Citation
Vitamin B12 status and absorption using holo-transcobalamin in young men and women

Material Information

Title:
Vitamin B12 status and absorption using holo-transcobalamin in young men and women
Creator:
Von Castel Roberts, Kristina ( Dissertant )
Bailey, Lynn B. ( Thesis advisor )
Kauwell, Gail P. A. ( Reviewer )
Gregory, Jesse F. ( Reviewer )
McDowell, Lee ( Reviewer )
Place of Publication:
Gainesville, Fla.
Publisher:
University of Florida
Publication Date:
Copyright Date:
2008
Language:
English

Subjects

Subjects / Keywords:
Biological markers ( jstor )
Blood ( jstor )
Dosage ( jstor )
Food ( jstor )
Genotypes ( jstor )
Money market accounts ( jstor )
Plasmas ( jstor )
Transcobalamins ( jstor )
Vegetarianism ( jstor )
Vitamins ( jstor )
Food Science and Human Nutrition thesis, Ph. D.
Dissertations, Academic -- UF -- Food Science and Human Nutrition

Notes

Abstract:
Vitamin B12 (B12) status of young adults has been considered adequate based on estimated intakes that met the RDA; however, few studies in the US have evaluated B12 status of young adults using a panel of B12 biomarkers. Vitamin B12 deficiency impairs neurological function and increases other health-related risks. Early detection and determination of whether B12 deficiency is due to dietary insufficiency, a genetic abnormality, or malabsorption are critical to effective treatment. The aims of the first study were to compare B12 status using numerous biomarkers in young adult non-supplement users consuming vegetarian and omnivorous diets, determine the level of intake associated with optimal B12 status, and determine if the transcobalamin (TC) 776C → G polymorphism affected B12 metabolism. Blood samples were collected for determination of holo-TC, B12, methylmalonic acid (MMA), and homocysteine (Hcy) (n = 388). Dietary B12 intake was assessed using a food frequency questionnaire. A surprisingly high incidence of B12 deficiency was observed in both vegetarians and omnivores. Relative to omnivores, vegetarians had a higher rate of B12 deficiency, with lower B12 and higher MMA concentrations. Vitamin B12 status improved with B12 intake above the RDA. No differences were detected between TC 776C → G genotypes for any biomarkers. In the second study the magnitude and patterns of post-absorption changes in several B12 biomarkers were assessed. Subjects (n = 21) had blood drawn at 17 intervals over three days with administration of three 9 microg doses of B12 at 6 hour intervals on day one. Mean B12, holo-TC, TC saturation, and the ratio of holo-TC to B12 increased significantly from baseline at hour 24 only. In conclusion, a high incidence of impaired B12 status was observed in otherwise healthy young adults. The data suggest that further assessment of the adequacy of the B12 RDA is warranted. Measurement of multiple B12 biomarkers may provide a more accurate assessment of B12 status than measurement of one biomarker alone. Holo-transcobalamin appears to be a sensitive indicator of B12 absorption and a holo-TC based absorption test should involve measurement at 0 and 24 hours. No effect of the TC 776C → G polymorphism was detected.
Subject:
absorption, B12, deficiency, holotranscobalamin, polymorphism, transcobalamin, vegetarian
General Note:
Title from title page of source document.
General Note:
Document formatted into pages; contains 103 pages.
General Note:
Includes vita.
Thesis:
Thesis (Ph. D.)--University of Florida, 2006.
Bibliography:
Includes bibliographical references.
General Note:
Text (Electronic thesis) in PDF format.

Record Information

Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
Copyright Von Castel Roberts, Kristina. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Embargo Date:
3/1/2007
Resource Identifier:
658230414 ( OCLC )

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Full Text





VITAMIN Bl2 STATUS AND ABSORPTION USING HOLO-TRANSCOBALAMIN IN
YOUNG MEN AND WOMEN





















By

KRISTINA VON CASTEL-ROBERTS


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

2006

































Copyright 2006

by

Kristina von Castel-Roberts


































To my father Gerard David von Castel-Dunwoody and my uncle Gimnter von Castel; they left
this world too early but will live in my heart forever.









ACKNOWLEDGMENTS

I would like to thank my committee members, doctors Lynn B. Bailey, Gail P.A. Kauwell,

Jesse F. Gregory III, and Lee McDowell for their guidance and support. I would particularly like

to thank Dr. Bailey and Dr. Kauwell for their daily encouragement during this exciting endeavor.

Their dedication and achievement in the field of science has given me high standards to follow

and has driven me to push myself to the best of my potential and beyond. I would like to thank

Dr. Gregory for the contribution of his scientific and technical knowledge and Dr. McDowell for

making me aware of how my animal nutrition education can help me better understand human

nutrition. I would like to express my gratitude to the members of our laboratory team, especially

David Maneval, Amanda Brown, Claire Edgemon, and Dr. Karla Shelnutt. It was their

combined effort that made it possible to successfully conduct two human studies, teaching me

the value of teamwork.

I owe tremendous thanks to my friends and family who supported me through the tough

spots, whether a few miles or a few hundred miles away. Their help and support allowed me to

keep my farm standing and my horses healthy, plan my wedding, get married, escape on my

honeymoon, and still have fun while pursuing my degree. I would particularly like to thank

those who have been with me from the start of my Gator career, Karen Knight, Christina Stortz

and Tiffany Tooley, and my friend turned-sister Revati Roberts. Special thanks go to my

mother and father Alice von Castel Dunwoody and Gerard von Castel Dunwoody, for their

unending and unconditional love and support, and for believing I could do anything I truly set

my mind to. Finally, my deepest love and appreciation go to my husband and best friend Nando

David Roberts for the continuous support, love, and devotion he has given me.












TABLE OF CONTENTS


page

ACKNOWLEDGMENTS .............. ...............4.....


LIST OF TABLES ................ ...............8............ ....


LIST OF FIGURES .............. ...............9.....


LI ST OF AB BREVIAT IONS ................. ............... 10......... ...


AB S TRAC T ............._. .......... ..............._ 12...


CHAPTER


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


Vitamin Bl2 2................ ...............14................
H history ................. ...............14.......... .....
Chem istry .............. ... ......... ......... ........1
Nomenclature for B l2 Binding Proteins ................. ......... ......... ..........1
Absorption ................ ............... ...............16.......
Transport and Cellular Uptake .............. ...............16....
Storage and Turnover ................. ...............17................
Biochemical Reactions ................. ...............18.......... .....

Daily Requirement ................. ...............18.................
Dietary and Supplemental Sources............... ...............19
Vitamin Bl2 Status Assessment ................. ...............21........... ...
Vitamin Bl 2 Concentration............... .............2
Holo-transcobalamin Concentration............... .............2

Methylmalonic Acid Concentration .............. ...............22....
Homocysteine Concentration .............. ...............23....
Vitamin Bl 2 Deficiency ..........._..._ ...............24......._ ....
Etiology .................. ...............24..
Clinical Abnormalities............... .............2

Vegetari ani sm ........._..._.._ ...._._. ...............25....
Gene-Nutrient Interactions .............. ...............27....
Malabsorption of Vitamin Bl2 ............ ......._.._ ...............28...
Overall Rationale ..........._...__........ ...............29.....

Hypothesis # 1 .............. ...............30....
Hypothesis #2 .........._..._._ ...............3_ 1....._.__...
Hypothesis #3 .............. ...............3 1....
Hypothesis #4 ..........._.._._ ...............3_ 1....._.__...












2 VITAMIN Bl2 STATUS IS IMPAIRED IN A SUBGROUP OF HEALTHY YOUNG
VEGETARIAN AND OMNIVOROUS ADULT MEN AND WOMEN .............. ..... ..........35


Subj ects and Methods .................. ....._ ...............35. ....
Subj ects and Subj ect Recruitment ........._....._ ...._.._......_. ...... .....3
Study Design and Data Collection .............. ...............36....
Sample Processing ........._...... .. .... _.._.. ....... .._._... .... ..............3
Competitive Binding Assays of Serum Holo-transcobalamin and Plasma Bl2..............37
Measurement of Serum Homocysteine and Methylmalonic Acid. ................ ...............38
D iet A analysis .................. ... .... .............3
Subj ect Dietary Intake Classification ......_. ................ ........._.._ ....... 3
Statistical M ethods. ............. ...............39.....
Re sults................ ...............40........ ......
Discussion ................. ...............41........ ......


3 VITAMIN Bl2 INTAKE AT THE CURRENT RDA LEVEL IS NOT OPTIMAL .............47


Subj ects and M ethods ............__..... ...._ ...............47...
Subj ects and Subj ect Recruitment ........._....._ ...._.._......_. ...... .....4
Study Design and Data Collection .............. ...............47....
Sample Processing and Analysis ........._....._ ...._.._......_._ ...........4
Diet Analysis .............. ...............48....
Statistical Analysis .............. ...............49....
Re sults........._..... ...._... ...............49.....
Discussion ........._..... ...._... ...............51.....


4 GENOTYPE FOR THE TRANSCOBALAMIN 776C+ G POLYMORPHISM IS NOT
ASSOCIATED WITH ABNORMAL VITAMIN Bl2 STATUS BIOMARKERS IN
HEALTHY ADULTS................ ...............57


Subj ects and M ethods .................. ........... ...............57. ....
Subj ects and Subj ect Recruitment ............... ...............57........._....
Study Design and Data Collection .............. ...............58....
Sample Processing and Analysis ........._....._ ...._.._......_._ ...........5
Genotype Determination .............. ...............59....
Diet Analysis .............. ...............59....
Statistical M ethods .............. ...............60....
Re sults ................ ...............60.................
Discussion ................. ...............61.................


5 HOLO-TRANSCOBALAMIN IS AN INDICATOR OF VITAMIN Bl2
ABSORPTION IN HEALTHY ADULTS WITH NORMAL VITAMIN Bl2 STATUS .....66


Subj ects and M ethods ................. ...............66................
Subj ects ............... .... .................. ...............66.......
Study Design and Data Collection .............. ...............67....
Biochemical Analysis............... ...............67













Sample Processing and Analysis ................. ...............68................
Statistical Methods .............. ...............69....
Re sults ................ ...............69.................
Discussion ................. ...............70.................


6 DI SCUS SSION ................. ...............79................


APPENDIX


A SUBJECT PHONE SCREENING FORM .............. ...............85....


B INTERVENTION DIET ................. ...............90................


LIST OF REFERENCES ................. ...............91........... ....


BIOGRAPHICAL SKETCH ................. ...............103......... ......











LIST OF TABLES


Table page

2-1 Characteristics of study groups ..........._...... ._ ...............44...

2-2 Mean dietary vitamin Bl2 intake and status of omnivorous and vegetarian adults. .........45

2-3 Cross-tabulation of vitamin Bl12 status of subj ects based on select biomarker
combinations ........... __..... ._ ...............46....

3-1 Subj ect Characteristics ........... __..... ._ ...............53..

3-2 Correlations between vitamin B l2 intake and concentrations of B l2 status
biomarkers .........._. ..... ._ __ ...............53......

3-3 Proportion of individuals with concentrations outside the normal range for select
vitamin Bl 2 status biomarkers............... ...............5

4-1 Demographic distribution of subj ects by genotype ................. .............................64

4-2 Mean concentrations of selected vitamin Bl12 biomarkers in all subj ects............._.._.. ......64

4-3 Mean concentrations of selected Bl2 biomarkers in subj ects with vitamin Bl2
deficiency ........._._. ._......_.. ...............64.....

4-4 Percentage of individuals within each TC 776 C+G genotype group with
concentrations outside the normal range for select B l2 biomarkers .............. .................65

5-1 Baseline concentrations of Bl2 status indicators............... ...............7

5-2 Mean concentrations of vitamin Bl2 status indicators at scheduled intervals ........._........75










LIST OF FIGURES


Figure page

1-1 Structure of vitamin Bl2 ................. ...............32...............

1-2 Overview of vitamin Bl12 ab sorption ................. ...............33........... .

1-3 Role of vitamin Bl12 in the remethylation of homocysteine. ........._._.. .. ......._... ........34

2-1 Percent of vegetarian and omnivorous adults with select Bl2 biomarker
concentrations outside the normal range............... ...............45.

2-2 Frequency of single versus combined vitamin Bl2 status biomarkers being outside
the normal range .............. ...............46....

3-1 Total daily vitamin Bl2 intake in individuals with select Bl2 biomarker
concentrations outside the normal range............... ...............54.

3-2 Relationship between vitamin B l2 intake and status. ............. ...............55.....

4-1 Melting curve plots for Dynamic Allele Specific Hybridization analysis of
polymorphism the TC 776C+G polymorphism.. ............ ...............63.....

5-1 Intervention protocol timeline............... ...............73

5-2 Change in vitamin Bl2 biomarkers during the 48 hour study period. .........._.... ..............76

5-3 Mean percent change in holo-transcobalamin and vitamin B l2 concentrations at
scheduled intervals after oral B l2 intake ................. ...............77........... ..

5-4 Mean percent change in transcobalamin saturation and holo-transcobalamin to
vitamin Bl2 ratio at scheduled intervals after oral Bl2 intake............... .................7









LIST OF ABBREVIATIONS

5 -methylterahy drofol ate

Adequate Intake

Analysis of variance

Free haptocorrin

Free transcobalamin

Vitamin B l2

Body mass index

Methyl

Cyano

day

National Cancer Institute Dietary History Questionnaire

Estimated Adequate Requirement

Ethylenediaminetetraacetic acid

Food frequency questionnaire

Haptocorrin

Holo-haptocorrin

Holo-transcobalamin

Hydrochloric acid

Homocysteine

Intrinsic factor

Intramuscular

Month

Minutes


5-CH3-THF

AI

ANOVA

Apo-HC

Apo-TC

Bl2

BMI

CH3

CN

d

DHQ

EAR

EDTA

FFQ

HC

Holo-HC

Holo-TC

HCI

Hcy

IF

IM

mo

min










MS Methionine synthase

nmol/L Nanomoles per liter

NTD Neural tube defect

OH Hydroxyl

OSC Optimal Solutions Corporation

pmol/L Picomoles per liter

RDA Recommended dietary allowance

TC Transcobalamin

TC-R Transcobalamin receptor

s Seconds

SAM S-adenosylmethionine

SD Standard deviation

SST Serum separator tube

UL Upper limit

US United States

Clmol/L Micromoles per liter

y year









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

VITAMIN Bl2 STATUS AND ABSORPTION USING HOLO-TRANSCOBALAMIN IN
ADULT MEN AND WOMEN

By

Kristina von Castel-Roberts

December 2006

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

Vitamin Bl12 (Bl12) status of young adults has been considered adequate based on

estimated intakes that met the RDA; however, few studies in the US have evaluated B l2 status

of young adults using a panel of B l2 biomarkers. Vitamin B l2 deficiency impairs neurological

function and increases other health-related risks. Early detection and determination of whether

Bl2 deficiency is due to dietary insufficiency, a genetic abnormality, or malabsorption are

critical to effective treatment.

The aims of the first study were to compare B l2 status using numerous biomarkers in

young adult non-supplement users consuming vegetarian and omnivorous diets, determine the

level of intake associated with optimal Bl2 status, and determine if the transcobalamin (TC)

776C & G polymorphism affected Bl2 metabolism. Blood samples were collected for

determination of holo-TC, Bl2, methylmalonic acid (MMA), and homocysteine (Hcy) (n = 388).

Dietary B l2 intake was assessed using a food frequency questionnaire. A surprisingly high

incidence of Bl2 deficiency was observed in both vegetarians and omnivores. Relative to

omnivores, vegetarians had a higher rate of Bl2 deficiency, with lower Bl2 and higher MMA

concentrations. Vitamin Bl2 status improved with Bl2 intake above the RDA. No differences

were detected between TC 776C G genotypes for any biomarkers.









In the second study the magnitude and patterns of post-absorption changes in several Bl2

biomarkers were assessed. Subjects (n = 21) had blood drawn at 17 intervals over three days

with administration of three 9 Clg doses of Bl2 at 6 hour intervals on day one. Mean Bl2, holo-

TC, TC saturation, and the ratio of holo-TC to Bl2 increased significantly from baseline at hour

24 only.

In conclusion, a high incidence of impaired Bl2 status was observed in otherwise healthy

young adults. The data suggest that further assessment of the adequacy of the Bl2 RDA is

warranted. Measurement of multiple Bl2 biomarkers may provide a more accurate assessment

of B l2 status than measurement of one biomarker alone. Holo-transcobalamin appears to be a

sensitive indicator of Bl2 absorption and a holo-TC based absorption test should involve

measurement at 0 and 24 hours. No effect of the TC 776C+G polymorphism was detected.









CHAPTER 1
INTTRODUCTION

Vitamin B12

History

Vitamin Bl12 (Bl12) is one of the thirteen essential vitamins that humans must obtain from

their diet. Vitamin Bl2 was the last vitamin to be discovered, due in part to the lack of a suitable

animal model in which to study the Bl2-related disease pernicious anemia (1). Pernicious

anemia, which literally means fatal anemia, has been reported in medical records as far back as

the early 1800's, although the condition was likely responsible for deaths well before then. The

earliest studies of patients with pernicious anemia led to the knowledge that the disease was due

to some ailment of the stomach; although no treatment was available and most patients died from

the disease (2). In the early 20th century, Minot and Murphy determined that feeding liver to

patients with pernicious anemia improved their condition, a discovery for which they received

the Nobel prize in 1934 (3). Castle conducted a series of experiments comparing the treatment

of pernicious anemia patients with partially digested beef, or beef incubated in gastric juice,

versus undigested beef. Patients receiving the pre-digested beef improved while those receiving

undigested beef did not, suggesting that some (intrinsic) factor within gastric juice interacted

with the unidentified (extrinsic) factor in the beef (4-7). The Einal identification of this extrinsic

factor was delayed until 1945 when it was discovered that the anti-anemia substance was

required by Lactobacillus locus, finally providing a useful laboratory model (8). Vitamin B l2

was crystallized in 1948 and was quickly identified as the illusive "extrinsic" anti-anemia factor

(9-12). After these discoveries, Bl2 research proceeded rapidly as did our understanding of the

vitamin's functions.









Chemistry

Vitamin Bl2 is a complex water-soluble molecule with a molecular weight of 1655.38

daltons. The molecule is comprised of a cobalt atom centered in a corrin ring, with two

coordinating ligands. The 5,6-dimethylbenzimidazole component is linked to the a-axial

position of the cobalt, and a variable ligand is linked to the p-axial position (Figure 1-1) (13).

Known ligands include CH3 (methylcobalamin), 5'-deoxyadenosyl (adenosylcobalamin), OH

(hydroxylcobalamin), and CN cyanocobalaminn). Cyanocobalamin, the synthetic form of Bl2

included in vitamin supplements and fortified foods, is converted to methylcobalamin or

adenosylcobalamin, the two coenzyme forms of the vitamin. Methylcobalamin is the primary

form found in human plasma making up 60 to 80% of total cobalamins (14).


Nomenclature for B12 Binding Proteins

The nomenclature for Bl2 binding proteins in the gastrointestinal tract and plasma has

changed over time, and both the new and old terms are used in current literature. The term R-

protein was originally used to differentiate Bl2 binding proteins, which move rapidly upon

electrophoresis, from intrinsic factor (IF), which moves slowly. Intestinal R-protein, now termed

haptocorrin (HC) because of its ability to bind corrins other than Bl2, is also found in saliva,

bile, and plasma. Transcobalamin (TC) I, II, and III were terms used to identify the plasma Bl2

binding proteins; however, further investigation proved that TC I and III were isoforms of HC,

which differed only by carbohydrate content. The term transcobalamin II was used to identify

the Bl2 binder that participated in delivery of Bl2 to cells, but now it is simply referred to as TC

(15).









Absorption

The absorption of Bl2 is primarily an active receptor mediated process that uses several

different transporters (Figure 1-2). Vitamin Bl2 is bound to proteins in foods and must be

liberated by the action of pepsin and hydrochloric acid (HCL) in the stomach in order for

absorption to occur. Reduced gastric pH, as often seen in adults over the age of 50 y and with

chronic antacid use, impairs breakdown of the protein matrix and ultimately results in reduced

Bl2 absorption (16, 17). Once Bl2 is released from the protein matrix, it binds to HC enabling

it to travel to the duodenum where pancreatic proteases degrade HC. In the duodenum, free Bl2

binds to IF, a glycoprotein synthesized and secreted from gastric parietal cells. The IF-Bl2

complex is resistant to attack from pepsin, chymotrypsin, and intestinal bacteria, allowing the

complex to travel to the ileum intact, where it is transferred across the ileal epithelium via-

receptor mediated endocytosis. This ileal receptor (cubilin) only recognizes the IF-Bl12 complex,

so that free Bl2 can not cross the membrane in this manner (18, 19). Although passive diffusion

of Bl2 across the epithelium does occur at a rate of 1% of any Bl2 dose, Bl2 is primarily

absorbed by active transport (14). Once in the enterocyte, IF is degraded by the lysosome.

Transcobalamin plays an essential role in Bl2 absorption, binding Bl2 at some point after

release from IF and appearance in the blood as holo-TC. The exact mechanism by which B l2

binds to TC is under debate, however, it is hypothesized that binding occurs in the enterocyte

(20-22). Holo-transcobalamin can be detected in the blood 3 hours after B l2 intake, with

maximum absorption occurring 8 to 12 hours after intake. Cellular uptake occurs within minutes

(23, 24).

Transport and Cellular Uptake

Transport of Bl2 in circulatory system and into the cells of target tissues is dependent on

two binding proteins, TC and HC. Each protein has only one binding site with a high affinity









(Kd = 10-10 to 10-17) for the various chemical forms of Bl2 (25). Transcobalamin is a 43 kDa

non-glycosylated protein found in plasma and in various cells including endothelial cells (22).

Numerous tissues contain TC mRNA, including kidney, heart, liver, and leukocytes; however the

specific cell type in which TC is synthesized is unknown (26). Transcobalamin is required for

transport of Bl2 into the cell since only the Bl2 bound to TC is taken up by cell surface

receptors. For this reason, the holo-TC fraction of serum Bl2 is the only component that is

considered biologically active (27-29). The TC receptor (TC-R) is a 50 kDa heavily

glycosylated protein, that binds both holo-TC and apo-TC (TC with no B l2 bound) (26). In the

cytoplasm, lysosomes break down the holo-TC complex making free Bl2 available for metabolic

processes. Holo-transcobalamin constitutes only 20% of plasma Bl2, the remaining 80% is

bound to HC (13, 30). Haptocorrin is a heavily-glycosylated, 70 kDa protein found in various

biological fluids including saliva, bile, and blood. Although the maj ority of plasma Bl12 is bound

to HC (holo-HC), holo-HC cannot be used by the cells as there are no receptors for this complex

(15). The function of HC is unclear, and is still debated (15, 31, 32).

Storage and Turnover

The main storage tissues for B l2 are the liver and muscle, which contain approximately

60% and 30%, respectively, of the body's total Bl2. High concentrations also are found in the

pituitary, kidney, heart, spleen and brain. Interestingly, although B l2 is enzyme bound in most

tissues, the kidney maintains a pool of free Bl2, which can be used to maintain plasma Bl2.

When Bl2 intake is high, uptake of Bl2 into the kidney increases; when plasma Bl2

concentrations are low, Bl2 is released first from the kidney (33, 34). Mean total body stores

range from 2 to 5 mg with a half life of 340 to 400 days (14).

Vitamin B l2 is excreted only in the free form in the urine and bile at a rate of 0. 1 to 0.2%

(2 to 5 Gig) of total body reserves per day (35). Enterohepatic recirculation is very efficient and










helps reduce total loss of Bl12 (13). Up to 75% of biliary Bl12 is actively reabsorbed in the ileum,

so that very little is excreted in the feces, effectively conserving this essential nutrient (14).

Additionally, because the kidney is rich in TC-R, Bl2 stored in the kidney is efficiently

reabsorbed back into circulation reducing urinary losses.

Biochemical Reactions

In humans and other higher animals, Bl2 serves as a coenzyme for two metabolic

processes, the conversion of methylmalonyl-CoA to succinyl-CoA as adenosylcobalamin and the

remethylation of homocysteine (Hcy) to methionine as methylcobalamin (13, 35). In succinyl-

CoA synthesis, adenosylcobalamin undergoes homolytic cleavage by the action of L-

methylmalonyl CoA mutase forming cob(II)alamin and a 5'-deoxyadenosyl radical. Radical

formation allows the rearrangement of the L-methylmalonyl-CoA molecule to form succinyl-

CoA.

The Hcy remethylation process is an important component of overall one carbon

metabolism. Methionine synthase (MS) catalyzes the remethylation of Hcy to methionine with

the associated Bl2 acting as a methyl carrier. Methionine synthase contains separate binding

domains for Hcy, 5 -methyltetrahydrofol ate (5 -CH3 THF), Bl12, and S -adenosylmethi onine

(SAM). Vitamin B l2 in its reduced active state as cob(I)alamin is remethylated by 5-CH3THF

and the methyl group can again be donated to Hcy (Figure 1-3). Methionine is of great

biological importance because it is the precursor of SAM the maj or methyl donor in over 100

biochemical reactions (36, 37).

Daily Requirement

The Dietary Reference Intakes (DRI) for essential nutrients are guidelines for estimating

the average vitamin and mineral intake needed to maintain health (38). The DRIs include

Estimated Average Requirement (EAR), Adequate Intake (AI), Recommended Dietary









Allowance (RDA) and Tolerable Upper Intake Level (UL). The RDA for nutrients is calculated

from the EAR and is defined as the average daily intake required to meet the needs of most

individuals in the defined age bracket (38). The daily requirement ofBl2 is relatively low in

comparison to other essential vitamins, with an EAR and RDA for adult men and women (19 to

50 y) of 2 Clg/d and 2.4 Clg/d, respectively. Estimates for the EAR for adults are based on the

level of intake needed to maintain normal hematological status and a serum Bl2 concentration

above 150 pmol/L minus the amount conserved in daily Bl2 turnover. Data were gathered

primarily from patients with pernicious anemia in remission who were receiving regular

intramuscular (IM) inj sections of B12. Studies of patients with pernicious anemia reported that

IM doses of 0.8 to 1.7 Clg/d were sufficient to maintain normal hematological parameters. From

these studies an average of 1.5 Clg/d was estimated to be the B l2 requirement. The final

calculation of the EAR in healthy adults with normal Bl2 absorption was calculated as 1.5

Gig/day minus 0.5 Clg/d (the estimated amount of Bl2 reabsorbed) with a correction to account

for an estimated bioavailability of 50% (3 8). The RDA was then calculated as the EAR (2.0

Clg/d) plus twice the coefficient of variation (CV) for 97 to 98% of the population, or 120% of

the EAR. Recommendations for children are based on Bl2 concentrations in milk for infants

and are extrapolated down from adult requirements for children up to age 18 y. Adults over the

age of 50 y have the same RDA as younger adults; however, due to an age-related reduction in

gastric pH, it is recommended that older adults obtain most of the RDA from fortified foods and

Bl2 supplements (23).

Dietary and Supplemental Sources

Vitamin B l2 is synthesized only by microorganisms in bacteria rich environments such as

the intestinal tracts of animals. Some species have sufficient microbial synthesis of Bl2 to meet









their biological need without any additional dietary Bl2. Although humans have Bl2

synthesizing bacteria in their intestinal tract, they are primarily found in the colon where little

absorption takes place making Bl2 an essential nutrient for humans. Natural dietary sources of

Bl2 are limited to foods of animal origin. The liver and kidney store large amounts of Bl2,

therefore, these organ meats are the richest dietary sources of Bl2 (24 to 122 Gig/100 g). Other

more commonly consumed food sources are red meat (0.55 to 3.64 Gig/100 g), poultry (0.32 to

0.379 Gig/100 g), fish (1.9 to 21.2 Gig/100 g), eggs (0.09 to 9.26 Gig/100 g), and milk products

(0.06 to 1.71 Gig/100 g) (14). Vitamin Bl2 also can be obtained in the following: (a)

supplements; (b) Bl2-fortified foods such as cereals and meal replacement bars and drinks; (c)

Bl2- fortified vegetarian products such as soy milk; and (d) Bl2-fortified meat substitutes and

frozen meal entrees made from wheat gluten or soybeans; and some fermented food products

(3 9).

In the US, foods of animal origin are a common part of the diet, and the estimated average

daily intake of Bl2 from food sources is 3 to 5 Clg/d on average (38). The largest percentage of

dietary B l2 in the US diet comes from mixed foods (16 to 19%) which includes non-beef meats,

poultry, and fish; a substantial portion also comes from beef (12 to 15%), and milk products (11

to 15%). In the case of vegetarians, who are estimated to represent up to 25% of US women of

reproductive age, dairy and egg products for lacoto-ovo-vegetarians and dairy products for lacto-

vegetarians, are the sole sources of Bl2 if supplements or fortified foods are not consumed.

Individuals who exclude some or all animal-derived foods and do not add Bl2 fortified

foods to their diet are at increased risk for developing Bl2 deficiency (40-42). Non-meat

animal-derived sources ofBl2 including dairy products and eggs can contribute significantly to

Bl2 intake (3 8), but are excluded from the diets of strict vegetarians (vegans). The extent to









which animal-derived foods are excluded from the diets of self-described vegetarians may

determine the effect on Bl2 status. For example, one serving (4 oz) of beef can provide the

RDA for Bl2 (2.4 Clg/d), while one serving of chicken (4 oz) provides only 12% of the RDA.

The Bl12 content of Eish varies by species; 1 serving (4 oz) of grouper provides 25% of the RDA,

while tuna and herring provide as much as 500% of the RDA per serving (1 to 4 oz). Dairy

products also provide variable amounts of Bl2 with 8 to 50% the RDA per serving (2 to 8 oz)

(3 9).


Vitamin B12 Status Assessment

Vitamin B12 Concentration

In clinical settings, serum Bl2 determination is the primary method for assessing Bl2

status (35). In the general US population, the mean serum Bl2 concentration for healthy

individuals over four years of age is 381 pmol/L (43). Reliance on serum Bl2 as the sole

diagnostic tool may lead to a false diagnosis since not all individuals with low values are

defieient and a "normal" serum concentration may or may not indicate adequate Bl2 status (44).

This is due in part to the manner in which B l2 is metabolized and stored in the body. Because

some tissues, such as the liver and kidney, can store a relatively large amount of Bl2, total body

depletion takes years; however, some cells with lower storage capacity may become Bl2

defieient while circulating Bl2 is still in the low normal range. In these cells, Bl2 dependant

enzyme function may become impaired causing elevated MMA and Hcy. Currently, clinical

Bl2 deficiency is classified as serum concentrations < 148 pg/mL; however, a significant

percentage of patients with clinical symptoms of Bl2 deficiency who respond to Bl2 therapy

have serum B l2 concentrations in the "low-normal" (148 to 221 pg/ml) range. To enhance the

diagnostic value of serum B l2 concentrations, additional status indicators should be evaluated.









Holo-transcobalamin Concentration

Measurement of holo-TC is considered a functional indicator of Bl2 status because only

the Bl2 bound to TC can be taken up by cell receptors for use in intracellular metabolic reactions

(27, 28, 45, 46). In contrast, serum Bl2 consists of holo-TC (~20%) and holo-HC (~80%), the

latter can not be used by cells since it lacks known cellular receptors. Evidence supports the

conclusion that holo-TC concentration responds more rapidly to changes in Bl2 intake than

other indices of Bl2 status (28). Bor et al. (47) reported that oral Bl2 treatment (400 Clg/d)

resulted in a highly significant maximal increase (+54%) in holo-TC after 3 days, in contrast to

serum B l2, which responded with a smaller initial change (+28%) and a slower graded increase

over time. Routine measurement of holo-TC as an index of B l2 status is now possible since

technical problems associated with the analytical procedure have been successfully addressed

(Holo-TC RIA, Axis-Sheild) (45, 48). Loikas et al. (49) confirmed the suitability of the holo-TC

RIA for use in a clinical laboratory, determined reference values for the method (37 to 171

pmol/L), and confirmed that low holo-TC concentrations (< 35 pmol/L) were associated with

other biochemical indicators of low Bl2 status.

Methylmalonic Acid Concentration

When Bl2 status is low the conversion of methylmalonyl-CoA to succinyl CoA is

impaired; as methylmalonyl-CoA accumulates, it is converted to methylmalonic acid. This

alteration in metabolism results in a measurable increase in MMA. Methylmalonic acid

concentration is a highly specific diagnostic indicator ofBl2 status because, unlike the MS

reaction that requires both Bl2 and folate, no other nutrient is required for the methylmalonyl

CoA mutase reaction (50-52). A normal serum MMA concentration is 5 271 nmol/L, with

reported reference ranges for serum MMA concentration of ~50 to 400 nmol/L (53, 54). Serum

MMA concentration also provides diagnostic information when it is obtained before and after









Bl2 supplementation in Bl2 defieient individuals. Moelby et al. (52), like previous

investigators, reported a marked decline in serum MMA concentration to normal one month after

treatment with Bl2 (55).

Homocysteine Concentration

Homocysteine concentration is inversely associated with Bl2 status, and may or may not

be elevated in individuals with low Bl2 status. Individuals that have elevated Hcy due to a Bl2

deficiency will respond to B l2 supplementation (55, 56). Traditionally the cut-off for normal

Hcy concentration has been < 14 Clmol/L; however, due to the implementation of mandatory

folate fortification in the US, Hcy concentrations within the population have decreased

significantly (57, 58). In a population-based study, Selhub et al. (56) reported that plasma Hcy

concentration was inversely associated with Bl2 status, and mean Hcy concentration was

significantly higher in individuals in the lowest compared to the highest decile for plasma B l2

concentration (15.4 and 10.9 Clmol/L, respectively). Mezzano et al. (59) evaluated plasma Hcy

concentrations and response to Bl2 therapy in a group of vegetarians with low Bl12 status

(baseline mean serum B l2 concentration 110 pmol/L) with elevated plasma Hcy concentration.

Following intramuscular inj section with Bl12, serum Bl12 concentration increased to 392 pmoL/L

and mean plasma Hcy concentration dropped significantly (12.4 to 7.9 Clmol/L). Unlike MMA,

Hcy concentration is not a specific indicator of Bl2 status. Because the folate derivative, 5-CH3-

THF is the methyl donor in the conversion of Hcy to methionine, low folate status also can lead

to an elevation in Hcy concentration.









Vitamin B12 Deficiency


Etiology

Vitamin Bl2 deficiency may occur due to dietary restriction, malabsorption, or

disturbances in transport or cellular uptake. Malabsorption of Bl2 can be caused by several

physiological and congenital defects. Pernicious anemia is a disease of the autoimmune system

in which antibodies to parietal cells and IF develop leading to a complete lack of IF and an

inability to absorb Bl2. Individuals with this condition can be given IM Bl2 inj sections to meet

Bl2 requirements, bypassing the absorption process (35). The elderly population is at a higher

risk of Bl2 malabsorption due to the age-related decrease in stomach acid. The acidic

environment in the stomach is required for the release of B 2 from food, and a significant

decrease in hydrochloric acid can impair the process leading to increased excretion and

decreased absorption. In such cases the daily Bl2 requirement must be met with supplemental

Bl2 (35).

Clinical Abnormalities

Depleted Bl2 status may take years to develop in individuals with impaired absorption or

inadequate intake and an individual may have marginal B l2 status prior to developing severe

clinical symptoms such as megaloblastic anemia and irreversible neurological damage (32).

Development of Bl2 deficiency begins with depletion of serum Bl2, followed by cellular

deficiency and biochemical changes including elevated Hcy and MMA concentrations (32).

Neurological abnormalities affecting physical reflexes, stamina, and mental attributes including

memory and behavioral changes may accompany a moderate Bl2 deficiency (60-62). In

addition, the risk of inadequate B l2 intake to a developing fetus, should pregnancy occur, is of

great concern for women of reproductive age. Infants born to mothers with a Bl2 deficiency

have been reported to suffer devastating symptoms including growth retardation, delayed









psychomotor development, and in some instances, permanent effects on the brain (63-66). A

Bl2 deficiency may increase the risk for birth defects as illustrated by the well documented

independent role for Bl2 in the etiology of neural tube defects (NTDs) (67-73). These studies

have provided evidence that even small reductions in serum B l2 concentrations within the

normal range may be associated with a significantly increased risk for NTDs. Afman et al. (74)

measured the plasma concentrations of Bl2, Hcy, and the apo- and holo- forms of TC in NTD

case mothers and in control women. Low plasma holo-TC concentration was associated with a

3-fold increased risk for having a child with an NTD, while a low percentage of Bl2 bound to

TC (TC saturation) was associated with a 5-fold increased risk.

Vegetarianism

Vegetarians are at increased risk for developing a Bl2 deficiency since Bl2 is only

naturally present in animal-derived products (i.e., meat, eggs, dairy). There is much variability

in the amount of B 2 consumed by individuals characterized as "vegetarians". This

classification includes those who consume diets completely devoid of all animal-derived

products (vegans), including meat, Eish, dairy, and eggs, as well as those who exclude meat but

consume either dairy products (lacto-vegetarians) or dairy and eggs (lacto-ovo-vegetarians) (75).

Approximately 2.5% of the entire US adult population (4.8 million people) report consumption

of vegetarian diets, and approximately 1% report consuming vegan diets (75-77). There is an

increasing trend for the younger segment of the population to consume vegetarian diets (75). In

a nationally-representative survey (75), the number of self-defined vegetarians who reported no

meat consumption was highest in the 20 to 29 year age group and was two to three times higher

than that of 50 to 59 and 60 to 69 year old individuals, respectively. This increasing trend in

consumption of vegetarian diets is especially prevalent among young adult women of










reproductive age, documented by survey data indicating that 20 to 25% of this group follow

some type of vegetarian diet (78).

Multiple reports provide evidence that all vegetarians including lacto-vegetarians and

lacto-ovo-vegetarians are at increased risk for developing a Bl2 deficiency compared to

omnivores (79-81), supporting the conclusion that a vegetarian does not have to be a strict vegan

for Bl2 status to be impaired (32, 81-85). Impaired Bl2 status may lead to elevated Hcy

concentrations and increased risk for cardiovascular disease, cancer and birth defect-affected

pregnancies. A number of studies indicate that vegetarians have significantly higher Hcy

concentrations than omnivores and that the consumption of a vegetarian diet may be associated

with elevated Hcy concentrations (59, 80, 86). For example, Mezzano et al. (59) reported that

the Hcy concentration was 41% higher in vegetarians than in omnivores and that Hcy was

inversely related to serum Bl2 concentration. In a study comparing Bl2 status of Taiwanese

vegetarians and non-vegetarians, Huang et al. (87) reported that vegetarians had higher plasma

Hcy concentrations than non-vegetarians (13.2 vs. 9.8 Clmol/L, respectively) and that serum Bl2

concentration was a strong predictor of plasma Hcy concentration. Another similar study

conducted in a European population reported that Hcy concentration was significantly higher

(1 1.6 Clmol/L) in a group of vegetarians compared to omnivorous controls (9.8 Clmol/L) and that

the Hcy concentration increased as the vegetarian diet became more restrictive, with vegans

having the highest values (86). In the only study reported to date in which the B l2 status of

young adult vegans in the US has been evaluated, Carmel et al. (88) found that elevated Hcy

concentration associated with dietary inadequacy of Bl2 was a maj or problem in young Asian

Indian medical students with hyperhomocysteinemia occurring in 25% of group. This study









illustrates that well-educated young adults are among the vegetarians in the US whose

consumption of a B l2-deficient diets has been associated with an elevation in Hcy concentration.

Gene-Nutrient Interactions


Transcobalamin 776C+G

The most common polymorphism affecting the TC Bl2 transport protein is a COG base

substitution in DNA at base pair 776, which results in the substitution of proline with arginine at

codon 259 (74, 89). The estimated prevalence of the TC 776 CC, CG and GG genotypes are

approximately 20%, 50%, and 30%, respectively (74, 89, 90). Our research group recently

estimated the distribution (27% CC; 49% CG; 24% GG) of the TC 776 polymorphism in a large

population group of young women for whom B l2 status was previously reported, which is in

agreement with previous studies (91).

The potential influence of the TC 776 C G polymorphism on indices of B l2 status has

been investigated by several research groups (74, 89, 90). Afman et al. (30) found lower holo-

TC, total TC, and holo-TC/total-TC ratios in individuals with either the TC 776 GG or CG

genotypes compared to those with the CC genotype. Miller et al. (89) reported a lower mean

holo-TC concentration, a lower percent of total Bl2 bound to TC, and a higher mean MMA

concentration in elderly subj ects with the TC 776 GG compared to the CC genotype. Our

research group evaluated Bl2 status in young women with all three TC 776 C+G genotypes

(CC, CG, GG) (91). Mean holo-TC concentration was significantly lower in TC 776 GG

compared to CC genotypes, and individuals with low (< 35 pmol/L) holo-TC had a significantly

higher mean Hcy concentration. Alternatively, some studies have reported no effect of the TC

776 C+ G polymorphism on Bl2 status or metabolism (92-94).









Any reduction in B l2 binding capacity, protein synthesis, or transport function caused by

the TC 776 C+G polymorphism could impair functional Bl2 status, leading to reduced cellular

availability of Bl2. If the polymorphism has a physiologically significant impact on Bl2

availability, it would likely be exacerbated by concurrently low Bl2 status due to insufficient

dietary B l2 intake. In a population where more individuals have marginal B l2 status, such as in

vegetarians, any negative effect of the TC 776 C+ G polymorphism would be expected to be

more apparent.

Malabsorption of Vitamin B12

The most common form of Bl2 malabsorption is often termed "food-bound

malabsorption"(95). Because only free Bl2 can bind to the transport proteins and be taken up

into the enterocyte or absorbed passively, any physiological condition that reduces the ability to

free Bl2 from the protein matrix will lead to malabsorption and can lead to a Bl2 deficiency.

Approximately 5 to 25% of adults over the age of 60 y are estimated to have some degree of

food-bound Bl2 malabsorption due to an age-related decrease in stomach acid, or achlorhydria

(96). Because achlorhydria is so prevalent in older adults it is recommended that the daily

requirement of Bl2 be met by consuming supplemental forms of Bl2 (97). Vitamin Bl2 found

in fortified foods and vitamin supplements is not protein bound and therefore, can be absorbed

with normal efficiency even if gastric pH is high.

Another less common, but often more severe form of Bl2 malabsorption, termed

pernicious anemia, can occur in all age groups, although incidence does increase with age.

Pernicious anemia is caused by a lack of IF resulting from an autoimmune response, atrophy of

the gastric mucosa, chronic gastritis, and in rare cases a congenital defect in the gene for IF.

Congenital defects may lead to synthesis of an altered, and therefore non-functional IF protein or

a complete lack of synthesis. Both conditions cause Bl2 deficiency at an early age and have









been reported to be caused by a variety of genetic mutations and post-translational defects. The

autoimmune-based Bl2 malabsorption condition is more prevalent in older adults but has been

observed in all age groups (98). In this case, the body recognizes either the IF itself or the

gastric parietal cells as foreign and synthesizes antibodies to the protein or cell eliciting an

immune response. Destruction of IF or the parietal cells by this autoimmune response may occur

to varying degrees resulting in variation in the severity of Bl2 malabsorption (98).

Currently the only available diagnostic tests for pernicious anemia are not clinically

practical. The Schilling test, which involves ingestion of radioactively-labeled Bl2, a flushing

dose of non-labeled Bl2, and collection of urine over a period of 24 hours requires meticulous

adherence to protocol making it error prone and costly (99-101). Presence of IF or parietal cell

antibodies can be measured to diagnose pernicious anemia; however, parietal cell antibodies can

occur in other autoimmune diseases, and both tests are only clinically meaningful in a subgroup

of patients with autoimmune conditions (102, 103). It has been hypothesized that changes in

holo-TC in response to a supplemental dose of Bl2 may be used to assess Bl2 absorption (28,

47, 104). Bor et al (20) reported a significant increase in holo-TC and TC saturation 24 and 48

hours after receiving three 9 Clg oral Bl2 doses. Since no blood was collected before 24 hours

(post baseline), the magnitude and pattern of change of holo-TC during the first 24 hours could

not be determined (47). In developing a clinical diagnostic test, it is important to know the

optimal time post dose at which to draw blood.

Overall Rationale

Vitamin Bl2 plays a central role in Hcy metabolism, and Bl2 deficiency has been

associated with numerous health risks, including birth defect-affected pregnancies. Few studies

have been designed to evaluate the relationship between dietary exclusion or limitation of










specific animal products and Bl2 status in individuals who do not take Bl2 supplements or

consume Bl2-fortified foods. The proposed study will evaluate the association between Bl2

status and intake of specific animal-derived food products among vegetarians who do not

consume Bl2-containing supplements.

Several studies, including one conducted by our research group (91), indicate that the TC

776 GG genotype results in decreased holo-TC concentrations, and could therefore be a risk

factor for a Bl2 deficiency. Although most studies have not found a correlation between TC 776

C+G genotype and Hcy or MMA, holo-TC and Hcy and MMA have been negatively correlated

(89, 105). It is hypothesized that Bl2 transport, and thus metabolic function, may be impaired in

individuals with the TC 776 GG genotype, and that an effect on Hcy and MMA concentrations

due to the TC 776 GG genotype may be evident in individuals with low Bl2 intake and status.

These data could be used for public health screening and intervention approaches for adults

whose combined dietary choices and genetic make-up may put them at higher risk for certain

diseases or poor pregnancy outcomes. Information generated from this study could benefit

individuals who exclude B l2-dense food sources from their diets for reasons related to health or

personal choice rather than religion, culture, or the environment, as well as for individuals who

consume strict vegan diets for religious/cultural or environmental reasons.



Hypothesis # 1

Moderate Bl2 deficiency will be more common in vegetarians not taking Bl2 supplements than

in their omnivorous counterparts.

Specific aim: To determine if non-supplement taking young adults who exclude animal-

based foods and are not taking Bl12-containing supplements are at a greater risk for a Bl2









deficiency than those who eat animal-based foods by comparing B l2 status indices between

groups.

Hypothesis #2

Vitamin B l2 intake at the current RDA may not be sufficient to maintain normal B l2 status.

Specific aim: To determine the level ofBl2 intake associated with optimal status as

defined by normal Bl2 status biomarkers.

Hypothesis #3

Genotype status for the TC 776C+G polymorphism will have a greater physiological impact on

individuals with low Bl2 status than those with normal Bl2 status.

Specific aim: To determine if genotype status for the TC 776C+G polymorphism further

impairs Bl2 status in individuals with low Bl2 intake by comparing Bl2 status indices among

genotype groups in individuals with low and normal B l2 status.

Hypothesis #4

Holo-transcobalamin concentration can be used to assess Bl2 absorption.

Specific aim: To determine if holo-TC concentration increases measurably in response to

Bl2 supplementation within a 24 hour time period.









Beta-IgandR



Corn n Ring H2N I HCTs" c


NH OH

Alpha-ligand



Figure 1-1 Structure of vitamin Bl2. Modified from Stabler (35) p. 22





Ileum


~1% diffusio

Enterocyte


Figure 1-2 Overview of vitamin Bl2 (Bl12) absorption. (1) Food bound Bl2 is released in the
acidic environment of the stomach. (2) Free Bl2 binds to haptocorrin and the
complex travels to the duodenum. (3) Pancreatic proteases degrade HC. (4) Free
Bl2 binds to intrinsic factor, which is synthesized in the gastric parietal cells. (5) The
Bl2 IF complex to travel to the ileum and is transferred across the ileal epithelium
via receptor mediated endocytosis, along with 1% passive diffusion. (6) In the
enterocyte, intrinsic factor is degraded by the lysosome. (7) Transcobalamin binds
Bl2 at some point after release from intrinsic factor, this may occur in the enterocyte.


Legend

~Dietary 812
Vitarnin B12
P Haptocorrin
SIntrinsic Factor
CbTranscobalar ni
~CCub~ulin


Portal blood




























Figure 1-3 Role of vitamin Bl2 in the remethylation of homocysteine. THF = tetrahydrofolate;
5 -CH3-THF = 5 -methyltetrahydrofol ate; MS = methionine synthase;
SAM = s-adenosylmethi onine; SAH = S -adenosylhomocy steine.









CHAPTER 2
VITAMIN Bl2 STATUS IS IMPAIRED IN A SUBGROUP OF HEALTHY YOUNG
VEGETARIAN AND OMNIVOROUS ADULT MEN AND WOMEN

Naturally occurring dietary sources of Bl2 are limited to foods of animal origin, which if

restricted in the diet may impair B l2 status (40-42). Vitamin supplements and fortified foods

can also contribute to Bl2 intake (97); however, it is estimated that ~70 % of the United States

(US) population does not take supplements (106). Vegetarians, individuals who avoid some or

all animal-derived foods, have limited dietary intake of Bl2 and may be at greatest risk for

developing a Bl2 deficiency compared to omnivores. Few data are available on Bl2 status in

young adult vegetarians in the United States, and further evaluation of B 2 status in this

subgroup of individuals is warranted to better determine relative risk of Bl2 deficiency and

related disease. Clinical determination of Bl2 deficiency relies on the availability of specific

and reliable biomarkers of Bl2 status. Biomarkers currently used to assess Bl2 status include

serum B l2, MMA, Hcy and holo-TC concentrations. Although holo-TC is not yet used

clinically, holo-TC is reported to be more sensitive than serum Bl2 and may be comparable to

MMA as a biomarker of Bl2 status. The objectives of this study were to determine if young

adult vegetarians who do not take Bl2 supplements are at a greater risk for Bl2 deficiency than

omnivores not taking Bl2 supplements, and to compare the various Bl2 status indicators within

these groups.

Subjects and Methods

Subjects and Subject Recruitment

Healthy adults (n = 388) from the Alachua county, FL community including university

students, faculty and staff were recruited by flyers and newspaper advertisements with

simultaneous recruitment for "healthy adult vegetarians" and "healthy adults". Subj ects were

screened by phone and selected based on the following exclusion criteria: (a) < 18 y & > 49 y (b)









maj or change in animal-product consumption (i.e. vegetarian or omnivore) habits during the past

3 years; (c) B l2-containing supplement use within 6 mo of screening; (d) chronic alcohol

consumption (>1 drink/d of any kind); (e) use of tobacco products; (f) use of prescription

medications other than oral contraceptives; (g) personal history of chronic disease; (h) regular

blood donations; and (i) pregnant or lactating women.

Potential subj ects were asked about their meat consumption habits during the phone

screening for initial classification as vegetarian or omnivore. Specifically subj ects were asked

"How often do you consume (a) beef, (b) chicken, (c) turkey, (d) pork, and (e) fish". Subj ects

who responded "never" to all questions were temporarily classified as vegetarian. This study

was approved by the University of Florida Institutional Review Board, and all subj ects signed an

informed consent prior to beginning the study.

Study Design and Data Collection

Between the hours of 7:00 am and 9:00 am subj ects were scheduled for fasting blood

sample collections. Subj ects were called 24 hours prior to their scheduled appointment to

remind them to fast overnight and the following morning. Following sample collection, subjects

were given a small meal and a comprehensive information session explaining how to complete

the National Cancer Institute Diet History Questionnaire (DHQ). Subjects were asked to

complete the questionnaire at home and return it within two weeks and to contact a designated

member of our recent team personnel if they had any questions or problems completing or

returning the questionnaire. For any unreturned DHQs, individuals were contacted by phone or

e-mail to determine if the questionnaires were lost in transit or if the subj ect had not had an

opportunity to complete the DHQ instrument. Subjects that chose not to return the DHQ were

not included in the final data analysis. The DHQ has been validated for the estimation and









quantification of dietary intake of all essential nutrients including Bl2 (107). Subj ects were

instructed to answer all questions based on their diet over the past 12 mo estimating the

frequency of intake and portion size of 125 different food items. A total of 70 mL of blood were

collected for analysis of the following indices: (a) serum holo-TC; (b) plasma Bl12; (c) serum

MMA; (d) serum Hcy (e) serum folate, and (f) hematocrit.

Sample Processing

Blood samples were collected in ethylenediaminetetraacetic acid (EDTA) and serum

separator clot activator (SST) tubes. EDTA tubes were centrifuged for 30 min at 2000 x g at 40C

to separate and collect plasma for Bl2 analyses. Serum separator tubes were centrifuged for 15

min at 650 x g at room temperature to separate and collect serum for holo-TC, MMA, Hcy, and

folate determination. All samples were stored frozen at -300C until analysis.

Competitive Binding Assays of Serum Holo-transcobalamin and Plasma B12.

Serum holo-TC concentration was determined by radioimmunoassay (holo-TC RIA

reagent kit; Axis Shield, Ulvenveien, Oslo, Norway) based on the method of Ulleland et al. (45).

Specifically, magnetic microspheres coated with anti-human TC monoclonal antibodies were

incubated with each sample for a period of one hour to isolate both holo-TC and apo-TC. Once

attached to the metal beads via antibody interaction, the TC protein and associated Bl2 were

magnetically separated from the sample. Next, isolated TC was incubated with 57Co-labeled Bl2

tracer plus reducing agent followed by a denaturing agent to free B l2 from the TC protein.

Finally, each sample was incubated with IF to which unlabeled and labeled Bl2 bind

competitively based on their relative concentrations. Remaining unbound B l2 was removed and

the relative radioactivity of each sample measured by gamma counter. Radioactivity of each









sample in counts per minute (CPM) was compared to a standard curve with serum holo-TC

concentration being inversely associated with CPM.

Plasma Bl2 was determined by RIA using a commercially available kit (Quantaphase II,

Bio-Rad). Specifically, samples were incubated with a 57Co labeled Bl2 tracer in a 100oC water

bath to convert all forms of B 2 to cyanocobalamin. Samples were brought to room temperature

after boiling for 20 min, and then mixed with purified porcine IF bound to polymer beads and

incubated for one hour. During incubation labeled and unlabeled Bl2 compete for binding to IF

at rates that match their relative concentrations. Finally, samples were centrifuged, and

supernatant containing unbound B l2 was removed. Sample radioactivity was measured by

gamma counter and Bl2 concentration was calculated using a standard curve on which the

radioactivity was inversely related to Bl2 concentration.

Measurement of Serum Homocysteine and Methylmalonic Acid.

Serum Hcy and MMA concentrations were determined by gas chromatography mass

spectrometry (Metabolite Laboratories, Inc. Denver, Colorado) (108, 109).

Diet Analysis

Daily Bl2 intake was assessed based on data obtained from the DHQ, which was modified

to include additional Bl2-containing foods including meats, mixed dishes, fortified foods and

meat substitutes. The original DHQ is available online at http ://appliedresearch.cancer.gov. The

DHQ was scanned by Optimal Solutions Corporation (OSC), Lynbrook, New York. Dietary data

obtained from scanning the questionnaires was sent to the University of North Carolina, Chapel

Hill as an ASCII text fie, and then analyzed using the Diet*Calc Analysis program. In order to

analyze the modified questionnaire, the Diet*Calc program was updated to include nutrient

values for all of the food items added based on data from the United States Department of










Agriculture (USDA) National Nutrient Database for Standard Reference and nutrition label

information when data was not available from the former USDA database Diet*Calc is a

freeware program and can be downloaded from the National Cancer Institute website

(www.ri skfactor. cancer.gov).

Subject Dietary Intake Classification

Individuals were classified as vegetarian or omnivore based on their responses to the DHQ.

Individuals classified as vegetarians were those who reported no consumption of any meat

products (i.e., beef, poultry, pork, lamb and seafood) or meat-based mixed dishes and who

reported consuming dairy products and eggs never to daily. Omnivores were defined as

individuals who consumed any meat, poultry or seafood products. In addition to the preexisting

questions in the DHQ that asked about all types of meat consumption, a new question was added

to more accurately classify subjects into specific dietary intake categories. This question

required subj ects to indicate the foods they never consumed including (a) beef, (b) chicken, (c)

turkey, (d) pork, (e) fish, (f) dairy products, and (g) eggs.

Statistical Methods.

Vitamin Bl2 status based on the measured Bl2 indicators and dietary Bl2 intake were

compared between groups using analysis of variance (ANOVA) with an alpha of 0.05.

Dependent variables also were classified as "normal" vs. "abnormal" according to whether they

were above or below established thresholds (Bl2, > 148 pmol/L; holo-TC, > 35 pmol/L; IVMA I

271 nmol/L; and < Hcy 12 Clmol/L) and comparisons with respect to dietary group were done by

Pearson Chi-Square tests. The distributions of all possible combinations of normal and abnormal

test results were calculated and the rate of each Bl2 indicator being abnormal when all others

were normal or abnormal was compared using a Chi-square test. Age distributions were










compared between the two dietary groups by ANOVA, while race and gender for the two dietary

groups were compared by Pearson Chi-Square tests.

Results

One hundred and twenty one vegetarians and 181 omnivores completed the study (total n =

305). Of the 388 enrolled subjects, 62 were excluded due to reporting supplement use, and 23

did not complete the DHQ. All results are reported as mean & SD unless otherwise noted. There

was a significant difference in age and BMI between groups, with vegetarians being older and

having a lower BMI. There were no significant differences in gender or ethnicity between diet

categories (Table 2-1).

Total Bl2 intake (Clg/d & SD) and Bl2 intake expressed as Gig/1000 kcals were

significantly lower (P < 0.001) in vegetarians than in omnivores (Table 2-2). Plasma Bl2

concentration was significantly lower (P < 0.01) in vegetarians than omnivores (Table 2-2).

Serum MMA concentration was significantly higher (P = 0.001) in vegetarians compared to

omnivores (Table 2-2). Mean holo-TC and Hcy concentrations were not significantly different

between groups (Table 2-2).

Vitamin B l2 deficiency, based on having a value outside the normal range for one or more

of the Bl2 status indicators, was twice as prevalent (P < 0.001) in vegetarians than omnivores

(42% and 23%, respectively). Impaired Bl2 status based on concentrations of plasma Bl2,

serum holo-TC and serum MMA combined also was significantly greater in vegetarians

compared to omnivores. Specifically, more than twice as many (P < 0.05) vegetarians had low

serum holo-TC (< 35 pmol/L), plasma Bl2 (< 148 pmol/L), and elevated serum MMA (> 270

nmol/L) concentrations than omnivores (Figure 2-1). There was no significant difference in the

percentage of vegetarians versus omnivores with elevated Hcy (> 12 Clmol/L).










Subj ects were cross-tabulated by Bl12 status as defined by having a value within (+) or

outside (-) the normal range for Bl2, holo-TC and MMA singly and in combination (Table 2-3).

Because of the small numbers of subj ects within each of the resulting 8 categories, statistical

analysis was not done; however, the likelihood of one Bl2 status indicator being abnormal while

the remaining tests were normal was conducted. In this analysis, Bl2, holo-TC and MMA were

more likely to be abnormal when one or more of the other indicators were abnormal (23, 3 8, and

36 % of the time, respectively) compared to when all others were normal (6, 11, 10 % of the

time, respectively) (Figure 2-2).

Discussion

The primary obj ective of this study was to assess and compare the Bl2 status and intake of

young adult vegetarians and omnivores who do not take Bl2-containing vitamin supplements to

determine if vegetarians are at greater risk for developing a Bl2 deficiency than their

omnivorous counterparts. Although long term adherence to a vegetarian diet can provide

substantial health benefits (1 10), limitation of most or all animal-based foods, particularly when

Bl2-fortified foods or supplements are not added to the diet, can increase the risk for developing

a Bl2 deficiency.

It has been estimated that Bl2 intake in the general US population is adequate (111),

however, data from this study suggest that a subgroup of healthy young, non-supplement using

adults may not be consuming adequate Bl2. Within the vegetarian groups, 43% were

determined to be potentially Bl2 deficient based on having a value outside the normal range for

one or more Bl2 status indicators, 61% of whom had elevated MMA, indicating metabolic

impairment. Surprisingly, of the omnivores, 23% were potentially B l2 deficient, with 48% of

them having an elevated MMA concentration, suggesting that even a meat-containing diet may

not provide sufficient Bl2.









This is of particular concern for young women of childbearing age, who might chose to

avoid meat in an attempt to lower fat and cholesterol intake, but do not consume other sources of

Bl2. Nutrient availability is crucial in the first 180 days of pregnancy, during a portion of which

a woman might not even know she is pregnant (112). This issue has been addressed in relation

to folic acid; however unlike folic acid, it is not well recognized that a Bl2 deficiency is an

independent risk factor for neural tube defects (57, 113, 114).

The DHQ used to assess dietary Bl2 intake asks subj ects to recall dietary intake over the

past 12 mo and answer questions based on their best estimate of food intake. Full analysis of

DHQ data was the focus of an investigation conducted by another member of the laboratory

group and will be reported separately. The data obtained from the analyses conducted for this

study were used to group individuals within a similar range of overall B l2 intake and to identify

foods eaten or excluded by each subj ect. This allowed for a very strict definition of "vegetarian"

subjects who reported no meat consumption and "omnivore" subj ects who reported some degree

of meat consumption. Previous studies, the maj ority of which were conducted in Europe, used a

similar approach to classify individuals as vegetarian. Vegetarians have previously been sub-

grouped into lacto-ovo-vegetarians, lacto-vegetarian and vegan categories, with the greatest

deficiency associated with the vegan diet relative to other vegetarian sub-groups (81). It is

widely believed in the US, that a Bl2 deficiency is not a problem in healthy young adults who

consume at least some animal-based products. The data from the present study do not support

this perception.

In addition to the moderate deficiency that was detected in the vegetarian and omnivorous

groups, a small subset (including both omnivores and vegetarians) was determined to be severely

Bl2 deficient as evidenced by reports of neurological problems including numbness and a









tingling sensation in the extremities. Of these subjects none had previously sought out medical

attention for these symptoms, which illustrates that Bl2 deficiencies may go undetected among

seemingly healthy young adult men and women. Early detection of a Bl2 deficiency is the most

effective way to prevent progression to serious health complications. Recent studies suggest that

measurement of holo-TC is superior to serum B l2 because only holo-TC is taken up into cells,

and therefore only that portion (< 20%) is biologically active (115). Additionally, holo-TC has

been reported to be more sensitive to changes in Bl2 intake than total serum Bl2 (28). Miller et

al. (1 16) reported that the use of holo-TC and serum Bl2 together as a ratio may be superior to

the use of either alone. Their data suggest (1 16) that use of combined holo-TC and serum B l2

measurement could lead to three possible diagnoses; normal, possible deficiency (only 1 low

indicator), and deficient (both indicators low). Finally MMA is still considered by many

researchers to be the gold standard (54, 117). In the current study, we cross tabulated subjects by

Bl2 status as defined by having a value within or outside the normal range for Bl2, holo-TC and

MMA singly and in combination with each other. Statistical analysis could not be conducted due

to limited sample size; however, a combined analysis of the two diet groups indicated that it is

more likely that when the value of one biomarker is outside the normal range at least one other

indicator is more likely to also be outside the normal range. Additionally, the length of time an

individual adheres to a Bl2-insufficient diet will have differential effects on specific Bl2

biomarkers (32). Considering the differences in the primary biomarkers of B 2 status, holo-TC

may be initially affected followed by a decrease in serum Bl2 (once Bl2 stores have been

depleted), and finally an elevation in MMA indicating impaired cellular Bl2-dependant enzyme

function. The data from the current study do not definitively support one B l2 biomarker as










being superior to another; however, in a clinical setting the more biomarkers that are outside the

normal range, the more likely a Bl2 deficiency exists.

In conclusion, the high incidence of impaired Bl2 status observed in these otherwise

healthy young adults was unexpected. These data indicate that dietary intake alone may not be

meeting the Bl2 needs of non-supplement using adults, especially vegetarians. Further research

focusing on B l2 status and intake in individuals consuming both vegetarian and low-meat

containing diets is warranted. Assessment of B l2 status by a combination of biomarkers may

provide a more definitive diagnostic approach prior to treatment.




Table 2-1 Characteristics of study groups
Vegetarians Omnivores
(n = 121) (n = 181)
Age (y; mean & SD) 28 & 9b 24 & 6
BMIa (mean & SD) 22.9 & 3.9b 23.9 & 4.1
Gender (count)
Female 67 98
Male 54 83
Race/Ethnicity (count)
White 69 119
African American 6 11
Asian 17 17
Asian Indian 12 4
Hispanic 12 26
Other 5 4
a~ody mass index (BMI); bSignlifcantly different from omnivores (P < 0.05)(ANOVA)












Table 2-2 Mean (a SD) dietary vitamin B l2 intake and status of omnivorous and vegetarian
adults .
Vegetarians Omnivores
Analysis a (n = 121) (n= 181)
Total Bl2 intake (Clg/d) 3.39 & 2.97 6.80 & 4.04b
Bl2 intake (Clg/1000 Kcal/d) 1.92 & 1.87 3.31 + 1.88b
Bl2 (pmol/L) 280 & 146 313 A 1240
Holo-TC (pmol/L) 83 & 84 87 & 55
MMA (nmol/L) 260 & 229 195 1 1160
Hcy (Clmol/L) 7.7 & 2.7 7.3 & 2.5
aVitamin Bl2 (Bl12); holo-transcobalamin (holo-TC); methylmalonic acid (MMA);
homocysteine (Hcy); b Different from vegetarians (P < 0.001); a Different from vegetarians (P <
0.01) (ANOVA)




SVegetarian
MOmnivore




O


30




o


0'


Holo-TC
< 35 pmol/L


B12
< 148 pmol/L


MMA
> 270 nmol/L


Hoy
> 14 Cpmol/L


Figure 2-1 Percent of vegetarian (n = 121) and omnivorous (n = 181) adults (18 to 49 y) with
concentrations outside the normal range for holo-transcobalamin (holo-TC, < 35
pmol/L), serum vitamin Bl2 (Bl12, < 148 pmol/L), methylmalonic acid (MMA, > 270
nmol/L), and homocysteine (Hcy, > 12 Clmol/L). Different from omnivores (P <
0.05) (ANOVA)












Table 2-3 Cross-tabulation of vitamin Bl2 status of subjects based on select biomarker
combinations


Status by biomarker measured
Bl2 Holo-TC MMA
S140 pmol/L > 35 pmol/L 5 270 nmol/L


Diet group
Omnivore
n =181
138
7


Vegetarian
n =121
68


Total
n = 302
206
12


+ -+ 11 12 23
-+ 2 2 4
+ + -14 13 27
-+ -3 1 4
+ --7 3 10
-8 3 10
a + Yes; No; vitamin Bl2 (Bl12); holo-transcobalamin (holo-TC); methylmalonic acid (MMA)


SAll other concentrations within normal range
SAt least one other concentration outside normal range


50-1


2 40-
e


O
c,20-

0-


B12 < 148 pmollL Holo-TC < 35 pmollL M MA> 270 pmollL


Figure 2-2 Frequency of single versus combined vitamin Bl2 (Bl2) status biomarkers being
outside the normal range including plasma Bl2, serum holo-transcobalamin (holo-
TC) and serum methylmalonic acid (MMA). *Significantly different from group with
all others normal (P < 0.001) (Pearson's Chi-squared test)









CHAPTER 3
VITAMIN B2 INTAKE AT THE CURRENT RDA LEVEL IS NOT OPTIMAL

The current RDA for B l2 was established based on data from patients who were being

treated for pernicious anemia. Specifically, the amount of Bl2 required in an inj ectable form to

normalize serum Bl2 in patients diagnosed with pernicious anemia was determined to be the

daily Bl2 requirement fro adults. The Bl2 RDA (2.4 Clg/d) was derived by adjusting the

estimated Bl2 requirement for bioavailability, enterohepatic recirculation, and the CV for 97 to

98 % of the population. It has been suggested that the RDA for Bl12 is not optimal, and that Bl2

status is improved with intakes up to 6 Clg/d (118, 119). The objective of this analysis was to

determine the level of dietary Bl2 intake associated with optimal Bl2 status as defined by Bl2

status biomarkers within the normal range.

Subjects and Methods

Subjects and Subject Recruitment

Healthy adults (n = 302) were recruited from the Alachua county, FL community including

university students, faculty and staff. Specifically, subjects were screened by phone and selected

based on the following inclusion criteria: (a) 18 to 49 y (b) no change in meat consumption

habits over the past 3 years; (c) no Bl2-containing supplement use within the past 6 mo; (d)

limited chronic alcohol consumption (<1 drink/d of any kind); (e) no use of tobacco products; (f)

no chronic use of prescription medications other than oral contraceptive agents; (g) no history of

chronic disease; (h) no chronic blood donations; and (i) non-pregnant and non-lactating. All 302

qualified subjects from the first part of the current study were included in this analysis. The

approved institutional review board informed consent form signed by the subj ects at the

beginning of the study included consent for all aspects of studies described in the manuscript.

Study Design and Data Collection









Subj ects were called the day before their scheduled study day to remind them to fast

overnight (8 hours) and the following morning prior to having their blood drawn. Between the

hours of 7:00 am and 9:00 am qualified subjects were scheduled for blood sample collection

followed by a comprehensive information session explaining how to complete the National

Cancer Institute Diet History Questionnaire (DHQ) used to assess dietary intake. A total of 70

mL of blood were collected for analysis of the following indices: (a) serum holo-TC; (b) plasma

Bl2; (c) serum MMA; (d) serum homocysteine (Hcy) (e) serum folate, and (f) hematocrit.

Sample Processing and Analysis

Blood samples were collected in EDTA and SST clot activator tubes. EDTA tubes were

centrifuged at 2000 x g at 40C for 30 min to obtain plasma for Bl2 analyses. SST tubes were

centrifuged at 650 x g at room temperature for 15 min to obtain serum for determination of holo-

TC, MMA, Hcy, and folate concentrations. Samples were stored at -300C until analysis. Serum

holo-TC concentration was determined by radioimmunoassay (holo-TC RIA reagent kit; Axis

Shield, Ulvenveien, Oslo, Norway) based on the method of Ulleland et al. (45) using magnetic

microspheres coated with anti-transcobalamin monoclonal antibodies to isolate both holo-TC and

apo-TC, and 57Co-labeled Bl2 as a tracer. Plasma Bl2 concentration was determined by RIA

using a commercially available kit (Quantaphase II, Bio-Rad). Serum Hcy and MMA

concentrations were determined by gas chromatography mass spectrometry(Metabolite

Laboratories, Inc. Denver, Colorado) (108, 109).

Diet Analysis

Daily Bl2 intake was estimated based on data obtained from the DHQ, which was

modified to include an extensive list ofBl2-containing foods including meat containing mixed

dishes, fortified foods, and meat substitutes. The unmodified DHQ is available for review online









at http ://appliedresearch.cancer.gov. The DHQ was scanned by Optimal Solutions Corporation

(OSC), Lynbrook, New York. Once scanned OSC sent the dietary data as an ASCII text file to

the University of North Carolina, Chapel Hill (UNC), where the data were analyzed using the

Diet*Calc Analysis program modified for this version of the DHQ. This freeware program can

be downloaded from the NCI web site (www.ri skfactor. cancer.gov). The Bl12 content of the food

items added to the DHQ was obtained from the USDA National Nutrient Database for Standard

Reference and nutritional labels (39).

Statistical Analysis

Results are reported in the text as mean & SD with an alpha = 0.05. The dependent

variables Bl2, holo-TC and MMA concentrations were classified as "normal" vs. "abnormal"

according to falling above or below an established threshold (B l2, 148 pmol/L; holo-TC, 35

pmol/L; MMA 271 nmol/L; and Hcy 12 Clmol/L) and comparisons with respect to dietary B l2

intake were performed using the Pearson Chi-Square test. Subj ects were divided into dietary

Bl2 intake quintiles, and Bl2 status based on plasma Bl2, serum holo-TC, MMA, and Hcy

concentrations were compared between groups using ANOVA with an alpha of 0.05. The "Least

Significant Difference" (LSD) method of multiple comparisons was used for assessment of

differences between quintiles. The LSD ensures every target population paired difference in

means will be within +/- LSD of the corresponding difference in sample means with 95%

confidence. The data were analyzed using EXCEL (Microsoft, Redmond, WA) and PRISM

software (Graph-Pad Software Inc. El Camino, CA).

Results

Three hundred and two healthy young adult (18 to 48 y) men and women were included in

this analysis. Subject characteristics are listed in Table 3-1. Seventy six subjects (25 %) had an

intake below the RDA of 2.4 Clg/d. Dietary Bl2 intake was significantly correlated (P < 0.05)









with B l2, Hcy, and MMA concentrations, but not with holo-TC concentration (Table 3-2).

Individuals with low plasma Bl2 (< 148 pmol/L) or holo-TC (< 35 pmol/L) concentrations and

those with elevated serum MMA (> 270 nmol/L) or Hcy (> 12 Clmol/L) concentrations had

significantly lower dietary B l2 intake than those with normal concentrations (Figure 3-1).

To further evaluate the influence of Bl12 intake on Bl2 status, subj ects were ranked and

grouped by quintile of Bl2 intake. The mean concentration for each Bl2 status indicator was

plotted against the mean Bl2 intake for each quintile group (Figure 3-2). Mean holo-TC, Bl2,

MMA and Hcy concentrations were significantly different (P < 0.05) among Bl2 intake quintile

groups (Figure 3-1). Specifically, mean holo-TC increased (P < 0.01) from quintile 1 through 3

and then maintained approximately the same value from quintiles 3 through 5, which was

associated with a mean Bl2 intake of 2 4.3 Clg/d. Mean plasma Bl2 concentration increased (P

< 0.001) from quintile 1 through 4, reaching a plateau from quintile 4 through 5 corresponding to

a Bl2 intake > 6.7 Clg/d. Mean MMA decreased (P < 0.001) from quintile 1 through 2 then

reached a plateau, which was associated with a Bl2 intake of 2 2.7 Clg/d. Homocysteine changed

to the smallest degree, but decreased (P < 0.05) from quintile 1 through 3 maintaining this

approximate value thought quintile 5, which corresponded to a B 2 intake > 4.3 Clg/d.

In the case of holo-TC, Bl2, and Hcy concentrations, the means across all quintiles of Bl2

intake were in the normal range; mean MMA concentration was elevated in quintile 1 and within

the normal range for all subsequent quintiles. The proportion of subjects with Bl2 deficiency as

defined by abnormal biomarkers within each quintile group (i.e. low B l2, low holo-TC, elevated

MMA or elevated Hcy) decreased significantly from the lowest to highest B l2 intake quintile for

all indices measured (Table 3-3). Specifically, in the group of subj ects who consumed > 3.4

Clg/d of Bl2 there was a significantly smaller percentage of subj ects with low holo-TC or










elevated IVMA concentrations than those consuming < 3.4 Clg/d. In the group of subj ects who

consumed at least the RDA for Bl2, there was a significantly lower number of individuals with a

low plasma B l2 concentration than in the group of individuals who consumed less than the RDA

(Table 3-3).

Discussion

In this study, the relationship between estimated Bl2 intake and a panel of Bl2 status

biomarkers were assessed in order to evaluate the adequacy of the current RDA for Bl2. It has

been suggested by another research group that a Bl2 intake of 6 Clg/d was associated with

improved concentrations of all Bl2 biomarkers compared to an intake of 2.4 Clg/d (1 18).

Although the findings in the current study vary depending on the specific biomarker, the data

indicate that an intake greater than the current RDA is associated with normal B l2 status.

Overall, no clear conclusion can be drawn from these data as to a specific intake level of Bl2

that might result in normalization of all Bl2 biomarkers; however, the data suggest that the

current RDA may not be optimal. In the first investigation of this study group, subjects were

classified as vegetarian or omnivore, and inadequate Bl2 status was detected in a surprising

number of individuals in both groups. Specifically, 40% of vegetarians and 1 1% of omnivores

were determined to have abnormal values for one or more of the four Bl2 biomarkers. The

mean Bl12 intake of both groups exceeded the RDA. In the current analysis, the third quintile

corresponded best to the current RDA for Bl2 with a mean and range of Bl2 intake at 2.7 Clg/d

and 2.0 to 3.4 Clg B l2, respectively. Beyond the second quintile, the mean concentrations of

holo-TC and Bl2 increased; mean Hcy decreased and overall rate of deficiency decreased

significantly. This suggests that B l2 status improves with intakes above the second quintile,










which in this study was represented as an intake level of 3.4 Clg/d. Little or no change was

observed in subsequent quintiles, suggesting that an intake level above 3 Clg/d may be required.

One limiting factor of the current study is the use of a FFQ to estimate Bl12 intake rather

than a 7-day weighed food record as was used in the study by Bor et al. (118). Because the DHQ

relies on subj ect recall and estimation of intake over the past 12 mo, it is more prone to error and

less precise than a direct measure. In addition, neither the one week weighed food record nor a

FFQ gives an estimate of duration of a particular diet, and because Bl2 status is slow to change

relative to changes in Bl2 intake, estimated intake over one week or even over one year may not

always correlate well with status at a given time. In a very large study using data of from the

Framingham Offspring population, which also used an FFQ to assess Bl2 intake, improvements

in Bl2 status were observed for intakes up to 10 Clg/d (119). Therefore, data from the current

study in addition to that from two previous studies agree with the conclusion that the RDA for

Bl2 is inadequate to maintain normal Bl2 status (39, 118). Further investigations focusing

specifically on changes in Bl2 status with increasing Bl2 intake are warranted to address this

issue and derive an estimate of Bl2 intake that is consistent with maintenance of normal Bl2

status. Because the current RDA was established using data from research conducted with

patients who had pernicious anemia and who were inj ected with Bl2 rather than in healthy

individuals consuming dietary B l2, there is clear justification for conducting controlled feeding

studies to obtain pertinent data necessary to revise the current RDA. The RDAs are not intended

to be therapeutic recommendations for individuals with disease conditions such as pernicious

anemia. Recommended intake of Bl2 for the adult population should apply to a majority of the

population, potentially with additional recommendations for some sub-groups such as vegetarian









groups and the elderly. In future studies, consideration must be made for potential differences in

bioavailability of Bl2 from foods and fortified products consumed by healthy young adults.

In conclusion, the previous assumption that the general US population has adequate Bl2,

status is not supported by data from this investigation in young healthy adult men and women

who consume either omnivorous or vegetarian diets. The data from this study support that from

two previous investigations including one in the US indicating that the current RDA for B l2 is

inadequate to maintain normal Bl2 status in healthy men and women. Further investigation of

the changes in B l2 status in response to controlled levels of B l2 intake is warranted to provide

data to support a revised RDA.



Table 3-1 Sub ect Characteristics
Mean (a SD) Range Reference interval
Age (y) 26 & 8 18 49
BMIa 24 & 4 16 48
Bl2 intake (Clg/d) 5.4 & 3.9 0.4 22.67
Bl2a (pmol/L) 300 & 134 40 937 148 444
Holo-TCa (pmol/L) 85 & 69 6 576 35 150
MMAa (nmol/L) 221 + 173 81 -1866 80 -270
Hcya (Clmol/L) 7.5 & 2.6 3.5 29.6 4.5 12.0
aBody mass index (BMI); vitamin Bl2 (Bl12); holo-transcobalamin (holo-TC); methylmalonic
acid (MMA); homocysteine (Hcy)


Table 3-2 Correlations (r) between vitamin Bl12 intake and concentrations of Bl2 status
biomarkers
B l2a Holo-TCa MMAa Hcya
r P r P r P r P
Bl2 intake 0.23 < 0.0001 0.11 0.06 -0.17 0.004 -0.12 0.04
aVitamin Bl2 (Bl12); holo-transcobalamin (holo-TC); methylmalonic acid (MMA);
homocysteine (Hcy)












Slow /elevated
Normal


0L


Holo-TC B12


Hcy MMA


Figure 3-1 Total daily vitamin Bl2 (Bl2) intake (mean + SD) in individuals with concentrations
outside the normal range for holo-transcobalamin (holo-TC; normal > 35 pmol/L),
plasma Bl2 (normal > 148 pmol/L), serum homocysteine (Hcy; normal < 12
Clmol/L), and serum methylmalonic acid (MMA; normal < 270 nmol/L). The current
RDA for Bl2 is represented (-----). *Different from normal (P < 0.01) (ANOVA,
Chi-square test)













































00 1 2 3 12 inta~ke(7mg) quint 1 1 3 e


250,


200



4100`
50


B12 intake (mg/d) quintile


B12 intake (mg) quintile


700-

600-

S500


2 00-

20-
100-


a, b
b,c
a, b, c


1 B12 intake(7mg)8qui til101 2 31


Figure 3-2 Relationship between vitamin Bl2 (Bl2) intake and status. Mean Bl2 (~ SD) intake
for each quintile (n = 60 for quintiles 1, 3 and 5; n = 61 for quintiles 2, and 4;
respectively), is plotted against concentrations (mean & SD) of Bl2, holo-
transcobalamin (holo-TC), methylmalonic acid (MMA), and homocysteine (Hcy).
Values with different superscript letters are significantly different (P < 0.001 for Bl2
and MMA, P < 0.01 for holo-TC and P < 0.05 for Hcy). The current RDA for Bl2 is
represented in each graph (----).


b b


b b b b










Table 3-3 Proportion (%) of individuals with concentrations outside the normal range for select vitamin B l2 status biomarkers
Bl2 intake quintile
Biomarkera Total 1 2 3 4 5
n (< 2.0 Clg/d) (< 3.4 Clg/d) (< 5.3 Clg/d) (< 8.5 Clg/d) (< 22.67 Clg/d)
Bl2
Low (< 148 pmol/L) 50 25b 8 10 7 5
Normal (> 148 pmol/L) 248 75 92 90 93 95
Holo-TC
Low (< 35 pmol/L) 33 350 23d 13e 5 7
Normal (< 35 pmol/L) 269 65 77 86 95 93
MMA
Elevated (> 270 nmol/L) 52 32c 23 10 10 12
Normal (< 270 ) nmol/L 248 68 77 90 90 88
Hcy
Elevated (> 14 ymol/L) 10 6d 2 2 0 0
Normal (< 14 pmol/L) 290 90 97 97 100 100
a Vitamin Bl2 (Bl12); holo-transcobalamin (holo-TC); methylmalonic acid (MMA); homocysteine (Hcy); data was not available for all
subjects fir some biomarkers; bSignificantly different from Q2, Q3, Q4, Q5; a Significantly different from Q3, Q4, Q5; d Significantly
different from Q4, Q5; e Significantly different from Q4. (P < 0.05) (Chi-square test)









CHAPTER 4
GENOTYPE FOR THE TRANSCOBALAMIN 776C+ G POLYMORPHISM IS NOT
ASSOCIATED WITH ABNORMAL VITAMIN Bl2 STATUS BIOMARKERS INT HEALTHY
ADULT S

Transcobalamin (TC), the B l2 transport protein required for cellular uptake is essential to

maintain B l2 metabolic function (26, 120). A common genetic polymorphism for TC (TC 776

C G) may impair the metabolic role of this protein (74, 89). It is hypothesized that Bl2

transport and thus metabolic function will be impaired in individuals with the homozygous

variant genotype (GG) for the TC 776 C G polymorphism. The metabolic and health-related

risks associated with this polymorphism are predicted to be exacerbated by the consumption of

low-Bl12 vegetarian diets that exclude specific animal-derived foods. The primary goals of this

study were to evaluate the effects of the TC 776 C G polymorphism on Bl2 metabolism in

young adult men and women who consume a low B l2 diet compared to those consuming

adequate B l2.

Subjects and Methods

Subjects and Subject Recruitment

Healthy adults (n = 302) were recruited from the Alachua county, FL community including

university students, faculty and staff. Subjects were initially screened by phone and selected for

the study based on the following inclusion criteria: (a) > 18 y & < 49 y (b) no change in meat

consumption habits during the last 3; (c) no B l2 containing supplement use within the past 6 mo;

(d) limited chronic alcohol consumption (<1 drink/d of any kind); (e) no use of tobacco products;

(f) no chronic use of prescription medications other than oral contraceptive agents; (g) no history

of chronic disease; (h) no chronic blood donations; and (i) non-pregnant and non-lactating. All

subjects from the first part of the research described in the manuscript were included in this









analysis after a second informed consent form approved by the University of Florida Institutional

Review Board that was specific for genetic analysis.

Study Design and Data Collection

Subj ects were called the day before their scheduled study day to remind them to fast

overnight (8 hours) and the following morning prior to having their blood drawn. Qualified

subj ects were scheduled for fasting blood sample collection to be performed between the hours

of 7:00 am and 9:00 am followed by a comprehensive information session explaining how to

complete the National Cancer Institute Diet History Questionnaire (DHQ), which was used to

assess dietary intake. A total of 70 mL of blood were collected for analysis of serum holo-TC;

plasma Bl2; serum MMA; serum homocysteine (Hcy), serum folate, and DNA extraction.

Sample Processing and Analysis

Blood samples were collected in EDTA and SST clot activator tubes. EDTA tubes were

centrifuged at 2000 x g at 40C for 30 min to obtain plasma for Bl2 analyses. SST tubes were

centrifuged at 650 x g at room temperature for 15 min to obtain serum for holo-TC, MMA, Hcy,

and folate determination. Samples were stored at -300C until analysis. Serum holo-TC was

determined by radioimmunoassay (holo-TC RIA reagent kit; Axis Shield, Ulvenveien, Oslo,

Norway) based on the method of Ulleland et al. (45) using magnetic microspheres coated with

anti-transcobalamin monoclonal antibodies to isolate both holo-TC and apo-TC, and 57Co-

labeled Bl2 as a tracer. Plasma Bl2 was determined by RIA using a commercially available kit

(Quantaphase II, Bio-Rad). Serum Hcy and MMA concentrations were determined by gas

chromatography mass spectrometry (108, 109).









Genotype Determination

DNA was extracted from blood as previously described (121) using a commercial kit

(Quantum Prep, BioRad, Hercules, CA) and standard laboratory procedures. Genotypes of

potential subj ects were determined using Dynamic Allele Specific Hybridization (DASH)

performed by DynaMetrix (Stockholm, Sweden). Briefly, a short PCR product was created

spanning the polymorphic position. One PCR primer was 5'-labeled with biotin for attachment

of the amplified targets to streptavidin-coated 96-well microtiter plates. Following denaturation

and a wash to remove the unbound strand, an allele-specific probe was hybridized to the bound

target DNA strand at low temperature in the presence of the double-strand specific intercalating

dye Sybr Green. Finally, the temperature was steadily increased while recording the probe-target

duplex melting temperature, as monitored by diminution of Sybr Green fluorescence with a

quantitative PCR analysis device.

Properly designed matched target-probe duplexes have higher melting temperatures than

those with single-base mismatches, enabling unambiguous allele discrimination. Heterozygous

samples show two separate phases of denaturation. For analysis, the negative derivatives of the

melting curves are plotted. A single peak at a lower temperature indicates homozygous allelic

mismatch to the probe, and a single peak at a higher temperature, a homozygous match. A

double peak is generated from a heterozygous sample (Figure 4-1).

Diet Analysis

Daily Bl2 intake was estimated based on data obtained from the DHQ, which was

modified to be inclusive of an extensive list of B l2-containing foods including meat containing

mixed dishes, fortified foods, and meat substitutes. The unmodified DHQ is available for review

online at http:.//appliedresearch. cancer.gov. The DHQ was scanned by Optimal Solutions

Corporation (OSC), Lynbrook, New York. Once scanned, OSC sent the dietary data as an









ASCII text file to the University of North Carolina, Chapel Hill (UNC). Data was analyzed

using the Diet*Calc Analysis program modified for this version of the DHQ. This freeware

program can be downloaded from the NCI web site (www.ri skfactor. cancer.gov). The Bl12

content of foods added to the DHQ were based on up-to-date information from the USDA

National Nutrient Database for Standard Reference and nutrition labels (39).

Statistical Methods

Results are reported in the text as mean + SD with an alpha = 0.05. Vitamin B l2 status,

based on measurement of plasma Bl2, serum holo-TC, the ratio of holo-TC to Bl2, MMA, and

Hcy, was compared among genotype groups with and without an adjustment for B l2 intake,

using ANOVA with an alpha of 0.05. Dependent variables also were classified as "normal" vs.

"abnormal" according to falling above or below an established threshold (Bl2 > 148 pmol/L;

holo-TC > 35 pmol/L; MMA I 271 nmol/L; and Hcy < 12 Clmol/L) and comparisons with

respect to genotype were performed using a Pearson Chi-Square test. Qualitative data including

gender, age, and ethnicity were compared using a Pearson Chi-Square test. The data were

analyzed using Statistical Analysis System Software (SAS Institute Inc. Cary, NC) and PRISM

software (Graph-Pad Software Inc. El Camino, CA).

Results

No significant differences were detected among genotype groups for gender, age, or BMI,

however there were differences in ethnic distribution (Table 4-1). There were no significant

differences among genotype groups, with or without adjustment for B l2 intake, for holo-TC,

MMA, and Hcy concentration whether considered alone (Table 4-2) or in combination with low

Bl2 status (Table 4-3). There was no significant difference in Bl2 among genotype groups,

though there was a trend for higher B l2 in the TC 776 GG group (Table 4-3; P < 0.01). In

addition, there were no significant differences among genotype groups in the number of









individuals with values outside the normal range for plasma B l2, holo-TC, MMA, or Hcy (Table

4-4).

Individuals with the TC 776 GG genotype had a significantly lower (P < 0.05) ratio of

holo-TC to plasma B l2 than individuals with the CC genotype (Table 4-2). Total TC was

significantly lower (P < 0.001) in the TC 776 GG genotype group compared to both the CG and

CC genotype groups (Table 4-2). Transcobalamin saturation was not significantly different

among groups.

Discussion

Studies investigating the effect of the TC 776 C & G polymorphism have resulted in

conflicting findings. In a previous study by our laboratory, significant differences were detected

in holo-TC concentration among the TC 776 genotype groups, however in the present larger

study, which included a wider range of Bl12 intake by subj ects, a significant difference was not

detected. In addition, there were no significant differences in Hcy or MMA concentration among

the genotype groups, further suggesting no real physiological impact of this single base pair

mutation of the TC gene on biochemical indexes of B l2 metabolism. Even when considering the

combined influence of the polymorphism and low Bl2 status, there were no significant

differences in any Bl2 status biomarkers among the genotype groups, indicating no effect on

Bl2 metabolism in Bl2 impaired individuals.

Interestingly, some significant differences were found among genotype groups, suggesting

a moderate effect of the polymorphism on TC protein synthesis or catabolism. Specifically,

total-TC concentration was lower in subjects with the TC 776 GG genotype. Transcobalamin

saturation, however, was not different among genotype groups, suggesting no effect on the

ability of TC to bind Bl2. The ratio of holo-TC to Bl2 also was significantly lower in subj ects

with the TC 776 GG genotype compared to the CC genotype, though there was no significant









difference in mean holo-TC among the groups. Because there was a significant difference in

total-TC among genotype groups but not in TC saturation there must have been some difference

in holo-TC as well, because TC saturation is the ratio of holo-TC to total-TC. Although the

differences were not significant, holo-TC was somewhat lower and TC saturation somewhat

higher in subjects with the TC 776 GG genotype compared to the CC genotype. Additionally,

because there was no difference in MMA concentration among genotype groups, even in

combination with low B l2 status, the reduced concentration of total-TC in the TC 776 CC

genotype likely has no important physiological effect on Bl2 metabolism or functional status.

Previous studies focusing on other higher risk groups, such as the individuals with low B l2

intake included in this study, also have not detected a significant effect of the TC 776 C+G

polymorphism (92, 93, 122). Fodinger et al (93) reported no significant difference in holo-TC or

Hcy concentration in end-stage renal disease patients with the TC 776 GG or CC genotypes.

Wans et al (92) compared holo-TC, Bl12, Hcy and MMA concentrations in elderly subj ects with

the TC 776 CC and GG genotypes, and reported a lower holo-TC concentration in subj ects with

the TC 776 GG genotype compared to the CC genotype, but no difference in Bl2 or MMA

concentrations. Comparing the absolute difference in mean values in the study by Wans et al. to

the current study, the differences were 4 pmol/L versus 51 pmol/L, respectively, for Bl2 (GG

mean CC mean); and 22 pmol/L versus 6 pmol/L respectively for holo-TC (GG mean CC

mean). Differences in initial Bl2 status could account for the discrepancies seen in the many

studies examining the relationship between genotype status for the TC 776 C+G polymorphism

and Bl2 status. Because such small differences in Bl2 status may overcome any negative effect

of the polymorphism, and because changes in metabolic indicators of B l2 status such as Hcy and










MMA are not consistently observed, it is unlikely that any Bl2-related metabolic change related

to this polymorphism is of clinical concern.

It is important to note that the developing embryo may be at risk of negative consequences

of metabolic changes associated with genetic polymorphisms that coexist with suboptimal

nutrient intake. A polymorphism affecting a key folate enzyme, methyl enetetrahydrofolate

reductase (MTHFR 776 C+T), is associated with a significant increase in risk for neural tube

defects, and the risk is exacerbated when folate intake is low (123-125). Again, reports of the

effect of the TC 776 C+G polymorphism on birth defect risk have been mixed (30, 94, 126,

127), but most results suggesting an increased risk for pregnant women with the TC 776 GG

genotype are not definitive. Potentially, combined effects of several polymorphisms that might

interfere with Bl2 absorption or metabolism could be physiologically important and further

investigation may be warranted based on findings from recent studies that considered several

types of birth defects (128-130).




A) TC 776 C G





35~~~ 41 4 2 57 6 8 74 7 5 9
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poyorhsmte C77C G oymrhim Ngtiedeiatvs fSyrGre
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double ~~ pek, etrzgossape










Table 4-1 Demographic distribution of subjects by genotype
TC 776 C+G genotype
CC CG GG P-value
(n = 94) (n = 139) (n = 65)
Gender > 0.05
Male 44 66 26
Female 50 73 39
Age (y) 20 & 10 26 & 7 26 & 7 > 0.05
BMIa 23.7 & 4.4 23.6 & 3.8 23.2 & 4.0 > 0.05
Ethnicity < 0.001
White 56 87 40
African American 12 3 2
Asian 5 22 15
Hispanic 17 15 4
Other 4 12 4
a Body mass index (BMI)


Table 4-2 Mean (a SD) concentrations of selected vitamin Bl12 biomarkers in all subj ects
TC 776 C+G genotype
CC CG GG
Biomarkera (n = 94) (n = 139) (n = 65)
Vitamin Bl2 (pmol/L) 280 & 119 298 & 137 331 + 145
Holo-TC (pmol/L) 87 & 56 87 & 68 81 & 85
Total-TC (pmol/L) 849 & 181 763 A 136 668 & 144a
Holo-TC/Bl2 0.34 & 0.18 0.31 & 0.22 0.25 & 0.17a
TC Saturation 0.10 + 0.07 0.11 & 0.08 0.13 & 0.13
MMA (nmol/L) 229 & 179 221 1187 205 A 128
Hcy (Clmol/L) 7.6 & 3.1 7.3 A 1.8 7.6 & 3.3
aVitamin Bl2 (Bl12); holo-transcobalamin (holo-TC); methylmalonic acid (MMA);
homocysteine (Hcy)


Table 4-3 Mean (a SD) concentrations of selected Bl2 biomarkers in subj ects with vitamin
Bl2 deficiency
TC 7776C+G genotype
Biomarkera Normal cutoff CC n CG n GG n
Bl2 (pmol/L) < 148 pmol/L 105 & 28 10 112 & 30 15 124 & 37 7
Holo-TC (pmol/L) < 35 pmol/L 28 & 8 14 24 & 7 19 23 & 7 14
MMA (nmol/L) > 270 nmol/L 505 & 280 17 461 & 340 25 420 & 240 9
aVitamin Bl2 (Bl12); holo-transcobalamin (holo-TC); methylmalonic acid (MMA);
homocysteine (Hcy)












Table 4-4 Percentage of individuals within each TC 776 C+G genotype group with
concentrations outside the normal range for select B l2 biomarkers
TC 776 C+G genotype


Biomarkera CC CG GG
Plasma Bl2 < 148 pmol/L 11 11 11
Holo-TC < 35 pmol/L 15 14 21
MMA > 270 nmol/L 18 18 14
Hcy > 12 Clmol/L 4 2 5
aVitamin Bl2 (Bl12); holo-transcobalamin (holo-TC); methylmalonic acid (MMA);
homocysteine (Hcy)


_









CHAPTER 5
HOLO-TRANSCOBALAMIN IS AN INDICATOR OF VITAMIN Bl2 ABSORPTION IN
HEALTHY ADULTS WITH NORMAL VITAMIN Bl2 STATUS

Circulating Bl2 is bound to one of two carrier proteins, haptocorrin (HC) or

transcobalamin (TC). Although the majority of Bl2 (~80%) is bound to HC (holo-HC), only TC

bound Bl2 (holo-TC) can be taken up by body cells (26). Depletion of total body Bl2 occurs

slowly, and is often a result of malabsorption, which is difficult to diagnose clinically (13, 81,

131, 132). Currently the only available diagnostic tests for vitamin Bl2 absorption are not

clinically practical. It has been hypothesized that changes in holo-TC in response to a

supplemental dose of orally administered Bl2 may be used to assess Bl2 absorption (28, 47,

104). Bor et al (20) reported a significant increase in holo-TC and TC saturation 24 and 48 hours

after receiving three 9 Clg oral Bl2 doses. Since no blood was collected before 24 hours (post

baseline), the magnitude and pattern of change of holo-TC during the first 24 hours could not be

determined (47). In developing a clinical diagnostic test, it is important to know the optimal time

post dose at which to draw blood. The objective of this study was to evaluate the post-absorption

response of holo-TC to oral Bl2 relative to other indicators of Bl2 status.

Subjects and Methods

Subj ects

Twenty one healthy adult men (n = 13) and women (n = 8) (18 to 49 y) from the

Gainesville, Florida community were selected based on the following inclusion criteria: (a)

serum B l2 concentration > 350 pmol/L at time of screening; (b) no B l2-containing supplement

use or Bl12 inj sections during past year; (c) no use of tobacco products; (d) no history of chronic

disease; (e) non-pregnant and non-lactating; (f) non-anemic (Hgb >11 g/dL [7.4 mmol/L],

females; >12 g/dL, [8.1 mmol/L] males); (g) normal blood chemistry profile; (h) BMI between

18 and 29; and (i) no blood donations within 30 days of the study.









Study Design and Data Collection

All participants signed an informed consent form approved by the University of Florida

Institutional Review Board prior to the initiation of the study. Individuals had a fasting blood

sample drawn at the University of Florida Shands General Clinical Research Center (GCRC).

Subj ects' heights and weights were measured and a medical history questionnaire was

completed. Blood analyses included serum Bl2, blood chemistry profile, hematological indices,

and a pregnancy test for women.

Eligible subj ects were admitted to the GCRC the evening before (day 0) the intervention.

The following morning (day 1) after an overnight fast, an indwelling catheter was inserted for all

blood collections during day 1. Blood samples were collected a total of 17 times starting on day

1 through day 3, and three 9 Clg B l2 doses were orally administered at six hour intervals on day

1 beginning after the baseline blood draw (Figure 1). Immediately after taking each B l2 dose,

subjects consumed a piece of bread and 236 ml (8 oz) of juice to improve absorption efficiency.

In addition to the bread and juice consumed with each Bl2 dose, subj ects were given a mid-

morning snack and lunch at 2 hours and 3.5 hours, respectively after dose 1. Dinner was fed 4

hours after dose 2, and an evening snack was provided 3 hours after dose 3. The RDA for Bl2

was provided in the diet on day 1 and on day 2. Take-home meals were provided on day 2 of the

study. Water and non-caffeinated, non-caloric beverages were allowed ad libitum. Subj ects

remained in the GCRC overnight and were allowed to leave on pass following the collection of a

fasting blood sample the morning of day 2. Subj ects returned on the morning of day 3 at which

time a final fasting blood sample was drawn.

Biochemical Analysis

At each blood collection, holo-TC, total-TC, Bl2, and plasma albumin concentrations were

determined. The ratios of holo-TC concentration to total-TC concentration (TC saturation) and









holo-TC concentration to Bl2 concentration (holo-TC/Bl2) were determined to assess changes

in these indicators in relation to one another. Additionally methylmalonic acid (MMA),

creatinine, serum folate, and homocysteine (Hcy) concentrations were measured at baseline. The

Bl2 supplement (9 Clg cyanocobalamin) was prepared by Westlab Pharmacy (Gainesville, FL).

The B l2 content of the supplement was validated by an independent laboratory (Analytical

Research Laboratories, Oklahoma City, OK).

Sample Processing and Analysis

Blood samples were collected in EDTA and SST clot activator tubes. EDTA tubes were

centrifuged at 2000 x g at 40C for 30 min to obtain plasma for Bl2 analyses. SST tubes were

centrifuged at 650 x g at room temperature for 15 min to obtain serum for holo-TC, MMA, Hcy,

and folate determination. Samples were stored at 800C in the GCRC until analysis.

Serum Bl2 and folate concentrations were assayed on the Advia Centaur automated

immunoassay system (Bayer A/S, Germany) with a total imprecision below 10%. Total TC

concentration was determined by a sandwich ELISA with a total imprecision of 4 to 6% (intra-

assay imprecision ~3%) (133). After removal of the apo-TC with Bl2 coated beads, holo-TC

was measured by the TC ELISA. The total imprecision for measurement of holo-TC was ~8%

(48), and the intra-assay imprecision was ~4% (134). Albumin and creatinine were measured on

the Cobas Integra 800 (Roche Diagnostics, Indianapolis). Total imprecision was ~2 % for

albumin and <3 % for creatinine.

Homocysteine concentration was measured by the immunological method on the

IMMUNLITE 2000 (Diagnostic Products Corporation, California) (total imprecision <6%) (135)

and MMA concentration was measured by slightly modified stable-i sotope-dilution capillary gas

chromatography mass-spectrometry (total imprecision <8%) (136).









Statistical Methods

Results are reported as mean & SD with an alpha = 0.05 unless otherwise noted. The

overall p-value for time was obtained by the F-test, which tests the null hypothesis that the

distribution of the dependent variable was the same at all time points. The Tukey method (137)

of multiple comparisons was utilized for assessment of differences between time periods. A

"Least Significant Difference" (LSD), as defined by the Tukey procedure, ensures that

simultaneously, in every target population, paired difference in means will be within +/- LSD of

the corresponding difference in sample means with 95% confidence.

Results

Mean baseline values for all analytes were within normal ranges, although some

individuals had values outside the normal range (Table 1). Plasma albumin concentration

fluctuated throughout the intervention period suggesting a change in hydration status throughout

day one and b etwe en the m boring s of day s 1, 2 and 3 (data not shown). Hol o-tran scob al ami n,

Bl2, and total-TC concentrations are reported as a ratio to albumin to adjust for diurnal changes

in overall body protein concentration due to changes in hydration status. Unadjusted means for

holo-TC, B l2, and total-TC concentrations are reported in Table 2. All time-points are reported

relative to baseline. Of all of the status indicator analytes, only holo-TC and TC saturation

changed significantly on day 1.

Mean holo-TC concentration increased steadily after baseline and fluctuated throughout

day 1. There were statistically significant increases in mean holo-TC concentration during the

first 24 hours of the intervention; however, these small increases were not maintained. Mean

holo-TC concentration reached a maximum value at hour 24, which was a significant increase

relative to baseline and all other time points (Figure 2A). The mean percent increase from

baseline also was greater at hour 24 than at all other all time points with a 49% increase relative









to baseline, and a 29% increase relative to hour 12 (Figure 3). This peak at hour 24 was

observed for almost all subj ects, with an increase of 22% or greater (22 to 85%) for all but one

subject. By hour 48, mean holo-TC concentration decreased significantly relative to hour 24

(33%); however, it was still significantly greater than baseline (Figure 5-2A).

Mean serum Bl2 concentration did not increase significantly relative to baseline on day 1,

although there were fluctuations in concentration throughout the day. At hour 24, mean serum

Bl2 concentration was significantly greater than baseline (Figure 5-2B). Overall, the percent

change in Bl2 concentration was smaller than for holo-TC throughout the intervention period

with ranges of -2 to 15% and -1 to 50%, respectively.

Mean total-TC concentration did not change significantly during the study varying less

than 6% from baseline at all time points (data not shown). Mean TC saturation began to increase

significantly relative to baseline at hour 12.5, with the most significant increase at hour 24

(Figure 2C). As observed with holo-TC concentration, the mean TC saturation and percent

change at hour 24 were significantly greater than at all other time-points with 48% and 15%

increases from baseline and hour 12.5, respectively (Figure 5-4). Among all subjects, the percent

change from baseline ranged from 7 to 104% with 19 of 21 subj ects having a value of 22% or

greater. The ratio of holo-TC to Bl2 did not increase significantly until hour 24 with absolute

and percent increases of 0. 15 and 32%, respectively. The range for percent change in this ratio

among all subj ects was -7 to 109% with 15 of 21 subj ects having an increase of 23% or greater at

hour 24.

Discussion

In this intervention study the changes in markers of Bl2 status were measured on an hourly

basis during and following administration three 9 Clg oral doses of Bl2. In previous studies the










changes in response to similar Bl2 doses were measured after 24 hours; however, no data were

collected prior to this time-point (28, 47, 104). The data from the present study indicate that a

series of three 9 Clg doses of oral Bl2, given over 12 hours, led to small fluctuations in holo-TC

concentration during the day 1 of the study followed by the previously observed maximum

increase in holo-TC concentration 24 hours after the first B l2 dose was given. There is a

similarity in the overall pattern of change in holo-TC, B l2 and TC saturation, with a gradual

increase over the first day and the most pronounced increase occurring 24 hours after the initial

Bl2 dose and 13 hours after the final Bl2 dose.

The timing of Bl12 absorption and metabolism may explain the pattern of change observed

in holo-TC concentration during the first 12 hours of the intervention. An increase in holo-TC

concentration is first measurable in the blood after 3 to 4 hours after ingestion and holo-TC can

be taken up by cells within minutes (23). It is hypothesized that until cells are saturated with

holo-TC, most of it is taken up so quickly that no maj or changes in blood levels would be

observed initially. When intake is sufficient to saturate the cells with Bl2, significant changes in

holo-TC can then be measured.

The absolute and percentage increases in Bl2 concentration were smaller, occurred later,

and were maintained longer than those for holo-TC. This finding is not surprising as total serum

Bl2 consists primarily of holo-HC, and the slower rate of HC metabolism relative to TC

metabolism leads to a slower overall turnover of serum Bl2 and a slower response to changes in

intake (26, 138). When comparing these two measures among the individual subjects, holo-TC

had the most consistent pattern with only 1 subj ect not having a change of 20% or greater at hour

24. Additionally, the mean percent change at hour 24 was three times that of Bl2. Holo-TC

concentration is clearly a more sensitive indicator of change in Bl2 intake and absorption than









serum B l2 concentration since it increases earlier after supplementation, increased relatively

more than serum B l2 and decreased earlier post-supplementation ceased.

Total-transcob alamin concentration did not change significantly during the intervention

period. Transcobalamin saturation increased in a similar manner to holo-TC (Figure 5-4). Both

holo-TC concentration and TC saturation had comparable results even when considering

individual subjects. Of all subj ects, 95% and 90% had increases of at least 22% at hour 24 for

holo-TC and TC saturation, respectively. In a previous study, a larger change in TC saturation

(at hour 24) than for holo-TC was observed, which was due to a drop in total TC at this time

point (47). No such conclusion can be made from our data since no significant difference was

observed. Since TC saturation is a calculated rather than a direct measure, the potential error in

this value is greater than that for holo-TC concentration. Therefore holo-TC concentration may

be the better indicator of Bl2 absorption.

This is the first study to monitor hourly changes in holo-TC concentration in response to

oral B l2 intake. The most significant change in holo-TC concentration occurred at hour 24,

indicating this is the optimal time post-dose at which to measure holo-TC. The three 9 Clg oral

vitamin dose sequence used in this study was used to minimize passive absorption and maximize

the amount of actively absorbed Bl2 (47, 104). This aspect of the protocol would be important

in a clinical Bl2 absorption test, because it is the capacity to actively absorb Bl2 that is being

assessed. Further studies evaluating the necessity of three doses and the exact timing of the

doses are warranted.

In conclusion, holo-TC increases measurably in response to administration of oral Bl2

within six hours with a maximum peak at 24 hours. Our results indicate that a Bl2 absorption










test based on measurement of holo-TC following three oral doses of 9 Clg Bl2 should run for 24

hours.




A Blood draw
*B12 dose


0 1 2 3 4 5 6 7 8 9 10 11 12 24 48
Time from baseline (h)

Figure 5-1 Intervention protocol timeline










Table 5-1 Baseline concentrations of B 2 status indicators
Variable Mean (a SD) Range Reference interval
Holo-TC (pmol/L) 85.2 & 38 41 208 40-150
Bl2 (pmol/L) 406.9 & 118 241 710 148-444
Transcobalamin saturation 0.12 0.05 0.27 0.05-0.20
Holo-TC/Bl12 0.22 0.08 0.44 0.15-0.51
Hcy (Clmol/L) 6.6 & 1.4 3.9 9.3 4.5-11.9
MMA (Clmol/L) 0.134 & 0.060 0.08 0.32 0.08-0.28
Folate (nmol/L) 32.7 & 7.3 22.2 54.4 >6.0
Creatinine (Clmol/L) 69 & 11.7 48 87 50 100
aVitamin Bl2 (Bl12); holo-transcobalamin (holo-TC); methylmalonic acid (MMA);
homocysteine (Hcy)










Table 5-2 Mean (a SD) concentrations of vitamin B l2 status indicators at scheduled intervals
Time from baseline (h)
Variable
0.5 1.5 2.5 3.5 4.5
Holo-TCa
84 138 85 139 89 143 91 143 96 143
(pmol/L)
Bl2
395 1 113 397 1 109 409 1 114 421 1 113 431 1 126
(pmol/L)
Total-transcob alamin
688 1 134 706 1 135 723 1 136 743 1 138 746 1 147
(pmol/L)
Time from baseline (h)
8.0 9.0 10.0 11.0 11.5
Holo-TC
95 141 96 138 96 138 97 139 99 141
(pmol/L)
Bl2
424 & 102 428 & 102 432 & 116 423 A 114 429 & 112
(pmol/L)
Total-transcob alamin
773 A 139 772 & 144 757 & 143 738 & 154 739 136
(pmol/L)
aVitamin Bl2 (Bl12); holo-transcobalamin (holo-TC); methylmalonic acid (MMA)


5.5

97 141

4141 109

763 1 144


12.5

100 139

411 +107

739 & 139


6.0

99 145

423 1 117

752 1 141


24

124 146

456 & 110

715 & 715


7.0

97 142

426 1 117

755 1 128


48

102 137

456 & 115

758 & 145











0.25


0.10
0 1 2 3 4 5 6 7 8 9 10 11 12 13 24 48

a 0.8










S1 2 3 4 5 6 7 8 9 10 11 12 13 24 48






-2 0.20




0.10
S1 2 3 4 5 6 7 8 9 10 11 12 13 24 48
B 12 dose 1 B 12 dose 2 B 12 dose 3
Time from baseline (h)


Figure 5-2 Change in vitamin Bl2 (Bl2) biomarkers during the 48 hour study period. (A) Mean
(A LSD) holo-transcobalamin (m, holo-TC) concentration relative to albumin at
scheduled intervals after oral Bl2 intake (n = 21). Holo-TC increased from baseline
at hours 6 to 7 and 11 to 48 (p < 0.001). Holo-TC increased significantly from all
other time-points at hour 24 (p < 0.001). (B) Mean (A LSD) Bl2 (*) concentration
relative to albumin at scheduled intervals after oral Bl2 intake (n = 21). Vitamin Bl2
increased significantly from baseline at hour 24 (p < 0.001). (C) Mean (A LSD)
transcobalamin (TC) saturation (V) at scheduled intervals after oral Bl2 intake
(n = 21). Transcobalamin saturation increased significantly at hours 12.5 48
relative to baseline, and hour 24 relative to all other time-points (p < 0.001).
(ANOVA, Tukey test)
































S1 2 3 4 5 6 7 8 9 10 11 12 13 24 48
B 12 dose 1 B 12 dose 2 B 12 dose 3
Time from baseline (h)

Figure 5-3 Mean (+ LSD) percent change in holo-transcobalamin (holo-TC; m) and vitamin Bl2
(Bl2; *) concentrations relative to albumin at scheduled intervals after oral Bl2
intake (n = 21). The increases in holo-TC and Bl2 from baseline to hour 24 were
significantly larger than changes at all other time-points (p < 0.001) (ANOVA,
Tukey's test)
























S15






S1 2 3 4 5 6 7 8 9 10 11 12 13 24 48
B 12 dose 1 B 12 dose 2 B 12 dose 3
Time from baseline (h)

Figure 5-4 Mean (+ LSD) percent change in transcobalamin (TC) saturation (r), and holo-
transcobalamin to vitamin Bl2 ratio (holo-TC/Bl2) (A) at scheduled intervals after
oral Bl2 intake (n = 21). There was a significantly larger percent increase in TC
saturation and holo-TC/Bl2 at hour 24 relative to all other time-points compared to
baseline (p < 0.001). (ANOVA, Tukey's test)









CHAPTER 6
DISCUSSION

Vitamin B l2 is an essential water soluble vitamin functioning as a coenzyme for two

metabolic processes, the conversion of methylmalonyl-CoA to succinyl-CoA as

adenosylcobalamin and the remethylation of Hcy to methionine as methylcobalamin (13, 35).

Absorption and utilization of Bl2 are dependent on adequate gastric HCI production to release

food-bound Bl2, IF in the intestine for active transport of Bl2 into the enterocyte, and TC for

uptake into body tissues. A deficiency of any of these components can impair Bl2 metabolism

and lead to deficiency even if dietary intake is sufficient (139).

The RDA for B l2 is 2.4 Clg for adults (140). Older adults ( > 60 y) have an increased risk

for Bl2 malabsorption due to an age related increased risk for achlorhydria and auto-immune

based pernicious anemia. The RDA for Bl2 in older adults is also 2.4 Clg/d, but it is

recommended that synthetic B l2 provided by supplements or fortified foods be the primary

source (140). Individuals with pernicious anemia are generally treated with IM Bl2 injections,

although it has been reported that passive absorption of megadoses of oral Bl2 may be sufficient

to meet dietary needs (141-143).

Vitamin B l2 is synthesized by microorganisms, present in the intestinal microflora and is

found naturally only in animal-derived foods. Consequently, individuals who restrict their intake

of some or all animal-derived foods limit their chances of consuming a diet that provides an

adequate amount of vitamin Bl2. Consumption of Bl12-fortified foods or Bl2- containing

vitamin supplements can provide sufficient B l2 for these individuals; however, it is estimated

that ~ 60 % of the US population does not take supplements (144). Although B l2 is required in

relatively small amounts, long term adherence to a Bl12-deficient diet can lead to a Bl2

deficiency and even moderate Bl2 deficiency can seriously impair health. Of greatest concern









are individuals who consume diets with restricted intakes of animal-based foods and who do not

take Bl2-containing vitamin supplements or consume Bl2 fortified foods. Studies comparing

the Bl2 status of vegetarians and omnivores have led to the conclusion that vegetarians are at

greater risk for developing a Bl2 deficiency compared to omnivores (40, 81, 86, 145); however

the maj ority of these studies have been conducted in Europe and therefore may not be applicable

to the US population. Additionally they have included both supplement users and nonusers,

making it difficult to interpret the effect of dietary Bl2 intake alone on status.

It is estimated that Bl2 intake in the US exceeds the current RDA (2.4 Clg/d) leading to the

conclusion that Bl2 dietary inadequacy is not a problem in the US (111, 146). The position of

the American Dietetic Association is that "appropriately planned vegetarian diets are healthful,

nutritionally adequate, and provide health benefits in the prevention and treatment of certain

diseases" (110). The key to this statement is that a meat-free diet must be well planned to ensure

that vitamin and mineral needs are met. The data from the current study, in addition to those

from the Framingham Offspring study, and an investigation by Bor at al. suggest that

consumption of the current RDA is insufficient to maintain normal Bl2 status in a significant

percentage of young healthy adults (118, 119). Although these data do suggest that the current

RDA is inadequate to maintain normal B l2 status, they are insufficient to provide a definitive

estimation of a new RDA. In the current study, a FFQ was used to estimate Bl2 intake. While

data generated from FFQs are adequate for obtaining information on relative frequency of

consumption of nutrients, contribution of food categories to overall intake, and estimating intake

of key nutrients, FFQs do not generate data precise or specific enough to estimate a nutrient

requirement. Future controlled metabolic studies designed to estimate the quantity of Bl2 intake









at which Bl2 status is optimal are needed since controlled metabolic studies have proven to be

highly useful in estimating other nutrient requirements (147, 148).

Future studies assessing Bl2 status need to measure multiple indicators of Bl2 status. One

strength of the current series of studies was that numerous biomarkers were used to asses B l2

status, rather than just one. Although the assessment of Bl2 status has traditionally been based

on plasma or serum B l2, approximately 5 to 10% of individuals with a plasma B l2

concentration between 148 to 221 pmol/L, have been reported to have hematological or

neurological abnormalities that responded to B l2 supplementation (44, 97). Assessment of

vitamin Bl2 status based on serum holo-TC concentration, a relatively new Bl2 status indicator,

has been reported to be an earlier marker of changes in Bl2 status than total plasma Bl2

concentration. It has been suggested that measurement of B 2 and holo-TC concentrations in

combination may be superior to either alone (27, 28, 81, 116, 149). Plasma homocysteine and

serum MMA concentrations are functional indicators of Bl2 status and are inversely related to

Bl2 concentration; however, only MMA concentration is specific for Bl2 status and is

considered by some to be the most reliable Bl2 status indicator (35, 54). There is no clear

consensus as to which particular Bl2 biomarker might be used as a "gold standard"; however,

data from the current set of studies suggest that a panel of B l2 biomarkers is preferable to any

one status indicator for Bl2 status assessment. Additionally measurements at multiple time

points over several days could help confirm a possible diagnosis ofBl2 deficiency, particularly

in the case of holo-TC, which has been reported to be highly sensitive to changes in dietary B l2

intake.

The sensitivity of holo-TC has also led to the hypothesis that it could be used to assess Bl2

absorption (47, 104). In a previous study conduced by Bor et al. (104), it was reported that









measurement of holo-TC 24 hours after administration of a series of three 9 Clg doses of oral B l2

identified individuals with Bl2 malabsorption. Individuals defined as Bl2 malabsorbers based

on the Schilling test for Bl2 absorption had no significant change in holo-TC in contrast to a

significant increase observed in normal controls. In the current set of studies, changes in holo-

TC and other markers of Bl2 status were measured hourly with administration of three 9 Clg oral

doses ofBl2, to determine whether any significant changes occur before 24 hours. A clear peak

in holo-TC concentration was observed at hour 24 for all but one of the 22 subj ects with only

small fluctuations in holo-TC prior to that. This was the first study to monitor hourly changes in

holo-TC in response to oral Bl2 suggesting a test of Bl2 absorption utilizing holo-TC should

involve measurement of holo-TC at baseline and 24 hours later. A limitation of the current study

was that only healthy individuals with normal Bl2 status were included in this investigation. It

is possible that saturation of cells might be necessary before an increase in holo-TC can be

measured even in an individual with no Bl2 absorption problems. Therefore, individuals with

low Bl2 status may need more oral Bl2 and may have a later peak increase in holo-TC

compared to individuals with normal Bl2 status. It is important to note that a Bl2 malabsorption

test would only be run in an individual with B l2 deficiency; therefore, future studies evaluating

holo-TC as a measure of Bl2 absorption needs to compare the efficacy of changes in holo-TC as

an index ofBl2 absorption in individuals with deficient versus normal Bl2 status.

A final obj ective of this series of studies was to determine the effect of specific gene-

nutrient interactions on Bl2 metabolism. Rare congenital defects known to impair Bl2

metabolism and status include various mutations and post-translational changes that result in

altering IF and TC protein structure or a total lack of protein synthesis. Congenital errors in IF

or TC lead to pernicious anemia; however errors evolving IF only impair intestinal Bl2









absorption and can be treated by lifelong IM administration of Bl2, while errors involving TC

lead to death early in life because Bl2 transport and uptake into body cells can not occur (150,

151). Perhaps less apparent than these severe genetic defects, are polymorphisms that also may

alter protein structure enough to impair function. One such polymorphism investigated in the

present investigation was the TC 776G4G polymorphism. In a previous study by our research

group a significant effect of the polymorphism on holo-TC concentration was observed but no

difference was detected in the current study. The small differences found between genotype

groups in total-TC but not TC saturation suggest some small effect of the polymorphism

however, there is likely to be no physiological impact of this polymorphism Previous studies

focusing only on the effect of the polymorphism on a developing fetus have resulted in mixed

Endings, though continued investigations related to the potential association of

Bl2-related polymorphisms and health-related consequences are warranted (128-130).

In conclusion, based on data from this series of investigations it is clear that healthy

individuals who do not take supplements may not be consuming adequate B l2 to meet biological

requirements, particularly those limiting some or all animal-based foods. Although moderate

Bl2 deficiency may not result in overt symptoms, the associated increased risk for disease and

birth defect-affected pregnancies provide an impetus for continued research focusing on

determining the optimal Bl2 intake to maintain normal status. Early findings of the potential

negative effect of the TC 776C+G polymorphism on Bl2 metabolism were not confirmed by

the current data, and any future investigations should focus on the combined effects of multiple

polymorphisms in genes involved in B l2 metabolism. Accurate detection and diagnosis of a

Bl2 deficiency and its cause will help in the prevention of related health problems including

abnormal pregnancy outcomes. Although there is yet no consensus on a single "gold standard"









test of Bl12 status, simultaneous measurement of two or more Bl2 biomarkers at several time

points may be the best diagnostic approach. If existence of a Bl2 deficiency is established,

further testing to determine if it is due to dietary insufficiency or malabsorption will aid in

determining an appropriate treatment, including changes in dietary B l2 intake and/or

supplementation. Data from the current investigations support the use of holo-TC as an indicator

of Bl2 absorption though further research is needed before a clinically reliable test could be

developed.









APPENDIX A
SUBJECT PHONE SCREENING FORM

Introduction

I am calling in regard to your interest in our nutrition study; do you have a few minutes right
now?

This is a UF Nutrition department study and involves coming in one morning for about I hour
for a fasting blood sample, we take about 1 V/2 Ounces of blood, and you only need to fast 8 hours.
We will give you a breakfast snack right afterward, and then give a brief explanation of a food
frequency questionnaire you will be taking home. You will be asked to mail it back in the
provided envelope, and once we receive the questionnaire you would get paid the $50. I just
have to ask you some questions to see if you are eligible for our study and to get background
information, OK?
How old are you? M~ust be 18-49
Do you smoke? M~ust answer no
Are you pregnant or breastfeeding? M~ust answer no
Do you take any prescription medications other than oral contraceptives? M~ust answer no

If not ~I ithrin the age range or if they answer yes to any above questions end call 11 irl.
I am very sorry, but you do not meet our exclusion criteria, but thank you for your interest.

Now I just have a few questions about your diet to see what specific category of our study
you would fit in to. Please answer as best you can, estimates are ok and consider all
instances of when you might eat the items I will ask about, even if only occasionally.

Do you take a multi-vitamin, complex, red star nutritional yeast, or any other supplement
or additive ever?

If they takettttt~~~~~~~tttttt a multivitamin,~~ttt~~~ttt~~~ B complex, red star nutritional yeast, complete the session through
all diet info only. Conclude by confirming thelir name and saysss~~~~~ssssing "This has been a preliminary
screening call, your information will be reviewed by the principal investigator based on need,
and our selection criteria at this time. If you are chosen you will be called again to schedule an
appointment over the next two weeks. Thank you very much for your interest and your time.

Do you eat breakfast cereals? (Ifso) What Kind do you eat mostly?

If they eat a 100% fortified cereal or eats a 50% cereal daily complete the through all diet info
but do not record. Conclude by confirming their name and saying "This has been a preliminary
screening call, your information will be reviewed by the principal investigator based on need,
and our selection criteria at this time. If you are chosen you will be called again to schedule an
appointment over the next two weeks. Thank you very much for your interest and your time.

If the interviewee fulfills all selection criteria continue with the questionnaire, record info on
moderate/non-fortified cereal consumption










Do you eat
o Yes
o No


breakfast cereals?


Name/Brand


Quantity


Frequency


Are you a vegan, vegetarian or meat eater?
Vegan this means you eat NO animal derived foods intentionally (if they eat small amt like in
cake then OK)
Vegetarian this means you eat NO beef, chicken, turkey, pork, or fish
How often do you eat ...


Never


Rarely
(<1 x/mo)


Occasionally
(1-4 x/mo)


Frequently
(2-4 x/wk)


Always
(5-7 x/wk)


Beef
Chicken
Turkey
Pork
Fish
Eggs
Cheese
Cow's Milk
Yogurt
Other Dairy


Do you follow a restricted diet such as


No red meat
Lactose-free
Kosher
Weight loss
Weight gain


o Low salt
0 Low fat
o Low cholesterol
o Low carbohydrate
0 Hypoallergenic


(If so) How long have you consumed this type of diet?


Have you made any major dietary changes within the last 3 years?

o No
0 Yes; How long ago did you make changes and what changes did you make?


NO YES


Do you consume alcoholic beverages?
> How often/quantity
Health Information










I am going to ask you a few questions about your health to determine if you are eligible for
our study. I will be recording this information, but it will be kept confidential and is this
ok with you?
Height: Weight:


Have you do you currently have any of the
following?
Alcoholism
Anemia
Blood clots
Bronchitis
Cystic Fibrosis
Dermatitis
Diabetes
Eating disorders/Chronic nausea or vomiting
Food allergy
Gall bladder disease
GI problems/ Lactose intolerance
Gout
Migraines
Hemorrhoids
Hepatitis/Liver disease
Heart disease/High cholesterol/High blood pressure
HIV
Kidney disease
Neurological disorder
Obesity
Seizures/Stroke
Thyroid problem
Tumors/Cancer
Ulcers
Other
Have you been hospitalized within the last 5 years?
> Cause


NO YES


Do you have a history of more than 1 miscarriage?
o Yes
o No

If you are selected to participate in this study are you willing to sign an informed consent
understanding we have access to medical information on you?
o Yes
o No


Demographic Information


What is your birth date?










Month Day Year


How would you describe your race or ethnic background?
o White
0 Black or African American
0 American Indian or Alaska Native
0 Hispanic or Latino
0 Asian
0 Native Hawaiian or Other Pacific Islander
o Other




What is the highest level of school or training that you have completed? [Circle only one
response]


Grade school
High school
Technical school or college
Graduate or professional
Don't know X


Marital status?
o Single/never married
o Married
o Separated
o Divorced
o Widowed


05 06 07 08


Are you a full-time or part-time student?
o Full time
o Part time
o Not a student


Are you employed?
o Yes
o No
o Student employee


































Name of person and phone number to call in case of an emergency if you are invited to
participate in this study:


Contact Information


Name


MI/F
Address


Last


First


Middle


Street


Apt. #


City



Day


Zip code


Phone



E-mail


Evening


Cell


If we need to contact you, and can not reach you where/with who can a message be left?






How did you hear about our study?










APPENDIX B
INTERVENTION DIET

Diet B12 Intervention Study


DAY 1
Brea kfast
3uice (apple or cranberry)
Bread


Snack
Graham crackers
Peanut butter
*Apple
*Coffee/tea
Lunch
Grilled cheese sandwich

Fruit cocktail
*Pudding
*Beverage*
Dinner
Bean burrito

Brown rice
Salad with dressing
Pears
*Beverage*
Snack
Sherbet
Pound cake
Beverage*


DAY 2
Brea kfast
Scrambled egg
Toast with jelly
Coffee/tea
uice (apple or cranberry)
Snack
Graham crackers
Peanut butter
*Apple
*Beverage*
Lunch
Pita sandwich with hummus and
veggies
Corn chips
Pineapple
*Beverage*
Dinner
Cheese tortellini with spaghetti
sauce
Green beans
Mandarin oranges
3ello
*Beverage*
Snack
*Pudding
Shortbread cookies
Beverage*


*Beverage may be Crystal Light, non-caffeinated soda, Gatorade, Hawaiian Punch
(selection to be made with the help of research staff)


Non-caffeinated, non-caloric beverages (Crystal Light, diet soda, water) available
throughout the day as desired









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Full Text

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1 VITAMIN B12 STATUS AND ABSORPTION USING HOLO-TRANSCOBALAMIN IN YOUNG MEN AND WOMEN By KRISTINA VON CASTEL-ROBERTS A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2006

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2 Copyright 2006 by Kristina von Castel-Roberts

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3 To my father Gerard David von Castel-Dunw oody and my uncle Gnter von Castel; they left this world too early but will live in my heart forever.

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4 ACKNOWLEDGMENTS I would like to thank my committee members, doctors Lynn B. Bailey, Gail P.A. Kauwell, Jesse F. Gregory III, and Lee McDowell for their guidance and support. I would particularly like to thank Dr. Bailey and Dr. Kauwell for their dail y encouragement during th is exciting endeavor. Their dedication and achievement in the field of science has given me high standards to follow and has driven me to push myself to the best of my potential and beyond. I would like to thank Dr. Gregory for the contribution of his scientif ic and technical knowledge and Dr. McDowell for making me aware of how my animal nutrition education can help me better understand human nutrition. I would like to express my gratitude to the members of our laboratory team, especially David Maneval, Amanda Brown, Claire Edgem on, and Dr. Karla Shelnutt. It was their combined effort that made it possible to succ essfully conduct two human studies, teaching me the value of teamwork. I owe tremendous thanks to my friends a nd family who supported me through the tough spots, whether a few miles or a few hundred miles away. Their help and support allowed me to keep my farm standing and my horses healt hy, plan my wedding, get married, escape on my honeymoon, and still have fun while pursuing my degree. I would partic ularly like to thank those who have been with me from the start of my Gator career, Karen Knight, Christina Stortz and Tiffany Tooley, and my friend turned-siste r Revati Roberts. Spec ial thanks go to my mother and father Alice von Castel Dunwoody and Gerard von Castel Dunwoody, for their unending and unconditional love an d support, and for believing I c ould do anything I truly set my mind to. Finally, my deepest love and a ppreciation go to my husba nd and best friend Nando David Roberts for the continuous support, love, and devotion he has given me.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........8 LIST OF FIGURES................................................................................................................ .........9 LIST OF ABBREVIATIONS........................................................................................................10 ABSTRACT....................................................................................................................... ............12 CHAPTER 1 INTRODUCTION..................................................................................................................14 Vitamin B12.................................................................................................................... ........14 History........................................................................................................................ .....14 Chemistry...................................................................................................................... ..15 Nomenclature for B12 Binding Proteins.........................................................................15 Absorption..................................................................................................................... ..16 Transport and Cellular Uptake........................................................................................16 Storage and Turnover......................................................................................................17 Biochemical Reactions....................................................................................................18 Daily Requirement...........................................................................................................18 Dietary and Supplemental Sources..................................................................................19 Vitamin B12 Status Assessment.............................................................................................21 Vitamin B12 Concentration.............................................................................................21 Holo-transcobalamin Concentration................................................................................22 Methylmalonic Acid Concentration................................................................................22 Homocysteine Concentration..........................................................................................23 Vitamin B12 Deficiency.........................................................................................................24 Etiology....................................................................................................................... ....24 Clinical A bnormalitie s.....................................................................................................24 Vegetarianism..................................................................................................................25 Gene-Nutrient Interactions.....................................................................................................27 Malabsorption of Vitamin B12...............................................................................................28 Overall Rationale.............................................................................................................. ......29 Hypothesis # 1.................................................................................................................30 Hypothesis #2..................................................................................................................31 Hypothesis #3..................................................................................................................31 Hypothesis #4..................................................................................................................31

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6 2 VITAMIN B12 STATUS IS IMPAIRED IN A SUBGROUP OF HEALTHY YOUNG VEGETARIAN AND OMNIVOROUS ADULT MEN AND WOMEN..............................35 Subjects and Methods........................................................................................................... ..35 Subjects and Subject Recruitment...................................................................................35 Study Design and Data Collection..................................................................................36 Sample Processing...........................................................................................................37 Competitive Binding Assays of Serum Ho lo-transcobalamin and Plasma B12..............37 Measurement of Serum Homocystei ne and Methylmalonic Acid..................................38 Diet Analysis.................................................................................................................. .38 Subject Dietary Intake Classification..............................................................................39 Statistical Methods..........................................................................................................39 Results........................................................................................................................ .............40 Discussion..................................................................................................................... ..........41 3 VITAMIN B12 INTAKE AT THE CURR ENT RDA LEVEL IS NOT OPTIMAL.............47 Subjects and Methods........................................................................................................... ..47 Subjects and Subject Recruitment...................................................................................47 Study Design and Data Collection..................................................................................47 Sample Processing and Analysis.....................................................................................48 Diet Analysis.................................................................................................................. .48 Statistical Analysis..........................................................................................................49 Results........................................................................................................................ .............49 Discussion..................................................................................................................... ..........51 4 GENOTYPE FOR THE TRANSCOBALAMIN 776C G POLYMORPHISM IS NOT ASSOCIATED WITH ABNORMAL VITA MIN B12 STATUS BIOMARKERS IN HEALTHY ADULTS.............................................................................................................57 Subjects and Methods........................................................................................................... ..57 Subjects and Subject Recruitment...................................................................................57 Study Design and Data Collection..................................................................................58 Sample Processing and Analysis.....................................................................................58 Genotype Determination.................................................................................................59 Diet Analysis.................................................................................................................. .59 Statistical Methods..........................................................................................................60 Results........................................................................................................................ .............60 Discussion..................................................................................................................... ..........61 5 HOLO-TRANSCOBALAMIN IS AN INDICATOR OF VITAMIN B12 ABSORPTION IN HEALTHY ADULTS WI TH NORMAL VITAMIN B12 STATUS.....66 Subjects and Methods........................................................................................................... ..66 Subjects....................................................................................................................... .....66 Study Design and Data Collection..................................................................................67 Biochemical Analysis......................................................................................................67

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7 Sample Processing and Analysis.....................................................................................68 Statistical Methods..........................................................................................................69 Results........................................................................................................................ .............69 Discussion..................................................................................................................... ..........70 6 DISCUSSION..................................................................................................................... ....79 APPENDIX A SUBJECT PHONE SCREENING FORM.............................................................................85 B INTERVENTION DIET.........................................................................................................90 LIST OF REFERENCES............................................................................................................. ..91 BIOGRAPHICAL SKETCH.......................................................................................................103

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8 LIST OF TABLES Table page 2-1 Characteristics of study groups..........................................................................................44 2-2 Mean dietary vitamin B12 intake and stat us of omnivorous and vegetarian adults..........45 2-3 Cross-tabulation of vita min B12 status of subjects based on select biomarker combinations................................................................................................................... ...46 3-1 Subject Characteristics.................................................................................................... ...53 3-2 Correlations between vitamin B12 inta ke and concentrations of B12 status biomarkers..................................................................................................................... .....53 3-3 Proportion of individuals with concentra tions outside the norm al range for select vitamin B12 status biomarkers...........................................................................................56 4-1 Demographic distributio n of subjects by genotype...........................................................64 4-2 Mean concentrations of selected vi tamin B12 biomarkers in all subjects.........................64 4-3 Mean concentrations of selected B12 biomarkers in subjects with vitamin B12 deficiency..................................................................................................................... ......64 4-4 Percentage of indivi duals within each TC 776 C G genotype group with concentrations outside the normal range for select B12 biomarkers.................................65 5-1 Baseline concentrations of B12 status indicators...............................................................74 5-2 Mean concentrations of vitamin B12 st atus indicators at scheduled intervals..................75

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9 LIST OF FIGURES Figure page 1-1 Structure of vitamin B12................................................................................................... .32 1-2 Overview of vitamin B12 absorption.................................................................................33 1-3 Role of vitamin B12 in the remethylation of homocysteine..............................................34 2-1 Percent of vegetarian and omnivorous adults with select B12 biomarker concentrations outside the normal range............................................................................45 2-2 Frequency of single versus combined v itamin B12 status biomarkers being outside the normal range............................................................................................................... .46 3-1 Total daily vitamin B12 intake in individuals with select B12 biomarker concentrations outside the normal range............................................................................54 3-2 Relationship between vitami n B12 intake and status........................................................55 4-1 Melting curve plots for Dynamic Allele Specific Hybridiz ation analysis of polymorphism the TC 776C G polymorphism...............................................................63 5-1 Intervention protocol timeline............................................................................................73 5-2 Change in vitamin B12 biomar kers during the 48 hour study period................................76 5-3 Mean percent change in holo-transcoba lamin and vitamin B12 concentrations at scheduled intervals af ter oral B12 intake...........................................................................77 5-4 Mean percent change in transcobalam in saturation and holo-transcobalamin to vitamin B12 ratio at scheduled in tervals after oral B12 intake..........................................78

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10 LIST OF ABBREVIATIONS 5-CH3-THF 5-methylterahydrofolate AI Adequate Intake ANOVA Analysis of variance Apo-HC Free haptocorrin Apo-TC Free transcobalamin B12 Vitamin B12 BMI Body mass index CH3 Methyl CN Cyano d day DHQ National Cancer Institut e Dietary History Questionnaire EAR Estimated Adequate Requirement EDTA Ethylenediaminetetraacetic acid FFQ Food frequency questionnaire HC Haptocorrin Holo-HC Holo-haptocorrin Holo-TC Holo-transcobalamin HCl Hydrochloric acid Hcy Homocysteine IF Intrinsic factor IM Intramuscular mo Month min Minutes

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11 MS Methionine synthase nmol/L Nanomoles per liter NTD Neural tube defect OH Hydroxyl OSC Optimal Solutions Corporation pmol/L Picomoles per liter RDA Recommended dietary allowance TC Transcobalamin TC-R Transcobalamin receptor s Seconds SAM S-adenosylmethionine SD Standard deviation SST Serum separator tube UL Upper limit US United States mol/L Micromoles per liter y year

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12 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy VITAMIN B12 STATUS AND ABSORPTION USING HOLO-TRANSCOBALAMIN IN ADULT MEN AND WOMEN By Kristina von Castel-Roberts December 2006 Chair: Lynn B. Bailey Major Department: Food Science and Human Nutrition Vitamin B12 (B12) status of young adults has been considered adequate based on estimated intakes that met the RDA; however, few studies in the US have evaluated B12 status of young adults using a panel of B12 biomarkers. Vitamin B12 deficiency impairs neurological function and increases other health -related risks. Early detecti on and determination of whether B12 deficiency is due to diet ary insufficiency, a genetic abno rmality, or malabsorption are critical to effective treatment. The aims of the first study were to compar e B12 status using numerous biomarkers in young adult non-supplement users consuming vegeta rian and omnivorous diets, determine the level of intake associated with optimal B12 status, and determine if the transcobalamin (TC) 776C G polymorphism affected B12 metabolis m. Blood samples were collected for determination of holo-TC, B12, methylmalonic aci d (MMA), and homocysteine (Hcy) (n = 388). Dietary B12 intake was assessed using a food fr equency questionnaire. A surprisingly high incidence of B12 deficiency was observed in bo th vegetarians and omnivores. Relative to omnivores, vegetarians had a higher rate of B 12 deficiency, with lower B12 and higher MMA concentrations. Vitamin B12 stat us improved with B12 intake a bove the RDA. No differences were detected between TC 776C G genotypes for any biomarkers.

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13 In the second study the magnitude and pattern s of post-absorption ch anges in several B12 biomarkers were assessed. Subjects (n = 21) had blood drawn at 17 intervals over three days with administration of three 9 g doses of B12 at 6 hour inte rvals on day one. Mean B12, holoTC, TC saturation, and the ratio of holo-TC to B12 increased significantly from baseline at hour 24 only. In conclusion, a high incidence of impaired B 12 status was observed in otherwise healthy young adults. The data suggest th at further assessment of the adequacy of the B12 RDA is warranted. Measurement of multiple B12 biomarkers may provide a more accurate assessment of B12 status than measurement of one biomarker alone. Holo-transcobalamin appears to be a sensitive indicator of B12 absorption and a holo-TC based absorption test should involve measurement at 0 and 24 hours. No effect of the TC 776C G polymorphism was detected.

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14 CHAPTER 1 INTRODUCTION Vitamin B12 History Vitamin B12 (B12) is one of the thirteen esse ntial vitamins that humans must obtain from their diet. Vitamin B12 was the last vitamin to be discovered, due in part to the lack of a suitable animal model in which to study the B12-related disease pernici ous anemia (1). Pernicious anemia, which literally means fatal anemia, has been reported in medical records as far back as the early 1800s, although the condi tion was likely responsible for deaths well before then. The earliest studies of patients with pernicious anemia led to the know ledge that the disease was due to some ailment of the stomach; although no treatm ent was available and most patients died from the disease (2). In the early 20th century, Minot and Murphy determ ined that feeding liver to patients with pernicious anemia improved thei r condition, a discovery for which they received the Nobel prize in 1934 (3). Castle conducted a series of experiments comparing the treatment of pernicious anemia patients with partially dige sted beef, or beef inc ubated in gastric juice, versus undigested beef. Patients receiving the pre-digested beef improved while those receiving undigested beef did not, suggesting that some (intrinsic) factor w ithin gastric juice interacted with the unidentified (extrinsic) factor in the beef (4-7). The final identification of this extrinsic factor was delayed until 1945 when it was discovered that the anti-anemia substance was required by Lactobacillus locus finally providing a useful labo ratory model (8). Vitamin B12 was crystallized in 1948 and was qui ckly identified as th e illusive extrinsic anti-anemia factor (9-12). After these discoveries, B12 research proceeded rapidly as did our understanding of the vitamins functions.

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15 Chemistry Vitamin B12 is a complex water-soluble mo lecule with a molecular weight of 1655.38 daltons. The molecule is comprised of a c obalt atom centered in a corrin ring, with two coordinating ligands. The 5,6-dimethylbenz imidazole component is linked to the -axial position of the cobalt, and a vari able ligand is linked to the -axial position (F igure 1-1) (13). Known ligands include CH3 (methylcobalamin), 5-deoxyade nosyl (adenosylcobalamin), OH (hydroxylcobalamin), and CN (cyanocobalamin). Cyanocobalamin, the synthetic form of B12 included in vitamin supplements and fortified foods, is converted to methylcobalamin or adenosylcobalamin, the two coenzyme forms of the vitamin. Methylcobalamin is the primary form found in human plasma making up 60 to 80% of total cobalamins (14). Nomenclature for B12 Binding Proteins The nomenclature for B12 binding proteins in the gastrointestinal tract and plasma has changed over time, and both the new and old terms are used in current literature. The term Rprotein was originally used to differentiate B12 binding proteins, which move rapidly upon electrophoresis, from intrinsic fact or (IF), which moves slowly. In testinal R-protein, now termed haptocorrin (HC) because of its ability to bind corrins other than B12, is also found in saliva, bile, and plasma. Transcobalamin (TC) I, II, and III were terms used to identify the plasma B12 binding proteins; however, further investigation pr oved that TC I and III we re isoforms of HC, which differed only by carbohydrate content. The te rm transcobalamin II was used to identify the B12 binder that participated in delivery of B 12 to cells, but now it is s imply referred to as TC (15).

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16 Absorption The absorption of B12 is primarily an active receptor mediated process that uses several different transporters (Figure 1-2). Vitamin B12 is bound to proteins in foods and must be liberated by the action of pepsin and hydrochlor ic acid (HCL) in the stomach in order for absorption to occur. Reduced gastric pH, as ofte n seen in adults over the age of 50 y and with chronic antacid use, impairs brea kdown of the protein matrix and ultimately results in reduced B12 absorption (16, 17). Once B12 is released fro m the protein matrix, it binds to HC enabling it to travel to the duodenum where pancreatic pr oteases degrade HC. In the duodenum, free B12 binds to IF, a glycoprotein synt hesized and secreted from gast ric parietal cells. The IF-B12 complex is resistant to attack from pepsin, c hymotrypsin, and intestinal bacteria, allowing the complex to travel to the ileum intact, where it is transferred across the ileal epithelium viareceptor mediated endocytosis. This ileal recep tor (cubilin) only recognizes the IF-B12 complex, so that free B12 can not cross the membrane in this manner (18, 19). Although passive diffusion of B12 across the epithelium does occur at a rate of 1% of any B12 dose, B12 is primarily absorbed by active transport (14). Once in the enterocyte, IF is degraded by the lysosome. Transcobalamin plays an essential role in B 12 absorption, binding B12 at some point after release from IF and appearance in the blood as holo-TC. The exact mechanism by which B12 binds to TC is under debate, however, it is hypot hesized that binding occurs in the enterocyte (20-22). Holo-transcobalamin can be detected in the blood 3 hours af ter B12 intake, with maximum absorption occurring 8 to 12 hours after intake. Cellular up take occurs within minutes (23, 24). Transport and Cellular Uptake Transport of B12 in circulatory system and into the cells of target tissues is dependent on two binding proteins, TC and HC. Each protein has only one bindi ng site with a high affinity

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17 (Kd = 10-10 to 10-17) for the various chemical forms of B12 (25). Transcobalamin is a 43 kDa non-glycosylated protein found in plasma and in various cells in cluding endothelial cells (22). Numerous tissues contain TC mR NA, including kidney, heart, live r, and leukocytes; however the specific cell type in which TC is synthesized is unknown (26). Transcobalamin is required for transport of B12 into the cell since only the B12 bound to TC is taken up by cell surface receptors. For this reason, the holo-TC fraction of serum B12 is the only component that is considered biologically active (27-29). Th e TC receptor (TC-R) is a 50 kDa heavily glycosylated protein, that binds both holo-TC and apo-TC (TC with no B12 bound) (26). In the cytoplasm, lysosomes break down the holo-TC co mplex making free B12 available for metabolic processes. Holo-transcobalamin constitutes on ly 20% of plasma B12, the remaining 80% is bound to HC (13, 30). Haptocorrin is a heavily-g lycosylated, 70 kDa protein found in various biological fluids including saliva, bile, and bloo d. Although the majority of plasma B12 is bound to HC (holo-HC), holo-HC cannot be used by the cells as there are no rece ptors for this complex (15). The function of HC is unclear and is still debated (15, 31, 32). Storage and Turnover The main storage tissues for B12 are the li ver and muscle, which contain approximately 60% and 30%, respectively, of the bodys total B12. High concentrations also are found in the pituitary, kidney, heart, spleen and brain. Inte restingly, although B12 is enzyme bound in most tissues, the kidney maintains a pool of free B12, which can be used to maintain plasma B12. When B12 intake is high, uptake of B12 in to the kidney increases; when plasma B12 concentrations are low, B12 is released first from the kidney (33, 34). Mean total body stores range from 2 to 5 mg with a half life of 340 to 400 days (14). Vitamin B12 is excreted only in the free form in the urine and bile at a rate of 0.1 to 0.2% (2 to 5 g) of total body reserves per day (35). Enterohepatic recirculatio n is very efficient and

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18 helps reduce total loss of B12 (13). Up to 75% of biliary B12 is actively re absorbed in the ileum, so that very little is excreted in the feces, effectively conservi ng this essential nutrient (14). Additionally, because the kidney is rich in TC -R, B12 stored in the kidney is efficiently reabsorbed back into circul ation reducing urinary losses. Biochemical Reactions In humans and other higher animals, B12 serves as a coenzyme for two metabolic processes, the conversion of me thylmalonyl-CoA to succinyl-CoA as adenosylcobalamin and the remethylation of homocysteine (Hcy) to methi onine as methylcobalamin (13, 35). In succinylCoA synthesis, adenosylcobalamin undergoe s homolytic cleavage by the action of Lmethylmalonyl CoA mutase forming cob(II)alami n and a 5'-deoxyadenosyl radical. Radical formation allows the rearrangement of the Lmethylmalonyl-CoA molecule to form succinylCoA. The Hcy remethylation process is an im portant component of overall one carbon metabolism. Methionine synthase (MS) catalyzes the remethylation of Hcy to methionine with the associated B12 acting as a methyl carrier. Methionine synthase c ontains separate binding domains for Hcy, 5-methyltetrahydrofolate (5-CH3THF), B12, and S-adenosylmethionine (SAM). Vitamin B12 in its reduced active st ate as cob(I)alamin is remethylated by 5-CH3THF and the methyl group can again be donated to Hcy (Figure 1-3). Methionine is of great biological importance because it is the precurs or of SAM the major methyl donor in over 100 biochemical reactions (36, 37). Daily Requirement The Dietary Reference Intakes (DRI) for esse ntial nutrients are guidelines for estimating the average vitamin and mineral intake needed to maintain health ( 38). The DRIs include Estimated Average Requirement (EAR), Ade quate Intake (AI), Recommended Dietary

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19 Allowance (RDA) and Tolerable Upper Intake Leve l (UL). The RDA for nut rients is calculated from the EAR and is defined as the average daily intake required to meet the needs of most individuals in the defined age br acket (38). The daily requirement of B12 is relatively low in comparison to other essential vitamins, with an EAR and RDA for adult men and women (19 to 50 y) of 2 g/d and 2.4 g/d, respectively. Estimates for the EAR for adults are based on the level of intake needed to maintain normal he matological status and a serum B12 concentration above 150 pmol/L minus the amount conserved in daily B12 turnover. Data were gathered primarily from patients with pernicious anem ia in remission who were receiving regular intramuscular (IM) injections of B12. Studies of patients with pernicious anemia reported that IM doses of 0.8 to 1.7 g/d were sufficient to ma intain normal hematological parameters. From these studies an average of 1.5 g/d was estim ated to be the B12 requirement. The final calculation of the EAR in hea lthy adults with normal B12 ab sorption was calculated as 1.5 g/day minus 0.5 g/d (the estimated amount of B12 reabsorbed) with a correction to account for an estimated bioavailability of 50% (38). The RDA was then calculated as the EAR (2.0 g/d) plus twice the coefficien t of variation (CV) for 97 to 98% of the population, or 120% of the EAR. Recommendations for children are based on B12 concentrations in milk for infants and are extrapolated down from adult requirement s for children up to age 18 y. Adults over the age of 50 y have the same RDA as younger adults ; however, due to an ag e-related reduction in gastric pH, it is recommended that older adults ob tain most of the RDA from fortified foods and B12 supplements (23). Dietary and Supplemental Sources Vitamin B12 is synthesized only by microorganism s in bacteria rich environments such as the intestinal tracts of animals. Some species ha ve sufficient microbial s ynthesis of B12 to meet

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20 their biological need without any additio nal dietary B12. Although humans have B12 synthesizing bacteria in their in testinal tract, they ar e primarily found in the colon where little absorption takes place making B12 an essential nut rient for humans. Natural dietary sources of B12 are limited to foods of animal origin. Th e liver and kidney stor e large amounts of B12, therefore, these organ meats are the richest dietary sources of B12 (24 to 122 g/100 g). Other more commonly consumed food sources are red meat (0.55 to 3.64 g/100 g), poultry (0.32 to 0.379 g/100 g), fish (1.9 to 21.2 g/100 g), eggs (0.09 to 9.26 g/100 g), and milk products (0.06 to 1.71 g/100 g) (14). Vitamin B12 al so can be obtained in the following: (a) supplements; (b) B12-fortified foods such as cereals and meal re placement bars and drinks; (c) B12fortified vegetarian products such as soy milk; and (d) B12fortified meat substitutes and frozen meal entrees made from wheat gluten or soybeans; and some fermented food products (39). In the US, foods of animal origin are a common part of the diet, and the estimated average daily intake of B12 from food sources is 3 to 5 g/d on average (38). The largest percentage of dietary B12 in the US diet comes from mixed foods (16 to 19%) which includes non-beef meats, poultry, and fish; a substantial por tion also comes from beef (12 to 15%), and milk products (11 to 15%). In the case of vegeta rians, who are estimated to repr esent up to 25% of US women of reproductive age, dairy and egg products for laco to-ovo-vegetarians and da iry products for lactovegetarians, are the sole sources of B12 if supplements or for tified foods are not consumed. Individuals who exclude some or all animal -derived foods and do not add B12 fortified foods to their diet are at increased risk fo r developing B12 deficiency (40-42). Non-meat animal-derived sources of B12 including dairy pr oducts and eggs can contribute significantly to B12 intake (38), but are excluded from the diets of strict vegeta rians (vegans). The extent to

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21 which animal-derived foods are excluded from the diets of self-described vegetarians may determine the effect on B12 status. For example, one serving (4 oz) of beef can provide the RDA for B12 (2.4 g/d), while one serving of chicken (4 oz) provides only 12% of the RDA. The B12 content of fish varies by species; 1 se rving (4 oz) of grouper provides 25% of the RDA, while tuna and herring provide as much as 500% of the RDA per serving (1 to 4 oz). Dairy products also provide variable amounts of B12 w ith 8 to 50% the RDA per serving (2 to 8 oz) (39). Vitamin B12 Status Assessment Vitamin B12 Concentration In clinical settings, serum B12 determin ation is the primary method for assessing B12 status (35). In the genera l US population, the mean serum B12 concentration for healthy individuals over four years of age is 381 pmol/L (43). Reliance on serum B12 as the sole diagnostic tool may lead to a false diagnosis since not all individuals with low values are deficient and a normal serum concentration may or may not indicate adequa te B12 status (44). This is due in part to the manner in which B 12 is metabolized and stor ed in the body. Because some tissues, such as the liver and kidney, can store a relatively large amount of B12, total body depletion takes years; however, some cells with lower storage capacity may become B12 deficient while circulating B12 is still in the low normal range. In these cells, B12 dependant enzyme function may become impaired causing elevated MMA and Hcy. Currently, clinical B12 deficiency is classified as serum c oncentrations < 148 pg/mL; however, a significant percentage of patients with cl inical symptoms of B12 defici ency who respond to B12 therapy have serum B12 concentrations in the low-nor mal (148 to 221 pg/ml) range. To enhance the diagnostic value of serum B12 concentrations, a dditional status indicators should be evaluated.

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22 Holo-transcobalamin Concentration Measurement of holo-TC is considered a functional indicator of B12 status because only the B12 bound to TC can be taken up by cell receptor s for use in intracellu lar metabolic reactions (27, 28, 45, 46). In contrast, serum B12 consists of holo-TC (~20%) and holo-HC (~80%), the latter can not be used by cells since it lack s known cellular receptors. Evidence supports the conclusion that holo-TC concentr ation responds more rapidly to changes in B12 intake than other indices of B12 status ( 28). Bor et al. (47) reported that oral B12 treatment (400 g/d) resulted in a highly significant maximal increase (+ 54%) in holo-TC after 3 days, in contrast to serum B12, which responded with a smaller initia l change (+28%) and a sl ower graded increase over time. Routine measurement of holo-TC as an index of B12 status is now possible since technical problems associated with the analytical procedure have been successfully addressed (Holo-TC RIA, Axis-Sheild) (45, 48). Loikas et al. (49) confirmed the su itability of the holo-TC RIA for use in a clinical laboratory, determin ed reference values for the method (37 to 171 pmol/L), and confirmed that low holo-TC concentrations (< 35 pmol/L) were associated with other biochemical indicators of low B12 status. Methylmalonic Acid Concentration When B12 status is low the conversion of methylmalonyl-CoA to succinyl CoA is impaired; as methylmalonyl-CoA accumulates, it is converted to methyl malonic acid. This alteration in metabolism results in a measur able increase in MMA. Methylmalonic acid concentration is a highly specific diagnostic i ndicator of B12 status because, unlike the MS reaction that requires both B12 and folate, no ot her nutrient is required for the methylmalonyl CoA mutase reaction (50-52). A nor mal serum MMA concentration is 271 nmol/L, with reported reference ranges for serum MMA concentr ation of ~50 to 400 nmol/L (53, 54). Serum MMA concentration also provides diagnostic info rmation when it is obtained before and after

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23 B12 supplementation in B12 deficient individua ls. Moelby et al. ( 52), like previous investigators, reported a marked decline in se rum MMA concentration to normal one month after treatment with B12 (55). Homocysteine Concentration Homocysteine concentration is inversely associated with B 12 status, and may or may not be elevated in individuals with low B12 status. Individuals that have el evated Hcy due to a B12 deficiency will respond to B12 supplementation (55, 56). Traditionally the cut-off for normal Hcy concentration has been 14 mol/L; however, due to the im plementation of mandatory folate fortification in the US, Hcy concentr ations within the popul ation have decreased significantly (57, 58). In a populat ion-based study, Selhub et al. ( 56) reported that plasma Hcy concentration was inversely associated with B12 status, and mean Hcy concentration was significantly higher in indi viduals in the lowest compared to the highest decile for plasma B12 concentration (15.4 and 10.9 mol/L, respectively). Mezzano et al. (59) evaluated plasma Hcy concentrations and response to B12 therapy in a group of vegetarians with low B12 status (baseline mean serum B12 concentration 110 pmo l/L) with elevated plasma Hcy concentration. Following intramuscular injection with B12, seru m B12 concentration incr eased to 392 pmoL/L and mean plasma Hcy concentration dropped significantly (12.4 to 7.9 mol/L). Unlike MMA, Hcy concentration is not a specific indicator of B12 status. Because the folate derivative, 5-CH3THF is the methyl donor in the conversion of Hcy to methionine, low folate status also can lead to an elevation in Hcy concentration.

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24 Vitamin B12 Deficiency Etiology Vitamin B12 deficiency may occur due to dietary restriction, malabsorption, or disturbances in transport or cellular uptake. Malabsorption of B12 can be caused by several physiological and congenital defects. Pernicious anemia is a dis ease of the autoimmune system in which antibodies to parietal cells and IF develop leading to a complete lack of IF and an inability to absorb B12. Individu als with this condition can be gi ven IM B12 injections to meet B12 requirements, bypassing the absorption process (35). The elderly popul ation is at a higher risk of B12 malabsorption due to the age-re lated decrease in stomach acid. The acidic environment in the stomach is required for th e release of B12 from food, and a significant decrease in hydrochloric acid can impair the process leadi ng to increased excretion and decreased absorption. In such cases the daily B12 requirement must be met with supplemental B12 (35). Clinical Abnormalities Depleted B12 status may take years to develop in individuals with im paired absorption or inadequate intake and an indivi dual may have marginal B12 stat us prior to developing severe clinical symptoms such as megaloblastic anemia and irreversible neurological damage (32). Development of B12 deficiency begins with depletion of serum B12, followed by cellular deficiency and biochemical changes including elevated Hcy and MMA concentrations (32). Neurological abnormalities affecting physical reflex es, stamina, and mental attributes including memory and behavioral changes may accompany a moderate B12 deficiency (60-62). In addition, the risk of inadequate B12 intake to a developing fetus, should pregnancy occur, is of great concern for women of repr oductive age. Infants born to mo thers with a B12 deficiency have been reported to suffer devastating symp toms including growth retardation, delayed

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25 psychomotor development, and in some instan ces, permanent effects on the brain (63-66). A B12 deficiency may increase the risk for birth defects as illustrated by the well documented independent role for B12 in the etiology of neur al tube defects (NTDs) (67-73). These studies have provided evidence that even small reducti ons in serum B12 concentrations within the normal range may be associated with a significan tly increased risk for NT Ds. Afman et al. (74) measured the plasma concentrations of B12, Hc y, and the apoand holoforms of TC in NTD case mothers and in control women. Low plasma holo-TC concentration wa s associated with a 3-fold increased risk for having a child with an NTD, while a low percentage of B12 bound to TC (TC saturation) was associated with a 5-fold increased risk. Vegetarianism Vegetarians are at increased risk for deve loping a B12 deficiency since B12 is only naturally present in animal-derived products (i.e., meat, eggs, dairy). Th ere is much variability in the amount of B12 consumed by individuals characterized as vegetarians. This classification includes those who consume diet s completely devoid of all animal-derived products (vegans), including meat, fish, dairy, an d eggs, as well as those who exclude meat but consume either dairy products (lacto-vegetarians) or dairy and eggs (lactoovo-vegetarians) (75). Approximately 2.5% of the entire US adult population (4.8 million people) report consumption of vegetarian diets, and approxi mately 1% report consuming vega n diets (75-77). There is an increasing trend for the younger segment of the popul ation to consume vegetarian diets (75). In a nationally-representative survey (75), the numbe r of self-defined vege tarians who reported no meat consumption was highest in the 20 to 29 y ear age group and was two to three times higher than that of 50 to 59 and 60 to 69 year old i ndividuals, respectively. This increasing trend in consumption of vegetarian diets is esp ecially prevalent among young adult women of

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26 reproductive age, documented by survey data in dicating that 20 to 25% of this group follow some type of vegeta rian diet (78). Multiple reports provide evidence that all ve getarians including lacto-vegetarians and lacto-ovo-vegetarians are at in creased risk for developing a B12 deficiency compared to omnivores (79-81), supporting the co nclusion that a vegetarian does not have to be a strict vegan for B12 status to be impaired (32, 81-85). Im paired B12 status may lead to elevated Hcy concentrations and increased risk for cardiovas cular disease, cancer and birth defect-affected pregnancies. A number of st udies indicate that vegetarians have significantly higher Hcy concentrations than omnivores a nd that the consumption of a vege tarian diet may be associated with elevated Hcy concentrations (59, 80, 86). For example, Mezzano et al. (59) reported that the Hcy concentration was 41% higher in vege tarians than in omnivor es and that Hcy was inversely related to serum B12 concentration. In a study compar ing B12 status of Taiwanese vegetarians and non-vegetarians, Hu ang et al. (87) reported that vegetarians had higher plasma Hcy concentrations than non-vegetarians (13.2 vs. 9.8 mol/L, respectively) and that serum B12 concentration was a strong pred ictor of plasma Hcy concentr ation. Another similar study conducted in a European population reported that Hcy concentration was significantly higher (11.6 mol/L) in a group of vegetarians compared to omnivorous controls (9.8 mol/L) and that the Hcy concentration increased as the vegetarian diet became more rest rictive, with vegans having the highest values (86). In the only study reported to date in which the B12 status of young adult vegans in the US has been evaluated, Carmel et al. (88) found that elevated Hcy concentration associated with dietary inade quacy of B12 was a major problem in young Asian Indian medical students with hyperhomocystein emia occurring in 25% of group. This study

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27 illustrates that well-educated young adults ar e among the vegetarians in the US whose consumption of a B12-deficient diets has been asso ciated with an elevation in Hcy concentration. Gene-Nutrient Interactions Transcobalamin 776C G The most common polymorphism affecting the TC B12 transport protein is a C G base substitution in DNA at base pair 776, which results in the substitution of pro line with arginine at codon 259 (74, 89). The estimated prevalence of the TC 776 CC, CG and GG genotypes are approximately 20%, 50%, and 30%, respectivel y (74, 89, 90). Our research group recently estimated the distribution (27% CC; 49% CG; 24% GG) of the TC 776 polymorphism in a large population group of young women for whom B12 stat us was previously reported, which is in agreement with previous studies (91). The potential influence of the TC 776 C G polymorphism on indices of B12 status has been investigated by several research groups (74, 89, 90). Afman et al. (30) found lower holoTC, total TC, and holo-TC/total-TC ratios in in dividuals with either the TC 776 GG or CG genotypes compared to those with the CC genotype. Miller et al. (89) reported a lower mean holo-TC concentration, a lower percent of to tal B12 bound to TC, and a higher mean MMA concentration in elderly subj ects with the TC 776 GG compar ed to the CC genotype. Our research group evaluated B12 status in young women with all three TC 776 C G genotypes (CC, CG, GG) (91). Mean holo-TC concentr ation was significantly lower in TC 776 GG compared to CC genotypes, and individuals with low (< 35 pmol/L) holo-TC had a significantly higher mean Hcy concentration. Al ternatively, some studies have reported no eff ect of the TC 776 C G polymorphism on B12 status or metabolism (92-94).

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28 Any reduction in B12 binding capacity, protein s ynthesis, or transport function caused by the TC 776 C G polymorphism could impair functional B 12 status, leading to reduced cellular availability of B12. If the polymorphism has a physiologically significant impact on B12 availability, it would likely be exacerbated by concurrently low B12 status due to insufficient dietary B12 intake. In a populati on where more individuals have ma rginal B12 status, such as in vegetarians, any negative effect of the TC 776 C G polymorphism would be expected to be more apparent. Malabsorption of Vitamin B12 The most common form of B12 mala bsorption is often termed food-bound malabsorption(95). Because only free B12 can bi nd to the transport prot eins and be taken up into the enterocyte or absorbed passively, any ph ysiological condition that reduces the ability to free B12 from the protein matrix will lead to malabsorption and can lead to a B12 deficiency. Approximately 5 to 25% of adults over the age of 60 y are estima ted to have some degree of food-bound B12 malabsorption due to an age-rela ted decrease in stomach acid, or achlorhydria (96). Because achlorhydria is so prevalent in older adults it is recommended that the daily requirement of B12 be met by consuming supplem ental forms of B12 (97). Vitamin B12 found in fortified foods and vitamin supplements is not protein bound and therefore, can be absorbed with normal efficiency even if gastric pH is high. Another less common, but often more severe form of B12 malabsorption, termed pernicious anemia, can occur in all age groups although incidence does in crease with age. Pernicious anemia is caused by a lack of IF re sulting from an autoimmune response, atrophy of the gastric mucosa, chronic gastriti s, and in rare cases a congenita l defect in the gene for IF. Congenital defects may lead to synthesis of an altered, and therefore non-f unctional IF protein or a complete lack of synthesis. Both conditions cause B12 deficiency at an early age and have

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29 been reported to be caused by a variety of geneti c mutations and post-translational defects. The autoimmune-based B12 malabsorption condition is more prevalent in older adults but has been observed in all age groups (98). In this case, the body recognizes either the IF itself or the gastric parietal cells as foreign and synthesizes antibodies to the protein or cell eliciting an immune response. Destruction of IF or the pari etal cells by this autoimmune response may occur to varying degrees resulting in variation in the severity of B12 malabsorption (98). Currently the only available di agnostic tests for pernicious anemia are not clinically practical. The Schilling test, wh ich involves ingestion of radio actively-labeled B12, a flushing dose of non-labeled B12, and collection of urin e over a period of 24 hours requires meticulous adherence to protocol making it erro r prone and costly (99-101). Pres ence of IF or parietal cell antibodies can be measured to diagnose pernicio us anemia; however, parietal cell antibodies can occur in other autoimmune diseases, and both te sts are only clinically meaningful in a subgroup of patients with autoimmune c onditions (102, 103). It has been hypothesized that changes in holo-TC in response to a supplemental dose of B 12 may be used to assess B12 absorption (28, 47, 104). Bor et al (20) reporte d a significant increase in holo-TC and TC saturation 24 and 48 hours after receiving three 9 g oral B12 doses. Since no blo od was collected before 24 hours (post baseline), the magnitude and pattern of ch ange of holo-TC during the first 24 hours could not be determined (47). In developing a clinic al diagnostic test, it is important to know the optimal time post dose at which to draw blood. Overall Rationale Vitamin B12 plays a central role in Hcy metabolism, and B12 deficiency has been associated with numerous health risks, including birth defect-affected pregnancies. Few studies have been designed to evaluate the relations hip between dietary excl usion or limitation of

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30 specific animal products and B12 status in indi viduals who do not take B12 supplements or consume B12-fortified foods. The proposed stud y will evaluate the association between B12 status and intake of specific animal-deriv ed food products among vegetarians who do not consume B12-containing supplements. Several studies, including one conducted by our research group (91), in dicate that the TC 776 GG genotype results in decreased holo-TC c oncentrations, and could therefore be a risk factor for a B12 deficiency. Although most st udies have not found a co rrelation between TC 776 C G genotype and Hcy or MMA, holo-TC and Hcy and MMA have been negatively correlated (89, 105). It is hypothesized that B12 transport, a nd thus metabolic function, may be impaired in individuals with the TC 776 GG ge notype, and that an effect on Hcy and MMA concentrations due to the TC 776 GG genotype may be evident in individuals with low B12 intake and status. These data could be used for public health screening and inte rvention approaches for adults whose combined dietary choices and genetic make -up may put them at higher risk for certain diseases or poor pregnancy outcomes. Inform ation generated from th is study could benefit individuals who exclude B12-dense food sources from their diets for reasons related to health or personal choice rather than religi on, culture, or the environment, as well as for individuals who consume strict vegan diets for religio us/cultural or environmental reasons. Hypothesis # 1 Moderate B12 deficiency will be more common in vegetarians not taking B12 supplements than in their omnivorous counterparts. Specific aim: To determine if non-supplement taking young adults who exclude animalbased foods and are not taking B12-containing su pplements are at a greater risk for a B12

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31 deficiency than those who eat animal-based foods by comparing B12 status indices between groups. Hypothesis #2 Vitamin B12 intake at the current RDA may not be sufficient to maintain normal B12 status. Specific aim: To determine the level of B12 intake associated with optimal status as defined by normal B12 status biomarkers. Hypothesis #3 Genotype status for the TC 776C G polymorphism will have a greater physiological impact on individuals with low B12 status th an those with normal B12 status. Specific aim: To determine if genotype status for the TC 776C G polymorphism further impairs B12 status in individuals with low B 12 intake by comparing B12 status indices among genotype groups in individuals with low and normal B12 status. Hypothesis #4 Holo-transcobalamin concentration can be used to assess B12 absorption. Specific aim: To determine if holo-TC concentration increases measurably in response to B12 supplementation within a 24 hour time period.

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32 Figure 1-1 Structure of vitamin B12. Modified from Stabler (35) p. 22

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33 Figure 1-2 Overview of vitamin B12 (B12) ab sorption. (1) Food bound B12 is released in the acidic environment of the stomach. (2) Free B12 binds to haptocorrin and the complex travels to the duodenum. (3) Panc reatic proteases degrade HC. (4) Free B12 binds to intrinsic factor, wh ich is synthesized in the gastric parietal cells. (5) The B12 IF complex to travel to the ileum a nd is transferred across the ileal epithelium via receptor mediated endocytosis, along w ith 1% passive diffusion. (6) In the enterocyte, intrinsic factor is degraded by the lysosome. (7) Transcobalamin binds B12 at some point after releas e from intrinsic factor, this may occur in the enterocyte.

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34 Figure 1-3 Role of vitamin B12 in the remethyl ation of homocysteine. THF = tetrahydrofolate; 5-CH3-THF = 5-methyltetrahydrofola te; MS = methionine synthase; SAM = s-adenosylmethionine; SAH = S-adenosylhomocysteine.

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35 CHAPTER 2 VITAMIN B12 STATUS IS IMPAIRED IN A SUBGROUP OF HEALTHY YOUNG VEGETARIAN AND OMNIVOROUS ADULT MEN AND WOMEN Naturally occurring dietary sources of B12 are li mited to foods of animal origin, which if restricted in the diet may impair B12 status ( 40-42). Vitamin supplements and fortified foods can also contribute to B12 inta ke (97); however, it is estimated that ~70 % of the United States (US) population does not take supplements (106). Vegetarians, individual s who avoid some or all animal-derived foods, have lim ited dietary intake of B12 and may be at greatest risk for developing a B12 deficiency compared to omnivor es. Few data are avai lable on B12 status in young adult vegetarians in the United States, and further evaluation of B12 status in this subgroup of individuals is warrant ed to better determine relative risk of B12 deficiency and related disease. Clinical determination of B 12 deficiency relies on the availability of specific and reliable biomarkers of B12 status. Biomarkers currently us ed to assess B12 status include serum B12, MMA, Hcy and holo-TC concentrati ons. Although holo-TC is not yet used clinically, holo-TC is reported to be more sensitive than serum B12 and may be comparable to MMA as a biomarker of B12 status. The objectiv es of this study were to determine if young adult vegetarians who do not take B12 supplements are at a greater risk for B12 deficiency than omnivores not taking B12 supplements, and to comp are the various B12 status indicators within these groups. Subjects and Methods Subjects and Subject Recruitment Healthy adults (n = 388) from the Alachua county, FL community including university students, faculty and staff were recruited by flyers and newspaper advertisements with simultaneous recruitment for healthy adult vegeta rians and healthy adults. Subjects were screened by phone and selected based on the follow ing exclusion criteria: (a) < 18 y & > 49 y (b)

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36 major change in animal-product consumption (i.e. vegetarian or omnivore) habits during the past 3 years; (c) B12-containing supplement use with in 6 mo of screening; (d) chronic alcohol consumption (>1 drink/d of any kind); (e) use of tobacco products; (f) use of prescription medications other than oral contraceptives; (g) pe rsonal history of chroni c disease; (h) regular blood donations; and (i) pregna nt or lactating women. Potential subjects were asked about their meat consumption habits during the phone screening for initial classificati on as vegetarian or omnivore. Specifically subjects were asked How often do you consume (a) beef, (b) chicken, (c) turkey, (d) pork, and (e) fish. Subjects who responded never to all questions were tem porarily classified as vegetarian. This study was approved by the University of Florida Institu tional Review Board, and all subjects signed an informed consent prior to beginning the study. Study Design and Data Collection Between the hours of 7:00 am and 9:00 am s ubjects were scheduled for fasting blood sample collections. Subjects were called 24 hou rs prior to their scheduled appointment to remind them to fast overnight and the following morning. Following sample collection, subjects were given a small meal and a comprehensive in formation session explaining how to complete the National Cancer Institute Di et History Questio nnaire (DHQ). Subjects were asked to complete the questionnaire at home and return it within two weeks and to contact a designated member of our recent team personnel if they had any questions or problems completing or returning the questionnaire. For any unreturne d DHQs, individuals were contacted by phone or e-mail to determine if the questionnaires were lost in transit or if the subject had not had an opportunity to complete the DHQ instrument. S ubjects that chose not to return the DHQ were not included in the final data analysis. The DHQ has been validated for the estimation and

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37 quantification of dietary intake of all essential nutrients including B12 (107). Subjects were instructed to answer all questions based on their diet over the past 12 mo estimating the frequency of intake and portion size of 125 different food items. A total of 70 mL of blood were collected for analysis of the following indices: (a) serum holo-TC; (b) plasma B12; (c) serum MMA; (d) serum Hcy (e) serum folate, and (f) hematocrit. Sample Processing Blood samples were collected in ethylenedi aminetetraacetic acid (EDTA) and serum separator clot activator (SST) tubes. EDTA tubes were centrifuged for 30 min at 2000 x g at 4 C to separate and collect plasma for B12 analyses Serum separator tube s were centrifuged for 15 min at 650 x g at room temperature to separa te and collect serum fo r holo-TC, MMA, Hcy, and folate determination. All samples were stored frozen at 30 C until analysis. Competitive Binding Assays of Serum Holo-transcobalamin and Plasma B12. Serum holo-TC concentration was determ ined by radioimmunoassay (holo-TC RIA reagent kit; Axis Shield, Ulvenve ien, Oslo, Norway) based on the method of Ulleland et al. (45). Specifically, magnetic microspheres coated with anti-human TC monoclonal antibodies were incubated with each sample for a period of one hour to isolate both holo-TC and apo-TC. Once attached to the metal beads via antibody inter action, the TC protein a nd associated B12 were magnetically separated from the sample. Next, isolated TC was incubated with 57Co-labeled B12 tracer plus reducing agent followed by a denatu ring agent to free B12 from the TC protein. Finally, each sample was incubated with IF to which unlabeled and labeled B12 bind competitively based on their relative concentr ations. Remaining unbound B12 was removed and the relative radioactivity of each sample meas ured by gamma counter. Radioactivity of each

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38 sample in counts per minute (CPM) was compared to a standard curve with serum holo-TC concentration being inversel y associated with CPM. Plasma B12 was determined by RIA using a co mmercially available kit (Quantaphase II, Bio-Rad). Specifically, samples were incubated with a 57Co labeled B12 tracer in a 100oC water bath to convert all forms of B12 to cyanocobala min. Samples were brought to room temperature after boiling for 20 min, and then mixed with purified porcine IF bound to polymer beads and incubated for one hour. During in cubation labeled and unlabeled B12 compete for binding to IF at rates that match their relative concentra tions. Finally, samples were centrifuged, and supernatant containing unbound B12 was removed. Sample radioactivity was measured by gamma counter and B12 concentration was calcu lated using a standard curve on which the radioactivity was inversely re lated to B12 concentration. Measurement of Serum Homocysteine and Methylmalonic Acid. Serum Hcy and MMA concentrations were determined by gas chromatography mass spectrometry (Metabolite Laboratories, Inc. Denver, Colorado) (108, 109). Diet Analysis Daily B12 intake was assessed based on data obtained from the DHQ, which was modified to include additional B12-containing foods incl uding meats, mixed dishes, fortified foods and meat substitutes. The original DHQ is available online at http://appliedresearch.cancer.gov The DHQ was scanned by Optimal Solutions Corporati on (OSC), Lynbrook, New York. Dietary data obtained from scanning the questionn aires was sent to the Universi ty of North Carolina, Chapel Hill as an ASCII text file, and then analyzed using the Diet*Calc Analysis program. In order to analyze the modified questionnaire, the Diet*C alc program was updated to include nutrient values for all of the food items added based on data from the United States Department of

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39 Agriculture (USDA) National Nu trient Database for Standard Reference and nutrition label information when data was not available from the former USDA database Diet*Calc is a freeware program and can be downloaded from the National Cancer Institute website (www.riskfactor.cancer.gov ). Subject Dietary Intake Classification Individuals were classified as vegetarian or omnivore based on their responses to the DHQ. Individuals classified as vege tarians were those who reporte d no consumption of any meat products (i.e., beef, poultry, pork, lamb and seaf ood) or meat-based mixed dishes and who reported consuming dairy products and eggs ne ver to daily. Omnivores were defined as individuals who consumed any meat, poultry or s eafood products. In addition to the preexisting questions in the DHQ that asked about all types of meat consumption, a new question was added to more accurately classify subjects into specif ic dietary intake categories. This question required subjects to indicate the foods they ne ver consumed including (a ) beef, (b) chicken, (c) turkey, (d) pork, (e) fish, (f) dairy products, and (g) eggs. Statistical Methods. Vitamin B12 status based on the measured B 12 indicators and dietary B12 intake were compared between groups using analysis of variance (ANOVA) with an alpha of 0.05. Dependent variables also were classified as n ormal vs. abnormal according to whether they were above or below esta blished thresholds (B12, 148 pmol/L; holo-TC, 35 pmol/L; MMA 271 nmol/L; and Hcy 12 mol/L) and comparisons with respect to dietary group were done by Pearson Chi-Square tests. The distributions of all possible combinations of normal and abnormal test results were calculated and the rate of each B12 indicator being abnormal when all others were normal or abnormal was compared using a Chi-square test. Age distributions were

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40 compared between the two dietary groups by ANOV A, while race and gender for the two dietary groups were compared by Pearson Chi-Square tests. Results One hundred and twenty one vegetarians and 18 1 omnivores completed the study (total n = 305). Of the 388 enrolled subjects, 62 were ex cluded due to reporting supplement use, and 23 did not complete the DHQ. All results are reporte d as mean SD unless otherwise noted. There was a significant difference in age and BMI betw een groups, with vegetarians being older and having a lower BMI. There were no significant di fferences in gender or ethnicity between diet categories (Table 2-1). Total B12 intake (g/d SD) and B12 in take expressed as g/1000 kcals were significantly lower (P < 0.001) in vegetarians than in omnivore s (Table 2-2). Plasma B12 concentration was significantly lower (P < 0.01) in vegetarians than omni vores (Table 2-2). Serum MMA concentration was significantly highe r (P = 0.001) in vegetarians compared to omnivores (Table 2-2). Mean holo-TC and Hcy concentrations were not significantly different between groups (Table 2-2). Vitamin B12 deficiency, based on having a valu e outside the normal range for one or more of the B12 status indicators, was twice as prev alent (P < 0.001) in vege tarians than omnivores (42% and 23%, respectively). Impaired B12 status based on concentrations of plasma B12, serum holo-TC and serum MMA combined also was significantly greater in vegetarians compared to omnivores. Specifically, more than twice as many (P < 0.05) vegetarians had low serum holo-TC (< 35 pmol/L), plasma B12 (< 148 pmol/L), and elevated serum MMA (> 270 nmol/L) concentrations than omnivores (Figure 2-1). There was no significant difference in the percentage of vegetarians versus omnivores with elevated Hcy (> 12 mol/L).

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41 Subjects were cross-tabulated by B12 status as defined by having a value within (+) or outside (-) the normal range for B12, holo-TC and MMA singly and in combination (Table 2-3). Because of the small numbers of subjects within each of the resulting 8 categories, statistical analysis was not done; however, the likelihood of one B12 status indicator being abnormal while the remaining tests were normal was conducted. In this analysis, B12, holo-TC and MMA were more likely to be abnormal when one or more of the other indicators were abnormal (23, 38, and 36 % of the time, respectively) compared to wh en all others were normal (6, 11, 10 % of the time, respectively) (Figure 2-2). Discussion The primary objective of this study was to asse ss and compare the B12 st atus and intake of young adult vegetarians and omnivores who do not take B12-containing vitamin supplements to determine if vegetarians are at greater risk for developing a B12 deficiency than their omnivorous counterparts. Although long term a dherence to a vegetarian diet can provide substantial health benefits (110), limitation of most or all animal -based foods, particularly when B12-fortified foods or supplements are not added to the diet, can increase the risk for developing a B12 deficiency. It has been estimated that B12 intake in the general US population is adequate (111), however, data from this study suggest that a subgroup of healthy young, non-supplement using adults may not be consuming adequate B12. Within the vegetarian groups, 43% were determined to be potentially B12 deficient base d on having a value outside the normal range for one or more B12 status indicators, 61% of whom had elevated MMA, indicating metabolic impairment. Surprisingly, of the omnivores, 23% were potentially B12 deficient, with 48% of them having an elevated MMA concentration, su ggesting that even a m eat-containing diet may not provide sufficient B12.

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42 This is of particular concern for young wome n of childbearing age, who might chose to avoid meat in an attempt to lower fat and choles terol intake, but do not consume other sources of B12. Nutrient availability is crucial in the fi rst 180 days of pregnanc y, during a portion of which a woman might not even know she is pregnant (112). This issue has been addressed in relation to folic acid; however unlike folic acid, it is no t well recognized that a B12 deficiency is an independent risk factor for ne ural tube defects (57, 113, 114). The DHQ used to assess dietary B12 intake asks subjects to recall di etary intake over the past 12 mo and answer questions based on their be st estimate of food intake. Full analysis of DHQ data was the focus of an investigation c onducted by another member of the laboratory group and will be reported separately. The data obtained from the analyses conducted for this study were used to group individual s within a similar range of overa ll B12 intake and to identify foods eaten or excluded by each subject. This allo wed for a very strict definition of vegetarian subjects who reported no meat consumption and omnivore subjects who reported some degree of meat consumption. Previous studies, the majo rity of which were conducted in Europe, used a similar approach to classify individuals as vege tarian. Vegetarians have previously been subgrouped into lacto-ovo-vegetarians, lacto-vegetarian and vegan cat egories, with the greatest deficiency associated with the vegan diet relative to other vegetarian sub-groups (81). It is widely believed in the US, that a B12 deficien cy is not a problem in healthy young adults who consume at least some animal-based products. The data from the present study do not support this perception. In addition to the moderate deficiency that was detected in the ve getarian and omnivorous groups, a small subset (including bo th omnivores and vegetarians) was determined to be severely B12 deficient as evidenced by reports of ne urological problems in cluding numbness and a

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43 tingling sensation in the extremities. Of thes e subjects none had previously sought out medical attention for these symptoms, which illustrates that B12 deficiencies may go undetected among seemingly healthy young adult men and women. Earl y detection of a B12 deficiency is the most effective way to prevent progressi on to serious health complications Recent studies suggest that measurement of holo-TC is superior to serum B1 2 because only holo-TC is taken up into cells, and therefore only that portion ( 20%) is biologically active (115). Addi tionally, holo-TC has been reported to be more sensitive to changes in B12 intake than total serum B12 (28). Miller et al. (116) reported that the use of holo-TC and serum B12 together as a ratio may be superior to the use of either alone. Their data suggest ( 116) that use of combined holo-TC and serum B12 measurement could lead to thr ee possible diagnoses; normal, po ssible deficiency (only 1 low indicator), and deficient (both indicators low). Finally MMA is still considered by many researchers to be the gold standard (54, 117). In the current study, we cross tabulated subjects by B12 status as defined by having a value within or outside the normal range for B12, holo-TC and MMA singly and in combination with each other. Statistical analysis co uld not be conducted due to limited sample size; however, a combined analys is of the two diet grou ps indicated that it is more likely that when the value of one biomarke r is outside the normal range at least one other indicator is more likely to also be outside the normal range. A dditionally, the length of time an individual adheres to a B12-in sufficient diet will have diffe rential effects on specific B12 biomarkers (32). Considering the differences in the primary biomarkers of B12 status, holo-TC may be initially affected followed by a decrease in serum B12 (once B12 stores have been depleted), and finally an elev ation in MMA indicating impaired cellular B12-dependant enzyme function. The data from the current study do not definitively support one B12 biomarker as

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44 being superior to another; however, in a clinical setting the more biomarke rs that are outside the normal range, the more likely a B12 deficiency exists. In conclusion, the high incidence of impaired B12 status observed in these otherwise healthy young adults was unexpected. These data i ndicate that dietary in take alone may not be meeting the B12 needs of non-supplement using adults especially vegetarians. Further research focusing on B12 status and inta ke in individuals consuming both vegetarian and low-meat containing diets is warranted. Assessment of B12 status by a combination of biomarkers may provide a more definitive diagnostic approach prior to treatment. Table 2-1 Characteristics of study groups Vegetarians (n = 121) Omnivores (n = 181) Age (y; mean SD) 28 9b 24 6 BMIa (mean SD) 22.9 3.9b 23.9 4.1 Gender (count) Female 67 98 Male 54 83 Race/Ethnicity (count) White 69 119 African American 6 11 Asian 17 17 Asian Indian 12 4 Hispanic 12 26 Other 5 4a Body mass index (BMI); b Significantly different from omnivores (P < 0.05)(ANOVA)

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45 Table 2-2 Mean ( SD) dietary vitamin B12 inta ke and status of omnivorous and vegetarian adults. Analysis a Vegetarians (n =121) Omnivores (n = 181) Total B12 intake (g/d) 3.39 2.976.80 4.04b B12 intake (g/1000 Kcal/d) 1.92 1.873.31 1.88b B12 (pmol/L) 280 146313 124c Holo-TC (pmol/L) 83 8487 55 MMA (nmol/L) 260 229195 116c Hcy (mol/L) 7.7 2.77.3 2.5 a Vitamin B12 (B12); holo-transcobalamin (holo-TC); methylmalonic acid (MMA); homocysteine (Hcy); b Different from vegetarians (P < 0.001); c Different from vegetarians (P < 0.01) (ANOVA) Holo-TC B12 MMA Hcy 0 10 20 30 40 50Vegetarian Omnivore < 35 pmol/L< 148 pmol/L> 270 nmol/L* *> 14 mol/LPercent subjects below/above cutoff value Figure 2-1 Percent of vegetarian (n = 121) and omnivorous (n = 181) adults (18 to 49 y) with concentrations outside the normal range for holo-transcobalamin (holo-TC, < 35 pmol/L), serum vitamin B12 (B12, < 148 pm ol/L), methylmalonic acid (MMA, > 270 nmol/L), and homocysteine (Hcy, > 12 mo l/L). Different from omnivores (P < 0.05) (ANOVA)

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46 Table 2-3 Cross-tabulation of vitamin B12 st atus of subjects based on select biomarker combinations Status by biomarker measureda Diet group B12 140 pmol/L Holo-TC 35 pmol/L MMA 270 nmol/L Vegetarian n = 121 Omnivore n = 181 Total n = 302 + + + 68138 206 + + 57 12 + + 1112 23 + 22 4 + + 1413 27 + 31 4 + 73 10 83 10a + Yes; No; vitamin B12 (B12); holo-trans cobalamin (holo-TC); methylmalonic acid (MMA) B12 < 148 pmol/L Holo-TC < 35 pmol/L MMA > 270 pmol/L 0 10 20 30 40 50 All other concentrations within normal range At least one other concentration outside normal range * *Percent Occurrence Figure 2-2 Frequency of single versus combined vitamin B12 (B12) status biomarkers being outside the normal range including plas ma B12, serum holo-transcobalamin (holoTC) and serum methylmalonic acid (MMA). *Significantly different from group with all others normal (P < 0.001) (Pearsons Chi-squared test)

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47 CHAPTER 3 VITAMIN B12 INTAKE AT THE CURR ENT RDA LEVEL IS NOT OPTIMAL The current RDA for B12 was established ba sed on data from patients who were being treated for pernicious anemia. Specifically, the am ount of B12 required in an injectable form to normalize serum B12 in patients di agnosed with pernicious anemia was determined to be the daily B12 requirement fro adults. The B12 RDA (2.4 g/d) was derived by adjusting the estimated B12 requirement for bioavailability, ente rohepatic recirculation, and the CV for 97 to 98 % of the population. It has been suggested that the RDA for B 12 is not optimal, and that B12 status is improved with intakes up to 6 g/d (118, 119). The object ive of this analysis was to determine the level of dietary B 12 intake associated with optimal B12 status as defined by B12 status biomarkers within the normal range. Subjects and Methods Subjects and Subject Recruitment Healthy adults (n = 302) were recruited from the Alachua county, FL community including university students, faculty and staff. Specifical ly, subjects were screened by phone and selected based on the following inclusion cr iteria: (a) 18 to 49 y (b) no change in meat consumption habits over the past 3 years; (c) no B12-containing supplement use within the past 6 mo; (d) limited chronic alcohol consumption (<1 drink/d of any kind); (e) no use of tobacco products; (f) no chronic use of prescription medications other than oral contraceptive agen ts; (g) no history of chronic disease; (h) no chronic blood donations; a nd (i) non-pregnant and n on-lactating. All 302 qualified subjects from the first pa rt of the current study were in cluded in this analysis. The approved institutional review board informed consent form signed by the subjects at the beginning of the study included cons ent for all aspects of studies described in the manuscript. Study Design and Data Collection

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48 Subjects were called the day before their sc heduled study day to remind them to fast overnight (8 hours) and the following morning prior to having their blood drawn. Between the hours of 7:00 am and 9:00 am qualified subjects were scheduled for blood sample collection followed by a comprehensive information session explaining how to complete the National Cancer Institute Diet History Qu estionnaire (DHQ) used to assess dietary intake. A total of 70 mL of blood were collected for analysis of the following indices: (a) serum holo-TC; (b) plasma B12; (c) serum MMA; (d) serum homocysteine (H cy) (e) serum folate, and (f) hematocrit. Sample Processing and Analysis Blood samples were collected in EDTA and SST clot activator tubes. EDTA tubes were centrifuged at 2000 x g at 4 C for 30 min to obtain plasma for B12 analyses. SST tubes were centrifuged at 650 x g at room temperature for 15 min to obtain serum fo r determination of holoTC, MMA, Hcy, and folate concentra tions. Samples were stored at 30 C until analysis. Serum holo-TC concentration was determined by radi oimmunoassay (holo-TC RIA reagent kit; Axis Shield, Ulvenveien, Oslo, Norway) based on the me thod of Ulleland et al (45) using magnetic microspheres coated with anti-transcobalamin m onoclonal antibodies to is olate both holo-TC and apo-TC, and 57Co-labeled B12 as a tracer. Plasma B12 concentration was determined by RIA using a commercially available kit (Quanta phase II, Bio-Rad). Serum Hcy and MMA concentrations were determined by gas ch romatography mass spectrometry(Metabolite Laboratories, Inc. Denver, Colorado) (108, 109). Diet Analysis Daily B12 intake was estimated based on data obtained from the DHQ, which was modified to include an extensiv e list of B12-containing foods including meat containing mixed dishes, fortified foods, and meat substitutes. The unmodified DHQ is available for review online

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49 at http://appliedresearch .cancer.gov. The DHQ was scanned by Optimal Solutions Corporation (OSC), Lynbrook, New York. Once scanned OSC sent the dietary data as an ASCII text file to the University of North Carolina, Chapel Hill (UNC), where the data were analyzed using the Diet*Calc Analysis program modified for this ve rsion of the DHQ. This freeware program can be downloaded from the NCI website (www.riskfactor.cancer.gov). Th e B12 content of the food items added to the DHQ was obtained from the US DA National Nutrient Database for Standard Reference and nutritional labels (39). Statistical Analysis Results are reported in the text as mean SD with an alpha = 0.05. The dependent variables B12, holo-TC and MMA c oncentrations were classified as normal vs. abnormal according to falling above or below an established threshold (B12, 148 pmol/L; holo-TC, 35 pmol/L; MMA 271 nmol/L; and Hcy 12 mol/L) and comparisons with respect to dietary B12 intake were performed using the Pearson Chi-Squa re test. Subjects were divided into dietary B12 intake quintiles, and B12 status base d on plasma B12, serum holo-TC, MMA, and Hcy concentrations were compared between groups using ANOVA with an alpha of 0.05. The Least Significant Difference (LSD) method of multiple comparisons was used for assessment of differences between quintiles. The LSD ensure s every target population paired difference in means will be within +/LSD of the corres ponding difference in sample means with 95% confidence. The data were analyzed usi ng EXCEL (Microsoft, Redmond, WA) and PRISM software (Graph-Pad Software Inc. El Camino, CA). Results Three hundred and two healthy you ng adult (18 to 48 y) men and women were included in this analysis. Subject characteristics are listed in Table 3-1. Seventy si x subjects (25 %) had an intake below the RDA of 2.4 g/d. Dietary B12 intake was si gnificantly correlated (P < 0.05)

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50 with B12, Hcy, and MMA concentr ations, but not with holo-TC concentration (Table 3-2). Individuals with low plasma B12 (< 148 pmol/L) or holo-TC (< 35 pmol/L) concentrations and those with elevated serum MMA (> 270 nmol/L) or Hcy (> 12 mol/L) concentrations had significantly lower dietary B12 in take than those with normal concentrations (Figure 3-1). To further evaluate the influence of B12 inta ke on B12 status, subjects were ranked and grouped by quintile of B12 intake. The mean co ncentration for each B12 status indicator was plotted against the mean B12 intake for each quintile group (Figure 3-2). Mean holo-TC, B12, MMA and Hcy concentrations were significantly different (P < 0.05) among B12 intake quintile groups (Figure 3-1). Specifically, mean holo-TC increased (P < 0.01) from quintile 1 through 3 and then maintained approximately the same value from quintiles 3 through 5, which was associated with a mean B12 intake of 4.3 g/d. Mean plasma B12 concentration increased (P < 0.001) from quintile 1 through 4, reaching a plat eau from quintile 4 through 5 corresponding to a B12 intake 6.7 g/d. Mean MMA decreased (P < 0.001) from quintile 1 through 2 then reached a plateau, which was associated with a B12 intake of 2.7 g/d. Homocysteine changed to the smallest degree, but decreased (P < 0.05) from quintile 1 through 3 maintaining this approximate value thought quintile 5, which corresponded to a B12 intake > 4.3 g/d. In the case of holo-TC, B12, and Hcy concentr ations, the means acros s all quintiles of B12 intake were in the normal range ; mean MMA concentration was elev ated in quintile 1 and within the normal range for all subsequent quintiles. Th e proportion of subjects with B12 deficiency as defined by abnormal biomarkers within each quin tile group (i.e. low B12, low holo-TC, elevated MMA or elevated Hcy) decreased significantly from the lowest to highest B12 intake quintile for all indices measured (Table 33). Specifically, in the group of subjects who consumed > 3.4 g/d of B12 there was a signifi cantly smaller percentage of subjects with low holo-TC or

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51 elevated MMA concentrations than those consuming 3.4 g/d. In the group of subjects who consumed at least the RDA for B12, there was a significantly lower number of individuals with a low plasma B12 concentration than in the group of individuals who cons umed less than the RDA (Table 3-3). Discussion In this study, the relationship between estim ated B12 intake and a panel of B12 status biomarkers were assessed in order to evaluate the adequacy of the current RDA for B12. It has been suggested by another research group that a B12 intake of 6 g/d was associated with improved concentrations of all B12 biom arkers compared to an intake of 2.4 g/d (118). Although the findings in the current study vary de pending on the specific biomarker, the data indicate that an intake greater than the current RDA is associ ated with normal B12 status. Overall, no clear conclusion can be drawn from these data as to a specific intake level of B12 that might result in normaliza tion of all B12 biomarkers; howev er, the data suggest that the current RDA may not be optimal. In the first investigation of this study group, subjects were classified as vegetarian or om nivore, and inadequate B12 status was detected in a surprising number of individuals in both groups. Specifi cally, 40% of vegetarian s and 11% of omnivores were determined to have abnormal values for on e or more of the four B12 biomarkers. The mean B12 intake of both groups exceeded the RDA. In the current analysis, the third quintile corresponded best to the current RDA for B12 with a mean and range of B12 intake at 2.7 g/d and 2.0 to 3.4 g B12, respectively. Beyond the second qui ntile, the mean concentrations of holo-TC and B12 increased; mean Hcy decrease d and overall rate of deficiency decreased significantly. This suggests that B12 status improves with inta kes above the second quintile,

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52 which in this study was represen ted as an intake level of 3.4 g/d. Little or no change was observed in subsequent qui ntiles, suggesting that an intake level above 3 g/d may be required. One limiting factor of the current study is the use of a FFQ to estimat e B12 intake rather than a 7-day weighed food record as was used in the study by Bor et al. (118). Because the DHQ relies on subject recall and estimation of intake over the past 12 mo, it is more prone to error and less precise than a direct measure. In additi on, neither the one week weighed food record nor a FFQ gives an estimate of duration of a particular diet, and because B12 status is slow to change relative to changes in B12 intake, estimated inta ke over one week or even over one year may not always correlate well with status at a given time. In a very large study using data of from the Framingham Offspring population, which also used an FFQ to assess B12 intake, improvements in B12 status were observed for intakes up to 10 g/d (119). Therefore, data from the current study in addition to that from two previous stud ies agree with the conclusion that the RDA for B12 is inadequate to maintain normal B12 st atus (39, 118). Further i nvestigations focusing specifically on changes in B12 status with increas ing B12 intake are warranted to address this issue and derive an estimate of B12 intake that is consistent with maintenance of normal B12 status. Because the current R DA was established using data from research conducted with patients who had pernicious anemia and who were injected with B12 rather than in healthy individuals consuming dietary B 12, there is clear just ification for conducting controlled feeding studies to obtain pertinent data necessary to re vise the current RDA. The RDAs are not intended to be therapeutic recommendations for individual s with disease conditions such as pernicious anemia. Recommended intake of B12 for the adu lt population should apply to a majority of the population, potentially with additio nal recommendations for some sub-groups such as vegetarian

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53 groups and the elderly. In future studies, consider ation must be made for potential differences in bioavailability of B12 from foods and fortif ied products consumed by healthy young adults. In conclusion, the previous assumption that the general US population has adequate B12, status is not supported by data from this in vestigation in young hea lthy adult men and women who consume either omnivorous or vegetarian diet s. The data from this study support that from two previous investigations including one in th e US indicating that the current RDA for B12 is inadequate to maintain normal B12 status in healthy men and women. Fu rther investigation of the changes in B12 status in response to controlle d levels of B12 intake is warranted to provide data to support a revised RDA. Table 3-1 Subject Characteristics Mean ( SD) Range Reference interval Age (y) 26 818 49 BMIa 24 416 48 B12 intakea (g/d) 5.4 3.90.4 22.67 B12a (pmol/L) 300 13440 937148 444 Holo-TCa (pmol/L) 85 696 57635 150 MMAa (nmol/L) 221 17381 186680 270 Hcya (mol/L) 7.5 2.63.5 29.64.5 12.0a Body mass index (BMI); vitamin B12 (B12); ho lo-transcobalamin (holo-TC); methylmalonic acid (MMA); homocysteine (Hcy) Table 3-2 Correlations (r) between vitamin B 12 intake and concentrations of B12 status biomarkers B12a Holo-TCa MMAa Hcya r P r P r P r P B12 intakea 0.23 < 0.00010.110.06-0.170.004 -0.120.04a Vitamin B12 (B12); holo-transcobalamin (holo-TC); methylmalonic acid (MMA); homocysteine (Hcy)

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54 2.4 Holo-TC B12 Hcy MMA 0 2 4 6 8 10 2.4normal low/elevated ** *Vitamin B12 intake g/d Figure 3-1 Total daily vitamin B12 (B12) intake (mean SD) in individuals with concentrations outside the normal range for holo-transc obalamin (holo-TC; normal > 35 pmol/L), plasma B12 (normal > 148 pmol/L), serum homocysteine (Hcy; normal < 12 mol/L), and serum methylmalonic acid (M MA; normal < 270 nmol/L). The current RDA for B12 is represented (-----). *D ifferent from normal (P < 0.01) (ANOVA, Chi-square test)

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55 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 0 50 100 150 200 250 300 350 400 450 500b b,c b a c 2.4 gB12 intake (mg) quintileB12 (pmol/L) 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 0 50 100 150 200 250a a b b b 2.4 gB12 intake (mg/d) quintileHolo-TC (pmol/L) 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 0 100 200 300 400 500 600 700b b b b a 2.4 gB12 intake (mg) quintileMMA (nmol/L) 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 0 2 4 6 8 10 12 14a a,b b,c c a,b,c 2.4 gB12 intake (mg) quintileHcy (mmol/L) Figure 3-2 Relationship between v itamin B12 (B12) intake and stat us. Mean B12 ( SD) intake for each quintile (n = 60 for quintiles 1, 3 and 5; n = 61 for quintiles 2, and 4; respectively), is plotted against conc entrations (mean SD) of B12, holotranscobalamin (holo-TC), methylmalonic acid (MMA), and homocysteine (Hcy). Values with different superscript letters are significantly different (P < 0.001 for B12 and MMA, P < 0.01 for holo-TC and P < 0.05 for Hcy). The current RDA for B12 is represented in each graph (----).

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56 Table 3-3 Proportion (%) of individuals with concentrations outside the normal range fo r select vitamin B12 status biomarkers B12 intake quintile Biomarkera Total n 1 (< 2.0 g/d) 2 (< 3.4 g/d) 3 (< 5.3 g/d) 4 (< 8.5 g/d) 5 (< 22.67 g/d) B12 Low (< 148 pmol/L) 5025b 81075 Normal ( 148 pmol/L) 2487592909395 Holo-TC Low (< 35 pmol/L) 3335c 23d 13e 57 Normal ( 35 pmol/L) 2696577869593 MMA Elevated (> 270 nmol/L) 5232c 23101012 Normal ( 270 ) nmol/L 2486877909088 Hcy Elevated (> 14 mol/L) 106d 2200 Normal ( 14 pmol/L) 290909797100100 a Vitamin B12 (B12); holo-transcobalamin (holoTC); methylmalonic acid (MMA); homocysteine (Hcy); data was not available for all subjects fir some biomarkers; bSignificantly different from Q2, Q3, Q4, Q5; c Significantly different from Q3, Q4, Q5; d Significantly different from Q4, Q5; e Significantly different from Q4. (P < 0.05) (Chi-square test)

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57 CHAPTER 4 GENOTYPE FOR THE TRANSCOBALAMIN 776C G POLYMORPHISM IS NOT ASSOCIATED WITH ABNORMAL VITAMIN B12 STATUS BIOMARKERS IN HEALTHY ADULTS Transcobalamin (TC), the B12 transport protein required for cellular uptake is essential to maintain B12 metabolic function (26, 120). A common genetic polymorphism for TC (TC 776 C G) may impair the metabolic ro le of this protein (74, 89). It is hypothesized that B12 transport and thus metabolic function will be impaired in individuals with the homozygous variant genotype (GG) for the TC 776 C G polymorphism. The metabolic and health-related risks associated with this polymorphism are pred icted to be exacerbated by the consumption of low-B12 vegetarian diets that ex clude specific animal-d erived foods. The primary goals of this study were to evaluate the effects of the TC 776 C G polymorphism on B12 metabolism in young adult men and women who consume a low B12 diet compared to those consuming adequate B12. Subjects and Methods Subjects and Subject Recruitment Healthy adults (n = 302) were recruited from the Alachua county, FL community including university students, faculty and staff. Subjects we re initially screened by phone and selected for the study based on the following inclusion criteria: (a) 18 y & 49 y (b) no change in meat consumption habits during the last 3; (c) no B12 containing supplement use within the past 6 mo; (d) limited chronic alcohol consumption (<1 drin k/d of any kind); (e) no use of tobacco products; (f) no chronic use of prescription medications other than oral cont raceptive agents; (g) no history of chronic disease; (h) no ch ronic blood donations; and (i) nonpregnant and non-lactating. All subjects from the first part of the research described in the manuscript were included in this

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58 analysis after a second informed consent form ap proved by the University of Florida Institutional Review Board that was specific for genetic analysis. Study Design and Data Collection Subjects were called the day before their sc heduled study day to remind them to fast overnight (8 hours) and the following morning prior to having their blood drawn. Qualified subjects were scheduled for fasting blood sample collection to be perf ormed between the hours of 7:00 am and 9:00 am followed by a comprehens ive information session explaining how to complete the National Cancer Institute Diet Hi story Questionnaire (DHQ), which was used to assess dietary intake. A total of 70 mL of blood were collected for analysis of serum holo-TC; plasma B12; serum MMA; serum homocysteine (Hcy), serum folate, and DNA extraction. Sample Processing and Analysis Blood samples were collected in EDTA and SST clot activator tubes. EDTA tubes were centrifuged at 2000 x g at 4 C for 30 min to obtain plasma for B12 analyses. SST tubes were centrifuged at 650 x g at room temperature for 15 min to obtain seru m for holo-TC, MMA, Hcy, and folate determination. Samples were stored at 30 C until analysis. Serum holo-TC was determined by radioimmunoassay (holo-TC RIA reagent kit; Axis Shield, Ulvenveien, Oslo, Norway) based on the method of Ulleland et al. (45) using magnetic microspheres coated with anti-transcobalamin monoclonal antibodies to isolate both holo-TC and apo-TC, and 57Colabeled B12 as a tracer. Plasma B12 was dete rmined by RIA using a commercially available kit (Quantaphase II, Bio-Rad). Serum Hcy and MMA concentrations were determined by gas chromatography mass spectrometry (108, 109).

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59 Genotype Determination DNA was extracted from blood as previously described (121) using a commercial kit (Quantum Prep, BioRad, Hercules, CA) and stan dard laboratory procedures. Genotypes of potential subjects were determined using D ynamic Allele Specific Hybridization (DASH) performed by DynaMetrix (Stockholm, Sweden ). Briefly, a short PCR product was created spanning the polymorphic position. One PCR primer was 5'-labeled with biotin for attachment of the amplified targets to streptavidin-coated 96-well microtiter plates. Following denaturation and a wash to remove the unbound strand, an alle le-specific probe was hybridized to the bound target DNA strand at low temperat ure in the presence of the doublestrand specific intercalating dye Sybr Green. Finally, the temperature was st eadily increased while recording the probe-target duplex melting temperature, as monitored by di minution of Sybr Green fluorescence with a quantitative PCR analysis device. Properly designed matched target-probe duplex es have higher melting temperatures than those with single-base mismatches, enabling una mbiguous allele discrimination. Heterozygous samples show two separate phases of denaturatio n. For analysis, the negative derivatives of the melting curves are plotted. A single peak at a lower temperature indica tes homozygous allelic mismatch to the probe, and a single peak at a higher temperature, a homozygous match. A double peak is generated from a he terozygous sample (Figure 4-1). Diet Analysis Daily B12 intake was estimated based on data obtained from the DHQ, which was modified to be inclusive of an extensive list of B12-containing foods in cluding meat containing mixed dishes, fortified foods, and meat substitute s. The unmodified DHQ is available for review online at http://appliedresearch.cancer.gov. The DHQ was scanned by Optimal Solutions Corporation (OSC), Lynbrook, New York. Once s canned, OSC sent the di etary data as an

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60 ASCII text file to the University of North Ca rolina, Chapel Hill (UNC). Data was analyzed using the Diet*Calc Analysis pr ogram modified for this version of the DHQ. This freeware program can be downloaded from the NCI website (www.riskfactor.cancer.gov). The B12 content of foods added to the DHQ were base d on up-to-date information from the USDA National Nutrient Database for Standard Reference and nutriti on labels (39). Statistical Methods Results are reported in the text as mean SD with an alpha = 0.05. Vitamin B12 status, based on measurement of plasma B12, serum hol o-TC, the ratio of holo-TC to B12, MMA, and Hcy, was compared among genotype groups with and without an adjustment for B12 intake, using ANOVA with an alpha of 0.05. Dependent vari ables also were classi fied as normal vs. abnormal according to falling above or below an established threshold (B12 148 pmol/L; holo-TC 35 pmol/L; MMA 271 nmol/L; and Hcy 12 mol/L) and comparisons with respect to genotype were performe d using a Pearson Chi-Square test Qualitative data including gender, age, and ethnicity were compared usi ng a Pearson Chi-Square test. The data were analyzed using Statistical Analysis System Soft ware (SAS Institute Inc. Cary, NC) and PRISM software (Graph-Pad Software Inc. El Camino, CA). Results No significant differences were detected among genotype groups for gender, age, or BMI, however there were differences in ethnic distri bution (Table 4-1). There were no significant differences among genotype groups with or without adjustment for B12 intake, for holo-TC, MMA, and Hcy concentration whether considered al one (Table 4-2) or in combination with low B12 status (Table 4-3). There was no signi ficant difference in B 12 among genotype groups, though there was a trend for higher B12 in th e TC 776 GG group (Table 4-3; P < 0.01). In addition, there were no significant differen ces among genotype groups in the number of

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61 individuals with values outside the normal range for plasma B12, holo-TC, MMA, or Hcy (Table 4-4). Individuals with the TC 776 GG genotype ha d a significantly lower (P < 0.05) ratio of holo-TC to plasma B12 than individuals with the CC genotype (Table 4-2). Total TC was significantly lower (P < 0.001) in the TC 776 GG genotype group co mpared to both the CG and CC genotype groups (Table 4-2). Transcobalami n saturation was not significantly different among groups. Discussion Studies investigating the effect of the TC 776 C G polymorphism have resulted in conflicting findings. In a previ ous study by our laboratory, significan t differences were detected in holo-TC concentration among the TC 776 genot ype groups, however in the present larger study, which included a wider range of B12 intake by subjects, a significant difference was not detected. In addition, there we re no significant differences in Hcy or MMA concentration among the genotype groups, further suggesting no real phys iological impact of this single base pair mutation of the TC gene on bioche mical indexes of B12 metabolism. Even when considering the combined influence of the polymorphism and low B12 status, there were no significant differences in any B12 status biomarkers among the genotype groups, indicating no effect on B12 metabolism in B12 impaired individuals. Interestingly, some significant differences were found among genotype groups, suggesting a moderate effect of the polymorphism on TC pr otein synthesis or catabolism. Specifically, total-TC concentration was lo wer in subjects with the TC 776 GG genotype. Transcobalamin saturation, however, was not different among ge notype groups, suggesting no effect on the ability of TC to bind B12. The ratio of holo-TC to B12 also was significantly lower in subjects with the TC 776 GG genotype compared to the CC genotype, though there was no significant

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62 difference in mean holo-TC among the groups. B ecause there was a significant difference in total-TC among genotype groups but not in TC satu ration there must have been some difference in holo-TC as well, because TC saturation is the ratio of holo-TC to total-TC. Although the differences were not significan t, holo-TC was somewhat lower and TC saturation somewhat higher in subjects with the TC 776 GG genotype compared to the CC genotype. Additionally, because there was no difference in MMA con centration among genotype groups, even in combination with low B12 status, the reduced concentration of total-TC in the TC 776 CC genotype likely has no important physiological effect on B12 me tabolism or functional status. Previous studies focusing on other higher risk groups, such as the in dividuals with low B12 intake included in this study, also have not detected a significant effect of the TC 776 C G polymorphism (92, 93, 122). Fodinger et al (93) reporte d no significant differe nce in holo-TC or Hcy concentration in end-stage renal disease patients with the TC 776 GG or CC genotypes. Wans et al (92) compared holo-TC, B12, Hcy a nd MMA concentrations in elderly subjects with the TC 776 CC and GG genotypes, and reported a lo wer holo-TC concentration in subjects with the TC 776 GG genotype compared to the CC genotype, but no differe nce in B12 or MMA concentrations. Comparing the absolute difference in mean values in the study by Wans et al. to the current study, the differences were 4 pmo l/L versus 51 pmol/L, respectively, for B12 (GG mean CC mean); and 22 pmol/L versus 6 pmol/L respectively for holo-TC (GG mean CC mean). Differences in initial B12 status coul d account for the discrepa ncies seen in the many studies examining the relationship betw een genotype status for the TC 776 C G polymorphism and B12 status. Because such small differences in B12 status may overcome any negative effect of the polymorphism, and because changes in metabo lic indicators of B12 st atus such as Hcy and

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63 MMA are not consistently observed, it is unlikely that any B12-re lated metabolic change related to this polymorphism is of clinical concern. It is important to note that the developing embr yo may be at risk of negative consequences of metabolic changes associat ed with genetic polymorphisms that coexist with suboptimal nutrient intake. A polymorphism affecting a ke y folate enzyme, met hylenetetrahydrofolate reductase (MTHFR 776 C T), is associated with a significant increase in risk for neural tube defects, and the risk is exacerbated when folate intake is low (123-125). Again, reports of the effect of the TC 776 C G polymorphism on birth defect ri sk have been mixed (30, 94, 126, 127), but most results suggesti ng an increased risk for preg nant women with the TC 776 GG genotype are not definitive. Potentially, combin ed effects of several polymorphisms that might interfere with B12 absorption or metabolism c ould be physiologically important and further investigation may be warranted based on findings from recent studies that considered several types of birth defects (128-130). A) TC 776 C G Figure 4-1 Melting curve plots for Dynamic A llele Specific Hybridization analysis of polymorphism the TC 776C G polymorphism. Negative de rivatives of Sybr Green fluorescence vs. time plots are shown for tw o samples of each allele combination. Single peak at a lower temperature ( ) indicates homozygous allelic mismatch to the preferred probe; single peak at a higher temperature ( ), a homozygous match; double peaks, a heterozygous sample.

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64 Table 4-1 Demographic distri bution of subjects by genotype TC 776 C G genotype CC (n = 94) CG (n = 139) GG (n = 65) P-value Gender > 0.05 Male 446626 Female 507339 Age (y) 20 1026 726 7> 0.05 BMIa 23.7 4.423.6 3.823.2 4.0> 0.05 Ethnicity < 0.001 White 568740 African American 1232 Asian 52215 Hispanic 17154 Other 4124 a Body mass index (BMI) Table 4-2 Mean ( SD) concentrations of se lected vitamin B12 biom arkers in all subjects TC 776 C G genotype Biomarkera CC (n = 94) CG (n = 139) GG (n = 65) Vitamin B12 (pmol/L) 280 119298 137 331 145 Holo-TC (pmol/L) 87 5687 68 81 85 Total-TC (pmol/L) 849 181763 136 668 144a Holo-TC/B12 0.34 0.180.31 0.22 0.25 0.17a TC Saturation 0.10 0.07 0.11 0.08 0.13 0.13 MMA (nmol/L) 229 179221 205 128 Hcy (mol/L) 7.6 3.17.3 1.8 7.6 3.3a Vitamin B12 (B12); holo-transcobalamin (holo-TC); methylmalonic acid (MMA); homocysteine (Hcy) Table 4-3 Mean ( SD) concentrations of sel ected B12 biomarkers in subjects with vitamin B12 deficiency TC 7776C G genotype Biomarkera Normal cutoff CC n CG n GG n B12 (pmol/L) < 148 pmol/L105 2810112 3015 124 377 Holo-TC (pmol/L) < 35 pmol/L28 81424 719 23 714 MMA (nmol/L) > 270 nmol/L505 28017461 34025 420 2409a Vitamin B12 (B12); holo-transcobalamin (holo-TC); methylmalonic acid (MMA); homocysteine (Hcy)

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65 Table 4-4 Percentage of indi viduals within each TC 776 C G genotype group with concentrations outside the normal range for select B12 biomarkers TC 776 C G genotype Biomarkera CC CG GG Plasma B12 < 148 pmol/L 111111 Holo-TC < 35 pmol/L 151421 MMA > 270 nmol/L 181814 Hcy > 12 mol/L 425 a Vitamin B12 (B12); holo-transcobalamin (holo-TC); methylmalonic acid (MMA); homocysteine (Hcy)

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66 CHAPTER 5 HOLO-TRANSCOBALAMIN IS AN INDICA TOR OF VITAMIN B12 ABSORPTION IN HEALTHY ADULTS WITH NORMAL VITAMIN B12 STATUS Circulating B12 is bound to one of two ca rrier proteins, haptocorrin (HC) or transcobalamin (TC). Although the majority of B12 (~80%) is bound to HC (holo-HC), only TC bound B12 (holo-TC) can be taken up by body cells (26). Depletion of total body B12 occurs slowly, and is often a result of malabsorption, which is difficult to di agnose clinically (13, 81, 131, 132). Currently the only ava ilable diagnostic tests for v itamin B12 absorption are not clinically practical. It has been hypothesized that changes in holo-TC in response to a supplemental dose of orally administered B12 may be used to assess B12 absorption (28, 47, 104). Bor et al (20) reported a significant increase in holo-TC and TC saturation 24 and 48 hours after receiving three 9 g oral B12 doses. Since no blood was collected before 24 hours (post baseline), the magnitude and pattern of change of holo-TC during the first 24 hours could not be determined (47). In developing a clinical diagnos tic test, it is important to know the optimal time post dose at which to draw blood. The objective of this study was to evaluate the post-absorption response of holo-TC to oral B12 relativ e to other indicators of B12 status. Subjects and Methods Subjects Twenty one healthy adult men (n = 13) a nd women (n = 8) (18 to 49 y) from the Gainesville, Florida community were selected ba sed on the following inclusion criteria: (a) serum B12 concentration > 350 pmol/L at time of screening; (b) no B 12-containing supplement use or B12 injections during past year; (c) no us e of tobacco products; (d) no history of chronic disease; (e) non-pregnant and nonlactating; (f) non-anemic (Hgb 11 g/dL [7.4 mmol/L], females; 12 g/dL, [8.1 mmol/L] males); (g) normal blood chemistry profile; (h) BMI between 18 and 29; and (i) no blood donations within 30 days of the study.

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67 Study Design and Data Collection All participants signed an informed consent form approved by the Un iversity of Florida Institutional Review Board prio r to the initiation of the study. Individuals had a fasting blood sample drawn at the University of Florida Shands General Clinical Resear ch Center (GCRC). Subjects heights and weight s were measured and a medi cal history questionnaire was completed. Blood analyses included serum B12, bl ood chemistry profile, hematological indices, and a pregnancy test for women. Eligible subjects were admitted to the GCRC th e evening before (day 0) the intervention. The following morning (day 1) after an overnight fa st, an indwelling catheter was inserted for all blood collections during day 1. Blood samples were collected a total of 17 times starting on day 1 through day 3, and three 9 g B12 doses were orally administ ered at six hour intervals on day 1 beginning after the baseline blood draw (Figure 1). Immediately after taking each B12 dose, subjects consumed a piece of bread and 236 ml (8 oz) of juice to improve absorption efficiency. In addition to the bread and juice consumed w ith each B12 dose, subjects were given a midmorning snack and lunch at 2 hours and 3.5 hours, respectively after dose 1. Dinner was fed 4 hours after dose 2, and an evening snack was pr ovided 3 hours after dose 3. The RDA for B12 was provided in the diet on day 1 and on day 2. Take-home meals were provided on day 2 of the study. Water and non-caffeinated, non-caloric beve rages were allowed ad libitum. Subjects remained in the GCRC overnight and were allowe d to leave on pass following the collection of a fasting blood sample the morning of day 2. Subj ects returned on the morning of day 3 at which time a final fasting blood sample was drawn. Biochemical Analysis At each blood collection, holo-TC, total-TC, B 12, and plasma albumin concentrations were determined. The ratios of holo-TC concentratio n to total-TC concentration (TC saturation) and

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68 holo-TC concentration to B12 concentration (holo-TC/B12) we re determined to assess changes in these indicators in relation to one anothe r. Additionally methylmalonic acid (MMA), creatinine, serum folate, and homocysteine (Hcy) c oncentrations were measured at baseline. The B12 supplement (9 g cyanocobalamin) was prepared by West lab Pharmacy (Gainesville, FL). The B12 content of the supplement was validated by an independent laboratory (Analytical Research Laboratories, Oklahoma City, OK). Sample Processing and Analysis Blood samples were collected in EDTA and SST clot activator tubes. EDTA tubes were centrifuged at 2000 x g at 4 C for 30 min to obtain plasma for B12 analyses. SST tubes were centrifuged at 650 x g at room temperature for 15 min to obtain seru m for holo-TC, MMA, Hcy, and folate determination. Samples were stored at 80 C in the GCRC until analysis. Serum B12 and folate concentrations were assayed on the Advia Centaur automated immunoassay system (Bayer A/S, Germany) w ith a total imprecision below 10%. Total TC concentration was determined by a sandwich ELISA with a total imprecision of 4 to 6% (intraassay imprecision ~3%) (133). After removal of the apo-TC with B12 coated beads, holo-TC was measured by the TC ELISA. The total im precision for measurement of holo-TC was ~8% (48), and the intra-assay imprecision was ~4% (1 34). Albumin and creatinine were measured on the Cobas Integra 800 (Roche Di agnostics, Indianapolis). Total imprecision was ~2 % for albumin and <3 % for creatinine. Homocysteine concentration was measur ed by the immunological method on the IMMUNLITE 2000 (Diagnostic Products Corporati on, California) (total imprecision <6%) (135) and MMA concentration was measured by slightly modified stable-isotope -dilution capillary gas chromatography mass-spectrometry (total imprecision <8%) (136).

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69 Statistical Methods Results are reported as mean SD with an alpha = 0.05 unless ot herwise noted. The overall p-value for time was obtained by the F-te st, which tests the nu ll hypothesis that the distribution of the dependent variable was the sa me at all time points. The Tukey method (137) of multiple comparisons was utilized for assessm ent of differences between time periods. A Least Significant Difference (LSD), as defi ned by the Tukey procedure, ensures that simultaneously, in every target population, paired di fference in means will be within +/LSD of the corresponding difference in sample means with 95% confidence. Results Mean baseline values for all analytes were within normal ranges, although some individuals had values outside the normal range (Table 1). Plasma albumin concentration fluctuated throughout the interven tion period suggesting a change in hydration status throughout day one and between the mornings of days 1, 2 and 3 (data not shown). Holo-transcobalamin, B12, and total-TC concentrations are reported as a ratio to albumin to adjust for diurnal changes in overall body protein concentration due to chan ges in hydration status. Unadjusted means for holo-TC, B12, and total-TC concen trations are reported in Table 2. All time-points are reported relative to baseline. Of all of the status i ndicator analytes, only hol o-TC and TC saturation changed significantly on day 1. Mean holo-TC concentration increased steadily after baseline and fluctuated throughout day 1. There were statistically significant increases in mean holo-TC concentr ation during the first 24 hours of the intervention; however, thes e small increases were not maintained. Mean holo-TC concentration reached a maximum value at hour 24, whic h was a significant increase relative to baseline and all other time points (F igure 2A). The mean percent increase from baseline also was greater at hour 24 than at all other all time point s with a 49% increase relative

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70 to baseline, and a 29% increase relative to hou r 12 (Figure 3). This peak at hour 24 was observed for almost all subjects, with an increas e of 22% or greater (22 to 85%) for all but one subject. By hour 48, mean holo-TC concentratio n decreased significantly relative to hour 24 (33%); however, it was still significantly greater than baseline (Figure 5-2A). Mean serum B12 concentration did not increas e significantly relative to baseline on day 1, although there were fluctuations in concentra tion throughout the day. At hour 24, mean serum B12 concentration was significantly greater than ba seline (Figure 5-2B). Overall, the percent change in B12 concentration was smaller than for holo-TC throughout the intervention period with ranges of -2 to 15% and -1 to 50%, respectively. Mean total-TC concentration did not change significantly during the study varying less than 6% from baseline at all time points (data not shown). Mean TC saturation began to increase significantly relative to baseli ne at hour 12.5, with the most significant increase at hour 24 (Figure 2C). As observed with holo-TC con centration, the mean TC saturation and percent change at hour 24 were signifi cantly greater than at all othe r time-points with 48% and 15% increases from baseline and hour 12.5, respectively (Figure 5-4). Among all subjects, the percent change from baseline ranged from 7 to 104% w ith 19 of 21 subjects having a value of 22% or greater. The ratio of holo-TC to B12 did not in crease significantly until hour 24 with absolute and percent increases of 0.15 and 32% respectively. The range for percent change in this ratio among all subjects was -7 to 109% with 15 of 21 subj ects having an increase of 23% or greater at hour 24. Discussion In this intervention study the changes in marker s of B12 status were measured on an hourly basis during and following administration three 9 g oral doses of B12. In previous studies the

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71 changes in response to similar B12 doses were measured after 24 hours; however, no data were collected prior to this time-point (28, 47, 104). Th e data from the present study indicate that a series of three 9 g doses of oral B12, given ove r 12 hours, led to small fluctuations in holo-TC concentration during the day 1 of the study followed by the previously observed maximum increase in holo-TC concentration 24 hours afte r the first B12 dose was given. There is a similarity in the overall pattern of change in holo-TC, B12 and TC saturation, with a gradual increase over the first day and the most pronounced increase occurring 24 hours after the initial B12 dose and 13 hours after the final B12 dose. The timing of B12 absorption and metabolism ma y explain the pattern of change observed in holo-TC concentration during th e first 12 hours of the interven tion. An increase in holo-TC concentration is first measurable in the blood af ter 3 to 4 hours after i ngestion and holo-TC can be taken up by cells within minutes (23). It is hypothesized that until ce lls are saturated with holo-TC, most of it is taken up so quickly that no major changes in blood levels would be observed initially. When intake is sufficient to sa turate the cells with B 12, significant changes in holo-TC can then be measured. The absolute and percentage increases in B12 concentration were smaller, occurred later, and were maintained longer than those for holo-TC This finding is not su rprising as total serum B12 consists primarily of holo-HC, and the sl ower rate of HC metabolism relative to TC metabolism leads to a slower overa ll turnover of serum B12 and a slower response to changes in intake (26, 138). When comparing these two me asures among the individual subjects, holo-TC had the most consistent pattern with only 1 subj ect not having a change of 20% or greater at hour 24. Additionally, the mean percen t change at hour 24 was three times that of B12. Holo-TC concentration is clearly a more sensitive indicato r of change in B12 inta ke and absorption than

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72 serum B12 concentration since it increases earl ier after supplementation, increased relatively more than serum B12 and decreased earlier post-supplementation ceased. Total-transcobalamin concentr ation did not change signifi cantly during the intervention period. Transcobalamin saturation increased in a similar manner to holo-TC (Figure 5-4). Both holo-TC concentration and TC saturation had comparable resu lts even when considering individual subjects. Of all subjects, 95% and 90% had increa ses of at least 22% at hour 24 for holo-TC and TC saturation, respectively. In a pr evious study, a larger ch ange in TC saturation (at hour 24) than for holo-TC was observed, which was due to a drop in total TC at this time point (47). No such conclusi on can be made from our data since no significant difference was observed. Since TC saturation is a calculated rather than a direct measure, the potential error in this value is greater than that for holo-TC concentration. Ther efore holo-TC concentration may be the better indicator of B12 absorption. This is the first study to mon itor hourly changes in holo-TC concentration in response to oral B12 intake. The most significant change in holo-TC concentra tion occurred at hour 24, indicating this is the optimal ti me post-dose at which to measure holo-TC. The three 9 g oral vitamin dose sequence used in this study was used to minimize passive absorption and maximize the amount of actively absorbed B12 (47, 104). This aspect of the protoc ol would be important in a clinical B12 absorption test, because it is the capacity to actively absorb B12 that is being assessed. Further studies evalua ting the necessity of three dose s and the exact timing of the doses are warranted. In conclusion, holo-TC increases measurably in response to administration of oral B12 within six hours with a maximum peak at 24 hour s. Our results indicate that a B12 absorption

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73 test based on measurement of holo-TC following three oral doses of 9 g B12 should run for 24 hours. 0 1 2 3 4 5 6 7 8 9 10 11 12 24 48B12 dose Blood draw Time from baseline (h) Figure 5-1 Intervention protocol timeline

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74 Table 5-1 Baseline concentra tions of B12 status indicators Variablea Mean ( SD) Range Reference interval Holo-TC (pmol/L) 85.2 38 41 208 40-150 B12 (pmol/L) 406.9 118 241 710 148-444 Transcobalamin saturation 0.12 0.05 0.27 0.05-0.20 Holo-TC/B12 0.22 0.08 0.44 0.15-0.51 Hcy (mol/L) 6.6 1.4 3.9 9.3 4.5-11.9 MMA (mol/L) 0.134 0.060 0.08 0.32 0.08-0.28 Folate (nmol/L) 32.7 7.3 22.2 54.4 >6.0 Creatinine (mol/L) 69 11.7 48 87 50 100 a Vitamin B12 (B12); holo-transcobalamin (holo-TC); methylmalonic acid (MMA); homocysteine (Hcy) a

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75 Table 5-2 Mean ( SD) concentrations of vitami n B12 status indicators at scheduled intervals Time from baseline (h) Variable 0.51.52.53.54.55.56.07.0 Holo-TCa (pmol/L) 84 3885 3989 4391 4396 4397 4199 45 97 42 B12 (pmol/L) 395 113397 109409 114421 113431 126414 109423 117426 117 Total-transcobalamin (pmol/L) 688 134706 135723 136743 138746 147763 144752 141755 128 Time from baseline (h) 8.09.010.011.011.512.52448 Holo-TC (pmol/L) 95 4196 3896 3897 3999 41100 39 124 46 102 37 B12 (pmol/L) 424 102428 102432 116423 114429 112411 107456 110 456 115 Total-transcobalamin (pmol/L) 773 139772 144757 143738 154739 136739 139715 715758 145a Vitamin B12 (B12); holo-transcobalamin (holo-TC); methylmalonic acid (MMA)

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76 Figure 5-2 Change in vitamin B12 (B12) biomar kers during the 48 hour study period. (A) Mean ( LSD) holo-transcobalamin ( holo-TC) concentration relative to albumin at scheduled intervals after oral B12 intake (n = 21). Ho lo-TC increased from baseline at hours 6 to 7 and 11 to 48 (p < 0.001). Holo-TC increased si gnificantly from all other time-points at hour 24 (p < 0.0 01). (B) Mean ( LSD) B12 ( ) concentration relative to albumin at scheduled intervals af ter oral B12 intake (n = 21). Vitamin B12 increased significantly from baseline at hour 24 (p < 0.001). (C) Mean ( LSD) transcobalamin (TC) saturation ( ) at scheduled intervals after oral B12 intake (n = 21). Transcobalamin saturation increased significantly at hours 12.5 48 relative to baseline, and hour 24 relative to all other time-points (p < 0.001). (ANOVA, Tukey test) 0 1 2 3 4 5 6 7 8 9 10 11 12 13 0.10 0.15 0.20 0.25 24 48Holo-TC/albumin 0 1 2 3 4 5 6 7 8 9 10 11 12 13 0.10 0.15 0.20 0.25 24 48 B12 dose 1 B12 dose 3 B12 dose 2Time from baseline (h)TC saturation 0 1 2 3 4 5 6 7 8 9 10 11 12 13 0.5 0.6 0.7 0.8 24 48Cobalamin/albumin A B C

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77 0 1 2 3 4 5 6 7 8 9 10 11 12 13 -15 0 15 30 45 60 75 24 48 B12 dose 1 B12 dose 3 B12 dose 2Time from baseline (h)Percent change Figure 5-3 Mean ( LSD) percent cha nge in holo-transcobalamin (holo-TC; ) and vitamin B12 (B12; ) concentrations relative to albumin at scheduled intervals after oral B12 intake (n = 21). The increases in holo -TC and B12 from baseline to hour 24 were significantly larger than changes at all other time-point s (p < 0.001) (ANOVA, Tukeys test)

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78 0 1 2 3 4 5 6 7 8 9 10 11 12 13 -15 0 15 30 45 60 75 24 48 B12 dose 2 B12 dose 1B12 dose 3Time from baseline (h)Percent change Figure 5-4 Mean ( LSD) percent change in transcobalamin (TC) saturation (), and holotranscobalamin to vitamin B12 ratio (holo-TC/B12) () at scheduled intervals after oral B12 intake (n = 21). There was a significantly larg er percent increase in TC saturation and holo-TC/B12 at hour 24 relative to all other time-points compared to baseline (p < 0.001). (ANOVA, Tukeys test)

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79 CHAPTER 6 DISCUSSION Vitamin B12 is an essential water soluble vitamin functioning as a coenzyme for two metabolic processes, the conversion of methylmalonyl-CoA to succinyl-CoA as adenosylcobalamin and the remethylation of Hcy to methionine as methylcobalamin (13, 35). Absorption and utilization of B12 are dependent on adequate gastric HC l production to release food-bound B12, IF in the in testine for active transport of B12 into the enterocy te, and TC for uptake into body tissues. A defici ency of any of these compone nts can impair B12 metabolism and lead to deficiency even if diet ary intake is sufficient (139). The RDA for B12 is 2.4 g for adults (140). Older adults ( > 60 y) have an increased risk for B12 malabsorption due to an age related in creased risk for achlorhydria and auto-immune based pernicious anemia. The RDA fo r B12 in older adults is also 2.4 g/d, but it is recommended that synthetic B12 provided by su pplements or fortified foods be the primary source (140). Individuals with pernicious anemia are generally treated with IM B12 injections, although it has been reported that passive absorption of megadoses of oral B12 may be sufficient to meet dietary needs (141-143). Vitamin B12 is synthesized by microorganisms, pr esent in the intestinal microflora and is found naturally only in animal-derived foods. Cons equently, individuals who restrict their intake of some or all animal-derived foods limit thei r chances of consuming a diet that provides an adequate amount of vitamin B12. Consumption of B12-fortified foods or B12containing vitamin supplements can provide sufficient B12 fo r these individuals; however, it is estimated that ~ 60 % of the US population does not take supplements (144). Alth ough B12 is required in relatively small amounts, long term adherence to a B12-deficient diet can lead to a B12 deficiency and even moderate B12 deficiency can seriously impair health. Of greatest concern

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80 are individuals who consume diets with restricted intakes of animal-based foods and who do not take B12-containing vitamin supplements or cons ume B12 fortified foods. Studies comparing the B12 status of vegetarians and omnivores have led to the conclusion th at vegetarians are at greater risk for developing a B12 deficiency compared to omnivores (40, 81, 86, 145); however the majority of these studies have been conducted in Europe and therefore may not be applicable to the US population. Additionally they have included both supplement users and nonusers, making it difficult to interpret the effect of dietary B12 intake alone on status. It is estimated that B12 intake in the US exceeds the current RDA (2.4 g/d) leading to the conclusion that B12 dietary inadequacy is not a problem in the US (111, 146). The position of the American Dietetic Association is that approp riately planned vegetarian diets are healthful, nutritionally adequate, a nd provide health benefits in the prevention and treatment of certain diseases (110). The key to this statement is that a meat-free diet must be well planned to ensure that vitamin and mineral needs are met. The da ta from the current st udy, in addition to those from the Framingham Offspring study, and an investigation by Bor at al. suggest that consumption of the current RDA is insufficient to maintain normal B12 status in a significant percentage of young healthy adults (118, 119). A lthough these data do suggest that the current RDA is inadequate to maintain normal B12 status they are insufficient to provide a definitive estimation of a new RDA. In the current study, a FFQ was used to estimate B12 intake. While data generated from FFQs are adequate for obtaining information on relative frequency of consumption of nutrients, contribution of food cate gories to overall intake and estimating intake of key nutrients, FFQs do not generate data prec ise or specific enough to estimate a nutrient requirement. Future controlled metabolic studies designed to estimate the quantity of B12 intake

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81 at which B12 status is optimal are needed since controlled metabolic studies have proven to be highly useful in estimating other nutrient requirements (147, 148). Future studies assessing B12 status need to m easure multiple indicators of B12 status. One strength of the current series of studies was that numerous biom arkers were used to asses B12 status, rather than just one. Although the asse ssment of B12 status has traditionally been based on plasma or serum B12, approximately 5 to 10% of individuals with a plasma B12 concentration between 148 to 221 pmol/L, have been reported to have hematological or neurological abnormalities that responded to B12 supplementation (44, 97). Assessment of vitamin B12 status based on serum holo-TC concentr ation, a relatively new B12 status indicator, has been reported to be an earlier marker of changes in B12 status than total plasma B12 concentration. It has been suggested that meas urement of B12 and holo-TC concentrations in combination may be superior to either alone (27, 28, 81, 116, 149). Plasma homocysteine and serum MMA concentrations are functional indicators of B12 status and ar e inversely related to B12 concentration; however, only MMA concentr ation is specific for B12 status and is considered by some to be the most reliable B12 status indicator (35, 54). There is no clear consensus as to which particular B12 biomarker might be used as a gold standard; however, data from the current set of studi es suggest that a panel of B12 bi omarkers is preferable to any one status indicator for B12 status assessment. Additionally measurements at multiple time points over several days could help confirm a possible diagnosis of B12 deficiency, particularly in the case of holo-TC, which has been reported to be highly sensitive to ch anges in dietary B12 intake. The sensitivity of holo-TC has also led to the hypothesis that it could be used to assess B12 absorption (47, 104). In a previous study conduc ed by Bor et al. (104), it was reported that

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82 measurement of holo-TC 24 hours after ad ministration of a series of three 9 g doses of oral B12 identified individuals with B12 malabsorption. Individuals defined as B12 malabsorbers based on the Schilling test for B12 absorption had no si gnificant change in holoTC in contrast to a significant increase observed in normal controls. In the current set of studies, changes in holoTC and other markers of B12 status were m easured hourly with administration of three 9 g oral doses of B12, to determine whether any significant changes occur before 24 hours. A clear peak in holo-TC concentration was observed at hour 24 for all but one of the 22 subjects with only small fluctuations in holo-TC prior to that. Th is was the first study to monitor hourly changes in holo-TC in response to oral B12 suggesting a te st of B12 absorption ut ilizing holo-TC should involve measurement of holo-TC at baseline a nd 24 hours later. A limita tion of the current study was that only healthy individuals with normal B12 status were incl uded in this investigation. It is possible that saturatio n of cells might be necessary before an increase in holo-TC can be measured even in an individual with no B12 abso rption problems. Therefore, individuals with low B12 status may need more oral B12 and may have a later peak increase in holo-TC compared to individuals with normal B12 status. It is important to note that a B12 malabsorption test would only be run in an i ndividual with B12 deficiency; ther efore, future studies evaluating holo-TC as a measure of B12 absorption needs to compare the efficacy of changes in holo-TC as an index of B12 absorption in individuals with deficient versus normal B12 status. A final objective of this series of studies wa s to determine the effect of specific genenutrient interactions on B12 metabolism. Rare congen ital defects known to impair B12 metabolism and status include various mutations and post-translational changes that result in altering IF and TC protein structur e or a total lack of protein synthesis. Congenital errors in IF or TC lead to pernicious anemia; however e rrors evolving IF only im pair intestinal B12

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83 absorption and can be treated by lifelong IM admi nistration of B12, while errors involving TC lead to death early in life because B12 tran sport and uptake into body cells can not occur (150, 151). Perhaps less apparent than these severe ge netic defects, are polymorphisms that also may alter protein structure enough to impair function. One such polymorphism investigated in the present investigation was the TC 776G G polymorphism. In a previous study by our research group a significant effect of the polymorphism on holo-TC concentration was observed but no difference was detected in the current study. The small differences found between genotype groups in total-TC but not TC saturation suggest some small effect of the polymorphism however, there is likely to be no physiological impact of this pol ymorphism Previous studies focusing only on the effect of the polymorphism on a developing fetus have resulted in mixed findings, though continued investigations re lated to the potentia l association of B12-related polymorphisms and health-relate d consequences are warranted (128-130). In conclusion, based on data from this series of investigations it is clear that healthy individuals who do not take supplements may not be consuming adequate B12 to meet biological requirements, particularly those limiting some or all animal-based foods. Although moderate B12 deficiency may not result in overt symptoms, the associated increased risk for disease and birth defect-affected pregnancies provide an impetus for continued research focusing on determining the optimal B12 intake to maintain normal status. Early findings of the potential negative effect of the TC 776C G polymorphism on B12 metabol ism were not confirmed by the current data, and any future investigations should focus on the combined effects of multiple polymorphisms in genes involved in B12 metabolis m. Accurate detection and diagnosis of a B12 deficiency and its cause will help in th e prevention of related health problems including abnormal pregnancy outcomes. Although there is yet no consensus on a single gold standard

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84 test of B12 status, simultaneous measurement of two or more B12 biomarkers at several time points may be the best diagnostic approach. If existence of a B12 deficiency is established, further testing to determine if it is due to diet ary insufficiency or mala bsorption will aid in determining an appropriate treatment, includ ing changes in dietary B12 intake and/or supplementation. Data from the current investigati ons support the use of holo-TC as an indicator of B12 absorption though further re search is needed before a clin ically reliable test could be developed.

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85 APPENDIX A SUBJECT PHONE SCREENING FORM Introduction I am calling in regard to your interest in our nutrition study; do you ha ve a few minutes right now? This is a UF Nutrition department study and involves coming in one morning for about 1 hour for a fasting blood sample, we take about 1 oun ces of blood, and you only need to fast 8 hours. We will give you a breakfast snack right afterward, and then give a brief explanation of a food frequency questionnaire you will be taking home. You will be asked to mail it back in the provided envelope, and once we receive the ques tionnaire you would get pa id the $50. I just have to ask you some questions to see if you are eligible for our st udy and to get background information, OK? How old are you? Must be 18-49 Do you smoke? Must answer no Are you pregnant or breastfeeding ? Must answer no Do you take any prescription medications other than oral contraceptives ? Must answer no If not within the age range or if they answe r yes to any above questions end call with: I am very sorry, but you do not meet our exclus ion criteria, but thank you for your interest. Now I just have a few questions about your diet to see what specific category of our study you would fit in to. Please answer as best you can, estimates are ok and consider all instances of when you might eat the items I will ask about, even if only occasionally. Do you take a multi-vitamin, complex, red star nutritional yeast, or any other supplement or additive ever? If they take a multivitamin, B complex, red star nutritional yeast, complete the session through all diet info only. Conclude by confirming their name and saying This has been a preliminary screening call, your information will be reviewed by the principal investigator based on need, and our selection criteria at this time. If you are chosen you will be called again to schedule an appointment over the next two weeks. Thank you very much for your interest and your time. Do you eat breakfast cereals? ( If so) What Kind do you eat mostly? If they eat a 100% fortified cer eal or eats a 50% cereal daily complete the through all diet info but do not record. Conclude by confirming their name and saying This has been a preliminary screening call, your information will be reviewed by the principal investigator based on need, and our selection criteria at this time. If you are chosen you will be called again to schedule an appointment over the next two weeks. Thank you very much for your interest and your time. If the interviewee fulfills all se lection criteria continue with the questionnaire, record info on moderate/non-fortified cereal consumption

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86 Do you eat breakfast cereals? o Yes o No Name/Brand Quantity Frequency Are you a vegan, vegetarian or meat eater? Vegan this means you eat NO animal derived foods intentionally (if they eat small amt like in cake then OK) Vegetarian this means you eat NO bee f, chicken, turkey, pork, or fish How often do you eat Never Rarely (<1 x/mo) Occasionally (1-4 x/mo) Frequently (2-4 x/wk) Always (5-7 x/wk) Beef Chicken Turkey Pork Fish Eggs Cheese Cows Milk Yogurt Other Dairy Do you follow a restricted diet such as o No red meat o Lactose-free o Kosher o Weight loss o Weight gain o Low salt o Low fat o Low cholesterol o Low carbohydrate o Hypoallergenic (If so) How long have you consumed this type of diet? ________________________________________________________________________ Have you made any major dietary ch anges within the last 3 years? o No o Yes; How long ago did you make cha nges and what changes did you make? ________________________________________________________________________ NO YES Do you consume alcoholic beverages? How often/quantity Health Information

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87 I am going to ask you a few questions about your health to determine if you are eligible for our study. I will be recording this information, but it will be kept confidential and is this ok with you? _______ Height: Weight: Have you do you currently have any of the following? NO YES Alcoholism Anemia Blood clots Bronchitis Cystic Fibrosis Dermatitis Diabetes Eating disorders/Chronic nausea or vomiting Food allergy Gall bladder disease GI problems/ Lactose intolerance Gout Migraines Hemorrhoids Hepatitis/Liver disease Heart disease/High cholesterol/High blood pressure HIV Kidney disease Neurological disorder Obesity Seizures/Stroke Thyroid problem Tumors/Cancer Ulcers Other Have you been hospitalized within the last 5 years? Cause Do you have a history of more than 1 miscarriage? o Yes o No If you are selected to participate in this study are you willing to sign an informed consent understanding we have access to medical information on you? o Yes o No Demographic Information What is your birth date? _______/_______/_________

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88 Month Day Year How would you describe your race or ethnic background? o White o Black or African American o American Indian or Alaska Native o Hispanic or Latino o Asian o Native Hawaiian or Other Pacific Islander o Other _________________________________________________ What is the highest level of school or training that you have completed? [Circle only one response] Grade school 01 02 03 04 05 06 07 08 High school 09 10 11 12 Technical school or college 13 14 15 16 Graduate or professional 17 18 19 20+ Dont know X Marital status? o Single/never married o Married o Separated o Divorced o Widowed Are you a full-time or part-time student? Are you employed? o Full time o Yes o Part time o No o Not a student o Student employee

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89 Contact Information Name of person and phone number to call in case of an emergency if you are invited to participate in this study: ______________________________________________________________________________ If we need to contact you, and can not reach you where/with who can a message be left? ______________________________________________________________________________ How did you hear about our study? _______________________________________________ Name M / F Last First Middle Street Apt. # Address City Zip code Phone Day Evening Cell E-mail

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90 APPENDIX B INTERVENTION DIET Diet B12 Inte rvention Study DAY 1 DAY 2 Breakfast Breakfast Juice (apple or cranberry) Scrambled egg Bread Toast with jelly Coffee/tea Juice (apple or cranberry) Snack Snack Graham crackers Graham crackers Peanut butter Peanut butter Apple Apple Coffee/tea Beverage* Lunch Lunch Grilled cheese sandwich Pita sandwich with hummus and veggies Fruit cocktail Corn chips Pudding Pineapple Beverage* Beverage* Dinner Dinner Bean burrito Cheese tortellini with spaghetti sauce Brown rice Green beans Salad with dressing Mandarin oranges Pears Jello Beverage* Beverage* Snack Snack Sherbet Pudding Pound cake Shortbread cookies Beverage* Beverage* *Beverage may be Crystal Light, non-ca ffeinated soda, Gatorade, Hawaiian Punch (selection to be made with th e help of research staff) Non-caffeinated, non-caloric beverages (Cryst al Light, diet soda, water) available throughout the day as desired

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91 LIST OF REFERENCES 1. McDowell LR. Vitamin B12. Vitamins in Animal and Human Nutrition. 2 ed. Ames: Iowa State University Press, 2000. 2. Farber K. Gastritis and its Consequences. Oxford: Oxford University Press, 1935. 3. Minot GR, Murphy WP. Landmark article (JAMA 1926). Treatment of pernicious anemia by a special diet. By George R. Minot and William P. Murphy. JAMA 1983;250:3328-35. 4. Castle WB. Observations on etological rela tionship of achylia gast rica to pernicious anemia II. Effect of administration to patient s with pernicious anem ia beef muscle after incubation with normal human gastric juice. Am. J. M. Sc. 1929;178:764-77. 5. Castle WB. Observations on etological rela tionship of achylia gast rica to pernicious anemia I. Effect of administ ration to patients with pernici ous anemia of contents of normal human stomach recovered after inge stion of beef muscle. Am. J. M. Sc. 1929;178:748-64. 6. Castle WB. Observations on etological rela tionship of achylia gast rica to pernicious anemia. IV. Site of interaction of food (extrinsic) and gastric (i ntrinsic) factor s: failure of in vitro incubation to produce thermostable hematopoietic princple. Am. J. M. Sc. 1929;178:748-64. 7. Castle WB. Observations on etological rela tionship of achylia gast rica to pernicious anemia III. Nature of reaction between nor mal human gastric juice and beef muscle leading to clinical improvement and increase d blood formation similar to effect of liver feeding. Am. J. M. Sc. 1930;180:305-35. 8. Shorb MS. Unidentified growth factors for Lactob acillus lactis in refined liver extracts. J. Biol. Chem. 1947;169:455-6. 9. Smith EL. Crystalline anti-pernicious -anaemia factor. Br Med J 1949;2:1367-9. 10. Berk L, Castle WB, Welch AD, Heinle RW Anker R, Epstein M. Observations on the etiologic relationship of achylia gastrica to pernicious anemia. X. Activity of vitamin B12 food (extrinsic) factor. 1 948. Nutr Hosp 2004;19:387-90. 11. Hodgkin DC. X-ray analysis and the struct ure of vitamin B12. Fortschr Chem Org Naturst 1958;15:167-220. 12. Hodgkin DC, Kamper J, Mackay M, Pickwort h J, Trueblood KN, White JG. Structure of vitamin B12. Nature 1956;178:64-6. 13. Carmel R. Cobalamin deficiency. In: Carm el R, Jacobson DW, eds. Homocysteine in Health and Disease: Cambridge University Press, 2001:289-305.

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92 14. Castle WB, Hale TH. Vitamin B12. In: Combs G, ed. The Vitamins: Fundamental Aspects in Nutrition and Health. Second ed. San Diego: Academic Press Limited, 1998:403-20. 15. Nexo E. Cobalamin binding proteins. In: Ke rautler B, Arigoni D, Golding B, eds. Vitamin B12 and B12-Proteins. Weinheim: Wiley-VCH, 1998:461-90. 16. Logan RF, Elwis A, Forrest MJ, Lawrence AC Mechanisms of vitamin B12 deficiency in elderly inpatients. Age And Ageing 1989;18:4-10. 17. Baik HW, Russell RM. Vitamin B12 defici ency in the elderly. Annual Review of Nutrition 1999;19:357-77. 18. Fyfe JC, Madsen M, Hojrup P, et al. The functional cobalamin (vit amin B12)-intrinsic factor receptor is a novel complex of cubilin and amnionle ss. Blood 2004;103:1573-9. 19. Kozyraki R. Cubilin, a multifunctional epit helial receptor: an overview. J Mol Med 2001;79:161-7. 20. Brada N, Gordon MM, Wen J, Alpers DH. Transf er of cobalamin from intrinsic factor to transcobalamin II. J Nutr Biochem 2001;12:200-6. 21. Chanarin I, Muir M, Hughes A, Hoffbra nd AV. Evidence for intestinal origin of transcobalamin II during vitamin B 12 absorption. Br Med J 1978;1:1453-5. 22. Quadros EV, Regec AL, Khan KM, Quadros E, Rothenberg SP. Transcobalamin II synthesized in the intestinal villi facilitates transfer of c obalamin to the portal blood. Am J Physiol 1999;277:G161-6. 23. Hom BL, Olesen HA. Plasma clearance of 57cobalt-labelled vitamin B12 bound in vitro and in vivo to transcobalamin I and II. Scand J Clin Lab Invest 1969;23:201-11. 24. Nexo E, Gimsing P. Turnover in huma ns of iodineand cobalamin-labeled transcobalamin I and of iodine-labeled al bumin. Scand J Clin Lab Invest 1975;35:391-8. 25. Cannon MJ, Myszka DG, Bagnato JD, Alpers DH, West FG, Grissom CB. Equilibrium and kinetic analyses of the interactions between vitamin B(12) binding proteins and cobalamins by surface plasmon res onance. Anal Biochem 2002;305:1-9. 26. Seetharam B, Li N. Transcobalamin II a nd its cell surface receptor. Vitam Horm 2000;59:337-66. 27. Herzlich B, Herbert V. Depletion of se rum holotranscobalamin II. An early sign of negative vitamin B12 balance. Lab Invest 1988;58:332-7.

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93 28. Nexo E, Hvas A-M, Bleie O, et al. Holo-trans cobalamin is an early marker of changes in cobalamin homeostasis. A randomized placebo-controlled study. Clin Chem 2002;48:1768-71. 29. Lindgren, Kilander, Bagge, Nexo. Holotransc obalamin a sensitive marker of cobalamin malabsorption. Eur J Clin Invest 1999;29:321-9. 30. Afman LA, Lievers KJ, van der Put NM, Trijbels FJ, Blom HJ. Single nucleotide polymorphisms in the transcobalamin gene : relationship with transcobalamin concentrations and risk for neural tu be defects. Eur J Hum Genet 2002;10:433-8. 31. Wickramasinghe SN, Fida S. Correlati ons between holo-transcobalamin II, holohaptocorrin, and total B12 in serum samples from healthy subjects and patients. J Clin Pathol 1993;46:537-9. 32. Herbert V. Staging vitamin B-12 (cobalamin) status in vegetarians. Am J Clin Nutr 1994;59:1213S-22S. 33. Birn H, Willnow TE, Nielsen R, et al. Me galin is essential for renal proximal tubule reabsorption and accumulation of transcobalamin-B(12). Am J Physiol Renal Physiol 2002;282:F408-16. 34. Moestrup SK. New insights into carrier binding and epithelial uptake of the erythropoietic nutrients cobalamin and folate. Curr Opin Hematol 2006;13:119-23. 35. Stabler SP. Vitamin B12. In: Bowman BA, Russell RM, eds. Present Knowledge in Nutrition. Washington, D.C.: ILSI Press, 2001:230-40. 36. Taylor RT, Weissbach H. N5-methyltetrahyd rofolate-homocysteine transmethylase. Role of S-adenosylmethionine in vitamin B12-depe ndent methionine synthesis. J Biol Chem 1967;242:1517-21. 37. Taylor RT, Weissbach H. Role of S-ade nosylmethionine in vitamin B12-dependent methionine synthesis. J Biol Chem 1966;241:3641-2. 38. Institute of Medicine. Dietary Reference Intakes: Thiamin, Riboflavin, Niacin, Vitamin B6, Folate, Vitamin B12, Pantothenic Aci d, Biotin, and Choline. Washington D.C: National Acadamy Press, 1998. 39. USDA National Nutrient Database for Sta ndard Reference, Rel ease 17. Nutrient Data Laboratory Home Page, 2004. http ://www.nal.usda.gov/fnic/foodcomp 40. Obeid R, Geisel J, Schorr H, Hubner U, Herrmann W. The impact of vegetarianism on some haematological parameters. Eur J Haematol 2002;69:275-9.

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94 41. Dagnelie PC. [Nutrition and health--potential health benefits and risks of vegetarianism and limited consumption of meat in th e Netherlands]. Ned Tijdschr Geneeskd 2003;147:1308-13. 42. Ritter MM, Richter WO. [Effect s of a vegetarian life styl e on health]. Fortschr Med 1995;113:239-42. 43. Wright JD, Bialostosky K, Gunter EW, et al. Blood folate and vitamin B12: United States, 1988-94. Vital Health Stat 11 1998:1-78. 44. Lindenbaum J, Allen R. Clinical spectrum a nd diagnosis of folate deficiency. In: Bailey L, ed. Folate in Health and Disease. New York, NY: Mar cel Decker, 1995:43-73. 45. Ulleland M, Eilertsen I, Quadros EV, et al. Direct Assay for Cobalamin Bound to Transcobalamin (Holo-Transcobalamin) in Serum. Clin Chem 2002;48:526-32. 46. Lindgren A, Kilander A, Bagge E, Nexo E. Holotranscobalamin a sensitive marker of cobalamin malabsorption. Eur J Clin Invest 1999;29:321-9. 47. Bor MV, Nexo E, Hvas AM. Holo-transc obalamin concentration and transcobalamin saturation reflect recent vitami n B12 absorption better than does serum vitamin B12. Clin Chem 2004;50:1043-9. 48. Nexo E, Christensen AL, Hvas AM, Peters en TE, Fedosov SN. Quantification of holotranscobalamin, a marker of vitamin B12 deficiency. Clin Chem 2002;48:561-2. 49. Loikas S, Lopponen M, Suominen P, et al RIA for serum holo-transcobalamin: method evaluation in the clinical la boratory and refere nce interval. Clin Chem 2003;49:455-62. 50. Lindenbaum J, Savage DG, Stabler SP, Allen RH. Diagnosis of cobalamin deficiency: II. Relative sensitivities of serum cobalamin, me thylmalonic acid, and total homocysteine concentrations. Am J Hematol 1990;34:99-107. 51. Savage DG, Lindenbaum J, Stabler SP, A llen RH. Sensitivity of serum methylmalonic acid and total homocysteine determinations for diagnosing cobalamin and folate deficiencies. Am J Med 1994;96:239-46. 52. Moelby L, Rasmussen K, Jensen MK, Peders en KO. The relationship between clinically confirmed cobalamin deficiency and se rum methylmalonic acid. J Intern Med 1990;228:373-8. 53. Rasmussen K, Nathan E. The clinical evaluation of cobalamin deficiency by determination of methylmalonic acid in serum or urine is not invali dated by the presence of heterozygous methylmalonic-acidaemia J Clin Chem Clin Biochem 1990;28:419-21.

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96 66. Centers for Disease Control. Recommendations for the use of folic acid to reduce the number of cases of spina bifida and other ne ural tube defects. Mo rb Mortal Wkly Rep, 1992:1-7. 67. Adams MJ, Jr., Khoury MJ, Scanlon KS, et al. Elevated midtrimester serum methylmalonic acid levels as a risk fact or for neural tube defects. Teratology 1995;51:311-7. 68. Dawson EB, Evans DR, Van Hook JW. Amniotic fluid B12 and folate levels associated with neural tube defects. Am J Perinatol 1998;15:511-4. 69. Schorah CJ, Smithells RW, Scott J. Vita min B12 and anencephaly. Lancet 1980;1:880. 70. Mills JL, Tuomilehto J, Yu KF, et al. Ma ternal vitamin levels during pregnancies producing infants with neural tube defects. J Pediatr 1992;120:863-71. 71. Wald NJ, Hackshaw AD, Stone R, Souria l NA. Blood folic acid and vitamin B12 in relation to neural tube defects. Br J Obstet Gynaecol 1996;103:319-24. 72. Magnus P, Magnus EM, Berg K. Transcobalamins in the etiology of ne ural tube defects. Clin Genet 1991;39:309-10. 73. Groenen PM, van Rooij IA, Peer PG, Goos kens RH, Zielhuis GA, Steegers-Theunissen RP. Marginal maternal vitamin B12 status in creases the risk of offspring with spina bifida. Am J Obstet Gynecol 2004;191:11-7. 74. Afman LA, Van Der Put NMJ, Thomas CMG, Trijbels JMF, Blom HJ. Reduced vitamin B12 binding by transcobalamin II increases the risk of neural tube defects. QJM 2001;94:159-66. 75. Haddad EH, Berk LS, Kettering JD, Hubbard RW, Peters WR. Dietary intake and biochemical, hematologic, and immune status of vegans compared with nonvegetarians. The American Journal Of Clin ical Nutrition 1999;70:586S-93S. 76. Vegetarian Resource Group. How many vege tarians are there? Veg J, 2000:Dec 2003;3:online http://www.vrg.org/journal/vj2003issue3/vj2003issue3poll.htm. 77. United States Department of Agricultur e. Continuing Survey of Food Intake by Individuals 1994. 1998. Online databa se http://www.ars.usda.gov/ 78. Ginsberg C, Ostrowski A. The market for vegetarian foods. Veg J 2002;4:25-9. 79. Mezzano D, Kosiel K, Martinez C, et al. Card iovascular risk factors in vegetarians: normalization of hyperhomocysteinemia with vitamin B12 and reduction of platelet aggregation with n-3 Fatty Acids. Thrombosis Research 2000;100:153-60.

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97 80. Krajcovicova-Kudlackova M, Blazicek P, Kopcova J, Bederova A, Babinska K. Homocysteine levels in vegetarians vers us omnivores. Ann Nutr Metab 2000;44:135-8. 81. Herrmann W, Schorr H, Obeid R, Geisel J. Vitamin B-12 status, particularly holotranscobalamin II and methylma lonic acid concentrations, and hyperhomocysteinemia in vegetarians. Am J Clin Nutr 2003;78:131-6. 82. Herrmann W, Obeid R, Schorr H, Za rzour W, Geisel J. Homocysteine, methylenetetrahydrofolate redu ctase C677T polymorphism and the B-vitamins: a facet of nature-nurture inte rplay. Clin Chem Lab Med 2003;41:547-53. 83. Hokin BD, Butler T. Cyanocobalamin (vitamin B-12) status in Se venth-day Adventist ministers in Australia. Am J Clin Nutr 1999;70:576S-8S. 84. Alexander D, Ball MJ, Mann J. Nutrient inta ke and haematological status of vegetarians and age-sex matched omnivores. Eur J Clin Nutr 1994;48:538-46. 85. Antony AC. Vegetarianism and vitamin B-12 (cobalamin) deficiency. Am J Clin Nutr 2003;78:3-6. 86. Herrmann W, Schorr H, Purschwitz K, Ra ssoul F, Richter V. Total homocysteine, vitamin B(12), and total antioxidant status in vegetarians. Clin Chem 2001;47:1094-101. 87. Huang YC, Chang SJ, Chiu YT, Chang HH, Cheng CH. The status of plasma homocysteine and related B-vitamins in h ealthy young vegetarians and nonvegetarians. Eur J Nutr 2003;42:84-90. 88. Carmel R. Measuring and Interpreting Ho lo-Transcobalamin (Holo-Transcobalamin II). Clin Chem 2002;48:407-9. 89. Miller JW, Ramos MI, Garrod MG, Flynn MA, Green R. Transcobalamin II 775G>C polymorphism and indices of vitamin B12 status in healthy older adults. Blood 2002;100:718-20. 90. McCaddon A, Blennow K, Hudson P, et al. Transcobalamin polymorphism and homocysteine. Blood 2001;98:3497-500. 91. von Castel-Dunwoody KM, Kauwell GP, Shel nutt KP, et al. Transcobalamin 776C->G polymorphism negatively affects vitamin B-12 metabolism. Am J Clin Nutr 2005;81:1436-41. 92. Wans S, Schuttler K, Jakubiczka S, Muller A, Luley C, Dierkes J. Analysis of the transcobalamin II 776C>G (259P>R) single nucleotide polymorphism by denaturing HPLC in healthy elderly: associations with cobalamin, homocysteine and holotranscobalamin II. Clin Chem Lab Med 2003;41:1532-6.

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98 93. Fodinger M, Veitl M, Skoupy S, et al. Eff ect of TCN2 776C>G on vitamin B12 cellular availability in end-stage renal di sease patients. Kidney Int 2003;64:1095-100. 94. Swanson DA, Pangilinan F, Mills JL, et al. Evaluation of transcobalamin II polymorphisms as neural tube defect risk fact ors in an Irish population. Birth Defects Res A Clin Mol Tera tol 2005;73:239-44. 95. Carmel R. Cobalamin, the stomach, and ag ing. The American Journal of Clinical Nutrition 1997;66:750-9. 96. Carmel R. Current concepts in cobala min deficiency. Annu. Rev. Medicine 2000;51:35775. 97. Institute of Medicine. Vitamin B12. Diet ary Reference Intakes. Washington, D.C.: National Academy Press, 1998:306-356. 98. Allen RH. Megaloblastic anemias. In: Go ldman L, Bennett JC, eds. Cecil textbook of medicine. Philadelphia: WB Saunders Co, 2000:859-67. 99. Ward PC. Modern approaches to the invest igation of vitamin B12 deficiency. Clin Lab Med 2002;22:435-45. 100. Zuckier LS, Chervu LR. Schilling evaluation of pernicious anemia: current status. J Nucl Med 1984;25:1032-9. 101. Nexo E, Hansen M, Rasmussen K, Lindgren A, Grasbeck R. How to diagnose cobalamin deficiency. Scand J Clin Lab Invest Suppl 1994;219:61-76. 102. Ardeman S, Chanarin I. Intrinsic factor an tibodies and intrinsic factor mediated vitamin B-12 absorption in pernicious anaemia. Gut 1965;6:436-8. 103. Oh R, Brown DL. Vitamin B12 defi ciency. Am Fam Physician 2003;67:979-86. 104. Bor MV, Cetin M, Aytac S, Altay C, Nexo E. Nonradioactive vitamin B12 absorption test evaluated in controls and in patients with inherited malabsorption of vitamin B12. Clin Chem 2005;51:2151-5. 105. Geisel J, Hubner U, Bodis M, et al. The ro le of genetic factors in the development of hyperhomocysteinemia. Clin Chem Lab Med 2003;41:1427-34. 106. Radimer K, Bindewald B, Hughes J, Er vin B, Swanson C, Picciano MF. Dietary supplement use by US adults: data from the National Health and Nu trition Examination Survey, 1999-2000. Am J Epidemiol 2004;160:339-49.

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99 107. Subar AF, Thompson FE, Kipnis V, et al. Co mparative validation of the Block, Willett, and National Cancer Institute food frequenc y questionnaires : the Eating at America's Table Study. Am J Epidemiol 2001;154:1089-99. 108. Stabler SP, Marcell PD, Podell ER, Allen RH Quantitation of total homocysteine, total cysteine, and methionine in normal serum a nd urine using capillary gas chromatographymass spectrometry. Analytical Biochemistry 1987;162:185-96. 109. Stabler SP, Marcell PD, Podell ER, Allen RH, Lindenbaum J. Assay of methylmalonic acid in the serum of patien ts with cobalamin deficiency using capillary gas chromatography-mass spectrometr y. J Clin Invest 1986;77:1606-12. 110. American Dietetics Associat ion. Position of the American Dietetic Association and Dietitians of Canada: Vegetarian diets. J Am Diet Assoc, 2003:748-65. 111. Ervin RB, Wright JD, Wang CY, Kennedy-Stephenson J. Diet ary intake of selected vitamins for the United States population: 1999-2000. Adv Data 2004:1-4. 112. Bailey LB, Moyers S, Gregory JF. Folate In: Bowman BA, Russell RM, eds. Present Knowledge in Nutrition. Washi ngton, D.C.: ILSI Press, 2001:214-29. 113. Wilson RD, Davies G, Desilets V, et al. The use of folic acid for the prevention of neural tube defects and other congenital anom alies. J Obstet Gynaecol Can 2003;25:959-73. 114. Scholl TO, Johnson WG. Folic acid: influen ce on the outcome of pregnancy. Am J Clin Nutr 2000;71:1295S-303S. 115. Lloyd-Wright Z, Hvas AM, Moller J, Sanders TA, Nexo E. Holotranscobalamin as an indicator of dietary vitamin B12 deficiency. Clin Chem 2003;49:2076-8. 116. Miller JW, Garrod MG, Rockwood AL, et al Measurement of to tal vitamin B12 and holotranscobalamin, singly and in combinati on, in screening for metabolic vitamin B12 deficiency. Clin Chem 2006;52:278-85. 117. Schneede J, Ueland PM. Novel and establ ished markers of cobalamin deficiency: complementary or exclusive diagnostic strategies. Semin Vasc Med 2005;5:140-55. 118. Bor MV, Lydeking-Olsen E, Moller J, Nexo E. A daily intake of approximately 6 micrograms vitamin B-12 appears to saturate all the vitamin B-12-related variables in Danish postmenopausal women. Am J Clin Nutr 2006;83:52-8. 119. Tucker KL, Rich S, Rosenberg I, et al. Pl asma vitamin B-12 concentrations relate to intake source in the Framingham Offspr ing study. Am J Clin Nutr 2000;71:514-22. 120. Leclerc D, Odievre M-H, Wu Q, et al Molecular cloning, ex pression and physical mapping of the human methionine syntha se reductase gene*1. Gene 1999;240:75-88.

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100 121. Bailey LB, Duhaney RL, Maneval DR, et al. Vi tamin B-12 status is inversely associated with plasma homocysteine in young women with C677T and/or A1298C methylenetetrahydrofolate reductase polymorphisms. J Nutr 2002;132:1872-8. 122. Anello G, Gueant-Rodriguez RM, Bosco P, et al. Homocysteine and methylenetetrahydrofolate redu ctase polymorphism in Alzhei mer's disease. Neuroreport 2004;15:859-61. 123. Bailey LB, Gregory JF, III. Polymorphisms of methylenetetrahydrof olate reductase and other rnzymes: metabolic significance, risk s and impact on folate requirement. J. Nutr. 1999;129:919-22. 124. van der Put NM, Gabreels F, Stevens EM et al. A second common mutation in the methylenetetrahydrofolate reductase gene: an additional risk factor for neural-tube defects? Am J Hum Genet 1998;62:1044-51. 125. Molloy AM, Mills JL, McPartlin J, Kirke PN Scott JM, Daly S. Maternal and fetal plasma homocysteine concentrations at birt h: the influence of folate, vitamin B12, and the 5,10-methylenetetrahydrofolate reductase 677C-->T variant. Am J Obstet Gynecol 2002;186:499-503. 126. Gueant JL, Gueant-Rodriguez RM, Anello G, et al. Genetic determinants of folate and vitamin B12 metabolism: a common pathway in neural tube defect and Down syndrome? Clin Chem Lab Med 2003;41:1473-7. 127. Zetterberg H. Methylenetetrahydrofolat e reductase and transcobalamin genetic polymorphisms in human spontaneous aborti on: biological and c linical implications. Reprod Biol Endocrinol 2004;2:7. 128. Zetterberg H, Zafiropoulos A, Spandidos DA, Rymo L, Blennow K. Gene-gene interaction between fetal MTHFR 677C>T and transcobalamin 776C>G polymorphisms in human spontaneous abor tion. Hum. Reprod. 2003;18:1948-50. 129. Alessio AC, Hoehr NF, Siqueira LH, Bydlowski SP, Annichino-Bizzacchi JM. Polymorphism C776G in the transcobalamin II gene and homocysteine, folate and vitamin B(12) concentrations. Associati on with MTHFR C677T and A1298C and MTRR A66G polymorphisms in healthy children. Th romb Res 2006;[Eprint ahead of pub]. 130. Martinelli M, Scapoli L, Pezzetti F, et al C677T variant form at the MTHFR gene and CL/P: a risk factor for mother s? Am J Med Genet 2001;98:357-60. 131. Baik HW, Russell RM. Vitamin B12 defici ency in the elderly. Annual Review of Nutrition 1999;19:357-77.

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101 132. Rauma AL, Torronen R, Hanninen O, Mykkane n H. Vitamin B-12 status of long-term adherents of a strict uncooke d vegan diet ("living food di et") is compromised. J Nutr 1995;125:2511-5. 133. Nexo E, Christensen AL, Petersen TE, Fedosov SN. Measurement of transcobalamin by ELISA. Clin Chem 2000;46:1643-9. 134. Morkbak AL, Heimdal RM, Emmens K, et al. Evaluation of the technical performance of novel holotranscobalamin (holoTC) assays in a multicenter European demonstration project. Clin Chem Lab Med 2005;43:1058-64. 135. Moller J, Ahola L, Abrahamsson L. Evaluation of the DPC IMMULITE 2000 assay for total homocysteine in plasma. S cand J Clin Lab Invest 2002;62:369-73. 136. Rasmussen KE, Tonnesen F, Thanh HH, Rogstad A, Aanesrud A. Solid-phase extraction and high-performance liquid chromatographic de termination of flumequine and oxolinic acid in salmon plasma. J Chromatogr 1989;496:355-64. 137. Longnecker MT, Ott L. Introduction to Stat istical Methods and Data Analysis. 6 ed. North Scituate: Duxbury Press, 2006. 138. Nexo E, Gimsing P. Turnover studies with ra dio-iodine-labelled tran scobalamin I. Scand J Gastroenterol Suppl 1974;29:17-8. 139. Dawson DW, Sawers AH, Sharma RK. Ma labsorption of protein bound vitamin B12. British Medical Journal (Clinical Research Ed.) 1984;288:675-8. 140. Institute of Medicine. Dietary Reference Intakes: Thiamin, Riboflavin, Niacin, Vitamin B6, Folate, Vitamin B12, Pantothenic Aci d, Biotin, and Choline. Washington D.C: National Academy Press, 1998. 141. Park S, Johnson MA. What is an adequate dos e of oral vitamin B12 in older people with poor vitamin B12 status? Nutr Rev 2006;64:373-8. 142. Vidal-Alaball J, Butler CC, Cannings-J ohn R, et al. Oral vitamin B12 versus intramuscular vitamin B12 for vitamin B12 deficiency. Cochrane Database Syst Rev 2005:CD004655. 143. Eussen SJ, de Groot LC, Clar ke R, et al. Oral cyanocoba lamin supplementation in older people with vitamin B12 deficiency: a dose-finding trial. Arch Intern Med 2005;165:1167-72. 144. Balluz LS, Kieszak SM, Philen RM, Mulinare J. Vitamin and mineral supplement use in the United States. Results from the third National Health and Nutrition Examination Survey. Arch Fam Med 2000;9:258-62.

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102 145. Larsson CL, Johansson GK. Dietary intake and nutritional status of young vegans and omnivores in Sweden. Am J Clin Nutr 2002;76:100-6. 146. Alaimo K, McDowell MA, Briefel RR, et al. Dietary intake of v itamins, minerals, and fiber of persons ages 2 months and over in the United States: Third National Health and Nutrition Examination Survey, Phase 1, 1988-91. Adv Data 1994:1-28. 147. Caudill MA, Cruz AC, Gregory JF, 3rd, Hutson AD, Bailey LB. Folate status response to controlled folate intake in pr egnant women. J Nutr 1997;127:2363-70. 148. O'Keefe CA, Bailey LB, Thomas EA, et al. Cont rolled dietary folate a ffects folate status in nonpregnant women. J Nutr 1995;125:2717-25. 149. Lloyd-Wright Z, Hvas A-M, Moller J, Sande rs TAB, Nexo E. Holotranscobalamin as an indicator of dietary vitamin B12 deficiency. Clin Chem 2003;49:2076-8. 150. Whitehead VM. Acquired and inherited disorder s of cobalamin and folate in children. Br J Haematol 2006;134:125-36. 151. Frater-Schroder M. Genetic patterns of transcoba lamin II and the relationships with congenital defects. Mo l Cell Biochem 1983;56:5-31.

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103 BIOGRAPHICAL SKETCH Kristina M von Castel Robert s was born in Camden, New Jersey in 1975. She lived in Glenside and Penllyn, Pennsylvania from 1981 until she graduated from Springside School for girls in 1993. She graduated from the University of Florida in 2000 with a bachelor of science degree in animal science, with a specialization in animal bi ology. She was employed by the University of Florida Racing Laboratory until she entered her Ph.D. program in nutritional sciences in the fall of 2002 under the Davis Alum ni fellowship. During her doctoral program she studied under the tutelage of Dr. Lynn B. Bailey in the field of folate and vitamin B12 nutrition and metabolism. Upon graduation she w ill continue her care er in academia.