (1986) examined the relationship between zinc

124
and folic acid. In one of these experiments, 30 cm segments
of rat small bowel were perfused in situ with a solution
containing folic acid and 65Zn with a zinc to folic acid molar
ratio of 1:4.5. Mucosal uptake of the labeled zinc was
significantly diminished when folic acid was present in the
lumen. The effect of folic acid on zinc appeared to be
related to the presence of zinc and folic acid together in the
intestinal lumen because parenteral administration of folic
acid had no effect on zinc transport. The significance of
these findings to humans is guestionable because the amount of
folic acid administered to the animals resulted in a zinc to
folic acid ratio that greatly exceeded that which would be
obtained with commonly prescribed levels of folic acid
supplementation.
Ghishan et al. (1986) also studied the effect of zinc on
folic acid absorption using an in vitro preparation of everted
rat jejunal segments. Mucosal-to-serosal transport of 0.1 jxM
and 0.5 /¿M radio-labeled folic acid was significantly
decreased in the presence of zinc chloride at concentrations
of 250 and 500 /¿M. In an attempt to understand the nature of
this mutual inhibitory effect, these investigators conducted
in vitro charcoal binding studies. They found that zinc and
folic acid formed insoluble complexes at pH 2.0, but at pH
6.0, these complexes dissolved. Since binding did not occur
at the normal pH of the intestine (i.e. ~ pH 6.0), it was
concluded that under normal physiological conditions the site

125
of the mutual inhibitory effect between zinc and folic acid
must occur at the intestinal membrane level.
Plasma zinc response to folic acid supplementation was
determined by Simmer et al. (1987), using the oral zinc
tolerance test, in ten pregnant women before and after two
weeks of daily supplementation with 100 mg iron and 350 /¿g
folic acid. Ten nonpregnant subjects supplemented with 350 /¿g
folic acid/d for two weeks were also studied. The oral zinc
tolerance test was conducted using 25 and 50 mg zinc loads for
the pregnant and nonpregnant subjects, respectively. The
areas under the plasma zinc concentration-time curves and the
peak heights of the curves were significantly decreased after
supplementation in both groups of subjects. Whether or not a
similar response would have been observed with a lower dose of
zinc sulfate or with dietary zinc is unknown. The fact that
there was a 24 h interval between the period of
supplementation and reassessment of zinc absorption makes it
unlikely that zinc absorption was impaired by the formation of
an insoluble folic acid/iron-zinc chelate in the lumen, as
suggested by Milne et al. (1984), and more likely that the
reported effect occurred at the level of the intestinal
membrane as proposed by Ghishan et al. (1986). As discussed
in an earlier section, use of the oral zinc tolerance test as
a tool for assessing the effect of other nutrients on zinc
absorption has been criticized because high levels of oral
zinc are used to produce the response and the test can be

126
influenced by factors other than absorption (Valberg et al.,
1985).
To circumvent some of the problems associated with the
oral zinc tolerance test, Milne (1989) examined the effect of
folic acid on zinc absorption by determining absorption of a
tracer dose of 65Zn from a breakfast meal. The percent of
labeled zinc absorbed from the meal with and without 800 /ug
folic acid was determined in 13 subjects (seven men and six
women) at weekly intervals using a whole-body counter. The
mean percent absorption of 65Zn was not significantly
different during the control and folic acid supplemented
periods. However, when subjects were divided into two groups
(i.e. those with control zinc absorption above and below 30%),
only the subjects with control zinc absorption above 30%
experienced a significant reduction in zinc absorption when
folic acid was fed with the meal.
In a separate study, the same subjects ingested 800 /Ltg
folic acid daily for two weeks. At the end of the
supplementation period they were fed the same Zn65-labeled
breakfast meal used for the previous study. Plasma zinc and
serum and erythrocyte folate concentrations were determined
before and after two weeks of folic acid supplementation, and
whole-body counting was performed at weekly intervals.
Similar to the results of the first study, the mean percent
zinc absorption after daily folic acid supplementation was not
significantly different from the control values unless the

127
subjects were divided into groups with control absorption
values above or below 30%. The subjects with control
absorption values greater than 30% had significantly lower
absorption values when they were receiving the supplement; no
effect was seen in the subjects with low control absorption
values. Plasma zinc and serum and erythrocyte folate values
were not significantly different between subjects with high
versus low control absorption values, nor was there a
significant difference in zinc turnover between the control
and supplemented periods. Although these studies suggested
that supplementation with folic acid did not impair zinc
absorption, Milne (1989) proposed that an interaction between
zinc and folic acid may only be manifested in conditions of
increased zinc need or low zinc intake.
Milne et al. (1990) confirmed the lack of an effect of
supplemental folic acid (i.e. 400 and 800 /¿g/d) on zinc
balance, zinc absorption and static indices of zinc status in
men consuming a zinc-adequate diet (i.e. 12.5 mg zinc/d).
However, folic acid supplementation impaired the mobilization
of zinc into the plasma following participation in continuous,
graded maximal exercise. It was suggested that in contrast to
the effects of supplemental folic acid in subjects consuming
a zinc-restricted diet (Milne et al., 1984), subjects
consuming a zinc-adequate diet could maintain zinc homeostasis
when given supplemental folic acid, but this homeostatic
mechanism was insufficient to prevent changes in zinc

128
mobilization during graded maximal exercise (Milne et al.,
1990).
The most recent report (Fuller et al., 1992) suggesting
that supplemental folic acid may adversely affect zinc status
was based on retrospective data collected from preterm infants
housed in a special care unit for up to the first 16 weeks of
life. The sample included 60 infants, 48 of whom received
oral supplementation with 1 mg folic acid/d. Statistical
analysis revealed a significant inverse relationship between
the maximum serum folate level attained and the minimum serum
zinc level attained for each infant. This relationship
remained significant after corrections for extraneous factors
such as gestational age at birth, diet, birth weight, gender,
fetal growth retardation, assisted ventilation and length of
time to full enteral feeding. The meaningfulness of these
results is guestionable, however, because the minimal plasma
zinc and maximal plasma folate values used for statistical
analysis did not necessarily occur on the same day. In some
cases, the lowest plasma zinc values occurred before the
highest plasma folate values. Additionally, there was no
control group; the data for the 12 infants who did not receive
folic acid supplements were analyzed together with the data
for those infants who received supplementation. Other factors
that could have influenced the results include the time at
which oral folic acid supplementation was started, the length
of time each subject received the supplement before being

129
discharged from the unit, and the fact that some infants
received supplemental zinc and/or folic acid parenterally for
different lengths of time prior to the initiation of oral
folic acid supplementation.
The first rat and human experiments suggesting that
supplementation with folic acid did not impair zinc absorption
or utilization were conducted by Keating et al. (1987).
Similar to the study by Simmer et al. (1987) , Keating and his
colleagues used the oral zinc tolerance test to assess zinc
absorption in human subjects. In this study, the serum zinc
concentration-time curve was determined for six healthy men
given a 25 mg oral dose of zinc as zinc sulfate. Eight days
later, the test was repeated with the addition of a 10 mg oral
dose of folic acid. In contrast to the Simmer study, there
were no significant differences in the areas under the serum
zinc concentration-time curves or peak heights with or without
10 mg folic acid. The discrepancy between these results and
the findings of Simmer et al. (1987) may be due to differences
in the experimental design. It is possible that a single
large dose of folic acid does not have the same effect as
smaller doses taken over a period of time.
Rat experiments performed by Keating et al. (1987)
examined zinc bioavailability using two different methods. In
the first study, an aqueous solution containing 13.0 [iq zinc
as zinc chloride plus 2 iiC i 65Zn or an infant formula
containing the same amount of unlabeled and radio-labeled

130
zinc, was administered to the rats via intragastric
intubation. These solutions were further supplemented with 0,
4.4 or 176 folic acid. The animals were sacrificed 5 h
after intubation, and the accumulation of 65Zn in the livers
and kidneys was measured. Zinc retention by the livers or
kidneys was not affected by either level of folic acid
supplementation. These results are surprising if one
considers that Ghishan et al. (1986) showed that zinc
transport was inhibited in rats when folic acid was present in
the intestinal lumen. This apparent inconsistency may be
explained in part by the fact that Ghishan et al. (1986)
introduced the folic acid into the intestinal lumen, whereas
Keating et al. (1987) introduced the supplement
intragastrically. Additionally, the molar ratios of
zinc:folic acid used were 1:4.5 in the former study and 20:1
and 1:2 in the latter experiment.
In the second part of the rat study conducted by Keating
et al. (1987), comparisons of total femur zinc and weight gain
were made in growing male rats fed a basal diet containing no
folic acid or zinc, or diets containing either 6 or 12 mg
zinc/kg diet supplemented with 0, 2 or 160 mg folic acid/kg
diet. The addition of 2 or 160 mg folic acid/kg diet had no
significant effect on weight gain of the animals or zinc
uptake by the femur at either level of zinc intake. The
results of this experiment substantiate the findings of the
other experiments conducted by Keating et al. (1987).

131
The opportunity to study the long-term effects of large
oral doses of folic acid in human subjects was provided by an
ongoing intervention trial designed to evaluate the possible
relationships between cervical dysplasia and nutritional
status in women. In this study (Butterworth et al., 1988),
fifty women with cervical dysplasia were randomly assigned to
receive supplementation with 10 mg/d oral folic acid or a
placebo. Dietary intake was not controlled or assessed during
this study. Erythrocyte folate and plasma and erythrocyte
zinc were evaluated at the initial visit and after 2 months of
treatment; 21 of the same subjects were evaluated after 4
months of treatment. Erythrocyte folate concentrations were
significantly higher in the folic acid-supplemented subjects
compared to the placebo-treated subjects after 2 and 4 months
of treatment suggesting compliance with the treatment;
however, there were no significant differences in plasma or
erythrocyte zinc concentrations between the groups. A
potential problem with the interpretation of these results is
that plasma and erythrocyte zinc concentrations tend to remain
fairly stable unless zinc status is severely compromised.
Consequently, it is difficult to assess whether the lack of an
effect of supplemental folic acid on plasma and erythrocyte
zinc is due to failure of these response variables to detect
the effect, or if indeed, no change occurred.
As reported in an abstract, Krebs et al. (1988) measured
the absorption of 70Zn administered with and without 30 mg of

132
folic acid in three healthy subjects. Similar to the findings
of Keating et al. (1987), this pharmacologic dose of folic
acid did not impair zinc absorption. These researchers also
reported that mean plasma, mononuclear and neutrophil zinc, as
well as serum alkaline phosphatase, erythrocyte delta-amino
levulinic acid dehydratase and prealbumin values of patients
with fragile X syndrome treated with 16 ± 5 mg folic acid/d
for 1 to 4 y, were not significantly different from values for
healthy control subjects. Thus, zinc status did not appear to
be affected by chronic ingestion of large doses of folic acid
in subjects with fragile X syndrome. Although it may be
inappropriate to make inferences from these data to a normal
population, this study appears to corroborate the study
conducted by Butterworth et al. (1988).
Fuller et al. (1988) were the first investigators to
examine the effect of supplemental folic acid in rats during
pregnancy and lactation. At 110 d of age, the rats were
allocated to receive a diet containing the four possible
combinations of: no additional folic acid or 100 jug folic
acid/g diet and 6.6 /¿g zinc/g diet or 20.2 Mg zinc/g diet.
The animals were acclimated to the diet for 21 d, after which
they were mated and further randomized to the pregnancy or
lactation study. Pups and dams were sacrificed on day 20 of
gestation or day 20 of lactation. Blood samples were analyzed
for plasma zinc, and maternal and pup livers and kidneys were
analyzed for zinc content. Regardless of the level of zinc in

133
the diet, supplemental folic acid did not compromise plasma
zinc or tissue zinc concentrations of pregnant rats, their
fetuses, lactating rats or suckling rats. The researchers
concluded that if these results are applicable to humans, then
some reassurance is provided that prenatal folic acid
supplementation does not necessarily cause zinc depletion in
pregnant women.
A subsequent experiment conducted by an independent group
of researchers (Southon et al., 1989) examined the effect of
supplementation with folic acid, calcium and iron in pregnant
and nonpregnant rats receiving either a low (8 ¿xg/g) or high
(60 ¿¿g/g) zinc diet. This diet was fed for 14 d before
mating. After mating, half of the rats in each of the two
diet groups were supplemented with iron, calcium and folic
acid. On the eighteenth day of gestation, 65Zn was fed along
with the usual diet. The animals were sacrificed on day 20 of
gestation. Although whole body radioactivity was
significantly lower in all groups fed the supplemented diet
compared with the unsupplemented groups, and plasma zinc
concentrations in the pregnant supplemented rats were
significantly lower than their unsupplemented counterparts,
supplementation had no effect on total femur or liver zinc
content in any of the groups. Fetal zinc concentration was
not affected by supplementation either. The authors concluded
that the risk of inducing either maternal or fetal zinc
depletion as a consequence of increased intakes of these

134
nutrients is very slight, since the differences in maternal
zinc status due to supplementation were small, and the fetuses
were apparently protected. Even if the authors had concluded
that supplementation with these nutrients had an adverse
effect during pregnancy or on pregnancy outcome, it would be
difficult to separate the potential contribution of folic acid
from that of iron and/or calcium.
Quinn et al. (1990) also used pregnant rats to study the
effects of supplemental folic acid on maternal and fetal zinc
status, pregnancy outcome and the incidence of fetal
malformations. In this study, pregnant rats were fed a zinc-
deficient (< 0.5 mg zinc/kg diet) or zinc-supplemented diet
(75 mg zinc/kg diet) from mating until the eighteenth day of
pregnancy. Half of the rats in each zinc group received the
basal level of folic acid provided by the diet (0.56 mg/kg
diet). The remaining rats were supplemented with 200 mg folic
acid/kg diet. Maternal plasma zinc and tibia zinc, placental
zinc, litter size, fetal weight and placental weight were not
significantly different in the folic acid-supplemented group
compared to the unsupplemented groups within the level of zinc
intake. Within the zinc-deficient groups, placental zinc
content was actually significantly higher in the folic acid-
supplemented animals. Examination of the fetuses for ten
different types of malformations revealed a significantly
higher incidence of clubbed foot in the fetuses from animals
fed the zinc-deficient folic acid-supplemented diet compared

135
to those from the dams fed the zinc-deficient unsupplemented
diet. Folic acid supplementation did not increase the
incidence of other deformities. Thus with the exception of
the potential influence on the development of clubbed foot,
this inordinately high level of folic acid supplementation did
not appear to adversely affect zinc status or pregnancy
outcome. The fact that none of the fetuses from rats fed the
zinc-supplemented diets (i.e. folic acid-supplemented or
unsupplemented) developed clubbed foot suggests that
expression of this developmental abnormality may be sensitive
to folic acid supplementation only when zinc deficiency is
present. This idea is congruent with Milne's (1989)
hypothesis that an interaction between supplemental folic acid
and zinc may only be manifested in conditions of zinc need or
low zinc intake.
Pigs have also been used as an animal model to study the
effect of supplemental folic acid. This animal model may be
better suited for investigating folic acid-zinc interactions
in humans because of the similarities between pigs and humans
in the enzymatic mechanism of absorption (Day and Gregory,
1984; Gregory et al., 1987; Wang et al., 1985). Tremblay et
al. (1989) studied the effect of supplemental folic acid in
sows from weaning to day 30 of gestation. The sows were fed
a commercial diet with or without supplemental folic acid (0
or 5 mg/kg diet). Serum folate, zinc, copper and iron were
measured at weaning, mating and day 30 of gestation. Folic

136
acid supplementation had no effect on serum copper and iron;
however, serum folate and plasma zinc concentrations were
significantly elevated between weaning and day 30 of gestation
in the supplemented group. Although this study suggests that
folic acid supplementation did not interfere with zinc status,
one must be careful in making conclusions since serum zinc
concentrations were used as the sole measure of zinc status.
To study the effect of folic acid on zinc uptake in pigs,
Turnbull et al. (1990) conducted an in vitro study in which
intestinal brush border membrane vesicles from porcine small
bowel were exposed to an incubation medium containing various
concentrations of folic acid (0.05, 0.5, 5.0 and 50.0 /xM) and
a 65Zn concentration of 5 Uptake of 65Zn was not
significantly changed by the addition of folic acid at any of
the concentrations tested. Although this study did not detect
a luminal interaction between zinc and folic acid, the
possibility that a mucosal effect occurred, as suggested by
Ghishan et al. (1986) and Simmer et al. (1987) , was not ruled
out.
Tamura et al. (1992) have recently published a study in
which they examined the relationship between pregnancy outcome
and maternal serum folate and zinc concentrations. These
investigators measured serum folate and zinc concentrations at
18 and 3 0 weeks gestation and correlated these data with birth
weight and Apgar scores of newborn infants and with the
incidence of maternal infections during the perinatal period.

137
The study population consisted of 285 women who delivered
full-term infants. Women with risk factors and a growth
retarded infant were matched to women with a normal-sized
infant by race, sex of infant, smoking status and a number of
other variables. All women had been offered daily folic acid
supplements of 1 mg/d during their pregnancy. Overall
compliance was estimated to be approximately 48% based on
serum folate concentrations of 46 nmol/L. Dietary intake was
not monitored. In contrast to the study by Mukherjee et al.
(1984), Tamura and associates (1992) did not find an
association between high serum folate and low serum zinc
concentrations. In fact, high serum folate concentrations
were associated with favorable effects on pregnancy outcome.
Thus, this study did not support the idea that supplementation
with folic acid has an adverse effect on maternal zinc status
and pregnancy outcome.
Tamura et al. (1992) have also recently reported the
results of an observational study in which they measured zinc
and folate concentrations in amniotic fluid. Samples of
amniotic fluid were obtained during the second trimester of
pregnancy from 221 women who gave birth to apparently healthy
infants and 8 women who delivered infants with neural tube
defects. Folate and zinc concentrations of amniotic fluid from
women who delivered infants with neural tube defects were not
significantly different from those of women who delivered
normal infants. These researchers also reported that there

138
were no significant differences in amniotic fluid nutrient
levels between women who took vitamin and/or mineral
supplements during pregnancy and those who did not. Although
the concentration of nutrients in amniotic fluid may not
necessarily reflect the concentrations found in fetomaternal
tissues, the results of this study may provide further
evidence for the lack of a folic acid-zinc interaction.
The fact that supplemental folic acid is frequently
prescribed for individuals undergoing chronic hemodialysis
prompted Reid et al. (1992) to examine zinc status in a small
sample of men (12) and women (9) who had been receiving
hemodialysis treatments for at least 6 months. The subjects
were classified into the following four groups based on their
prescribed supplementation regimen: no folic acid or zinc; no
folic acid and 22.5 mg zinc/d; 5 mg folic acid/d and no zinc;
or 22.5 mg zinc/d and 5 mg folic acid/d. Average daily food
folate and zinc intakes were estimated using a food frequency
questionnaire. Serum and erythrocyte folate and serum zinc
concentrations were also measured. There were no significant
differences in dietary folate or zinc intakes between the
groups. Erythrocyte folate concentrations were significantly
higher in the folate-supplemented groups, but serum zinc
concentrations were unaffected by any combination of
supplementation. Although no evidence of an adverse effect of
folic acid on zinc status was noted in this study,
generalization of these findings is limited because of the

139
descriptive nature of the study, the use of a chronically-ill
population and the question of the validity of serum zinc as
a valid index of zinc status in these subjects.
In summary, the results of animal and human studies
investigating the effect of folic acid supplementation on zinc
status are equivocal. The diversity of research designs and
protocols used to investigate this question may explain, to
some extent, the discrepant results. Within the human
subjects studies alone, factors that may have contributed to
the disparity in outcomes include: differences in the
metabolic states of the subjects (i.e. healthy, chronically
ill, pregnant, etc.); administration of widely varying levels
of supplemental folic acid; failure to control dietary intake
in most studies; and the lack of a satisfactory index to
assess zinc status. Milne's (1989) hypothesis that folic
acid-zinc interactions may only be manifested in conditions of
increased zinc need or low zinc intake may also explain the
contradictory results, since most studies did not control for
differences in zinc intake or prior status.

CHAPTER 3
RATIONALE FOR RESEARCH PROTOCOL
It is apparent from the literature review pertaining to
folic acid-zinc interactions that neither the question of the
effect of impaired zinc status on the utilization of folic
acid, nor the question of the effect of supplemental folic
acid on zinc status in human subjects, has been adequately
resolved. The possibility that folic acid supplementation may
interfere with zinc absorption and status is disconcerting
because supplementation with this vitamin is frequently
$
recommended for many population groups, including: patients
with various gastrointestinal/malabsorption syndromes; cancer
patients receiving anti-folate medications; users of oral
contraceptive agents; individuals with cervical dysplasia;
renal patients undergoing hemodialysis treatments; and the
majority of prenatal patients. Folic acid supplements may
also be recommended to individuals with documented
deficiencies such as the elderly. Use of supplemental doses
of this nutrient is likely to become even more widespread as
a result of recent recommendations suggesting that women of
childbearing age who are capable of becoming pregnant increase
their intake of folic acid (Anon., Morbidity and Mortality
Weekly, 1992). These recommendations were prompted by the
140

141
results of studies demonstrating the positive correlation
between the use of folic acid supplements during the
periconceptional period and a reduction in the risk of
occurrence/recurrence of neural tube defects. Beyond
supplementation, if the Food and Drug Administration's, Food
Advisory Council's recommendation to enrich flour with folic
acid is accepted (Anon., Food Chemical News, 1993), the
majority of the population will be exposed to higher intakes
of this nutrient. Consequently, research directed at
resolving the issue of whether folic acid supplementation
exerts a deleterious effect on zinc status, and if so, under
what conditions, is very timely and warranted. Impaired zinc
status due to injudicious recommendation/use of folic acid
supplements may have untoward consequences given the
multiplicity of physiologic functions associated with zinc.
Likewise, considering the central role of folate in DNA
synthesis and one-carbon metabolism, impaired utilization of
folate due to zinc deficiency or marginal zinc intake may also
have damaging consequences.
In an attempt to provide more definitive information
about the interrelationship between folic acid and zinc, the
present study was designed to determine if supplemental folic
acid affected zinc status in human subjects consuming zinc-
adequate or zinc-restricted diets; and to determine if
utilization of supplemental folic acid was affected in human
subjects consuming either a zinc-restricted or zinc-adequate

142
diet. To overcome some of the limitations of previous
studies, the subjects were fed a constant diet containing 14.5
or 3.5 mg zinc/d for two, 28 day study periods. The study
periods were separated by an 80-day washout period. During
the study periods, subjects were assigned to receive
supplemental folic acid and placebo treatments in a crossover
fashion, so that subjects served as their own control on and
off the supplement. Supplementation was provided at a level
comparable to that used in many prenatal vitamin supplements.
In addition to traditional measures used to assess zinc
status, erythrocyte metallothionein concentrations were
evaluated as a new and potentially more responsive index of
zinc status (Grider et al., 1990). The responsiveness of
erythrocyte metallothionein concentrations to dietary zinc
intake has been confirmed more recently by Thomas et al.
(1992) .
The practical constraints of conducting a feeding study
(i.e. space, facilities, meal preparation, cost, etc.) made it
necessary to limit the number of subjects who could be
included in each treatment group. To minimize heterogeneity
within this small group of subjects, the study was restricted
to Caucasian males. The racial restriction was imposed
because differences in folate status and hematological
parameters have been observed in population studies (Bailey
1982a; 1982b). Males were selected over females because
erythrocyte metallothionein concentrations have not been

143
measured or standardized for this group. Also, previous
feeding studies examining the effect of folic acid
supplementation on zinc status in subjects fed a zinc-
restricted (Milne et al., 1984) or zinc-adequate (Milne, 1989)
diet included only male subjects. Thus, by limiting the
present study to males, the potential question of whether
differences in outcome measures were due to gender differences
was avoided.
Data from the study conducted by Grider et al. (1990)
were used to determine the length of the study periods. These
researchers found a significant decrease in erythrocyte
metallothionein concentrations within seven days after
consuming a diet containing less than 1 mg zinc/d. Since the
response to a diet containing 3.5 mg zinc/d had not yet been
determined, it was assumed that a longer period of time might
be needed to observe any changes due to the effect of
supplemental folic acid. Consequently, the study periods were
increased by a factor of 3.5. This also provided the
opportunity to examine erythrocyte metallothionein response
over a longer time interval.
The length of the washout phase of the study was planned
using data from Heseker and Schmitt (1987) . These researchers
determined the average daily increase in erythrocyte folate
concentrations after 17 weeks of supplementation with 1 mg
folic acid/d, as well as the average daily rate of decline
after cessation of supplementation. Based on these

144
calculations, it was estimated that an 80-day washout period
would be more than sufficient to eliminate any residual
effects due to folic acid supplementation.
As intimated by the objectives outlined above, this study
was also designed to take into account the possibility that
folic acid-zinc interactions may only be manifested in
conditions of low zinc intake. This was achieved by randomly
assigning the subjects to either a zinc-restricted diet (3.5
mg zinc/d) or an identical diet supplemented with 11 mg of
zinc as zinc sulfate (14.5 mg zinc/d total intake). A zinc-
restricted diet, rather than a zinc-deficient diet, was
selected because this level of intake is more representative
of the zinc content of diets consumed by individuals with
marginal zinc intakes and/or status.
Another unique aspect of this study was the use of
deuterium-labeled folic acid monoglutamate to examine the
effect of zinc-adequate and zinc-restricted diets on folate
utilization over time. The availability of stable
isotopically-labeled folic acid provided the opportunity to
quantify the excretion of supplemental folic acid without the
risks associated with the use of radio-isotopes in human
subjects.

CHAPTER 4
SUBJECTS, EXPERIMENTAL DESIGN, MATERIALS AND METHODS
Subjects
Recruitment and Selection
The University of Florida Institutional Review Board
approved the screening and experimental protocols for this
study, and informed consent was obtained from all prospective
subjects. Subjects were recruited from the University of
Florida campus and the city of Gainesville through
advertisements. Interested individuals completed a
questionnaire (Appendix A) and food records (Kauwell
Methodology, 1993, Food Science and Human Nutrition Archives)
which were evaluated by the investigator to determine
eligibility. Fifty of the original prospects were selected
for further screening, which included an interview with the
investigator and donation of a blood sample for routine
laboratory analysis (i.e. 25-item blood chemistry profile and
a complete blood count with differential).
Subjects were selected after reviewing the information
obtained from the questionnaires, food records, interviews and
blood chemistry profiles. This information was used to select
Caucasian males between the ages of 20 to 35 with no history
145

146
or current diagnosis of epilepsy, liver disease, renal
disease, diabetes mellitus, alcoholism, gastrointestinal
diseases/surgery, malabsorptive states or genetic disorders
such as acrodermatitis enteropathica and sickle cell disease.
Subjects were also excluded if they routinely used
prescription/nonprescription medications; admitted to the use
of recreational drugs or tobacco/tobacco products; had blood
chemistry or hematological profiles that were not within the
normal range; and/or were over/under-weight. Food records and
questions related to adherence to special diets, food
habits/beliefs/taboos, supplement use, fluctuations in body
weight and concerns about body weight were used to select
subjects for whom it was thought that compliance would be
high.
Description of Subjects
Twelve adult (20-34 y) Caucasian males with an average
(mean ± SD) weight of 71.3 ± 10.6 kg were selected. None of
the subjects reported a history or current diagnosis of any of
the medical conditions listed in the exclusion criteria. All
subjects denied the use of prescription medications,
recreational drugs and tobacco/tobacco products. Blood
chemistry and hematological profiles were normal for all
subjects, and review of the food records revealed that the
subjects routinely consumed a typical nonvegetarian, Western
diet.

147
Experimental Design
The purposes, procedures, risks and benefits of the study
were explained in detail to each of the subjects, and a copy
of the informed consent form was provided to each of them for
their perusal. Upon signing the informed consent, the
subjects agreed to adhere to the study protocol which
included: consuming all of, and only those foods and beverages
provided by the researcher; avoiding oral contact with nonfood
items; complying with urine and blood collection protocols and
schedules; restricting use of personal hygiene products to
those provided or approved by the investigator (Kauwell
Methodology, 1993, Food Science and Human Nutrition Archives);
conforming to the metabolic style of eating; completing a
daily checklist (Kauwell Methodology, 1993, Food Science and
Human Nutrition Archives); limiting exercise to one hour each
day; abstaining from the use of nonprescription medications
unless approved by the investigator; and informing the
investigator of any changes in health status or unusual
symptoms. Subjects were informed that if they were unable or
unwilling to comply with any of these conditions they would be
released from the study.
The study design was a single-blind crossover protocol as
depicted in Figure 4-1. Subjects were fed a controlled
constant diet for two, 28-day periods. No treatments were
introduced during the first three days of each feeding period
to allow the subjects time to adjust to the diet and to their

PERIOD I
PERIOD II
ZINC-RESTRICTED
DIET (3.5 mg/d)
0 Folic Acid 0 Folic Acid
0 Folic Acid 0 Folic Acid
1 3 28 1 3 28
DAYS
0FA
Acclimation period; zinc-adequate diet; no supplemental folic acid.
0 /ug supplemental folic acid/d; +FA = 800 ng supplemental folic acid/d.
Figure 4-1. Experimental design.

149
new eating environment. A baseline 24-h urine collection was
started after the first morning void on the third day of the
study, and thereafter, collections were started on days 11, 18
and 27. Fasting blood samples were obtained on the morning of
the fourth day of the study, and additional fasting blood
samples were obtained on days 8, 11, 15, 21, 24 and 29.
Beginning with breakfast on the fourth day of the study, the
subjects were randomly assigned to receive a diet containing
either 3.5 or 14.5 mg zinc/d. (Subjects assigned to the zinc-
restricted or zinc-adequate diets were designated #1 through
#6 and #7 through #12, respectively.) Half of the subjects in
each zinc group were also randomly assigned to receive 800 /xg
of deuterium-labeled folic acid monoglutamate administered
daily in apple juice. The remaining subjects received plain
apple juice served in an identical container. Adequacy of
caloric intake was monitored by weighing subjects three times
a week.
Upon completion of the first phase of the study, folic
acid supplementation was discontinued and the subjects
consumed self-selected diets for 80 days. The restrictions
imposed on the subjects during the treatment phases of the
study were lifted during the washout period; however, the
subjects were not permitted to take vitamin/mineral
supplements or engage in excessive alcohol consumption during
this time. During the washout period, blood samples and five-
day food records were obtained every three to four weeks. At

150
the end of the 80-day washout period, the study was repeated.
During study period two, subjects consumed the same level of
zinc to which they had been randomized during study period
one; however, the folic acid/placebo treatments were switched
so that subjects who did not receive supplemental folic acid
during the first phase of the study received it during the
second study period. The time intervals at which weights and
blood and urine specimens were obtained were the same as those
for study period one.
Blood samples were analyzed to determine the
concentrations of plasma and erythrocyte zinc; serum and
erythrocyte folate; erythrocyte metallothionein; serum
alkaline phosphatase; and serum ferritin. The packed cell
volume and the protein concentration of erythrocyte lysates
were also determined. A complete blood count with
differential, and a 25-item blood chemistry profile were
performed by SmithKline Beecham Clinical Laboratories
(Gainesville, FL) at three time points during each of the
study periods. Twenty-four hour urine collections were
analyzed for total urinary zinc and folate concentrations.
The amounts of labeled and unlabeled folate excreted in the
urine were also quantified. Aliquots of meal composites
prepared on two separate occasions during both study periods
were analyzed to determine the total zinc content of the diet.
Food records collected during the prestudy and washout periods
were evaluated by computer (Practorcare 4000, San Diego, CA)

151
to determine the average intake of calories, protein, folate
and zinc from self-selected diets.
Materials and Methods
Description of Diet and Supplements
A three-day cycle menu containing a daily average of 2985
kilocalories (42 kcal/kg body weight), 80 g protein (l.l g/kg
body weight), 290 /¿g folate, and by actual analysis, 3.5 mg
zinc, was developed for this study (Appendix B). The three-
day average nutrient content for other nutrients, as
determined by computer analysis (Practorcare 4000, San Diego,
CA) , is listed in Table 4-1. In addition to the diet,
supplements were provided so that the experimental diet would
more closely approximate the 1989 RDA for all nutrients.
Consistency in nutrient content for repeated days of the diet
was maintained by purchasing foods according to case lot
number. The menu was the same for both study periods and
consisted of conventional foods fed as three meals and one
snack. In order to provide enough protein in the diet, the
subjects also received two "protein shakes" per day (Table
4-2) . The diet consumed by subjects in the zinc-adeguate
group was identical to that consumed by the zinc-restricted
group except that 5.5 mg zinc, as zinc sulfate (VWR
Scientific, Atlanta, GA), was added to each protein shake to
provide a total zinc intake of 14.5 mg/d.

152
Table 4-1. Average nutrient
without supplements.
content of three-day diet with and
Diet
Supplements Diet+
Nutrient
alone
alone
supplement
kilocalories
2,985
same
same
protein, g
80
same
same
carbohydrate, g
465
same
same
fat, g
90
same
same
vitamin A, IU
12,387
same
same
vitamin C, mg
420
same
same
thiamin, mg
1.2
0.7
1.9
riboflavin, mg
2.0
0.7
2.7
niacin, mg
21
9
30
vitamin B6, mg
pantothenic
1.7
0.9
2.6
acid, mg
2.8
same
same
folate, /¿g
290
*
*
vitamin B12, M9
0.9
2.0
2.9
calcium, mg
482
200
682
phosphorous, mg
869
same
same
sodium, mg
3,363
same
same
potassium, mg
2,564
same
same
iron, mg
12
same
same
magnesium, mg
165
81
246
zinc, mg
3.5
*
*
copper, mg
0.9
2.0
2.9
selenium, /¿g
56
same
same
biotin, /¿g
300
300
*Zinc and folic acid supplementation
provided as per the
experimental design
and randomization
scheme.

153
Table 4-2. Composition of protein shakes.
Ingredients
Amount per serving
powdered, sweetened, instant
drink mix
67.5 g
egg whites, (frozen, pasteurized)
150.0 g
frozen nondairy creamer
52.5 g
doubly deionized, distilled water
90.0 g
360.0 g
All meals were prepared in the University of Florida,
College of Medicine, Clinical Research Center Metabolic
Kitchen. Standardized recipes, food preparation techniques
and food service and sanitation procedures (Kauwell
Methodology, 1993, Food Science and Human Nutrition Archives)
were developed and followed to ensure uniformity. Doubly
deionized, distilled water, hereafter referred to as water,
was used during all aspects of food preparation, service and
sanitation. Other procedures followed in order to avoid trace
mineral contamination are discussed in a succeeding
subsection.
With the exception of ready-to-consume foods available in
prepackaged individual portion sizes (i.e. potato chips, corn
chips, fruit juices, salad dressings and breakfast cereals),
all foods and ingredients used in meal preparation were
weighed before they were served to the subjects. During the
first three days of the study (i.e. prior to initiation of the

154
treatments), subjects were given the opportunity to decide if
they wished to include up to one gram of salt and/or one
package of instant coffee reconstituted with water. Once the
subjects committed to either or both of these options, they
were required to consume these foods daily for the duration of
both treatment periods. Subjects were also offered the option
of including up to 32 oz of Diet 7UP® (Pepsico, Inc., Somers,
NY), three sticks of spearmint or peppermint Carefree®
Sugarless Gum (Planter Lifesavers Co. , Winston Salem, NC) ,
and/or one package of Equal® (NutraSweet Co., Deerfield, IL)
on a daily basis. Water was provided to all subjects ad
libitum.
Subjects consumed breakfast and dinner at the same time
every day in the Clinical Research Center. Breakfast and
dinner trays were checked for accuracy prior to meal service
to avoid omission of any foods/beverages and to ensure that
each subject received the appropriate treatments. Subjects
consumed their meals using the eating techniques in which they
had been instructed, which included using a rubber spatula and
a rinse bottle to loosen any food particles or residues
remaining on their dishes or beverage containers. Bread,
which was served at both meals, was used to absorb any liquid
remaining on the serving dish or container. At the end of
each meal, the subjects' trays were checked for completeness
of consumption. A bag lunch and an evening snack were
distributed to subjects after they completed their breakfast

155
and dinner meals, respectively. Subjects consumed this meal
and snack on their own at noon and 10:00 pm, respectively,
using the eating techniques in which they had been instructed.
No treatments (i.e. additional zinc or deuterium-labeled folic
acid) were administered at lunch or as part of the evening
snack.
The supplements provided to the subjects included calcium
citrate (Citracal 950®, Mission Pharmacal Co., San Antonio,
TX), magnesium gluconate (Willner Chemists, Inc., New York,
NY) and vitamin B complex (Squibb®, Princeton, NJ) in tablet
form. Copper, as cupric sulfate (VWR Scientific, Atlanta,
GA), was dispensed into the protein shakes. Biotin tablets
(Puritan's Pride®, Bohemia, NY) were provided as a safeguard
to protect against the possible development of a biotin
deficiency due to the use of egg whites in the protein shakes.
All supplements were consumed in the presence of the
investigator.
Supplemental folic acid was provided as 3',5' deuterium-
labeled folic acid monoglutamate. This stable isotope of
folic acid was synthesized by Dr. J.F. Gregory, III, using an
improved labeling procedure that yields complete labeling of
the 3' and 5' positions of the folic acid molecule (Gregory,
1990). In this procedure, unlabeled folic acid is brominated
at the 35'-positions, followed by catalytic debromination
with deuterium gas and a palladium/carbon catalyst. The
labeled folic acid is purified chromatographically, and the

156
degree of purity is established using analytical HPLC.
Finally, the site and degree of deuterium labeling is
confirmed by mass spectrometry and proton nuclear magnetic
resonance.
To prepare the supplement for consumption by the
subjects, the amount of deuterium-labeled folic acid needed to
make enough 800 ¿xg dose portions for the first study period
was weighed; dissolved in a small amount of 1.0 M sodium
hydroxide (NaOH); diluted with 0.1 M phosphate-buffered saline
(PBS) (pH 7.0); adjusted to pH 7.0 with HC1; and brought to
volume with more buffer. The volume of the deuterium-labeled
folic acid solution to be dispensed into each tube of apple
juice was calculated after determining the folic acid
concentration spectrophotometrically at 280 nm. The labeled
folic acid was dispensed into 50 mL conical centrifuge tubes
(Corning®, Corning, NY) containing 45 mL of apple juice. The
tubes were flushed with nitrogen, sealed and stored at -20° C.
Precautions were taken during all phases of preparation,
storage and service to protect the folic acid from light.
Plain apple juice served in identical containers was used as
the placebo.
Procedures Used to Foster. Monitor and Assess Compliance
Compliance was fostered by maintaining close personal
interaction with the subjects and reinforcing the importance
of adherence to all aspects of the protocol. Subjects were

157
also asked to sign contracts (Appendix C) that detailed their
responsibilities. Methods used to monitor and assess
compliance to the protocol included: 1) observing the subjects
during meal times and checking their trays to make sure that
all foods and beverages were consumed; 2) completion and
signing of a daily checklist addressing compliance issues
(Kauwell Methodology, 1993, Food Science and Human Nutrition
Archives); and 3) completion of an anonymous guestionnaire at
the end of the study to identify any deviations from the
protocol.
Procedure Used to Assess Adequacy of Blinding
In order to determine if subjects had been adequately
blinded to the level and order of treatments they received,
subjects were asked to complete a questionnaire addressing
these issues at the end of the study. The questionnaire
listed the possible combinations of treatments the subjects
could have received during each of the study periods and asked
them to identify which combination of treatments they thought
they had received.
Procedures Used to Prevent Zinc Contamination
Hair restraints and powder-free gloves were worn during
all aspects of food handling, preparation and sanitation in
order to prevent incidental zinc contamination. All work
surfaces and food service areas where sanitized with a diluted

158
solution of bleach, after which they were rinsed with water.
The lids of canned foods were treated in the same manner
before they were opened to prevent incidental zinc
contamination.
Samples of all disposable containers and utensils used
for food preparation and/or service were tested for zinc
contamination before the study was begun. Cups, dishes,
bowls, plastic utensils, polypropylene conical centrifuge
tubes (Corning®, Corning, NY), plastic bags and other
disposable items were filled/soaked in 0.1 N hydrochloric acid
(HC1) overnight. Duplicate samples of HC1 from each
disposable item were analyzed for the presence of zinc by
air/acetylene flame atomic absorption spectrophotometry (AAS)
(Perkin-Elmer® 2600, Norwalk, CT). Only containers/utensils
determined to be zinc-free were used. Vials and containers
used to collect and/or store blood or urine samples to be
analyzed for their zinc content were tested in the same
manner.
Food service equipment, containers and utensils to be
used repeatedly throughout the study were also tested for zinc
contamination. Since it was important to know if the
procedures to be used for sanitizing these items would be a
source of incidental zinc contamination, all items were
sanitized before they were tested. The sanitation process
included washing the items in a household automatic dishwasher
with a prescribed amount of dish washing detergent, rinsing

159
them three times with water (manually, while wearing powder-
free gloves) and allowing them to air dry. Gallon and half¬
gallon plastic beverage containers used to supply the subjects
with drinking water, were sanitized manually since the
dishwasher would not accommodate these items. These
containers were also rinsed three times with water and air
dried. Only equipment, containers and utensils for which
there was no evidence of zinc contamination were used in the
study. Every time these items were used, they were cleaned
using the same procedures.
The protein shakes consumed by the subjects were served
in sixteen ounce, reusable, plastic drinking cups with lids
(Packard Plastics, Lawrence, KS) . Since zinc sulfate was
added to some of the shakes, the cups and lids were washed in
an automatic dishwasher and then submerged in a solution of
Radiacwash® (Atomic Products Corporation, Shirley, NY) , a
metal-chelating agent, in order to prevent incidental zinc
contamination. After soaking in the Radiacwash® solution, the
cups and lids were vigorously rinsed six times (powder-free
gloves were worn) and then air dried. Containers used for
collecting urine were processed in a similar manner except
that they were soaked in bleach before being washed by hand.
Separate soaking tubs were used for drinking cups and urine
containers.

160
Urine Collection and Processing Procedures
Opaque urine containers with capacities of 2.5 L and
0.5 L were cleaned in the manner described above. Free
ascorbate (1.5 g/L) was added to each container to protect
urinary folates. Subjects were provided with written and
verbal instructions on how to collect their urine. Urine was
collected after the first void of the day for a period of
24 h. Subjects were instructed to void directly into the
container, but to avoid touching the inside of the container;
to completely fill one container before starting a new
container; to keep the urine they collected cool at all times;
and to return all containers to the investigator at the end of
the 24 h collection period.
Precautions were taken to avoid zinc contamination while
processing the urine. The volume of the urine excreted by
each subject was measured using acid-washed graduated
cylinders. Powder-free gloves were worn during the processing
operation, and samples were stored in zinc-free containers.
Aliquots of well-mixed urine, to be used to determine the
concentrations of urinary zinc, urinary folate and deuterium-
labeled folates, were protected from light and stored at
-20° C.
Blood Collection and Processing Procedures
Fasting morning blood samples were drawn by a trained
phlebotomist. A description of the equipment, supplies and

161
procedures used to obtain, collect and process the specimens
is outlined below. Doubly deionized, distilled water,
hereafter referred to as water, was used exclusively for all
aspects of sample processing and analysis.
Plasma/erythrocyte zinc and erythrocyte metallothionein.
Precautions were taken to avoid zinc contamination during
sample collection and processing. All equipment, solutions
and vials used for blood collection and processing were tested
and found to be zinc-free. A blood collection set with
multiple sample luer adapter (Vacutainer®, Becton Dickinson,
Rutherford, NJ) was used in combination with two, 10 mL
polypropylene syringes (Sarstedt, Princeton, NJ) to draw blood
for determination of plasma zinc, erythrocyte zinc and
erythrocyte metallothionein concentrations. Sodium heparin
(Lyphomed, Inc., Rosemont, IL) was added to each syringe
before the blood samples were drawn. Samples were held on
ice, and processing was begun within one hour after the
samples were obtained.
Duplicate samples were centrifuged in the original
container at 1500gr for 12 minutes at 4° C. The plasma
fraction was separated from each sample and stored at -20° C.
The remainder of each blood specimen was used to prepare
duplicate samples of erythrocyte lysates. The buffy coat was
removed from the erythrocyte pellet, and an equal volume of
ice-cold 0.9% sodium chloride (NaCl) was added. The samples
were centrifuged for 5 minutes, after which the supernatant

162
was removed from the pellet. This process was repeated two
times. After the final wash, the supernatant was removed and
discarded, and 2.5 mL of packed red blood cells were mixed
with 1 mL of ice cold water. The samples were frozen at
-70° C.
Serum/ervthrocvte folate. serum ferritin and serum
alkaline phosphatase. Precautions were taken to protect
samples from light during collection, processing and storage.
Samples used to determine serum concentrations of analytes
were collected in 13 mL silica coated tubes (Vacutainer®,
Becton Dickinson, Rutherford, NJ) and held at room temperature
for 30 to 60 minutes to allow time for clotting. The clotted
portion of each sample was removed, after which the samples
were centrifuged. The serum samples were removed, placed in
clean centrifuge tubes and centrifuged again before preparing
separate aliquots of the serum to be analyzed for serum
ferritin and serum folate concentrations. Ascorbic acid
(1 mg/mL) was added to vials to be analyzed for serum folate
to ensure stability during storage. All serum samples were
stored at -20° C. On days 4, 15 and 29 of the study, serum
samples were dispensed into a third set of vials and sent to
a clinical laboratory (SmithKline Beecham Clinical
Laboratories, Gainesville, FL) for routine laboratory tests.
Serum alkaline phosphatase activity was determined as a
component of this routine blood chemistry profile using the
method of Bowers and McComb (1966; 1975).

163
Samples for whole blood analysis were collected in 10 mL
ethylenediamine tetraacetic acid (EDTA) tubes (Vacutainer®,
Becton Dickinson, Rutherford, NJ) . Prior to processing,
duplicate samples of blood were drawn into capillary tubes and
centrifuged for 5 minutes at approximately 12,000 rpm.
Hematocrit levels were determined using a microhematocrit tube
reading device. The remaining sample was diluted 1:10 with
0.1 % sodium ascorbate and allowed to incubate at room
temperature for 3 0 minutes. After incubation, PBS (0.2 M) was
added to a final dilution of 1:20, and this whole blood lysate
was stored at -20° C.
Preparation of Diet Composites
All foods and beverages served to the subjects on a given
day were assembled. Foods that required cooking were prepared
according to the procedures established for this study. A
rinse bottle was filled with hot water, and the water was
sprayed around the edges of dishes containing cooked foods as
soon as they were removed from the oven to prevent the food
from sticking to the cooking dish. Solid foods were
transferred to a stainless steel blender, before transferring
liquids, to avoid the loss of liquids due to splashing.
Crumbs remaining in the original serving container were
loosened with a spatula and subsequently emptied into the
blender. Beverages were poured into the blender, and the
serving carton/cup was rinsed with water and scraped dry with

164
a spatula. A portion of the daily bread allowance was used to
wipe any liquid remaining in the serving containers. Bread
was also used to wipe residues left in the serving containers
from oil-based foods such as salad dressing and mayonnaise.
After all foods and beverages had been added to the blender,
the spatula was rinsed over the top of the blender and dried
with a small piece of bread. The diet was blended on low
speed for two minutes and high speed for two minutes.
A preweighed, gallon size, grip lock plastic bag was
tared on a scale and half of the blended diet was poured into
it. The remaining blended diet was poured into a second bag.
The weights of both bags were recorded. The bags were sealed
and placed flat on a tray in a -4° C freezer. After the bags
were frozen solid, they were freeze-dried. The bags of
freeze-dried diet were reweighed to determine the dry weights
of the diets for each day of the cycle menu. Each day of the
menu was prepared in duplicate during both study periods.
Powder-free gloves were worn throughout the entire process to
prevent zinc contamination.
Determination of Dietary Zinc Content
Unused, 100 mL high-form porcelain crucibles were soaked
in 10% nitric acid, dried in an oven at 105° C overnight and
transferred to desiccators containing silica dioxide. After
cooling, approximately 6 g of freeze-dried diet were added to
each labeled crucible. The exact weight of the empty crucible

165
and the weight with the sample were noted. Triplicate samples
were prepared from each bag of freeze-dried diet, resulting in
a total of six samples for each day of the cycle menu. The
crucibles containing the samples, and an empty crucible to be
used as a blank, were then returned to the 105° C oven for
overnight drying. In the morning, crucibles were removed from
the drying oven, placed in desiccators to cool, reweighed and
transferred to a muffle furnace with an initial temperature of
200° F. The temperature was increased by 100° F every hour
until the internal oven temperature reached 550° F. This
temperature was held overnight and cooled to 2 00° F in the
morning, after which, the crucibles were transferred to
desiccators for cooling.
After cooling, the ashed samples were dampened with water
and transferred to a prewarmed hot plate. Approximately 10 mL
of 50% HC1 were added to each crucible. The liguid was
evaporated to half volume and then the crucibles were filled
to two-thirds of their capacity with 10% HC1. The HCl was
evaporated to a level representing approximately 10 mL, after
which the crucibles were filled to two-thirds of their
capacity with water. This was evaporated to about 5 mL of
liquid, at which time the samples were removed from the hot
plate for cooling.
Each solubilized sample was filtered into a labeled 25 mL
volumetric flask using an extended neck glass funnel and 11 cm
Whatman filter paper. After filtering, a plastic paddle and

166
water were used to loosen and solubilize residues that had
accumulated on the internal surfaces of the crucibles. The
rinse water was filtered, and this process was repeated two
more times. (All glassware and paddles were acid washed in
10% nitric acid and thoroughly rinsed with water before they
were used.) When the funnels ran dry, the filter papers and
the funnels were rinsed with a small amount of water. Each
flask was brought up to volume with water. The flasks were
covered, inverted and mixed by agitation. The samples were
transferred into labeled zinc/trace element-free polypropylene
tubes (Sarstedt, Princeton, NJ).
Standards were made in a matrix containing calcium,
magnesium, phosphorous, potassium and sodium in proportions
similar to those in the diet (as determined by computer
analysis). The matrix solution also contained 250 mL of 10%
HC1 and water. The matrix was added to 100 mL volumetric
flasks containing 1, 3 and 6 mL, respectively, of 100 ppm zinc
standard prepared from 1000 ppm Certified Zinc Reference
Solution (Fisher Scientific Co. , Pittsburgh, PA) . These
solutions were mixed well and poured into labeled zinc/trace
element-free polypropylene tubes. The National Institute of
Standards and Technology's standard reference material #1572
(i.e. citrus leaves) was dried and dissolved following the
manufacturer's directions.
The absorbance readings of the blank, standards and
samples were determined by air/acetylene flame AAS using a

167
Perkin-Elmer® 5000 atomic absorption spectrophotometer
(Perkin-Elmer Corporation, Norwalk, CT) set at 213.9 nm.
Linear regression analysis was used to calculate the average
zinc concentration for the reference standard and each day of
the cycle menu.
Biochemical Analyses
Plasma and Urine Zinc Concentrations
Plasma zinc concentrations were measured by air/acetylene
flame AAS at a wavelength of 213.9 nm, using a Perkin-Elmer®
2380 (Perkin-Elmer Corporation, Norwalk, CT) flame atomic
absorption spectrophotometer. Duplicate plasma samples were
diluted five-fold with water. Metal-free pipette tips and
metal-free 1.5 mL polypropylene test tubes (Biorad, Richmond,
CA) were used to prepare these samples. Pooled samples were
prepared in the same manner. A sample of the National
Institute of Standards and Technology's standard reference
material #1598 (bovine serum) was prepared for recovery to
evaluate the accuracy of the analytical method. Certified
Zinc Reference Solution (1000 ppm; Fisher Scientific Co.,
Pittsburgh, PA) was used to make the zinc standards. These
standards were prepared in duplicate in 5% glycerol to
minimize differences due to viscosity (Smith and Butrimovitz,
1979) . A solution of 5% glycerol was used for the blank. All
glassware used to prepare the standards and blank had been
acid-washed (10% nitric acid solution) and thoroughly rinsed

168
in water. Each sample/standard was vortexed just before
aspiration. A pooled sample was aspirated every fifth sample
and the standards were aspirated every tenth sample.
Hydrochloric acid (0.1 N) , followed by water were aspirated
between each sample/standard. Sample concentrations were
calculated using linear regression from the standard
concentration line.
Duplicate urine samples were vortexed and aspirated
directly using the same spectrophotometer used for the plasma
samples. Standards for urine zinc were prepared in 0.1 N HC1.
As a control, lyophilized Gilford Urine Metal Control II (Ciba
Corning Diagnostics Corporation, Irvine CA) was dissolved in
water and diluted with 0.1 N HC1. Standards and the control
were checked every tenth sample. Sample concentrations were
calculated using linear regression from the standard
concentration line.
Erythrocyte Lysate Zinc Concentration
The zinc concentrations of erythrocyte lysates were
measured by air/acetylene flame AAS at a wavelength of 213.9
nm, using a Perkin-Elmer® 5000 (Perkin-Elmer Corporation,
Norwalk, CT) flame atomic absorption spectrophotometer.
Duplicate erythrocyte lysate samples were diluted to a
fourteen-fold final dilution with water. Metal-free vials and
pipette tips (Biorad, Richmond, CA) were used. Pooled samples
were prepared in the same manner. Certified Zinc Reference

169
Solution (1000 ppm; Fisher Scientific Co., Pittsburgh, PA) was
used to make the zinc standards. Zinc standards were prepared
in duplicate in 5% glycerol. A solution of 5% glycerol was
used for the blank. All glassware used to prepare the
standards and blank had been acid-washed (10% nitric acid
solution) and thoroughly rinsed in water. Each
sample/standard was vortexed just before aspiration. A pooled
sample was aspirated every seventh sample and the standards
were aspirated every fifteenth sample. Hydrochloric acid
(0.1 N) , followed by water were aspirated between each
sample/standard. Sample concentrations were calculated using
linear regression from the standard concentration line. The
final zinc concentrations were expressed per milliliter of
erythrocyte lysate and per gram of protein.
Protein Determination
Protein concentrations of erythrocyte lysates were
determined by the Folin phenol reagent method of Lowry et al.
(1951). One milliliter of Lowry Reagent (0.58 mM Na2CuEDTA,
0.18 M Na2C03 and 0.1 M NaOH) was added to duplicate samples
diluted 1:700 and to standards prepared using bovine serum
albumin (BSA) (Sigma® Chemical Co., St. Louis, MO). Samples
and standards were vortexed and incubated for 10 minutes,
after which 0.1 mL of phenol reagent (Folin-Ciocalteu Phenol
Reagent, Sigma® Chemical Co., St. Louis, MO) was added. After
3 0 minutes, absorbance was read at 500 nm (Beckman DU-64

170
Spectrophotometer, Palo Alto, CA) . Sample concentrations were
calculated using linear regression from the standard
concentration line.
Erythrocyte Metallothionein Concentration
Metallothionein (MT) concentrations of erythrocyte
lysates were measured using an ELISA for human
metallothionein-1 (Grider et al., 1989). In this assay,
primary antibody (i.e. sheep anti-human metallothionein-1
immunoglobulin G) directed against human metallothionein-1
binds in a competitive fashion to human metallothionein-1
coated to the wells of a microtiter plate and to free
metallothionein present in the sample or standard. The
antigen-antibody complex formed between the primary antibody
and the free metallothionein in the sample/standard is
removed, leaving behind only the primary antibody bound to the
human metallothionein coated to the wells of the plate.
Enzyme-linked secondary antibody (donkey anti-sheep
IgG/alkaline phosphatase conjugate, Sigma® Chemical Company,
St. Louis, MO) is added to the plate, and this binds with the
antigen-antibody complex. Enzyme-linked secondary antibody
that does not bind to the antigen-antibody complex is removed.
Following incubation with para-nitrophenyl phosphate, color
development occurs, and absorbance is measured
spectrophotometrically at 405 nm. Metallothionein-1
concentrations of unknown samples are determined by linear

171
regression of the standard curve after logit Y transformation.
A schematic presentation of this assay is presented in Figure
4-2.
Purification of human metallothionein. Human
metallothionein-1 used for the coating antigen and standard
were provided by Dr. R.J. Cousins. Pooled human liver samples
obtained from the College of Medicine at the University of
Florida, Gainesville Florida, were homogenized and
centrifuged. The supernatant was subjected to gel filtration
chromatography, and the zinc-containing fractions that
comprised the metallothionein peak were fractionated by anion
exchange HPLC. The metallothionein isoforms were separated
using a step gradient generated by a gradient pump. All
metallothionein-1 fractions were pooled and concentrated by
ultrafiltration. The purity of metallothionein-1 was
determined by amino acid analysis (Grider et al., 1989).
Production of sheep anti-human metallothionein-1
immunoglobulin G. Sheep anti-human metallothionein-1
immunoglobulin G (primary antibody) was provided by Dr. R.J.
Cousins. Purified human metallothionein-1 was incubated
overnight with purified rat immunoglobulin G (Sigma® Chemical
Co., St. Louis) in PBS. Prior to injection, this solution was
combined with an equal amount of a 1:1 mixture of Freund's
complete/incomplete adjuvant. Sheep received multiple
intradermal and intramuscular injections of this solution,
with booster injections given at 30, 50 and 106 days following

172
the initial injection. Serum was harvested seven days after
each booster injection. The sheep anti-serum was applied to
a protein A agarose column (Bethesda Research Laboratories,
Gaithersburg, Md.), and fractions containing peak absorbance
at 280 nm (anti-human metallothionein-1 immunoglobulin G) were
pooled and assayed for protein concentration (Grider et al.,
1989) .
Preparation of standard, pooled control and samples.
Purified human metallothionein-1 and a pooled erythrocyte
lysate with known concentrations of metallothionein-1 were
provided by Dr. R.J. Cousins. The purified human
metallothionein-1 was diluted 1:20 with a solution of 10
mmol/L PBS, 1% BSA, 0.5% polyoxyethylene sorbitan monolaureate
(i.e. Tween 20) (Sigma® Chemical Company, St. Louis, MO) and
0.02% sodium azide (NaN3), pH 7.2. The unknown samples were
diluted 1:1 with the same solution to which 2-mercaptoethanol
was added at a level of 2 /¿L/mL of diluent.
ELISA procedure. Coating antigen (1 mL human
metallothionein-1 antigen, 100 ng/mL) was added to 9.5 mL of
PBS (10 mmol/L, pH 7.2) and 10 jiiL of 2-mercaptoethanol.
Aliquots (100 iih) of this solution were dispensed into the
wells of a microtiter plate (NUNC, USA Scientific, Ocala, FL).
The plate was covered and stored at 4° C for 16 h, after which
the coating solution was discarded. The plate was washed
three times with a washing solution consisting of 10 mmol/L
PBS, plus 0.5% Tween 20. To reduce nonspecific binding,

173
Colored
Product
Substrate abs at 405 nm
Sample or
Standard
Primary
Antibody
human metallothionein-1
Figure 4-2. Schematic of enzyme-linked immunosorbent assay
used for determination of erythrocyte metallothionein
concentrations.

174
300 /XL of buffer solution (10 mmol/L PBS, 1% BSA, 0.5%
Tween 20, 0.02% NaN3, pH 7.2) were added to all wells of the
plate, and the plate was incubated at room temperature for 30
minutes. The buffer solution was discarded and the plate was
washed with the washing solution and tapped dry. Fifty
microliters of the buffer solution, and 100 iih of the
standard, pooled control and samples were added to designated
wells of the plate. The samples, pooled control and standard
were added in duplicate. A multi-channel pipette was used to
perform a total of four serial dilutions, after which, buffer-
diluted primary antibody (i.e. 3 fiL antibody plus 10.5 mL
buffer) was dispensed (50 ¿¿L/well) into the appropriate wells.
The plate was covered and incubated at room temperature for
4 h. The solution in the wells of the plate was discarded at
the end of the incubation period, and the plate was washed and
tapped dry. Buffer-diluted secondary antibody (20 /¿L of
secondary antibody plus 10.5 mL buffer) was added
(100 ¿¿L/well) to all wells of the plate and incubated for 30
minutes at room temperature. The solution was discarded
again, and the plate was washed and tapped dry. A substrate
solution of para-nitrophenyl phosphate (i.e. 4 mol/L in
carbonate buffer; one tablet of para-nitrophenyl phosphate
disodium mixed with 41 mL of carbonate buffer, pH 9.6) was
added to all wells of the plate (200 /xL/well) and incubated in
the dark at 37° C for 1 h. Absorbance at 405 nm was measured
using a microtiter plate reader (Molecular Devices Corporation

175
UV Max, Menlo Park, CA) interfaced with a computer.
Metallothionein concentrations of unknown samples were
determined by linear regression of the standard curve after
logit Y transformation. The final results were expressed as
/Ltg/g protein.
Determination of Serum Ferritin Concentration
Serum ferritin concentrations were determined using a
sandwich-type ELISA (Flowers et al., 1986). Rabbit, anti¬
human ferritin (Dako Corporation, Carpintería, CA) diluted
1:500 in 0.1 M bicarbonate buffer (pH 9.6) was used to coat
the wells of a 96-well microtiter plate (NUNC, USA Scientific,
Ocala. FL) . The plate was covered and incubated at room
temperature for 6 h, after which the coating solution was
discarded and the plate was washed three times with a solution
of 0.05% Tween 20/Dulbecco's phosphate-buffered saline (DPBS).
Ferritin standard (10 /ng/mL in NaCl; ICN Biochemicals,
Cleveland, OH) , diluted with 1% BSA/DPBS, was added to the
plate and serially diluted with additional 1% BSA/DPBS. The
samples, diluted 1:5 with the same solution as the standards,
were added to the plate in triplicate. The plate was covered,
refrigerated overnight and then washed with 1%BSA/DPBS.
Horseradish peroxidase conjugated ferritin (Dako Corporation,
Carpinteria CA) , diluted with 1%BSA/DPBS to a final
concentration of 1:4000, was added to the plate. The plate
was covered, incubated for 6 h at room temperature and then

176
washed. A substrate solution consisting of 1,2-ortho-
phenylenediamine, dihydrochloride (Dakopatts, Denmark)
dissolved in 0.1 M citric acid-phosphate buffer with 30%
hydrogen peroxide (HRP Color Reagent B, BioRad Laboratories,
Richmond, CA) was added to the plate, and the plate was placed
in the dark for 15 minutes to allow for color development.
Absorbance was read at 490 nm using a Molecular Devices
Corporation UV Max microtiter plate reader (Menlo Park, CA) .
Sample concentrations were calculated using linear regression
from the standard concentration line.
Determination of Serum. Whole Blood and Urinary Folate
Concentrations
Total folate concentrations of serum, whole blood and
urine samples were determined microbiologically using the 96-
well microtiter plate assay (Newman and Tsai, 1986) . The
assay organism for the microbiological assay was Lactobacillus
casei (L. casei) grown in ATCC 7469 (Difco Laboratories,
Detroit, MI) growth media. Preparation of the standard,
samples and culture organism, as well as performance of the
assays, were done under controlled lighting.
Preparation of media and reagents. Dehydrated folic acid
casei medium (Difco Laboratories, Detroit, MI) , was
reconstituted, filter sterilized using a Corning®
filter/storage system with a 0.22 micron cellulose acetate
filter (Corning Glass, Corning, NY) and stored at 4° C. The
same type of filtration system was used to sterilize a 0.1 M

177
phosphate buffer solution (pH 6.3) containing 1 mg/mL ascorbic
acid, which was prepared immediately before each assay was
begun.
Microorganism. Freeze-dried cultures of L. casei 7469
were obtained from American Type Culture Collection
(Rockville, MD) . Dry culture was suspended in a sterile
solution containing 25 mL of media and an equal volume of
0.1 M phosphate buffer solution (pH 6.3) containing 1 mg/mL
ascorbic acid. Folinic acid (Sigma® Chemical Company, St.
Louis, MO; 1 ng/mL) was added to the suspended culture, and
the culture was incubated at 37° C for 24 h.
After demonstrating that the assay organism provided an
adequate folate response curve, the cryoprotected assay
organism was prepared. Approximately, 0.5 mL of the previous
day's growth was added to a 200 mL sterile receptacle (Corning
Glass, Corning, NY) containing 25 mL of sterile medium, 25 mL
of 0.1 M potassium phosphate buffer (pH 6.3), 50 ng folinic
acid and 50 mL of sterile 80% glycerol. The solution was
mixed well and 0.5 mL aliquots were pipetted into sterile
vials (Sarstedt, Princeton, NJ). The vials were stored at
-20° C. Prior to inoculating the samples and standards, an
aliquot of the cryoprotected microorganism was allowed to come
to room temperature, and for each plate to be assayed, 45 /¿L
of the microorganism were diluted with 15 mL of sterile
medium.

178
Preparation of standard. Folinic acid (150 mg; Sigma®
Chemical Company, St. Louis, MO) was added to a 50 mL sterile
conical centrifuge tube (Corning Glass, Corning, NY) and
dissolved in sterile water (15 mL) by adding 0.1 N NaOH. The
pH was adjusted to 7.0 with 0.1 N HC1, and the volume was
adjusted to 25 mL with sterile water. After filtration, a
1:100 dilution of the standard was made. One milliliter of
this standard solution was diluted with 2 mL 2N NaOH, and the
absorbance at 282 nm was determined using a Gilford 250
spectrophotometer (Ciba Corning Gilford Systems, Oberlin, OH) .
The concentration of the standard was calculated using an
extinction coefficient of 28,200. Aliquots of the standard
(1:100 dilution) were stored at -20° C. Prior to performing
the assay, the standard was defrosted and diluted with the
ascorbate-phosphate buffer (pH 6.3) described above. The
concentration of the folate standard used to construct the
standard curve ranged from 0.023 to 0.375 pmoles/well.
Assay procedure using 96-well microtiter plate. Falcon®
(Becton Dickinson, Rutherford, NJ) , 96-well, flat bottomed,
sterile, low evaporation tissue culture plates with lids
(Becton Dickinson, Lincoln Park, NJ) were used for this assay.
The assay was performed under a laminar flow hood using
sterile equipment, supplies and solutions.
For each assay, water was added (300 ¡jlL) to the wells of
the plate that were designated as blanks. One hundred and
thirty microliters of the ascorbate-phosphate buffer and 20 nL

179
of appropriately diluted standard, pooled controls and samples
were added in duplicate to selected wells of the plate.
(Whole blood samples were diluted with buffer to a final
concentration of 1:30 or 1:40, and urine samples were diluted
1:2, 1:4 or 1:8 before being added to the plate; serum samples
were not diluted prior to being added to the plate.) One
hundred and fifty microliters of buffer were dispensed into
all other wells of the plate. A total of five serial
dilutions of the samples, standard and pooled control were
made using a multi-channel pipette. All wells were inoculated
with 150 ijlL of the medium containing the assay organism, and
the plates were covered and placed in an incubator at 37° C
for 16 h.
After incubation, the contents of each well were
resuspended by repeated aspiration and flushing using a multi¬
channel pipette. A flame was guickly passed over the surface
of the plate to eliminate air bubbles. Growth of the
microorganism was measured by reading the turbidity of each
well at 650 nm using a microtiter plate reader (Molecular
Devices UV Max, Menlo Park, CA) interfaced with a computer.
The output generated was used to calculate the folate
concentration of the original serum, whole blood or urine
sample. Once serum and whole blood folate concentrations were

180
determined, erythrocyte folate concentrations were calculated
using the following formula:
iwhole blood folatel-rserum folate x (1 - hematocrit/100)1
hematocrit
100
Determination of Urinary Deuterium-Labeled Folate
Urinary excretion of deuterium-labeled folate (D2~FA) was
determined using the method of Gregory and Toth (1988). This
method involves the separation of folates using affinity
chromatography, chemical cleavage of the folates to para-
aminobenzoyl glutamate (pABG), purification of the pABG
fragments using HPLC and quantification by GCMS.
Affinity chromatography column preparation. The columns
used for affinity chromatography were provided by Dr. J.F.
Gregory, III. These 2 mL columns were packed with Affigel-10®
(Biorad, Richmond, CA), and folate binding protein isolated
from bovine whey was bound to the Affigel-10®. Five
milliliters of 0.1 N HC1 were added to each column to remove
any residual folates, and each column was equilibrated with
15 mL of 1.0 M potassium phosphate (pH 7.0). The capacity of
each Affigel-10® folate-binding protein (FBP) column (Affigel-
FBP columns) was determined by applying ten, 1 mL aliquots of
10 nmol/mL folic acid standard (Sigma® Chemical Co. , St.
Louis, MO) in 0.1 M potassium phosphate buffer (pH 7.0) at a
flow rate of 0.3 mL/minute. Each 1 mL fraction was collected
and saved. Five milliliters of 0.1 N HC1 were added to each

181
column and the fractions were collected in 10 mL volumetric
flasks. The fractions collected were brought to volume with
additional 0.1 N HC1. The columns were flushed with an
additional 5 mL of 0.1 N HC1, and these fractions were
discarded. The columns were equilibrated with 0.1 M potassium
phosphate buffer (pH 7.0) and stored in the refrigerator with
the column bed filled with the same buffer solution.
The absorbance of each 1 mL fraction was measured
spectrophotometrically at a wavelength of 282 nm. The 10 mL
fractions were measured at 296 nm. This information was used
to determine the amount of folate that would saturate the
columns and the amount of folate that would bind to the
columns, respectively.
Purification and treatment of urine samples for HPLC.
Urine samples were defrosted, mixed well and adjusted to pH
7.0 with 5 N NaOH. The urine was filtered using Buchner
funnels, #1 Whatman filter paper and a vacuum pump. Forty to
85 mL of urine were applied to an Affigel-FBP column that had
been previously flushed with 5 mL of 0.1 N HCl and
equilibrated with 0.1 M potassium phosphate buffer (pH 7.0).
(The amount of urine used varied depending on the expected
urinary folate concentration.) A peristaltic pump was used to
maintain a constant flow rate of 0.3 mL/min. After all of the
sample had been applied to the column, the column was rinsed
with 5 mL of 0.25 M potassium phosphate buffer containing

182
1.0 M NaCl (pH 7.0), followed by 5 mL of the buffer without
NaCl. The folate bound to the column was eluted with 0.1 N
HC1, with the first milliliter discarded. The eluted folate
was brought up to a volume of 5 mL with 0.1 N HC1. The
Affigel-FBP column was flushed with 5 mL of 0.1 N HC1,
equilibrated with 0.1 M potassium phosphate buffer (pH 7.0)
and stored in the refrigerator with the column bed filled with
the same buffer.
Chemical cleavage of folate to pABG. A 2 mL portion of
the folate-containing solution eluted from the Affigel-FBP
column was gently agitated and exposed to air for 30 minutes.
Under these conditions, tetrahydrofoíate is spontaneously
cleaved by oxidation. Potassium acetate (2 N) was added to
the sample, followed by the addition of 1 N NaOH, to bring the
sample to a pH of 6.0. Five percent (v/v) hydrogen peroxide
at 0.05 volume was added to oxidize N5-methyl-tetrahydrofoíate
to N5-methyl-dihydrofoíate. Thirty seconds later, 0.1% bovine
hepatic catalase (Sigma Chemical Co., St. Louis, MO) was added
at 0.1 volume to decompose the remaining hydrogen peroxide.
The N5-methyl-dihydrofoíate was cleaved by acidifying the
sample with 5 N HC1 (0.1 volume). Cleavage of the folic acid
present in the sample was achieved by the addition of a 0.1 mL
zinc dust suspended in gelatin (1 g zinc dust in 4 mL 0.5%
aqueous gelatin). The cleaved product was centrifuged to
sediment the zinc. Finally, the sample was placed in an
Amicon® MPS filtration unit with a YMT membrane (Amicon

183
Division, W.R. Grace & Co., Danvers, MA) and centrifuged at
lOOOg for 20 minutes to remove the gelatin and catalase.
HPLC Detection of pABG. High-performance liguid
chromatography separation was performed using a Beckman
Ultrasphere column and 0.1 M formic acid/2.5% acetonitrile
mobile phase. Para-aminobenzoyl-L-glutamic acid (Sigma®
Chemical Co., St Louis, MO), dissolved in 0.1 N NaOH to a
final concentration of 5 ¿¿M, was used as the standard.
Detection was achieved with an absorbance monitor set at
280 nm. Two milliliters of each filtered, cleaved sample were
loaded onto the HPLC column. Elution of the pABG fractions
occurred in about 10 to 15 minutes, at which time the
fractions were collected in 5 mL reaction vials. The pABG
fractions were flushed with nitrogen gas and stored at -20° C.
Derivatization of pABG for GCMS. The pABG fractions were
dried under nitrogen gas in a Reacti-Therm heating block and
incubated with trifluoroacetic anhydride and trifluoroethanol
at 90° C for one hour. The reagents were then evaporated
under nitrogen gas and the derivatized pABG samples were
stored in reaction vials at -20° C.
GCMS detection of derivatized pABG. The derivatized pABG
samples were defrosted and dissolved in 100 /¿L ethyl acetate.
The samples were injected into the instrument and separated by
gas chromatography. The carrier gas was helium. Derivatives
of unlabeled and labeled pABG were analyzed by mass spectral
monitoring (mass to charge ratios of 426 and 428,

184
respectively) using the negative-ion electron-capture chemical
ionization mode with methane at 0.4 Torr as the reagent gas.
The ratios of unlabeled/labeled pABG, as measured by GCMS, and
the total urinary folate concentrations determined by the
microbiological assay, were used to calculate the total
urinary excretion of unlabeled and labeled folates.
Statistical Analysis
Since the primary
objective of
this
study
was
to
determine the effect
of
supplemental
folic
acid
on
zinc
status, computations
for
statistical
power
were
based on
univariate information of interindividual variation for plasma
zinc and erythrocyte metallothionein concentrations. Using
Student's two-sided t-test and assuming independence between
successive measurements on the same individual, it was
determined that a sample size of six subjects per group would
provide 80% power to detect a meaningful change in plasma zinc
concentrations. This same sample size provided 75% power in
the case of erythrocyte metallothionein concentrations. These
power calculations represented a conservative estimate since
independence was assumed in a setting in which it was likely
that positive correlations between successive measurements on
a given subject would be observed.
Student's T-test was used to test for differences in age;
weight; and usual intake of calories, protein, folate and zinc
between subjects fed the zinc-adequate or zinc-restricted

185
diet. Analysis of variance for repeated measures was used to
test for significant differences in mean values and slopes
within and between the diet groups due to the effect of folic
acid supplementation and level of zinc intake over time. The
Statistical Analysis System (SAS Institute, Inc., 1989, Cary,
NC) computer package was used for all analyses.

CHAPTER 5
RESULTS
The results of this study are reported in three sections.
The first section describes results that are relevant to both
objectives of the study. This is followed by a description of
the findings pertaining to the first and second objectives of
the study, respectively.
No significant difference (P>0.05) was detected in the
mean (± SD) age of subjects in the zinc-restricted (28.3 ±
3.8 y) versus the zinc-adequate diet groups (25.0 ± 4.0 y) ,
nor were significant differences (P>0.05) detected in the
average baseline weights of subjects (Table 5-1) within or
between the study periods. Comparisons of the change in
average weights, within or between study periods one and two,
failed to detect significant differences (P>0.05) as well.
The results of the complete blood counts with differentials
and the 25-item blood chemistry profiles obtained at the
beginning of both phases of the study were within normal
limits for each subject.
Analysis of data obtained from food records (Table 5-2)
did not reveal a significant difference (P>0.05) in the usual
mean intake of calories or folate between the zinc diet
groups; however, there were significant differences in the
186

187
usual mean intakes of protein (P<0.05) and zinc (P<0.05). The
difference in the mean protein intake (i.e. 18 g) was
equivalent to the protein content of a 2.5 oz portion of meat.
If this was a 2.5 oz serving of red meat, it would essentially
account for the difference in mean usual zinc intake
(i.e. 2.8 mg) between the zinc diet groups. These differences
Table 5-1. Mean weights (± SD) of subjects on zinc-restricted
or zinc-adequate diets.
Studv period
I
Dav
1
Dav
29
Zn-restricted
68.7
± 6.4
69.5
+
6.2
Zn-adequate
74.0
± 13.7
73.7
+
12.4
Studv period
II
Zn-restricted
68.9
± 5.9
69.4
+
5.2
Zn-adequate
74.4
± 13.0
74.2
+
11.4
Table 5-2. Mean (± SD) usual calorie, protein, folate and
zinc (Zn) intakes of subjects on zinc-restricted or zinc-
adequate diets.
Zn-restricted
Zn-adeauate
Calories (kcal/d)
2313
+
215
2556
+
257
Protein (g/d)
98.5
+
15.2
116.8
+
11.7
Folate (/¿g/d)
306
+
202
243
+
49
Zinc (mg/d)
11.4
+
0.9
14.2
+
2.2

188
were not considered to be clinically significant since the
mean protein intake of the zinc-restricted (i.e. 1.4 g/kg body
weight) and zinc-adequate (i.e. 1.6 g/kg body weight) groups
exceeded the RDA, and the mean usual zinc intakes exceeded 75%
of the RDA for both groups. Additionally, the group with the
lower mean usual zinc intake was the same group that received
the zinc-restricted diet.
All subjects completed both phases of the study, and the
results of the daily checklists, as well as the anonymous
guestionnaire completed at the end of the study revealed
satisfactory compliance to the feeding regimen and study
protocol. None of the subjects correctly identified the
combination and order of treatments they received suggesting
that they were adequately blinded to the treatments.
The mean (± SD) zinc content of the diet fed to the
subjects during study periods one and two, as determined by
direct analysis, was 3.5 ± 0.2 mg zinc/d. The accuracy and
precision of the method used to determine the zinc content of
the diet were verified by concurrent analysis of citrus leaves
certified for zinc content by the National Institute of
Standards and Technology. The published reference value for
this material is 29 ± 2 /¿g/g, and the mean laboratory value
obtained was 30 ± 1 ¿xg/g. The interassay coefficient of
variation was 3.3%.
The molar ratio of zinc to folic acid provided by the
various treatment combinations is listed in Table 5-3. In all

189
cases, the molar ratios were lower than the ratio obtained
using the 1989 RDA (Food and Nutrition Board, 1989a; 1989b)
for zinc and folate (i.e. 511:1).
Table 5-3. Molar ratios of zinc (Zn) to folic acid (FA) for
each treatment combination.
Treatment combination
Molar ratio (Zn:FA)
Zn-restricted, no supplemental FA 82:1
Zn-restricted, 800 nq supplemental FA 30:1
Zn-adequate, no supplemental FA 338:1
Zn-adequate, 800 [iq supplemental FA 123:1
Effect of Supplemental Folic Acid on Zinc Status
The response variables measured to determine if
supplemental folic acid affects zinc status in human subjects
consuming zinc-restricted or zinc-adequate diets included:
plasma zinc, erythrocyte zinc, erythrocyte metallothionein,
serum alkaline phosphatase and urinary zinc. Serum ferritin
concentrations were also measured in order to determine if the
erythrocyte metallothionein response was specific or due to
changes in iron metabolism. Analysis of variance for repeated
measures was used to test for differences in the means for
each response variable, within and between the zinc diet
groups, due to the effect of the supplement (i.e. the response
on the supplement minus the response off the supplement) . The
same statistical procedure was used to test for differences in

190
the mean response over time due to the effect of the
supplement.
Plasma Zinc
The accuracy and precision of the method used to
determine plasma zinc concentrations were evaluated by
concurrent analysis of National Institute of Standards and
Technology bovine serum and pooled plasma samples. The mean
(± SD) value for the bovine serum was 93 ± 0 /¿g/dL compared
with the published value of 92 ± 6 /¿g/dL. The interassay
coefficient of variation was 4.0%, and the average intraassay
coefficient of variation was 3.4%.
No significant differences (P>0.05) were detected in mean
plasma zinc values (Table 5-4) within or between the zinc diet
groups due to the effect of the supplement, and plasma zinc
values were within the normal range (i.e. >70 /¿g/dL) (Gibson,
1990) for all but one subject. The subnormal plasma zinc
values reported for subject #5 occurred during phase two of
the study when he was consuming the zinc-restricted diet
without supplemental folic acid. The initial baseline value
for this subject during study period two was 62 /¿g/dL. This
value declined to a minimum of 41 /¿g/dL about midway through
the study period and then increased to a final value of 58
/¿g/dL. The mean plasma zinc values for this subject during
study periods one and two (89 ± 4 versus 53 ± 9 /¿g/dL,
respectively) were significantly different (P<0.05). This

191
subject also displayed behavior suggestive of depression
and/or irritability.
Table 5-4. Overall mean (± SD) values of response variables
used to assess the effect of supplemental folic acid (FA) on
zinc status in subjects consuming zinc-restricted or zinc-
adeguate diets.
Resoonse variables
Zn intake
FA
treatment
+FA
-FA
Plasma zinc
Restricted
82
+
5
81
±
14
(Mg/dL)
Adeguate
86
+
6
86
+
10
Erythrocyte zinc
Restricted
37
+
3
40
+
3
(/xg/g protein)
Adeguate
38
+
5
38
+
6
Serum alkaline
Restricted
87
+
11
92
±
15
phosphatase (U/L)
Adeguate
93
+
21
100
+
27
Erythrocyte MT
Restricted
14
+
2
12
+
2
(/¿g/g protein)
Adeguate
13
+
3
12
+
2
Serum ferritin
Restricted
101
+
45
109
+
58
(ng/mL)
Adeguate
128
+
40
114
+
72
Urinary zinc
Restricted
384
+
138
539
+
281
(Mg/24 h)
Adeguate
652
+
406
597
+
446
The effect of supplemental folic acid on plasma zinc in
subjects consuming the zinc-restricted or zinc-adeguate diets
over time is illustrated in Figure 5-1. No significant
(P>0.05) time x supplement x diet interaction was detected.
Comparison of the slopes of the lines for the zinc-restricted
and zinc-adeguate groups, due to the effect of the supplement
(i.e. on minus off the supplement), revealed the lack of a

Figure 5-1. Effect of supplemental folic acid on plasma zinc in subjects
consuming zinc-restricted or zinc-adequate diets.
192

193
statistically significant difference (P>0.05) as well. This
finding suggests the lack of a supplemental effect due to the
level of zinc intake.
Within the zinc-restricted group, no significant
difference (P>0.05) was detected in the slopes of the lines
when subjects received the supplement versus the placebo.
Mean plasma zinc concentrations on and off the supplement were
similar, as can be seen by the fact that the line representing
the mean difference values for this group was almost super¬
imposed on the zero axis (Figure 5-1). The same was true for
the zinc-adequate group.
Erythrocyte Zinc
No significant differences (P>0.05) in mean erythrocyte
zinc values (Table 5-4) were detected within or between the
zinc diet groups due to the effect of the supplement. Unlike
plasma zinc, significant differences (P>0.05) in mean
erythrocyte zinc concentrations were not detected for subject
#5 during study periods one and two (i.e. 39 ± 4 versus 36 ±
3 M9/g protein).
As illustrated by the lack of contrast in the patterns of
change over time (Figure 5-2), a significant time x supplement
x diet interaction was not detected (P>0.05) for this response
variable. Also, no significant differences (P>0.05) in the
change in erythrocyte zinc concentrations were detected

Figure 5-2. Effect of supplemental folic acid on erythrocyte zinc in subjects
consuming zinc-restricted or zinc-adequate diets.
194

195
between or within the zinc diet groups due to the effect of
the supplement.
The precision of the method used to determine erythrocyte
zinc concentrations was assessed by analyzing a pooled
erythrocyte lysate sample. The interassay coefficient of
variation of 3.4%, and the average intraassay coefficient of
variation was 1.8%.
Serum Alkaline Phosphatase
No significant differences (P>0.05) were detected in mean
serum alkaline phosphatase values (Table 5-4) within or
between the zinc diet groups, and with the exception of
subject #11, serum alkaline phosphatase values were within
normal limits (20 to 140 U/L, SmithKline Beecham Clinical
Laboratories, Gainesville, FL) during both study periods.
During study period two, subject #11 had a baseline value of
151 U/L, which declined to 136 and 139 U/L on days 15 and 29,
respectively. The latter two values were comparable to his
prestudy value of 138 U/L. During study period one, subject
#ll,s baseline value was 122 U/L. This value declined to 118
and 98 U/L on days 15 and 29, respectively. The larger
decline in values observed from days 15 to 29 during study
period one may have been related to the low grade fever (99.7
°F to 101.3 °F) experienced by this subject on days 23 through
27, for which he was treated with acetaminophen.

196
The effect of supplemental folic acid on serum alkaline
phosphatase over time is shown in Figure 5-3. Although it was
observed that some individuals in both diet groups had larger
baseline difference values, their difference values at
subsequent time intervals tended to be smaller and were
clustered near the zero axis. Exceptions to this phenomenon
were subjects #4 and #11. Subject #4 had large difference
values throughout the study; however, his pattern of change
was similar to the overall pattern for the zinc-restricted
group. As discussed above, subject #11 responded much more
negatively to the effect of the supplement between days 15 and
29 compared to the rest of the subjects in the zinc-adequate
group. Consequently, the line connecting the mean serum
alkaline phosphatase difference values for subjects consuming
the zinc-adequate diet had a downward trend at the conclusion
of the study. This line would have been parallel to the line
representing the means for the zinc-restricted group if
subject #11's data for day 29 had been omitted. Despite these
somewhat different patterns of change, no significant (P>0.05)
time x supplement x diet interaction was detected. No
significant difference (P>0.05) in the change in serum
alkaline phosphatase values due to the effect of the
supplement were detected between the zinc diet groups; nor
were significant differences detected (P>0.05) when the change
in this response variable on versus off the supplement was
compared within each of the diet groups.

d -H
+j C3
cd g
o &
«5
Dh 5~
CD ?
c S
3o.o :
20.0 :
A
a Zinc-Adequate Diet
o Zinc-Restricted Diet
- a â–  Mean Zinc-Adequate Diet
- «- Mean Zinc-Restricted Diet
-40.0
-50.0
0 4
j.
x
Treatment Initiated
J I I I 1 I I I L
15
Day of Study
A
#4
O
j i i i i i i i i i i
29
Figure 5-3. Effect of supplemental folic acid on serum alkaline phosphatase in
subjects consuming zinc-restricted or zinc-adequate diets.
197

198
Erythrocyte Metallothionein
Baseline, and all successive values for erythrocyte
metallothionein were below the mean value (± SD) previously
reported for a group of 44 subjects (i.e. 47.5 ± 30.0 /xg/g
protein; Thomas et al., 1992). The reason for these
differences is unknown, although it is possible that the
variation in results occurred due to the use of standards
prepared at different points in time.
The precision of the method, using a pooled erythrocyte
lysate sample, could not be adequately evaluated because the
values obtained for this sample could not be read within the
limits of the standard curve. The absorbance readings
suggested that the erythrocyte metallothionein concentrations
of the pooled sample were very low. It is possible that the
concentration of metallothionein in this sample declined
during storage due to protein degradation (Davies and
Goldberg, 1987). Unlike the pooled sample, it was possible to
read the subjects' samples within the limits of the standard
curve.
In an attempt to ascertain the approximate degree of
interassay variability, the interassay coefficient of
variation was calculated using the adjusted absorbance
readings of the pooled sample (i.e. absorbance reading minus
the blank). This value was 18.6%. The coefficients of
variation, based on the slope and intercept of the standard

199
curve for each run of the assay, were 14.0% and 8.3%,
respectively.
There was a significant (PcO.OOl) time x supplement x
diet interaction for this response variable as illustrated in
Figure 5-4. For subjects consuming the zinc-restricted diet,
the supplemental effect resulted in a gradual increase in
erythrocyte metallothionein difference values, followed by a
decline. The effect of folic acid supplementation in the
zinc-adequate group resulted in a slight decrease in
erythrocyte metallothionein difference values over time. The
continued negative direction of the line connecting the mean
erythrocyte metallothionein difference values for the zinc-
adequate group (i.e days 22 through 29) was due to the large
difference value observed on day 29 for subject #8. In
comparison, by day 29, the rest of the subjects in the zinc-
adequate group had difference values clustered around the zero
axis. No significant differences (P>0.05) were detected when
the slopes of the lines on the supplement versus the slopes of
the lines on the placebo were compared within the zinc-
restricted and zinc-adequate diet groups. Comparisons of the
mean erythrocyte metallothionein concentrations (Table 5-4)
within and between the zinc diet groups failed to produce
significant differences (P>0.05) as well.

Figure 5-4. Effect of supplemental folic acid on erythrocyte metallothionein in
subjects consuming zinc-restricted or zinc-adequate diets.
200

201
Serum Ferritin
Individual serum ferritin concentrations tended to
decline over the course of each study period, and compared
with study period one, baseline values were observed to be
lower during study period two. These trends may have been due
to the frequency of phlebotomy and the volume of blood
obtained. Despite these trends, serum ferritin concentrations
were within normal limits (i.e. 18 to 300 ng/mL) (Young, 1987)
for all but one subject during both study periods. The
exception was subject #5 whose serum ferritin concentrations
during study period two (19, 15 and 27 ng/mL) were suggestive
of borderline deficiency. Corresponding values during study
period one were 175, 136 and 106 ng/mL. When mean serum
ferritin values (Table 5-4) within and between the zinc diet
groups were compared, no significant differences (P>0.05) were
detected.
Figure 5-5 shows the effect of supplemental folic acid on
serum ferritin. Compared to the zinc-restricted group, a
slight upward trend in the line connecting the mean difference
values for the zinc-adequate group was observed between days
15 and 29; however, no significant (P>0.05) time x supplement
x diet interaction was detected.
Despite the slight upward trend noted for the zinc-
adequate group, no significant difference (P>0.05) was
detected due to the effect of the supplement between the zinc
diet groups. This trend was due to the large difference


203
value obtained for subject #11 on day 29. This subject's
serum ferritin values were 80, 50 and 209 ng/mL when he
received the supplement and 37, 31 and 18 ng/mL when he
received the placebo (i.e. study periods one and two,
respectively) . The large increase from day 15 to day 29
during study period one, which was not replicated during study
period two, may have been related to the low grade fever
experienced by this subject rather than to the effect of the
supplement, since serum ferritin concentrations are known to
increase with infection (Reeves and Haurani, 1980). When the
effect of the supplement versus the placebo was compared
within each level of zinc intake, there were no significant
differences (P>0.05) in the slopes of the lines for either of
the groups.
Urinary Zinc
The accuracy and precision of the method used to
determine urinary zinc concentrations were assessed by
simultaneous analysis of Gilford Urine Metal Control (Ciba
Corning Diagnostics Corporation, Irvine, CA). The mean value
obtained for this control was 100 ± 0.6 /xg/dL, compared to the
published target concentration of 100 jug/dL. The interassay
coefficient of variation was 0.6%, and the average intraassay
coefficient of variation was 0.5%.
No significant differences (P>0.05) in mean urinary zinc
values (Table 5-4) were detected within or between the zinc
diet groups due to the effect of the supplement. Baseline

204
urinary zinc values ranged from 164 to 1235 ¡iq/24 h, which is
similar to the normal range (150 to 1200 ¡j.g/24 h) (Young,
1987). Urinary zinc concentrations dropped below the lower
limit of normal for subject #5 during study period two. This
coincided with the timing of the reduction observed in his
plasma zinc concentrations. Below normal urinary zinc
concentrations were also noted for two subjects consuming the
zinc-adequate diet. Subject #10 had consistently low, yet
relatively stable concentrations of zinc in his urine,
regardless of whether he received the folic acid supplement or
the placebo. Borderline low urinary zinc concentrations were
observed in subject #7 only when he received the placebo. This
was probably related to the fact that his baseline value was
also much lower during the placebo period.
The effect of the supplement on urinary zinc in subjects
consuming zinc-restricted or zinc-adequate diets over time is
shown in Figure 5-6. No significant (P>0.05) time x
supplement x diet interaction was detected. No significant
difference (P>0.05) in the change in urinary zinc difference
values was detected between the zinc diet groups either,
suggesting the lack of an effect due to the supplement.
Subjects consuming the zinc-restricted diet responded to
the reduction in zinc intake with significant decreases
(placebo: P<0.01; supplement:P<0.05) in mean urinary zinc
concentrations from baseline. However, no significant
difference (P>0.05) was detected in the change in mean values

600 “
400 '
O
c

U
o
C
c
(U
a
JU
Oh
Oh
0
CO
200
R §
b-c
-200 "
S3
•G BP
-400;
-600;
-800
0
a Zinc-Adequate Diet
o Zinc-Restricted Diet
A
O
—*i Mean Zinc-Adequate Diet
- •- Mean Zinc-Restricted Diet
O
#4
J 1 1 I I I I I I I I I I 1 L
11 18
i
A
J I L
28
j
Treatment Initiated Day of Study
Figure 5-6. Effect of supplemental folic acid on urinary zinc in subjects
consuming zinc-restricted or zinc-adequate diets.
M
O
<

206
on the supplement compared to the change on the placebo.
Despite the lack of a statistically significant difference,
two subjects (i.e. subjects #4 and #5) appeared to respond to
the effect of the supplement differently than the others. The
greater reduction in urinary zinc excretion that occurred when
subject #4 received the supplement is illustrated in Figure
5-6 (days 11, 18 and 28). In contrast, subject #5 excreted
less zinc in his urine when he received the placebo; however,
compared to baseline, little difference in the change in his
values on versus off the supplement was observed at any time
point. This suggests that he responded similarly to the
effects of a zinc-restricted diet during both phases of the
study, but that his zinc status was more severely compromised
at the beginning of study period two than study period one.
In contrast to the zinc-restricted group, no significant
(P>0.05) change from baseline was detected for urinary zinc
when subjects in the zinc-adeguate group received the
supplement or when they received the placebo. Furthermore, a
significant difference (P>0.05) was not detected in the change
in mean urinary zinc concentrations on the supplement compared
to the change on the placebo.
Effect of Level of Zinc Intake on Folate Utilization
To determine the effect of zinc intake on folate
utilization, total urinary, serum and erythrocyte folate
concentrations, and urinary excretion of deuterium-labeled and

207
unlabeled folate were determined in subjects consuming zinc-
restricted or zinc-adequate diets. Analysis of variance for
repeated measures was used to test for differences in mean
values and slopes when subjects in the zinc-restricted and
zinc-adequate diet groups were receiving supplemental folic
acid.
Serum Folate
The precision of the method used to determine serum
folate concentrations was evaluated by concurrent analysis of
pooled serum samples with known concentrations. The
interassay coefficients of variation were 5.8 and 8.2%, and
the average intraassay coefficients of variation were 9.6 and
9.2% for the low and high pooled samples, respectively.
There was no evidence to suggest (P>0.05) that the mean
serum folate response to folic acid supplementation (Table
5-5) was influenced by the level of zinc intake. As shown in
Figure 5-7, both groups experienced a significant increase
(P<0.001) in serum folate concentrations over the course of
the study; however, no significant difference (P>0.05) was
detected when the slope of the line for the zinc-restricted
group was compared to the slope for the zinc-adequate group.
Erythrocyte Folate
No significant difference (P>0.05) in the mean
erythrocyte folate response to folic acid supplementation

40 1
a Zinc-Adequate Diet
o Zinc-Restricted Diet
* Mean Zinc-Adequate Diet
- â– #- Mean Zinc-Restricted Diet
O
O
O
O
§
Treatment Initiated
i i i i i i i i
11 15 18
Day of Study
Figure 5-7. Serum folate response to folic acid
consuming zinc-restricted or zinc-adequate diets.
8
i i i i i i i i i i i i
22 25 29
supplementation in subjects
208

209
Table 5-5. Overall mean (±SD) values of response variables
used to assess the response to folic acid (FA) supplementation
in subjects consuming zinc-restricted or zinc-adequate diets.
ResDonse variables
+ /-FA
Zn
intake
3.5
ma
14.5
mq
Serum folate
+FA
23.8
+
6.1
18.0
+
4.5
(ng/mL)
-FA
17.7
+
2.6
15.3
±
4.0
Erythrocyte
+FA
375
+
90
297
+
98
folate (ng/mL)
-FA
348
+
77
300
+
98
Urinary folate
+FA
494
+
300
424
+
293
(total) (Mg/24 h)
-FA
128
+
96
119
+
84
Deuterium-labeled
+FA
290
+
80
259
+
108
urinary folate
-FA
not
applicable
(Mg/24 h)
(Table 5-5) was detected due to the level of zinc intake. The
overall response to the supplement was positive (Figure 5-8),
with both groups achieving statistically significant increases
(P<0.05, zinc-restricted; p<0.01, zinc-adequate) in mean
erythrocyte folate concentrations over the course of the
study. However, no significant difference (P>0.05) was
detected in the change in the mean erythrocyte folate response
due to the level of zinc intake.
A pooled whole blood sample with a known folate
concentration was used to assess the precision of the method.
The interassay coefficient of variation was 8.1%, and the
average intraassay coefficient of variation was 5.7%.

o
600 1
500 "
400 _
300 '
200 "
100 “
O
O
0
J L
0
Treatment Initiated
i i i i i i i I L
11 15 18
Day of Study
22
Figure 5-8. Erythrocyte folate response to folic acid
subjects consuming zinc-restricted or zinc-adequate diets.
j i i i i i i
25 29
supplementation
in
to
o

211
Urinary Folate
Total urinary folate. Pooled serum samples with known
folate concentrations were used to assess the precision of the
method used to determine urinary folate concentrations. The
interassay coefficients of variation were 6.1 and 8.0%, and
the average intraassay coefficients of variation were 9.4 and
12.2% for the low and high pooled samples, respectively.
There was no evidence (P>0.05) to suggest that the mean
urinary folate response to folic acid supplementation (Table
5-5) was influenced by the level of zinc intake, and mean
urinary folate concentrations for both groups of subjects were
significantly higher (PcO.OOl) when they received the
supplement versus the placebo. The large standard deviations
obtained for both zinc diet groups during supplementation were
due to the fact that folate excretion was much lower at
baseline, after which it steadily increased in response to
folic acid supplementation.
As depicted in Figure 5-9, there was no evidence (P>0.05)
to support a difference in urinary folate response to folic
acid supplementation due to the level of zinc intake. Within
each of the zinc diet groups, the subjects experienced
significantly greater increases (P<0.001) in urinary folate
concentrations when they received the supplement versus the
placebo.
Subject #9 tended to excrete less folate (both labeled
and unlabeled) in his urine than other subjects, although the

Urinary Folate (ng/24 h)
Figure 5-9. Urinary folate response to folic acid supplementation in subjects
consuming zinc-restricted or zinc-adequate diets.
212

213
overall change in his excretion pattern was similar to that of
the other subjects. This subject also tended to have low
urinary folate concentrations when he received the placebo.
Urinary deuterium-labeled folate. Excretion of
deuterium-labeled folate in response to folic acid
supplementation is illustrated in Figure 5-10. Similar to the
data for total urinary folate, there was no evidence to
support a difference (P>0.05) in the urinary deuterium-labeled
folate response to folic acid supplementation due to the level
of zinc intake. There was also no significant difference
(P>0.05) in the average amount of deuterium-labeled folate
excreted (Table 5-5) due to the level of zinc intake. There
was, however, a significant increase for both groups (PcO.OOl)
in the amount of labeled folate excreted between days 11 and
18.
When the data were expressed as percentages of total
folate intake (i.e. 290 ng/d dietary folate, plus 800 nq/d.
supplemental folic acid), no significant difference (P>0.05)
was detected in the mean value (Table 5-6) obtained for the
zinc-restricted versus the zinc-adeguate diet groups. There
was also no evidence to suggest (P>0.05) that the change in
the percent of total folate intake excreted with the deuterium
label was different due to the level of zinc intake (Figure
5-11) . The same was true when the data were expressed as
percentages of total urinary folate (Table 5-6; Figure 5-12)
or as percentages of the oral dose (Table 5-6; Figure 5-13).


215
As shown in Figure 5-12, approximately 45% of the folate
excreted in the urine was excreted as deuterium-labeled folate
by day 28. Since 74% of the total folate intake was
deuterium-labeled, equilibrium was not reached within the time
frame of this study.
Table 5-6. Overall mean (±SD) deuterium-labeled folate (D2FA)
excreted expressed as a percentage of total folate intake,
total urinary folate and oral dose.
Response variables
D2FA/total folate
intake (%)
D2FA/total urinary
folate (%)
D2FA/oral dose (%)
Zn intake
3.5mg
26.7 ± 7.3
47.0 ± 5.1
36.3 ± 10.0
14.5mg
23.8 ± 9.8
46.6± 6.5
32.4 ± 13.3

Urinary Deuterium-Labeled Folate/
Oral Labeled Dose + Dietary Folate
Co 40.0
30.0
20.0
a Zinc-Adequate Diet
o Zinc-Restricted Diet
^ Mean Zinc-Adequate Diet
- Mean Zinc-Restricted Diet
O
A
O
10.0 '
A
0.0
Treatment Initiated
l L
11
J I I I 1 I L
18
Day of Study
i i i i
27
Figure 5-11. Percent of oral labeled folic acid plus dietary folate excreted as
deuterium-labeled folate in subjects consuming zinc-restricted or zinc-adequate diets.

217


CHAPTER 6
DISCUSSION
Effect of Supplemental Folic Acid on Zinc Status
The present study used traditional indices of zinc
status, and a newer index which has been shown to respond
quickly to dietary zinc intake (Grider et al., 1990), to
examine the effects of supplemental folic acid on zinc status
in subjects consuming zinc-restricted or zinc-adequate diets.
The lack of significant differences in both mean values and
the change in mean values for these response variables
suggests that short-term supplementation with 800 nq folic
acid/d does not adversely affect zinc status in healthy young
men consuming zinc-restricted or zinc-adequate diets.
In the present study, significant changes in plasma zinc
concentrations in response to the zinc-restricted diet without
supplemental folic acid were not expected because of the
relatively short duration of each study period (i.e. 25 days).
It was reasoned, however, that if the purported effect of
folic acid supplementation on zinc absorption was severe
enough, a significant change in plasma zinc concentrations
might be detected within this time interval when subjects
consuming the zinc-restricted diet received the supplement.
This type of response was not observed, which suggests that
219

supplemental
folic
acid
does
not
affect
220
plasma zinc
concentrations
or
that
the
level
and/or
duration of
supplementation was insufficient to elicit this change.
Despite the fact that the zinc-restricted diet was fed
for a relatively short period of time, and folic acid
supplementation did not appear to affect zinc status adversely
during this time interval, it was surprising that subject #5
developed biochemical and behavioral symptoms of zinc
deficiency while receiving the placebo. It is possible that
consumption of the zinc-restricted diet for 25 days, combined
with weight-lifting activity and a variable zinc intake during
the washout phase (i.e. range of 8.9 to 19.7 mg zinc/d; mean
of 13.3 ± 5.6 mg/d), contributed to this subject's lowered
zinc status during study period two. Alternatively, the
provision of supplemental folic acid during study period one
may have conferred a protective effect that resulted in the
maintenance of plasma zinc concentrations during this phase of
the study. Evidence against the latter suggestion was
reported by Milne et al. (1990). These researchers found that
zinc mobilization during exercise was impaired when folic acid
supplements were fed.
Similar to plasma zinc, erythrocyte zinc and serum
alkaline phosphatase concentrations are less sensitive and
specific to changes in zinc status. Consequently, changes in
these indices would not be expected in subjects consuming
marginally zinc-deficient intakes for relatively short periods

221
of time unless the effect of the supplement was profound.
Apparently, the effect of folic acid supplementation, if any,
was not sufficient to produce significant changes in this
study. A possible exception was subject #11, whose serum
alkaline phosphatase difference value on day 29 was much more
negative than that of the other subjects. In addition to
being different from the response observed for any of the
other subjects, this type of response was also different than
the response observed when Milne et al. (1990) administered
supplemental folic acid (i.e. 400 and 800 /ig/d) to subjects
consuming a zinc-adeguate diet (i.e. 12.5 mg/d) for six weeks.
These findings suggest that the decline in this subject's
serum alkaline phosphatase activity may have been related to
the fever he developed rather than ingestion of the
supplement. Since infection and inflammation have been
associated with changes in zinc mobilization and distribution
(Beisel et al., 1974; Pekarek et al., 1978; Solomons et al.,
1978), it is possible that zinc associated with serum alkaline
phosphatase may have been mobilized and redistributed to be
used for more critical functions.
By using a more sensitive and specific indicator of zinc
status that responds quickly to changes in zinc intake (i.e.
erythrocyte metallothionein; Grider et al., 1990; Thomas et
al., 1992), it was predicted that the effect of supplemental
folic acid on zinc status could be evaluated within the time
frame of this study. The fact that significant differences in

222
mean erythrocyte metallothionein values or in the change in
mean erythrocyte metallothionein values on versus off the
supplement were not detected for either of the zinc diet
groups, suggests that supplemental folic acid does not
adversely affect zinc status. If folic acid supplementation
had adversely affected zinc status, the concentration of
erythrocyte metallothionein would have been significantly
lower when the subjects received the supplement, particularly
in subjects consuming the zinc-restricted diet.
Although the primary metallothionein isoform expressed in
the bone marrow of rats is metallothionein-1 (Huber and
Cousins, 1993), the various metallothionein isoforms and the
relative proportions of these isoforms in human erythrocytes
have not been determined. Since human metallothionein-1 (i.e.
the form of the antibody used in this study) does not appear
to cross-react with metallothionein-2, only the concentration
of erythrocyte metallothionein-1 was determined in this study.
Consequently, it is possible that changes in the concentration
of metallothionein-2 due to the effect of the supplement
and/or level of zinc intake could have occurred without being
detected.
The reason for the significant difference between the
patterns of change in erythrocyte metallothionein for the
zinc-restricted and zinc-adequate diet groups is unknown. The
fact that no significant time x supplement x diet interaction
was detected for serum ferritin suggests that the changes

223
observed in erythrocyte metallothionein were specific and not
due to an indirect effect on protein/iron metabolism. Since
the erythrocyte is a cellular target of folate, it is possible
that in response to continued low zinc intake, folate
temporarily increased cellular zinc uptake through some
unknown mechanism, and this increased concentration of
intracellular zinc enhanced the induction and transcription of
the metallothionein gene. Thus, by day 22, the zinc-
restricted group appeared to have a notable positive response
to the supplement. In support of this idea, mean plasma zinc
difference values for the zinc-restricted group declined
slightly from baseline through day 22, after which they began
to increase. Alternatively, it is possible that the
dissimilarities in the patterns of change between the zinc-
restricted and zinc-adequate diet groups were due to the order
in which the samples were analyzed. Conceivably, the
variability in standard curves was large enough to produce
erythrocyte metallothionein concentrations that were randomly
lower or higher than their actual values depending on sampling
order. This type of random error could have produced
artificially lower or higher difference values that
subsequently resulted in divergent patterns of change.
Nevertheless, it is important to remember that within the time
frame of this study, there was no evidence to suggest that
erythrocyte metallothionein concentrations responded

224
differently to the effect of the supplement versus the
placebo, regardless of the level of zinc intake.
Urinary zinc excretion has been shown to respond rapidly
to changes in dietary zinc intake (Baer and King, 1984) , but
the usefulness of this response variable as an indicator of
zinc status is limited because the amount of zinc excreted is
highly variable (King, 1986) and can be altered by certain
diseases and/or conditions (Prasad, 1983) . To reduce the
impact of these factors on the outcome and interpretation of
urinary zinc data, this study used healthy subjects who served
as their own control. As expected, subjects consuming the
zinc-restricted diet, but not the zinc-adeguate diet,
responded to the reduction in zinc intake by excreting less
zinc in their urine, regardless of the folic acid treatment.
The fact that urinary zinc concentrations responded to a
reduction in zinc intake, but not to the presence or absence
of the supplement, provides further support for the conclusion
that supplemental folic acid does not adversely affect zinc
status under the conditions of this study.
As a caveat, it is possible that for some individuals,
supplemental folic acid may affect tissue distribution and/or
excretion of zinc. Supplemental folic acid may cause a
reduction in urinary zinc excretion by: enhancing tissue
uptake; enhancing endogenous zinc secretion and excretion; or,
interfering with intestinal absorption, resulting in increased
fecal excretion. These possibilities could explain why

225
subject #4 had a greater reduction in urinary zinc excretion
when he was on the supplement. It is also conceivable that
this subject had a large decrease in urinary zinc excretion
due to greater dermal losses during the supplementation period
(July/August) compared to the placebo period
(October/November).
The only other controlled feeding studies that have
examined the effect of supplemental folic acid on zinc status
and absorption in human subjects were conducted by Milne et
al. (1984; 1990). Based on differences in fecal and urinary
zinc excretion (Milne et al., 1984; 1990), as well as
absorption studies using labeled zinc isotopes (Milne, 1989;
Milne et al., 1990), these researchers have concluded that
supplemental folic acid adversely affects zinc absorption and
may subseguently impair zinc status in individuals with low
zinc intakes or increased zinc need.
In comparison to the present study, subjects in the study
by Milne et al. (1984) experienced a significant decrease in
mean plasma zinc concentrations when they consumed a diet
containing 3.5 mg zinc/d for 16 weeks. The difference in
plasma zinc concentrations between the folic acid-supplemented
and placebo-treated groups was not significant. Apparently,
the duration and extent of the reduction in zinc intake was
sufficient to cause a notable reduction in mean plasma zinc
concentrations in both groups, but further reductions due to
the effect of the supplement were not observed. Although

226
plasma zinc is not a sensitive and specific indicator of
marginal zinc status (King, 1990), the longer duration of
these studies and the fact that plasma zinc concentrations
responded to a reduction in zinc intake but not to folic acid
supplementation seem to support the view that supplemental
folic acid does not adversely affect zinc status, even when
zinc intake/need is low. In a more recent study, Milne et al.
(1990) did not find significant differences in plasma zinc
concentrations when subjects consuming zinc-adequate diets
received two six-week periods of folic acid supplementation
alternated with two six-week periods without supplementation.
One might argue that the lack of a significant difference
in mean plasma zinc concentrations between the folic acid-
supplemented and placebo-treated groups in the study by Milne
et al. (1984) was due to greater homeostatic adaptation in the
former group. Urinary zinc data obtained during the zinc-
depletion stage of their study support this idea. However,
the fact that supplemented subjects also had significantly
lower mean urinary zinc concentrations after consuming a diet
containing 33.5 mg zinc/d for four weeks raises the question
of whether the response was due to the supplement or to some
other difference(s) in the subjects assigned to each of the
treatment groups. It seems unlikely that such a small amount
of folic acid, relative to the level of zinc intake (i.e.
zincrfolic acid ratio = 647:1), could interfere with zinc
absorption. In fact, at this level of zinc intake no

227
significant difference in fecal zinc excretion between
subjects in the folic acid-supplemented versus the placebo-
treated groups was detected, suggesting that zinc absorption
was not adversely affected by supplemental folic acid. Given
the small number of subjects in this study (n=8) it would have
been more informative if each subject had served as his own
control on and off the supplement. Significant differences in
absorption and excretion patterns due to the effect of
supplemental folic acid were not detected in subjects
consuming a zinc-adeguate diet (Milne et al., 1990).
Effect of Level of Zinc Intake on Folate Utilization
Urinary excretion of deuterium-labeled and unlabeled
folate, as well as serum and erythrocyte folate data, support
the conclusion that under the conditions of this study, the
bioavailability of supplemental folic acid monoglutamate was
not influenced by the level of zinc intake. The findings of
Tamura et al. (1978) were similar except that they used
severely zinc-deficient subjects and determined the response
to folic acid supplementation by measuring the rise in serum
folate concentrations after administering a single oral dose
of folic acid monoglutamate.
Although the results of the present study suggest that
the level of zinc intake did not adversely affect the
utilization of supplemental folic acid monoglutamate, it is
possible that the amount of folate in the diet (i.e. 290 /ig/d)

228
and supplement (i.e. 800 /¿g/d) was sufficient to overcome
subtle changes in folate metabolism that may have occurred
secondary to a marginal zinc intake. Thus, it is possible
that a marginal zinc intake may adversely affect folate
metabolism when smaller doses of folic acid monoglutamate are
consumed.
In contrast to the effect of zinc intake/status on
absorption of folic acid monoglutamate, Tamura et al. (1978)
found that absorption of folic acid polyglutamate was
compromised in severely zinc-deficient subjects. Whether or
not this is a problem in individuals with marginal zinc
intakes remains to be determined. It is possible that the
defect in hydrolysis and/or absorption of pteroyl-
polyglutamates only occurs in severely zinc-deficient
individuals, or impaired absorption may only become evident
when large amounts of pteroylpolyglutamates are consumed.
Alternatively, the reduction in cellular proliferation and
growth associated with mild zinc deficiency may decrease the
metabolic demand for folate sufficiently to offset any
impairment in absorption of this nutrient.
The presence of deuterium-labeled folate in the subjects'
urine showed that the folic acid supplement was absorbed
across the intestinal mucosa, transported in the plasma and
excreted by the kidneys. The lack of significant differences
in urinary excretion of labeled folate metabolites and serum
and erythrocyte folate concentrations in subjects consuming

229
zinc-restricted versus zinc-adequate diets suggests that
overall tissue uptake and utilization of folate was similar
between the groups. However, whether or not there were
differences between tissues in folate uptake and metabolism in
subjects consuming zinc-restricted versus zinc-adequate diets,
as suggested by previous animal studies (Tamura et al., 1987;
Williams and Mills, 1973), is unknown. As noted earlier, one
of the subjects consuming the zinc-adequate diet (i.e. subject
#9) had lower urinary folate concentrations compared to
subjects in either of the zinc diet groups. A possible
explanation for this finding is that this subject absorbed
folic acid less efficiently. However, the fact that his serum
folate concentration more than doubled during supplementation
(i.e. baseline: 7.5 ng/mL; final 18.7 ng/mL) argues against
this idea. Interestingly, the increase in his erythrocyte
folate concentration during supplementation was modest
compared to the average increase for the zinc-adequate group.
This raises the possibility that folate was being directed to
some other tissue(s). It is also possible that this subject
catabolized folate more extensively, and the products of
folate catabolism were not quantified, resulting in under¬
estimation of urinary folate excretion.
The total folate intake of subjects in this study
consisted of 74% deuterium-labeled folic acid and 26%
unlabeled dietary folate. Based on these percentages, the
isotopic enrichment of tissue and excreted folates at complete

230
equilibrium should be approximately 74%. By day 29, only 45%
of the folate excreted in the urine was deuterium-labeled,
which suggests that equilibrium had not been achieved within
the time frame of the study. Research currently being
conducted suggests that a long period of time is required to
reach equilibrium.

CHAPTER 7
SUMMARY AND CONCLUSIONS
The concept that zinc and folic acid metabolism may be
related was first suggested in the early 1970's when Williams
and Mills (1973) observed a reduction in the mean hepatic
folate concentration in rats fed zinc-deficient diets. The
discovery that bovine hepatic folate conjugase was a zinc-
dependent enzyme (Silink et al., 1975) prompted further
research regarding the potential effect of zinc deficiency on
folate metabolism, and in 1978, Tamura et al. reported that
absorption of folate polyglutamate, but not folate
monoglutamate, was adversely affected in zinc-deficient males.
In addition to the effect of zinc deficiency on folate
absorption, a subseguent animal study (Tamura et al., 1987)
suggested that impaired zinc status may alter folate
metabolism.
The hypothesis that folic acid may adversely affect zinc
absorption and homeostasis was first proposed by Milne and
colleagues in 1984. Milne (1989) subsequently revised this
hypothesis, suggesting that impaired zinc absorption/status
may only be manifested in conditions of elevated zinc need or
low zinc intake. Although numerous animal and human studies
have examined the effect of supplemental folic acid on zinc
231

232
absorption/status, the results of these investigations have
been equivocal. The lack of agreement among these studies may
be due in part to differences in study designs, the lack of a
sensitive and reliable index to assess zinc status and/or
failure to control dietary intake.
Determination of the relative safety of supplemental
doses of folic acid has important public health implications
since folic acid supplements are commonly recommended for
individuals with chronic diseases and disorders, pregnant
women and individuals with a documented folate deficiency.
Supplemental doses of this nutrient have also recently been
recommended for women of childbearing age as a means for
reducing the occurrence/recurrence of neural tube defects.
Since folate and zinc are important in protein and nucleic
acid metabolism and genetic expression, changes in the
bioavailability and/or metabolism of either or both of these
nutrients can have deleterious effects.
The objectives of the present study were to determine if
supplemental folic acid affects zinc status in subjects
consuming zinc-restricted or zinc-adequate diets and if folate
utilization was affected by the level of zinc intake.
Subjects were fed a constant diet containing either 3.5 or
14.5 mg zinc/d for two 25-day periods, and deuterium-labeled
folic acid (i.e. 800 /ig/d) was consumed during one of the
study periods. The effect of supplemental folic acid on zinc
status was determined using traditional measures of zinc

233
status (i.e. plasma, erythrocyte and urinary zinc
concentrations; serum alkaline phosphatase activity), as well
as a newer method (i.e. erythrocyte metallothionein) that
responds quickly to changes in zinc intake (Grider et al.,
1990). The response of serum, erythrocyte and urinary folate
(i.e. total folate and labeled folate) concentrations to folic
acid supplementation were used to determine the effect of
level of zinc intake on folate utilization.
The present study did not detect a difference in zinc
status as a result of short-term supplementation with 800 ¿¿9
of folic acid/d in subjects consuming zinc-restricted or zinc-
adequate diets, nor did it find a difference in folate
utilization due to the level of zinc intake. The safety and
effectiveness of supplementation in other population subgroups
(i.e. women, pregnant women, individuals taking anticonvulsant
medications, population groups at risk for developing vitamin
B12 deficiency, etc.) remains to be determined. Thus, further
research in this area seems warranted since folic acid
supplements are already being used by certain segments of the
population, and if the Food and Drug Administration - Food
Advisory Council's (Anon., Food Chemical News, 1993)
recommendation to enrich flour with folic acid is accepted,
the population as a whole will be consuming additional amounts
of this nutrient.
This study represents the first diet-controlled human
study to be conducted since Milne (1989) first suggested that

234
supplemental folic acid may adversely affect zinc status in
subjects with low zinc intakes or increased zinc needs.
Although additional well-controlled and designed studies are
needed in order to bring closure to this issue, this study
serves as a starting point toward this goal.

APPENDIX A
SUBJECT SELECTION SCREENING TOOL
Food Science and Human Nutrition
University of Florida
359 Food Science Building
Gainesville, FL 32611
904-392-1991
Demographic Information
Date
Mo.
Name
Day Yr.
Last
First
Middle
ss#
Phone
Day Evening
Aqe
Date of
Birth
Mo. Day
Yr.
ADDRESS
Sex
Male
Female
Race
Caucasian
Hispanic
Black
Other
Asian
Religion
(optional)
Catholic
Hindu
Protestant Muslim
Jewish None
Jehovah Witness
Other
235

236
Education Completed
High School Graduate
Bachelor's Degree
Master's Degree
Doctoral Degree
Present Work/Student Status (Check all that apply):
Working Full-time Full-time Student
Working Part-time Part-time Student
Not Employed
Medical History
Indicate if you have had or currently have any of the
following medical problems (Check all that apply):
Past Now
Alcoholism
Allergies
Anemia
Arthritis
Asthma
Blood Clots
Bronchitis
Cardiovascular Disease
(Atherosclerosis/Heart Attack)
Claudication
Cystic Fibrosis
Dermatitis
Emotional Disorder
Eye Problems

237
Past
Now
Gall Bladder Disease
Glaucoma
Gout
Hair Loss (excessive)
Headaches
Hemorrhoids
Hernia
Hypercholesterolemia/Hyperlipidemia
Hypertension
Intestinal Disorders
Kidney Disease
Liver Disease
Lung Disease
Mental Illness
Neurologic Disorder
Obesity/Overweight
Prostate Trouble
Rheumatic Fever
Seizure Disorder
Stomach Disease
Stroke
Thyroid Disease
Tumors/Cancer - List Type:
Ulcers
Other - Specify:

238
Indicate if you have had any of the following surgeries, and
if so, the approximate date (Check all that apply):
Month/Year
Cardiovascular Surgery
Gastric Surgery
Gall Bladder Surgery
Intestinal Surgery
Kidney Surgery
Lung Surgery
Pancreatic Surgery
Prostate Surgery
Thyroid Surgery
Other - Specify:
Indicate the prescription/nonprescription medicines you
currently use on a regular basis (Check all that apply):
Allergy Medicines/Antihistamines
Antacids
Antibiotics
Anti-arrhythmics
Anti-inflammatory Agents (i.e. ibuprofen)
Aspirin
Asthma Medicines
Beta Blockers
Blood Pressure Medicines
Blood Thinners (i.e. anticoagulants)
Cortisone
Decongestants

239
Diabetes Medicines/Insulin
Diuretics
Gout Medicines
Heart Medicines
Hormones
Laxatives
Nitroglycerin
Pain Medicines
Psychiatric Medicines/Anti-depressants
Sedatives/Sleeping Pills
Seizure Medicines
Thyroid Medicines
Tranquilizers
Other - Specify:
Do you take any type of nutritional supplement (i.e. vitamin
and/or mineral pill; protein supplement; rose hips; other
supplements; etc.)?
Yes No
If you answered "yes" to the last question, indicate the
brand(s), type of supplement(s), frequency and amount(s).
If you currently use supplements, would you be willing to
discontinue use of your usual supplements for 5 to 6 months?
Yes No
Do you use any of the following tobacco products (check all
that apply):
Cigarettes Chewing Tobacco
Cigars Snuff
Pipe

240
Activity Status
Indicate your usual activities, frequency per month and
minutes per session by placing a check mark in the appropriate
columns (check all that apply):
Frequency/Month Minutes/Session
1-4 5-8 9-12 13-16 0-20 20-40 40-60
Badminton
Baseball/
Softball
Basketball
Boating
Bowling
Cycling(motor)
Cycling (road)
Cycling
(stationery)
Dancing
(aerobic)
Dancing
(social)
Fishing
Golf (ride)
Golf (walk)
Gymnastics
Hiking
Horseback
Riding
Hunting
Jogging/
Running

241
Frequency/Month
1-4 5-8 9-12 13-16
Martial Arts
Racquetball/
Handball
Rope Jumping
Rowing/
Canoeing
Sailing
Scuba Diving/
Snorkeling
Skating
Skiing
(cross country)
Skiing
(downhill)
Skiing (water)
Soccer/
Football
Swimming
Table Tennis
Tennis
Volleyball
Walking
Weight Training
Yard Work
Gardening
Other - Specify
Minutes/Session
0-20 20-40 40-60

242
Does your
activity?
usual job/schoolwork
require sustained
physical
Yes
No
How would
you rate your physical fitness/endurance (check
one) ?
Low
Medium
High
How would
you rate your strength
(check one)?
Low
Medium
High
Diet History
Height (without shoes) inches
Usual weight (dressed without shoes) lbs
Current weight (dressed without shoes) lbs
Is your weight fairly stable? Yes No
Are you satisfied with your current weight? Yes No
If not, please explain:
Do you weigh yourself frequently? Yes No
Have you gained or lost weight in the past year?
Yes No
If yes, how much did you gain/lose? lbs
Was this weight change intentional? Yes No
How many times (meals and snacks) do you eat each day?
Are you allergic to any foods? Yes No
If yes, please list all foods:
Are there any foods you cannot or will not eat?
Yes
If yes, please list these foods:
No

243
Which of the following words best describes your appetite
(check only one)?
Small
Medium
Large
Do you follow any of the modified diets listed below (check
all that apply)?
Diabetic Weight Reduction
Low Sodium Weight Gain
Renal Gastric Banding
Ulcer/Bland Kosher
Vegetarian Other:
Low Cholesterol/Low Fat
Do you drink coffee or tea? Yes No
If yes, could you limit your intake or these to one cup per
day for a month without difficulty?
Yes No
Do you consume alcoholic beverages (i.e. beer, wine, wine
coolers, hard liguor, etc.)
Yes No
If yes, could you discontinue your intake of these for a
month?
Yes No
Indicate the amount and frequency with which you consume the
following:
Amt. Never/ Occas. Freq. Always
Rarely
Beef
Beer (regular)
Beer (light)
Bread
Cake

244
Amt. Never/ Occas. Freq.
Rarely
Candy
Cereal
Chicken
Coffee (regular)
Coffee (decaf)
Cola (regular)
Cola (diet)
Cookies
Eggs
Fish (& tuna)
Fruit
Fruit Juice
Hard Liquor
Legumes
Milk
Pork
Shellfish
Snack Foods
Starches
Tea
Turkey
Vegetables
Wine
Always
Wine Coolers

245
Indicate if you disagree (DA), moderately agree (MA) or (SA)
strongly agree with each of the following statements by
placing a check in the appropriate column.
DA MA SA
Food is very important to me.
Eating is one of my favorite
pastimes.
I consider myself to have a lot
of will power.
I don't usually pay much attention
to what I eat.

APPENDIX B
ZINC RESTRICTED METABOLIC DIET
Day 1
Breakfast
Corn flakes
White bread
Margarine
Grape jelly
Coffee Rich®
Apple juice
Apple juice, special
Protein shake, orange
Morning supplements
Amount
38 g
50 g
10 g
24 g
120 g
120 g
45 g
360 g
1 vitamin B complex
1 calcium citrate
1 magnesium gluconate
Lunch
Grape juice 236 g
Turkey breast 30 g
White bread 50 g
Mayonnaise 12 g
Potato chips 30 g
Vanilla wafers 16 g
Pineapple chunks 100 g
246

247
Dinner
Apple juice
120 g
Chicken, boneless breast
60 g
Barbecue sauce
16 g
Margarine
15 g
Anellini®
120 g
Green beans
100 g
White bread
12 g
Salt, iodized
2 60 mg
Cupcake, white (prebaked)
40 g
Blueberries
30 g
Cool Whip®
5 g
Protein shake, tropical
360 g
Evening supplements
1 biotin
2 magnesium gluconate
Snack
Twinkie®
43 g
Cranberry juice
Dav 2
120 g
Breakfast
Amount
Trix®
21 g
White bread
50 g
Margarine
10 g
Apple jelly
24 g
Coffee Rich®
120 g

248
Apple juice
Apple juice, special
Protein shake, lemon
Morning supplements
120 g
45 g
360 g
1 vitamin B complex
1 calcium citrate
1 magnesium gluconate
Lunch
Orange juice 236 g
Turkey breast 30 g
White bread 50 g
Mayonnaise 12 g
Fritos® corn chips 50 g
Lorna Doones® 40 g
Applesauce 120 g
Dinner
Chicken, boneless breast 60 g
Corn flakes 5 g
Margarine 22.7 g
Mashed potatoes, dry mix 19 g
Coffee Rich® 20 g
Salt, iodized 700 mg
Rusks 10 g
Carrots, canned 100 g
White bread 12 g
Strawberry banana gelatin 120 g
Protein shake, orange 360 g

249
Evening supplements
1 biotin
2 magnesium gluconate
Snack
Angel food cake
30 g
Grape juice
118 g
Day 3
Breakfast
Amount
Corn flakes
38 g
White bread
50 g
Margarine
10 g
Grape jelly
24 g
Coffee Rich®
120 g
Apple juice
120 g
Apple juice, special
45 g
Protein shake, orange
360 g
Morning supplements
1 vitamin B complex
1 calcium citrate
1 magnesium gluconate
Lunch
Cranberry juice
236 g
Turkey breast
30 g
White bread
50 g
Mayonnaise
12 g
Fritos® corn chips
50 g
Pound cake
25 g
Pears
100 g

250
Dinner
Chicken, boneless breast
60 g
White rice, dry
30 g
Salt, iodized
260 mg
Lettuce
28 g
French dressing
12 g
Rusks®
10 g
White bread
12 g
Margarine
15 g
Applesauce
100 g
Cinnamon crumb topping
20 g
Protein shake, tropical
360 g
Evening supplements
1 biotin
2 magnesium gluconate
Sugar cookie
Orange juice
118 g

APPENDIX C
CONTRACT
I, , agree to comply with the
following rules as part of my participation in the study being
conducted to investigate the interrelationships between zinc
and folic acid:
1. Report on time for meals and blood draws.
2. Eat all foods and beverages served to me by the
researcher, including vitamin and mineral supplements.
3. Avoid eating/drinking anything except the foods and
beverages provided to me by the researcher. (Remember NO
ALCOHOL.)
4. Eat my meals and snack using metabolic style eating
techniques, which requires scraping, rinsing and licking
clean all food containers and utensils.
5. Avoid the use of prescription and over the counter
medications. If the use of medication is essential, I
will consult the researcher first, if possible. If the
use of prescription medication is unavoidable, I
understand that I may need to withdraw from the study.
6. Collect 24 hour urine specimens in the containers
provided; keep specimens refrigerated; and return all
containers on the appropriate dates.
7. Limit exercise to a MAXIMUM of one hour per day.
(This is not the same as an average of one hour per day.)
8. Use only approved personal hygiene products.
9. Complete the "Morning Checklist" on a daily basis.
10. Wash hands thoroughly before eating.
11. Return empty water bottles to be sanitized and
refilled.
12. Report any unusual symptoms.
251

252
13. Report any food losses or spills.
14. Complete food records, when requested, during study
periods one and two.
15. Comply with weight check schedule.
16. Avoid the use of tobacco products such as cigarettes,
cigars, pipes, chewing tobacco and snuff.
In return for my complete cooperation and compliance with
the above, I understand that I will be provided with all of my
meals for a total of 56 days. I will also receive financial
compensation in the amount of $800.00. I recognize that if I
fail to comply with the above, I will be dropped from the
study and will only receive compensation for the time that I
actually participated in the study.
Signed this second day of July, 1990:
Witnesses:

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BIOGRAPHICAL SKETCH
Gail Patricia Abbott Kauwell was born in New York, NY, on
August 30, 1952. She is the eldest of Robert and Audrey
Abbott's six children. Gail graduated from East Brunswick
High School, East Brunswick, NJ, in 1970. She attended the
University of Maine, Orono, and graduated with highest
distinction in 1974 and 1975, earning a B.A. in psychology and
a B.S. in nutrition, respectively. As an undergraduate, she
was inducted into the honor societies of Phi Kappa Phi, Phi
Beta Kappa and Omicron Nu. She completed a dietetic
internship at Perth Amboy Medical Center in 1976 and became a
registered dietitian that same year. She was employed as a
registered dietitian for two years and then returned to school
after being awarded a scholarship from the Bureau of Health
Manpower. In 1979, she graduated from the University of
Florida with a master's degree in nutrition. Upon graduation,
she worked as a registered dietitian for several more years
and then accepted an academic appointment at the University of
Florida in the College of Health Related Professions' Program
in Clinical and Community Dietetics.
During her employment with the University, Gail has been
promoted to the rank of Assistant Professor, has received the
College of Health Related Professions' Teacher of the Year
286

287
Award and the Faculty Research Award, was awarded tenure in
1987 and has served as Interim Director for the Program in
Clinical and Community Dietetics (1983-1985; 1991-1992). In
1986, she received permission from the Graduate School of the
University of Florida to pursue a doctoral degree in the
Department of Food Science and Human Nutrition. She began her
studies on a part-time basis and met her residency requirement
while on sabbatical/leave of absence without pay from the
University. During this time she was awarded a fellowship
from the Center for Nutritional Sciences. She was also the
recipient of scholarship awards from the Florida Dietetic
Association and the American Dietetic Association and was
inducted into the honor society of Gamma Sigma Delta in 1988.
Her research was funded in part by competitive grants that she
was awarded through the University of Florida's Division of
Sponsored Research.
Gail has been the recipient of many other awards and
honors, including Florida's Distinguished Dietitian, the
Florida Dietetic Association President's Award and the
American Dietetic Association Outstanding Service Award. She
is listed in several biographical texts including Who's Who in
the South and Southwest. She has been active in several
professional associations and has served as president of the
Florida Dietetic Association. After completion of her
doctorate, Gail will continue as a faculty member at the
University of Florida.

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
LynnJ B. Bailey, Chair
Professor of Food Science
and Human Nutrition
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Robert J. Cousins
Boston Family Professor of
Human Nutrition
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Jeése F. Gregory, ifll
Professor of Food Science
and Human Nutrition
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
s' VS 6
Rachel Shiremen
Professor of Food Science
and Human Nutrition

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Claudia Probart
Assistant Professor of
Health Science Education
This dissertation was submitted to the Graduate Faculty
of the College of Agriculture and to the Graduate School and
was accepted as partial fulfillment of ¿he requirements for
the degree of Doctor of Philosophy. (j w? //
'¿uk . \TA
August, 1993
De'ah, College of
Agriculture
Dean, Graduate School

UNIVERSITY OF FLORIDA
3 1262 08556 9415




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