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Relationship between Beef Consumption and Vitamin B12 Intake and Status in Healthy Men and Women


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RELATIONSHIP BETWEEN BEEF CONSUMPTION AND VITAMIN B12 INTAKE AND STATUS OF HEA LTHY MEN AND WOMEN By AMANDA L. BROWN A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2006

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Copyright 2006 by Amanda L. Brown

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iii ACKNOWLEDGMENTS I would like to thank my supervisory committee members Lynn B. Bailey, PhD, Gail P.A. Kauwell, PhD, and Anna-Maria Sieg a-Riz, PhD. I especially would like to thank my committee chair Lynn B. Bailey, PhD. Her continuous guidance and encouragement were amazing. She is a remarkable mentor and role model. I would like to thank Dave Maneval for his direction while working in the laboratory. I also would like to thank Karla Shelnutt, PhD, Kristina von-Castel-Roberts, and Claire Edgemon. All of these women we re amazing support systems and were always willing to listen. In addition, I would like to thank my fam ily. They truly are my foundation. They are remarkable listeners and always provide th e best advice. I also would like to thank Danny Wittmann. Everyday he reminded me of my hard work and of my accomplishments. He always told me how proud he was of me and what a good job I was doing. I would not have been able to complete this progra m without his support.

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iv TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iii LIST OF TABLES............................................................................................................vii LIST OF FIGURES.........................................................................................................viii LIST OF ABBREVIATIONS............................................................................................ix ABSTRACT....................................................................................................................... xi CHAPTER 1 INTRODUCTION........................................................................................................1 Hypotheses....................................................................................................................2 Specific Objectives.......................................................................................................2 2 LITERATURE REVIEW.............................................................................................3 Vitamin B12..................................................................................................................3 Chemistry..............................................................................................................3 Dietary Sources.....................................................................................................4 Absorption.............................................................................................................6 Transport................................................................................................................7 Storage...................................................................................................................7 Excretion................................................................................................................8 Biochemical Functions..........................................................................................8 Dietary Reference Intakes...................................................................................10 Vitamin B12 Deficiency......................................................................................11 Etiology........................................................................................................11 Clinical symptoms........................................................................................14 Health related risks of a vitamin B12 deficiency.........................................15 Vitamin B12 Status Assessment.................................................................................20 Serum Vitamin B12 Concentration.....................................................................20 Methylmalonic Acid Concentration....................................................................20 Holotranscobalamin Concentration.....................................................................21 Serum Homocysteine Concentration...................................................................22 Megaloblastic Anemia.........................................................................................22

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v Dietary Intake Assessment in Adults..................................................................22 Twenty-four hour recall method..................................................................22 Multiple-day food record.............................................................................24 Food frequency questionnaire......................................................................25 Assessment of vitamin B12 intake...............................................................26 Vitamin B12 Status in the United States....................................................................30 Vegetarianism.............................................................................................................31 Definitions of Vegetarianism and Prevalence in the United States.....................31 Assessment of Dietary Adequ acy of Vegetarian Diets.......................................32 Assessment of Beef Consumption and Health............................................................33 Assessment of Dietary Adequacy of Beef Consumption...........................................35 Research Rational and Potent ial Application of Findings..........................................35 3 MATERIALS AND METHODS...............................................................................38 Study Design and Methods Overview........................................................................38 Diet History Questionnaire.........................................................................................39 Overview.............................................................................................................39 Modifications Made to the Paper Versi on of the Diet Hist ory Questionnaire....39 Modifications Made to Diet *Calc Analysis Software.........................................40 Data Generation...................................................................................................41 Diet History Questionnair e Instruction Pretest...........................................................41 Human Subjects Procedures.......................................................................................42 Diet History Questionnaire Instructions.....................................................................42 Sample Collection and Processing..............................................................................43 Processing of Plasma for Vitamin B12 Analysis................................................43 Processing the Diet Hi story Questionnaire..........................................................44 Analytical Methods.....................................................................................................44 Formation of Dietary Groups..............................................................................44 Identification of Food Groups from the Diet History Questionnaire..................45 Analysis of Additional Nutrients.........................................................................45 Plasma Vitamin B12 Concentration....................................................................46 Statistical Methods......................................................................................................47 4 RESULTS...................................................................................................................49 Demographic Characteristics of the Study Population...............................................49 Subjects................................................................................................................49 Demographic Characteristics...............................................................................49 Dietary Vitamin B12 Intake........................................................................................50 Beef, Poultry, Pork, Seafood, and Mixed Foods.................................................52 Dairy and Eggs....................................................................................................53 Cereal...................................................................................................................53 Fortified Soy Products, Meal Repl acements, and Other Sources........................54 Naturally-Occurring and Fortified Sources.........................................................54 Dietary Intake of Macronut rients and Micronutrients................................................58 Energy Intake.......................................................................................................58

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vi Protein Intake.......................................................................................................58 Carbohydrate Intake............................................................................................59 Fat Intake.............................................................................................................59 Saturated Fat........................................................................................................59 Folate Intake........................................................................................................60 Vitamin B6 Intake...............................................................................................60 Iron Intake...........................................................................................................60 Zinc Intake...........................................................................................................60 Plasma Vitamin B12 Concentration...........................................................................61 Vitamin B12 Status..............................................................................................61 Vitamin B12 Intake and Status............................................................................64 5 DISCUSSION AND CONCLUSION........................................................................65 APPENDIX A SUBJECT DATA CO LLECTION FORM.................................................................73 B DIET HISTORY QUESTIONNAIRE........................................................................79 C SCRIPT FOR DIET HISTORY QUE STIONNAIRE INSTRUCTIONS................119 D DIRECTIONS AND SURVEY FO R DIET HISTORY QUESTIONNAIRE PRETEST.................................................................................................................125 E ADDITIONAL INSTRUCTIONS PACKET...........................................................128 LIST OF REFERENCES.................................................................................................134 BIOGRAPHICAL SKETCH...........................................................................................143

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vii LIST OF TABLES Table page 2-1 Dietary vitamin B12 sources......................................................................................6 3-1 Daily and weekly beef intake frequency..................................................................44 3-2 Foods categorized within each food group..............................................................46 4-1 Demographic characteristics of dietary groups........................................................50 4-2 Daily total dietary in take of vitamin B12.................................................................51 4-3 Daily mean vitamin B12 c ontribution by dietary sources........................................55 4-4 Daily mean dietary intake of macronutrients...........................................................59 4-5 Daily mean dietary intake of micronutrients............................................................61 4-6 Plasma vitamin B12 concentration...........................................................................61 4-7 Plasma vitamin B12 concentration among non-vegetarians and vegetarians..........63

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viii LIST OF FIGURES Figure page 2-1 Structure of cobalamin...............................................................................................4 2-2 Vitamin B12 function in the remet hylation pathway of homocysteine to methionine..................................................................................................................9 2-3 Role of cobalamin in th e formation of succinyl-CoA..............................................10 4-1 Mean vitamin B12 intake compared to recommended intakes................................52 4-2 Percent of total vitamin B12 intake from food sources............................................56 4-3 Percent of total vitamin B 12 intake from cereal sources.........................................56 4-4 Percent of total vitamin B12 intake from shellfish and fish.....................................57 4-5 Percent of total vitamin B12 intake from naturally-occurring and fortified sources of vitamin B12.............................................................................................58 4-6 Plasma vitamin B12 concentration...........................................................................62 4-7 Vitamin B12 status among dietary groups...............................................................63 4-8 Percent of individuals deficient among non-vegetarians and vegetarians...............64

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ix LIST OF ABBREVIATIONS Abbreviation Meaning AMPM automated-multiple-pass method ANOVA analysis of variance BMI body mass index CARDIA coronary artery risk development in young adults CNS central nervous system CSFII Continuing Survey of Food Intakes by Individuals DHQ Diet Histor y Questionnaire DNA deoxyribonucleic acid DTT dithiothreitol EAR Estimated Average Requirement EDTA ethylenediaminetraacetic acid EPIC-Oxford European prospective in vestigation into cancer and nutritionOxford FFQ food frequency questionnaire FIN food identification number g gram IF intrinsic factor kcal kilocalories kg kilogram L liter LDL low-density lipoprotein m meter MCV mean corpuscular volume mg milligram mL milliliter MMA methylmalonic acid MS methionine synthase NCI National Cancer Institute NDS-R Nutrient Data System for Research NFCS Nationwide Food Consumption Survey NHANES National Health and Nutrition Examination Survey nmol nanomole NTD neural tube defects OSC Optimal Solutions Corporation oz ounce pmol picomole RDA Recommended Dietary Allowance

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x SAH s -adenosylhomocysteine SAM s -adenosylmethionine SD standard deviation TC transcobalamin THF tetrahydrofolate UL Tolerable Upper Intake Level UNC University of North Carolina, Chapel Hill USDA United States Department of Agriculture wk week g microgram mol micromole *qdd Questionnaire Data Dictionary 125I iodine-125 57Co cobalt-57

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xi Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science RELATIONSHIP BETWEEN BEEF CONSUMPTION AND VITMAIN B12 INTAKE AND STATUS OF HEA LTHY MEN AND WOMEN By Amanda L. Brown May 2006 Chair: Lynn B. Bailey Major Department: Food Science and Human Nutrition Vitamin B12 is an essential nutrient requi red in the diet to ensure normal cell division and nervous system f unction. Dietary sources of naturally-occurring vitamin B12 are limited to those of anim al origin; therefore diets re stricting animal products are likely to be vitamin B12 deficient. Beef, a highly concentrated source of vitamin B12 that is often restricted in the diet of both meat-consumers and vegetarians for health reasons, is hypothesized to be a major cont ributor to dietary vitamin B12 intake among meat-consumers. It is important to assess th e effect of beef consumption on total dietary intake of vitamin B12 and plasma vitamin B 12 concentration, which is the focus of this investigation. Vitamin B12 intake and plasma concentr ation were assessed among frequent beef consumers (beef intake >1 time/wk) (n=97) seldom beef consumers (beef intake 1 time/wk) (n=42), and never beef consumers (n =42), and compared to a vegetarian group (n=121). The dietary vitamin B12 intake (m ean SD g/1000 kcal/d ) of the never beef

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xii group (2.0 1.4) and vegetarian group (1.9 1.5) was both lower (p < 0.0001) than the frequent beef group (3.3 1.4) and the sel dom beef group (3.7 2.2). In the frequent beef group, the largest contribu tors to vitamin B12 intake were seafood (30%), beef (28.5%), and dairy products (16%). Among the seldom beef group, the largest contributors to vitamin B12 intake were seafood (33.5%), cereal (20%), and dairy (13.8%). Sources in the never beef group th at contributed the la rgest proportion of vitamin B12 were seafood (41%), dairy (21.3%), and soy products (1 2%). There were no differences (p = 0.70) in mean plasma vitamin B12 concentration (range 263 to 289 pmol/L) among the four dietary groups. Th e percentage of individuals who were deficient based on plasma vitamin B12 con centration (<148 pmol/L) was approximately two-fold higher in the non-b eef consumers [never beef ( 12%); and vegetarians (17%)] compared to beef consumers [freque nt beef (6%); and seldom (5%)]. The present study was the firs t to stratify subjects ba sed on their frequency of consumption of beef products and to compar e the dietary vitamin B12 intake and food sources to vitamin B12 concentration among beef consumption groups. This study also was the first to use a dietary history questi onnaire modified specifi cally for vitamin B12containing foods and to use a nutrient database where 100% of foods have information on vitamin B12 content. These data indicate that consumption of beef at least one time per week plays an important role in providing adequate amounts of vitamin B12 in the diet of meat consumers. In addition to beef, seaf ood and dairy products were major contributors to dietary vitamin B12 intake. Dietetics practitioners and nutrition educators can use these data to promote beef, seafood, and da iry consumption for optimizing vitamin B12 status through dietary means.

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1 CHAPTER 1 INTRODUCTION Vitamin B12, a water-soluble vitamin, plays an important role in the remethylation of homocysteine to methionine (1) and the formation of succinyl CoA, a Kreb’s cycle intermediate, from L-methylmalonyl CoA (2). V itamin B12 is an essent ial nutrient that is naturally present only in dietary sources of an imal origin. Foods that are naturally rich sources of vitamin B12 are seafood, organ m eats (especially liver), beef, poultry, pork, dairy products (e.g., milk, cheese, yogurt), and eggs (especially egg yolk) (3). Compared to other meat products (e.g., poultry, pork, and so me types of fish), beef has the highest concentration of vitamin B12 per 3 ounce (oz) serving. Dairy products and eggs have smaller amounts of vitamin B12 per serving than meat products (4). Consumption of a vegetarian diet, which restricts animal sources, may limit dietary intake of vitamin B12 and impair vitamin B 12 status (5). Vitamin B12 deficiency has been related to elevated homo cysteine concentration, a risk factor for cardiovascular disease, impaired fetal development, and neurological abnormalities (6-8). Dietary intake of vitamin B12 has b een assessed in meat-consumers and vegetarians by using a number of different assessment methodologi es including 24-hour food recalls, multiple-day food records, a nd food frequency questionnaires (FFQ). Dietary vitamin B12 intake has been reported to be consisten tly lower in vegetarians than meat-consumers when the vegetarian group does not consume supplements or vitamin B12-fortified products (9-11). The impact of frequent beef consumption on total dietary

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2 vitamin B12 intake and plasma vitamin B12 c oncentration relative to other food sources among meat-consumers has not been pr eviously investigated. Hypotheses 1. High frequency of beef intake is associated with a greater intake of dietary vitamin B12 and higher plasma vitamin B12 concentr ation compared to other food sources. 2. Among meat eaters, beef and beef-containing foods are th e largest contributors to dietary vitamin B12 intake re lative to other food sources. Specific Objectives The overall goal of this study was to assess vitamin B12 intake a nd status in healthy individuals who consumed beef more fre quently compared to those who consumed vitamin B12 from other food sources. The objectives of this research study were as follows: 1. To determine the differences in mean diet ary vitamin B12 intake [micrograms (g) per 1000 kilocalories (kcals)] among all dietary groups. 2. To characterize the daily contribution of dietary vitamin B12 intake from beef sources (g/1000 kcals) relativ e to the total dietary vita min B12 intake from all food sources and other types of animal-based foods. 3. To assess the relationship between dietary vitamin B12 intake and plasma vitamin B12 concentration within each dietary group.

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3 CHAPTER 2 LITERATURE REVIEW Vitamin B12 Chemistry The chemical properties of vitamin B 12, also known as cobalamin, have been previously reviewed (12). Cobalamin, a wa ter-soluble vitamin has a molecular mass of 1,580 daltons. Cobalamin is a general term fo r a group of corrinoids that are cobaltcontaining compounds (12). The structure of co balamin consists of a macrocyclic corrin ring, a 5,6-dimethylbenzimidazole nucleotide, an d an aminopropanol group (Figure 2-1). The macrocyclic corrin ring c onsists of four reduced pyrrole rings linked to one cobalt atom at the center. Attached to the central cobalt atom is a lower (alpha) ligand 5,6dimethylbenzimidazole nucleotide (13). Numer ous upper (beta) ligands such as –CN, OH, -H2O, -NO2, 5’-deoxyadenosyl, and -CH3 can be covalently bound to the central cobalt atom. Addition of a ligand creates various forms of cobalamin, such as cyanocobalamin, hydroxocobalamin, aquocobalamin, nitritocobalamin, adenosylcobalamin, and methylcobalamin, respectively (12). Methylcobalamin and adenosylcobalamin are active coenzymes and can be derived from other cobalamin forms (2). Cyanocoba lamin is the commercial form produced by industry (12). In tissue, a “coenzyme synt hetase” system converts cyanocobalamin and hydroxocobalamin to adenosylcobalamin. In this reaction, ATP provides a 5’-deoxy-5’adenosyl moiety that is moved to the coba lamin. Hydroxocobalamin can be methylated in the cytosol to form methylcobalamin (2).

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4 Co2+N N N N N O P N O O-O O NH O O O O O H O O O C H3C H3C H3CH3NH2N H2N H2CH3NH2N H2NH2C H3CH3CH3O H CH3CH3C H3H H H H X Corrin Ring Alpha Ligand Beta Ligand Figure 2-1. Structure of cobalamin. Adapted from Martens et al (2002), p. 276 (12). During exposure to light, methyl cobalamin, adenosylcobalamin, and cyanocobalamin are unstable. Light di srupts the carbon-coba lt bond and decreases enzyme activity (12). Cyanocobalamin is stab le in aqueous solutions, low pH, and high temperatures (14). Dietary Sources Cobalamin, which will be referred to subse quently as vitamin B12, is an essential nutrient that is naturally presen t only in dietary sources of an imal origin (3). Only certain microorganisms (members of Archea and some eubacteria) can synthesize vitamin B12 as reviewed by Raux et al. (2000) (15). Animal sources rich in vitamin B12 have obtained the vitamin from these microorganism s. Sources rich in vitamin B12 are seafood (e.g., fish and shellfish), organ meats (especia lly liver), beef, poultry, pork, dairy products (e.g., milk, cheese, yogurt), and eggs (especiall y egg yolk) (16). It may be possible to

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5 acquire vitamin B12 from plant sources if they are contaminated with vitamin B12 producing-microorganisms (12). Beef has the potential to pr ovide a large amount of diet ary vitamin B12 since one 3 oz serving contains approximately 2.2 g of vitamin B12 (4), almost providing the Recommended Dietary Allowance (RDA) for vitamin B12 (RDA = 2.4 g; non-pregnant, non-lactating adults). Compared to all ot her meat products (e.g., poultry, pork, and some types of fish), beef has the highest concentr ation of vitamin B12 per 3 oz serving (Table 2-1) (4). Dairy products and eggs have smaller amounts of vitamin B12 per serving compared to meat. Seafood provides a higher amount of vitamin B12 in one 3 oz serving than beef. Specifically, one 3 oz serving of shellfish (e.g., clams, mussels, and oysters) provides approximately 20 g per serving of vitamin B12 (4). Although, according to a report by the United States Department of Commerce, seafood, incl uding shellfish, is consumed in much smaller amounts per year (per capita 16.6 pounds) compared to beef (per capita 65 pounds) (17-19). Adenosylcobalamin and hydroxocobalamin are the predominant vitamin B12 forms found in meat, fish, poultry, pork, and eggs (16). Methylcobalamin and hydroxocobalamin are the predominant forms of vitamin B12 found in dairy products (16). The form of vitamin B12 in commercia l supplements and fortified foods such as breakfast cereals, soy products, and meal replacement formulas is predominately cyanocobalamin (20).

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6 Table 2-1. Dietary vitamin B12 sources Dietary Source Vitamin B12 Content (g) per 100 grams (g) Vitamin B12 Content per serving (g) Serving Size Beef 2.50 2.2 3 ounce Chicken 0.34 0.29 3 ounce Turkey 0.34 0.29 3 ounce Pork 0.75 0.64 3 ounce Milk 0.45-0.53 1.1-1.3 1 cup Cheese 0.35-1.55 0.10-0.44 1 ounce Yogurt 0.47 1.07 8 ounce Egg 1.42 0.64 1 large Seafood Shellfish Clams, mussels, oysters Shrimp, lobster Fish Cod, tuna, salmon 24-49 1.4-3.1 1.05-2.8 20-42 1.2-2.6 0.89-2.38 3 ounce 3 ounce 3 ounce Adapted from United States Department of Agricu lture, National Nutrient Database for Standard Reference, Release 17 (2005) (4). Absorption The mechanism of dietary vitamin B12 absorption has been reviewed previously (21). In the stomach, dietary vitamin B12 bound to protein is disasso ciated by pepsin and hydrochloric acid. The acid environment incr eases the vitamin B12 binding affinity for R proteins (i.e., cobalophilins or haptocorrins), which are se creted by salivary glands and the gastric mucosa (3, 21). These R proteins prevent denaturation of the vitamin from chemicals produced in the stomach. Intrinsic factor (IF), a glycopr otein secreted by the parietal cells of the stomach in response to stimulation by food, is released but does not bind to free vitamin B12 in the stomach (21). The vitamin B12-R protein complex and IF then travel to the small intestine. Vitamin B12 is released from the R protein complex in the duodenum by pancreatic proteases. These proteases hydrolyze the R proteins thereby releasing vitamin B12 (21). The alkaline en vironment enhances the binding of vitamin B12 to IF, and the IF-vitamin B12 comple x then travels to the ileum for receptormediated uptake (21). The intestinal cells of the ileum have receptors for vitamin B12

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7 called cubilins. It is unclear whether both IF and vitamin B12 are absorbed during this process or if only vitamin B12 is absorbed. It is known that this is a calcium dependent process (14). Vitamin B12 is absorbed througho ut the ileum, but the greatest amount is in the distal third. Most absorption of vitami n B12 is receptor-mediated, but approximately 1% is absorbed by passive diffusion (22). Transport Once vitamin B12 is internalized into the en terocyte it can take 3 to 4 hours before the vitamin is in circulati on (2). Vitamin B12 is bound in the plasma to two proteins: haptocorrin and transcobalamin (TC) (23). Haptocorrin accounts for 80% of all vitamin B12 bound in circulation, while TC account s for 20% of all vitamin B12 bound in circulation. Transcobalamin carries vitamin B12 in a one-to-one ratio (24). Tissue cells have receptors only for TC, making it the ma jor transport protein for cellular uptake. Once vitamin B12 crosses the cell membrane of peripheral tissu es, vitamin B12 is released from TC into the cytosol (3). Storage Unlike most water-soluble vitamins, vitamin B12 can be stored in the body for a considerable length of time due to enterohepa tic circulation via cont inuous bile secretion (25). The liver is the main storage tissu e followed by the muscles accounting for 60% and 30% of total body stores, respectively. Vi tamin B12 also can be stored in small amounts in other tissues such as bone, kidneys, heart, muscle, brain, and spleen (26). Approximately 2 to 5 milligrams (mg) of vitamin B12 can be stored in the body (2) predominately in the form of adenosylcobalamin and methylcobalamin (26).

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8 Excretion Vitamin B12 is excreted primarily th rough feces originating primarily from unabsorbed bile (27). Excess vitamin B12 losses through urine occur when the serum vitamin B12 concentration surpasses the bindi ng ability of the transport proteins in the blood (22). There is an inverse relationship betw een fecal and urinary losses and storage. If storage of vitamin B12 decreases, then excretion of vitamin B12 will decrease and more will be conserved. Approximately, 0.1% to 0.2% of stored vitamin B12 is lost each day (28). Biochemical Functions Vitamin B12 plays an important role in tw o enzymatic processes. The first is a methylcobalamin dependent pathway that re methylates homocysteine to methionine (Figure 2-2). This reaction occurs in the cytoplasm of the cell. Cobalamin bound to the enzyme methionine synthase (MS) acts as an acceptor of a met hyl group donated from 5methyltetrahydrofolate (5-methyl THF). Meth ionine synthase acts as a catalyst during this remethylation reaction by removing the methyl group from 5-methyl THF and adding it to cob(I)alamin, forming methylcobalami n and regenerating THF. Methylcobalamin donates the methyl group to homoc ysteine, resulting in remet hylation of homocysteine to methionine (1). Methionine can further be converted to S-adenosylmethionine (SAM). SAM functions to provide methyl groups fo r over 100 methylation reactions including deoxyribonucleic acid (DNA) protein, and phospholipid synthesis (29). In a vitamin B12 deficiency, the human body lacks sufficient am ounts of cobalamin to accept the methyl group from 5-methyl THF. Folate is trappe d in the 5-methyl THF form preventing the regeneration of THF. Since THF is re quired to produce 5,10 methylene THF used for DNA synthesis, a vitamin B12 deficiency leads to decreased DNA synthesis and

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9 impaired cell division (30). Homocysteine cannot be remethylated to methionine, resulting in a build-up of ho mocysteine in the blood (5). MS -B12 Methionine SAM SAH Homocysteine 5,10, CH3-THF THF 5-CH3-THF DNA Synthesis DNA Methylation Figure 2-2. Vitamin B12 function in the reme thylation pathway of homocysteine to methionine. Adapted from Scott (1999) p. 442 (21). THF = tetrahydrofolate; 5,10-CH3-THF = 5, 10 methylenetet rahydrofolate; 5-CH3-THF = 5methyltetrahydrofolate; MS = methionine synthase; SAM = s adenosylmethionine; SAH = s -adenosylhomocysteine. The second vitamin B12-dependent reaction is an adenosylcobalamin dependent pathway, in which L-methylmalonyl CoA is c onverted to succinyl CoA (Figure 2-3). This reaction occurs in the mitochondria. The oxidation of me thionine, threonine, isoleucine, and odd chain fatty acids result s in the formation of propionyl CoA. Propionyl CoA is then converted to D-me thylmalonyl CoA by a biotin, ATP, and magnesium dependent enzyme, propionyl CoA carboxylase. Methylmalonyl CoA racemase converts D-methylmalonyl CoA to L-methylmalonyl CoA. Methylmalonyl CoA mutase is adenosylcobalamin depende nt and requires two adenosylcobalamin molecules to transform L-methylmalonyl Co A to succinyl CoA (2). Succinyl CoA is a key Kreb’s cycle intermediate. In a defi ciency of adenosylcobalamin, methylmalonyl

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10 CoA builds up, is hydrolyzed to methylmalonic acid (MMA), and MMA ultimately accumulates in the blood (12). Isoleucine, Valine, Methionine, Threonine Odd-chain Fatty Acids Propionyl-CoA (D)-Methylmalonyl-CoA (L)-Methylmalonyl-CoA Methylmalonyl-CoA-Mutase Succinyl-CoA Citric Acid Cycle Propionyl-CoA Carboxylase Figure 2-3. Role of cobalamin in the forma tion of succinyl-CoA. Adapted from Horster and Hoffmann (2004), p. 1073 (31). Dietary Reference Intakes The RDA for vitamin B12 is based on the amount of vitamin B12 required for maintenance of normal hematological stat us and serum vitamin B12 concentration considering estimates of intake and turnover (20). The RDA for male and female adults is 2.4 g/day (non-pregnant, non-lactating) (20). During pregnancy and lactation, the vitamin B12 RDA increases to 2.6 and 2.8 g/da y, respectively (20). A dults greater than 51 years of age frequently malabsorb food-bound vitamin B12, therefore, it is recommended that their vitamin B12 RDA (2.4 g/day) be derived from consumption of foods fortified with vita min B12 or from vitamin B12 supplements (20).

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11 Dietary data from the National Health and Nutrition Examination Survey (NHANES) 1999-2000 indicated that Americans of all age groups are consuming well above the RDA for vitamin B12. The mean vitamin B12 intake for males and females aged 20 to 39 years was 6.1 and 4.0 g/day, re spectively, and the intakes for ages 40 to 59 years was 6.1 and 4.1 g/day, respectively. Individuals over 60 years of age had a slightly lower vitamin B12 intake, although the in take still surpassed th e RDA. In this age group, reported intake of vitamin B12 for males was 5.3 g/day and for females was 3.9 g/day (32). Data from the Continuing Survey of Food Intakes by Individuals (CSFII) 1995 was used to estimate the percent that speci fic dietary sources contributed to vitamin B12 intake. For men, the sources that contri buted the greatest pe rcentage of dietary vitamin B12 were mixed foods (18.5%), beef (15%), and milk and milk drinks (10%). For women, the greatest percentage of total dietary vitamin B12 intake was from mixed foods (16.4%), milk and milk drinks (14.6%), and beef (12%) (20). To date, no adverse effects of large doses of vitamin B12 have been reported from food or supplements. No adverse or toxic effect s have been reported in studies of patients who receive very large doses of vitamin B12 for the treatment of pernicious anemia (33). There is no Tolerable Upper Intake Level (UL) established for vitamin B12 (20). Vitamin B12 Deficiency Etiology In addition to dietary inadequacy of v itamin B12, there are numerous other causes contributing to a vitamin B12 deficiency including pernicious anemia, food-bound malabsorption, pancreatic insufficiency, jejuna l bacterial overgrowt h, tropical sprue, or gastric or ileal resection ( 22). Drugs such as nitrous oxide, metformin, and stomach acid blockers can also impair vitamin B12 stat us (34-36). Only vitamin B12 deficiency

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12 associated with pernicious anemia, foodbound malabsorption, and dietary deficiency will be discussed in the following sections. Pernicious anemia Pernicious anemia is an auto immune disease that occurs when the body produces auto-antibodies against the parietal cells. This causes loss of parietal cells and a subsequent decrease in IF. Other auto-antibodies can bind to IF and block the vitamin B12 binding site. Lack of IF due to the absence or reduction of parietal cell mass or blockage of the IF-B12 binding site resu lts in vitamin B12 malabsorption (21). The exact number of Americans with pernic ious anemia is unknown. Although, based on studies of elderly it has been estimated th at 1.9% of the populati on over the age of 65 years has pernicious anemia (37). Historically, a Shilling’s test was the pr imary test used to diagnose pernicious anemia. This test involves giving an individual a radioa ctive oral dose and a flushing dose of crystalline cobalamin and measur ing the amount of radioactive cobalamin excreted in the urine over time (38). This test is not used as widely by clinicians as it was in the past (39). Alternative methods for diagnosing pernicious anemia include testing an individual’s serum for IF antibodies or te sting for parietal cel l antibodies (38). In 1926, Minot and Murphy used raw liver to cure pernicious anemia in humans. Vitamin B12 was eventually isolated and termed the “anti-pernicious anemia factor” (40). Individuals with pernicious anemia can be initially treated with weekly intramuscular injections of 1,000 g of cyanocobalamin for up to 2 months, preceded by monthly injections for life (29). Kuzmin ski et al. (1998) reported that 2,000 g per day of oral cobalamin may be an e ffective alternative to intramuscular injections (41).

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13 Food-bound malabsorption. Vitamin B12 absorption requires secretion of stomach acid to disassociate the vitamin from any bound components. With inadequate amounts of gastric acid, vitamin B12 will not be released from the protein moiety resulting in decreased vitamin absorption and increased excreti on (21). Vitamin B12 consumed in a crystalline form is not bound, so regardless of stomach acid secretion, crystalline vitamin B12 will be absorbed normally (42). Drugs such as antacids and proton-pump inhibitors may contribute to food-bound malabsorption of vitamin B12 by decreasing gastric acid production (34). To determine if vitamin B12 deficien cy is due to food-bound malabsorption, research laboratories use a protein-bound cobalamin absorption test (43). In this technique, radiolabelled vitamin B12 is mixe d with powdered egg yolk and made into an omelet. The omelet is consumed by the pati ent and 1.5 hours later the patient is given an unlabelled dose of hydroxocobalamin. Urinary excretion of vitamin B12 is then measured (43). Individuals over the age of 51 are at risk for food-bound vitamin B12 malabsorption due to the agerelated decrease in stomach acid or achlorhydria (42, 44). As a part of the RDA recommendations, i ndividuals older than 51 years of age are advised to obtain vitamin B12 from for tified foods sources or from vitamin B12 supplements (20). These sources are not pr otein bound, therefore th ese sources can be absorbed through the normal mechanism (45). Dietary deficiency. Those who restrict their intake of vitamin B12 containing foods, such as vegetarians or vegans, are at risk for vitamin B12 deficiency, unless they consume vitamin B12 from fortified foods or supplements (5). Since vitamin B12 can be

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14 stored in the body, a decrease in dietary intake may not affect vitamin B12 concentration initially (9). Those who restri ct their intake of vitamin B 12 containing foods (naturally occurring, fortified, or supplements) for longer durations are at higher risk for a vitamin B12 deficiency due to deplet ion of body stores (9, 10). Clinical symptoms Megaloblastic anemia. Vitamin B12 deficiency occurs in stages as previously reviewed (5, 38). Vitamin B12 deficiency begi ns with a reduction in serum concentration followed by decreased cellular vitamin B12 c oncentration. Metabolic abnormalities occur next as indicated by increased homocystein e and MMA concentra tions and decreased DNA synthesis (5, 38). The last stage is indicated by the presen ce of megaloblastic anemia (5). Megaloblastic anemia results in large abnormally shaped erythrocytes and elevated mean corpuscular volume (MCV). Si milar to a folate deficiency, leukocytes become enlarged and hypersegmentation of the nuclei occurs. Treatment with vitamin B12 can reverse changes in the erythroc ytes and leukocytes (2). Presence of megaloblastic anemia may indicate a possibl e deficiency of vitamin B12, although it is not a strong diagnostic tool since a folate deficiency results in the same type of anemia (38). Neurological disorders. According to a review by the Institute of Medicine (1998), neurological abnormalities occur in 75 to 90% of individuals with vitamin B12 deficiency. They generally a ppear long after the deficiency has occurred making it a poor assessment tool for vitamin B12 status ( 20). Neurological abnormalities are caused by demyelination of the central nervous system (CNS). The my elin sheath and spinal cord near the brainstem are affected first. Degrad ation of the CNS can occur before a vitamin B12 deficiency is recognized in an indivi dual. Lindenbaum et al (1988) found that 28%

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15 of individuals with neurological abnormalities presented with normal hematological indices. Tingling and numbness in extremities, memory loss, disorientation, and dementia are common neurological signs of a deficiency (46). These symptoms are not specific for a vitamin B12 deficiency since they also can be present among individuals who have other neurological conditions. Therefore, an individual can not be diagnosed with a vitamin B12 deficiency based only on pres entation of neurological symptoms (38). Health related risks of a vitamin B12 deficiency As a person restricts more animal food s ources, the risk of in adequate intake of vitamin B12 increases (5). Since vegetari ans restrict various animal food sources, thereby restricting natu rally-occurring food sources of vitamin B12, they are at risk for a deficiency. This is especially the case if vitamin B12-for tified foods or supplements are not consumed. Data continue to support a relationship between ve getarian diets and increased risk for birth defects, neurological abnormalities, and elevated homocysteine concentration related to a vita min B12 deficiency (6-8). Birth defects. Women of childbearing age who consume a vitamin B12 deficient diet are at an increased risk for impaired fe tal development if they are pregnant. Folic acid is generally the vitamin associated with bi rth defects, specifically neural tube defects (NTD) such as spina bifida. There is, howev er, strong evidence supporting a role for low vitamin B12 status as an NTD risk factor, whic h may be related to the interdependent role of folic acid and vitamin B12 in one car bon metabolism (8, 47-49). Kirke et al. (1993) compared maternal folate and vitamin B12 c oncentrations before the birth of a child. After delivery, these women were grouped base d on presence of an NTD in the child. Both plasma folate and vitamin B12 concen trations were lower in women who had a child with an NTD compared to those who did not give birth to a child with an NTD.

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16 Low vitamin B12 status was considered an independent risk factor for NTD (49). Groenen et al. (2004) investig ated maternal vitamin B12 status and the risk for spina bifida in offspring. In this study, serum vitamin B12 concentr ations of mothers and their children who had spina bifida were compared to those of control mothers. The mothers with children who had spina bifida had a m ean serum vitamin B12 concentration that was 21% lower [<185 picomoles per liter (pmol/L)] than that of the control mothers and was associated with a three a nd a half-fold increase in spina bifida risk (8). van Rooij et al. (2003) evaluated vitamin B12 status of mothers and infants with nonsyndromic orofacial clef ts. Mothers who had decr eased serum vitamin B12 concentration (<185 pmol/L) had a three-fold increased risk of ha ving offspring with a nonsyndromic orofacial cleft. There were no differences between median serum vitamin B12 concentrations in the aff ected versus the control infant s (48). Afman et al. (2001) investigated the association between vitami n B12 binding by TC and the risk of NTD. They found that mothers with children who ar e affected with an NTD had a significantly higher mean homocysteine concentration and lower TC and vitamin B12 concentrations than mothers with normal children. They hypot hesized that decreased binding of vitamin B12 by transcobalamin, which resulted in a vitamin B12 deficien cy as indicated by increased homocysteine concentration, was a ssociated with increased risk for birth defects. They concluded that vitamin B 12 supplementation may be needed along with folic acid supplementation in wome n of childbearing age (47). Neurological abnormalities. Neurological abnormalities can occur in individuals with a vitamin B12 deficiency (20). Depe nding on the time at which a neurological disorder persists, some can be reversed. Pitto ck et al. (2002) discu ssed a case-report of a

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17 34-year old man with reversible myelopathy who suffered from pernicious anemia. He was treated with intramuscular injections of cyanocobalamin every two weeks. After one month of treatment, all clinical symptoms ha d completely reversed to normal (7). Louwman et al. (2000) inves tigated the effect of marginal cobalamin status on adolescent cognitive functioning when following a macrobiotic diet. Data from 28 adolescents on vegan diets and 24 adolescents on non-vegetarian diets indicated that adolescents on macrobiotic diet s performed worse on psychologi cal tests than adolescents on non-vegetarian diets (50). There are numerous published studies that show neurological abnormalities in infants who are exclusively brea st-fed from mothers following a vegetarian or vegan diet and who have poor maternal vitamin B12 status (51-55). The first re port (Jadhav et al. 1962) included six cases occurring in India. None of the infants ha d absorption problems, but all had megaloblastic anemia attributed to poor vitamin B12 intake due to low maternal vitamin B12 breast milk concentratio ns. Half of the mo thers had inadequate vitamin B12 breast milk concentrations because of their restriction of animal products, the other half suffered from vitamin B12 malabsorption. All infants were born without complications, but began to display a bnormal skin pigmentation, apathy, anemia, involuntary movements, and developmental regr ession around the ages of 7 to 12 months. Upon diagnosis, they were treated with oral cobalamin and the symptoms were reversed (56). Elevated homocysteine concentration. As discussed previous ly, with inadequate amounts of vitamin B12, homocysteine cannot be remethylated and homocysteine can build up in the blood (5). There are many pr oposed mechanisms by which an elevated

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18 homocysteine concentration may increase the risk for cardiovascular disease, as reviewed by Ueland et al. (2000) (57). One hypothe sis is that homocysteine may increase endothelial permeability by decreasing tumor n ecrosis factor and increasing thrombin formation, which may exacerbate the development of atherosclerotic plaque and contribute to the progression of cardiovascular disease (58). Since vegetarians limit or exclude animal products, wh ich are the only naturallyoccurring sources of dietary vitamin B12, they are at an increased risk for a vitamin B12 deficiency and hyperhomocysteinemia (59) Data from NHANES III showed that twothirds of United States men and women with elevated homocystein e concentrations had low serum vitamin B12 concentrations (60). Ga o et al. (2003) used an FFQ to investigate dietary patterns and serum homocysteine concentration in a Chinese population. Individuals were categorized in to three dietary groups based on greater intake of calories from: (1) milk and fruit, (2) red meat, and (3) unbleached, refined cer eals (rice and flour products). Forty percent of subj ects in the refined cereals gr oup had a high homocysteine concentration and 67% had a low serum vitami n B12 concentration. This group was four to five times more likely than individuals in the milk and fruit gr oup or red meat group to have these hematological factors. They concluded that dietary patterns where the majority of calories were from low vitamin B12 sources contributed to the increase in homocysteine concentration in Chinese adults (61). Obeid et al. (2002) measured homocys teine and MMA concentrations among individuals following various types of vegeta rian diets. It wa s shown that vegans, followed by the combined lacto/lactoovove getarian group, had th e highest median homocysteine concentration, lowest medi an serum vitamin B12 concentration, and

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19 highest median MMA concentration (62). A report by Herrmann et al. (2003) further supported these findings of an association between vitamin B12 in take and increased homocysteine concentration in vegetarians. They reported that 67% of vegans, 38% of vegetarians, and 16% of omnivores had elev ated homocysteine concentrations. These researchers concluded that th e vitamin B12 status of vegetarians and vegans could be described as marginal to severely defici ent compared to a more normal vitamin B12 concentration in the omnivore control group. Th ese data indicated th at decreased serum vitamin B12 concentration was directly corre lated with elevated homocysteinemia in vegetarians (63). Krajcovivova-Kudlackova et al. (2000) measured homocysteine concentration in vegetarians a nd reported that 53% of vega ns, 29% of vegetarians, and 5% of omnivores had hyperhomocysteinemia. Serum vitamin B12 concentrations were significantly lower in 28% of vegetarians, 78% vegans, and none of the omnivores (64). Research findings support the conclusion that vitamin B12 supplements can correct hyperhomocysteinemia. Mezzano et al. (2000) studied vegetarians with poor serum vitamin B12 status. After one intramus cular injection of cyanocobalamin (10,000 g), serum vitamin B12 concentration increased significantly above normal from 110 46 pmol/L to 393 151 pmol/L) and mean tota l homocysteine con centration decreased significantly [from 12.4 4.7 micromoles per liter ( mol/L) to 7.9 31 mol/L)] (6). Recently, Bor et al. (2006) reported th at a daily intake of at least 6 g of vitamin B12 (from food or supplements) reduced blood homocysteine, MMA, and vitamin B12 concentrations in postmenopausal women (65).

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20 Vitamin B12 Status Assessment Serum Vitamin B12 Concentration Serum vitamin B12 is generally the first diagnostic test performed in a clinical setting on patients suspected of having a v itamin B12 deficiency (2). Established standards that have been used in both clinical and resear ch settings are defined as follows: (a) deficient (<148 pmol/L); (b) marg inally deficient (148-221 pmol/L); and (c) normal (>221 pmol/L) (66); although depending on the method of analysis, the normal range can vary from laboratory to labora tory. Ranges of vitamin B12 values using radioassay methods are found to be higher a nd less accurate compared to microbiological assays, which are generally lo wer and more accurate (2). The use of serum vitamin B12 concentration as an indicator of vitamin B12 status is currently under much scrutiny because of the potential for false normals (2 ). Lindenbaum et al. (1990) found that MMA concentration predicted a vitamin B12 defici ency two times more than serum vitamin B12 concentration, and five percent of indivi duals who were diagnos ed with a clinical vitamin B12 deficiency had a normal serum v itamin B12 concentrati on (67). In contrast, Bolann et al. (2000) reported th at serum vitamin B12 concentr ation was the best indicator of vitamin B12 status and should be used first in determining if the symptoms experienced by an individual are vitamin B12 re lated (68). Currently other metabolites of vitamin B12 metabolism such as MMA, are co nsidered a better determinate of vitamin B12 status. Holo-TC is under investigation as a potentially more sensitive vitamin B12 status assessment indicator. Methylmalonic Acid Concentration Serum MMA concentration is considered a functional measure of intracellular vitamin B12 status (20) Vitamin B12 is re quired to convert Lmethylmalonyl CoA to

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21 succinyl CoA. In the event of a vitamin B12 deficiency, this pathway is disrupted due to decreased enzyme activity. Consequently, th e concentration of MMA increases in the blood and in response to a vitamin B12 defici ency in the urine (12). The normal serum concentration range for MMA is 73 to 271 na nomoles per liter (nmol/L) (2). Urinary MMA may be an earlier status indicator than other stat us indicators since MMA can become elevated before serum vitamin B12 concentration drops below 148 pmol/L (2). Renal insufficiency is associated with an increase in the c oncentration of MMA independent of vitamin B12 deficiency (38). MMA concentration also can be elevated in the elderly regardless of vitamin B12 status (69). The photometric methods used to determine serum MMA concentration are ve ry costly. For this reason serum MMA concentration is not used routin ely as a clinical diagnostic te st (2). Gultepe et al. (2003) developed a modified procedure for measuri ng urinary MMA concentration that is more sensitive than the older photometric method (70). Holotranscobalamin Concentration Serum holo-TC is a biologically ac tive complex of vitamin B12 and transcobalamin. Since serum vitamin B12 c oncentration may not always be a reliable indicator of vitamin B12 stat us, holo-TC is currently bei ng investigated as a more sensitive test for vitamin B12 assessment (71). Our research group is currently investigating the influence of the polymorphism transcobalamin 776C G (TC 776C G) on the concentration of serum holo-TC, and the reliability of holo-TC as an indicator of vitamin B12 status. von Castel -Dunwoody et al. (2005) reported an inverse relationship between plasma homocysteine and serum holo-TC, and plasma vitamin B12 concentrations. These findings correlated to previous studie s investigating the relationship between homocystei ne and holo-TC concentrati ons. Holo-TC concentration

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22 may be helpful in characterizing vitamin B12 status in marginally deficient individuals (72). Serum Homocysteine Concentration Vitamin B12 and folate are required for the remethyla tion of homocysteine to methionine (1), and vitamin B6 is required for the transulfurati on of homocysteine to cystathionine (12). Serum ho mocysteine concentration ma y provide an indication of intracellular vitamin B12 status (20). Se rum homocysteine concentration below 12 mol/L is considered normal (60). In a v itamin B12 deficiency, serum homocysteine concentration can be elevated. Since there are three vitamins (e.g., vitamin B12, folate, and vitamin B6) that play a role in convert ing homocysteine to other compounds in the body, homocysteine concentration is not a specific determinant for vitamin B12 deficiency (73). Megaloblastic Anemia Megaloblastic anemia is diagnosed when hematocrit and hemoglobin are low, but MCV is high. Vitamin B12 and folate deficien cies, among other disorders, can cause this type of anemia. Therefore, megaloblastic anemia is not a definitive status indicator of a vitamin B12 deficiency. If folic acid is given to treat the condition, the anemia will reverse to normal, even if the underlying cause is due to vitamin B12 deficiency. However, the neurological complications of a deficiency will not resolve and can result in further damage (38). Dietary Intake Assessment in Adults Twenty-four hour recall method The twenty-four hour recall method of dietary intake assessment can be administered in–person or over the phone a nd can be used for large epidemiological

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23 studies. This method is inexpensive, requi res no literacy, and has a high response rate. Twenty-four hour recalls rely heavily on memory and individu als may omit food items or even report food items not consumed that day (74). The three-pass method is generally used to obtain complete dietary recalls (75) In this method, an individual will simply start listing all the foods they ate without interruption. Afte r the individual has given a brief list of the foods they ate, the interv iewer asks specific questions regarding each food item eaten, portion size, and the meal time. Fo r example, if the person said they had a piece of bread, the interviewer should ask, the type of bread (e.g., multi-grain, white, etc), additional products added, the amount, and time of day consumed. Next, the interviewer repeats the foods that were reportedly consum ed at that meal or snack, to see if any additional foods were omitted. The interv iewer should address snacks between meals, beverages, and food consumed during the night. Individuals generally think of their main meals and disregard daytime or evening snack s. Food models and measuring cups can be used with the individual to help them co rrectly identify their portion size (75). The United States Department of Agri culture (USDA) recently revised their method from three-steps to five-steps for interviewing during a 24hour dietary recall. They have developed a very comprehensive automated-multiple-pass method (AMPM) (75). It was developed to keep the attenti on of individuals during the interview and to correct for under-reporting (75). This five -step method has more passes and increased opportunities to add forgotten foods. The computerized method uses a database with predetermined questions, possible followup questions, and instructions for the interviewer. The AMPM contains 2,400 que stions and 21,000 response options (75, 76). The AMPM is directly linked to the USDA Food and Nutrient Database for Dietary

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24 Studies for nutrient analysis. Moshfegh et al. (2001) pilot test ed this method in 700 individuals and found a higher av erage daily caloric intake an d more foods reported than the CSFII (1996) that used a three-step method (77). Multiple-day food record The multiple-day food record involves in struction of an individual to write everything they ate on a food reco rd that is provided (78). A food record can be selfadministered or administered by an interviewer. If self-administered, instructions should be provided with the diet record. Exampl e instructions would include information on food description, preparation methods, brand name s, and ingredients in mixed foods (78). Kolar et al. (2005) provided a 12-page serving size booklet with pictures to illustrate and quantify serving sizes during their study. R ecords are reviewed for completeness and entered into a nutrient database program such as Nutrient Data System for Research (NDS-R), University of Minnesota (78) Since they require review and manual correction, multiple-day diet records are gene rally discouraged for large epidemiological studies (78). Dietary records do not rely on memory, but can be tedious for the individual completing the record (79). The reliability of the nutrien t data from the food record increases with the numbers of days recorded ( 80). Craig et al. (2000) measured the effect of atypical days, as described by particip ants, on nutrient intake estimates in 1,090 women. Those who reported an atypical da y as less than normal or higher than normal had lower intakes or higher intakes, respectiv ely, of all nutrients. They concluded that atypical days in an individual’ s diet can affect estimated nutrient intakes in research studies that use the multiple -day food record (80).

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25 Food frequency questionnaire A FFQ is used to determine usual dietary in take (81). This type of questionnaire is commonly used for large epidemiological studi es (81). A FFQ can be self-administered and is less expensive than other dietary a ssessment methods (82). Twenty-four hour recall methods and multiple-day food records ar e more detailed than a FFQ. A FFQ uses general food groupings, such as high-fiber cereal, whereas a 24-hour recall method or multiple-day food record provides the specific brand name of cereal that was consumed. A FFQ has preset questions and portion sizes to which an individual responds. These types of questionnaires are developed to be scanned by a computer and uploaded into a software package (81). There generally is no option for the individual to add a personal response not listed on the questionnaire. F ood frequency questionnaires need to be validated before using them for a large ep idemiological study to ensure that the questionnaire is capturing true dietary intake (82). The National Cancer Institute (NCI) has va lidated a FFQ referred to as the Diet History Questionnaire (DHQ) (81). Frequency of inta ke and portion size for 124 individual food items is captured in this FFQ. Forty-four of these questions have embedded questions that ask about seasonal in take, type of fats added, and whether the food item was low-fat or fat free (81). The original DHQ also includes ten questions regarding dietary supplements, and four su mmary questions (81). Portion size choices were determined from analyses of data from the CSFII (1994-1996). Based on responses from 10,019 adults during this study, por tion size choices were changed in the questionnaire. Instead of using portion sizes such as small, medium, and large, this questionnaire provides portion sizes specific to the food item such as “less than 1 cup,” “1-2 cups,” and “more than 2 cups”. Als o, based on data from the CSFII (1994-1996),

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26 5,261 individual foods were categorized into 170 food groups (83). These food groups are the basis of the 124 food items used in the DHQ (81). Nutrient estimations are calculated for each food item listed in the DHQ by multiplying the daily frequency by a nutrient value specific for the gender of th e individual and the portion size selected. The NCI DHQ was validated in 2001 against repeated 24-hour recalls and compared to the Block and Willett FFQ (81). The Block FFQ is an eight-page questionnaire that covers 106 food items. Portion size choices are described as small, medium, or large (81). The questionnaire has thirteen dietary supplement questions, eight questions regarding use of added fats, five summary questions, and six questions regarding restaurant eating. The Willett FFQ is a four pa ge questionnaire with 126 food items. The questionnaire is unique in th at it does not have a separate portion size question. Individuals select one response in which the fr equency and portion size are combined together in one response. Th e questionnaire also includes ten dietary supplement questions and ten questions about added fats (81). The validation study for the DHQ consisted of 1,301 men and women who completed four telephone 24-hour recalls during one session. The subjects we re then randomized to receive the DHQ and Block FFQ or the DHQ and Wille tt FFQ. Compared to the 24-hour recall method, it was found that the DHQ was more accu rate in determining nutrien t intake than either the Block or Willett FFQs (81). Assessment of vitamin B12 intake The assessment of vitamin B12 intake among vegetarians and non-vegetarians has generally been assessed along w ith other nutrients to provide an overall profile of dietary adequacy of a vegetarian diet in contrast to a non-vegetarian diet Studies where only

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27 vitamin B12 intake has been assessed (generally in relationship to serum concentration status) have largely been conducted with vegans compared to non-vegetarians. Haddad and Tanzman (2003) used CSFII 1994-1996 data to compare the dietary intake of various nutrients including vita min B12 among self-defined vegetarians and non-vegetarians in the United States. Diet ary data was collected using two 24-hour dietary recalls. Men and women ages 20 y ears and above who reported consuming no meat had a vitamin B12 intake lower than t hose who consumed meat (84). Two separate studies by Barr and Broughton (2000) and Jane lle and Barr (1995) re ported findings in Canadian women that were similar to those reported in a United States study. Both studies compared nutrient intake, includi ng vitamin B12, among ve getarian and nonvegetarian women in Canada. The study by Barr and Broughton ( 2000) assessed dietary intake using the 24-hour recall method, a nd the study conducted by Janelle and Barr (1995) assessed dietary in take using 3-day food freque ncy questionnaires. Among women, vitamin B12 intakes of the vegetarian s were lower than non-vegetarians (85, 86). Haddad et al. (1999) also assessed vitamin B12 intake and status of vegans and non-vegetarians. To measure dietary inta ke, a 24-hour recall and 4-day food records were used. Vitamin B12 intake among the fe male vegans was lower than the vitamin B12 intake among the female non-vegetarians. No differences in vitamin B12 intake were reported among the male vegan and nonvegetarian groups. Serum vitamin B12 concentration (male and female combined) did not differ among the vegan and nonvegetarian groups (87). Miller et al. (1991) assessed vitamin B12 intake in relation to serum vitamin B12 status among individuals who consumed a macrobiotic diet but sometimes included dairy,

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28 eggs, and seafood in their diet. Dietar y intake was obtained although the specific assessment tool used (i.e., food record, FFQ, or dietary recall) was not indicated. To determine dietary vitamin B12 intake, the frequency of intake was multiplied by the vitamin B12 content of the foods. This numbe r was then added to a frequency score that was based on how often specific foods (e.g., dairy, eggs, seafood) were consumed. This study compared the frequency intake of dair y, eggs, and seafood in relation to serum vitamin B12 concentration. Those who consumed dairy products 1 time/week or > 1 time/week had a significantly higher serum vitamin B12 c oncentration than those who never consumed dairy products. No differe nces were found in frequency of egg or seafood intake on serum vitamin B12 concen tration between intake groups. Serum vitamin B12 concentration was then compared to tertiles of vitamin B12 intake score. Vitamin B12 intake scores of 0, 0.01 to 0.15, and 0.16 to 1.7 were defined as low, medium, and high, respectively. Adults who had a low vitamin B12 intake score had significantly lower serum vitamin B12 concentr ation than those with a score of medium or high. (9). Rauma et al. (1995) used 5-day food reco rds to determine vitamin B12 intake among vegans who followed a strict uncooked diet and non-vegetarians. The mean vitamin B12 intake from the vegan group was significantly lower than the non-vegetarian group (1.8 versus 6.2 g/day, respectively). Although, the authors stated that dietary assessment of vitamin B12 of the vegan gr oup was underreported because their database did not contain data for the foods consumed on the “uncooked diet”. Serum vitamin B12 concentration was correlated (r = 0.63; p < 0.01) with dietary vita min B12 intake among

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29 the vegans. Contribution of total vitamin B 12 from various food sources consumed were not determined in this study (10). Larsson and Johansson (2002) compared dietary intake of various nutrients including vitamin B12 among 16 to 20 year old ve gans and omnivores in Sweden. In this study, participants were interviewed for 1 to 2-hours, 1 to 2 weeks apart to determine dietary intake. Dietary vitamin B12 intakes for female and male vegans (0.0 0.1, 0.1 0.03, respectively) were signifi cantly lower (p<0.0001) than female and male omnivores (5.0 2.5, 5.9 1.5, respectively) (88). La rsson and Johansson (2005) published additional data comparing dietary sources of vi tamin B12, in addition to other nutrients. Among the vegans, 100% of vitamin B12 intake was from dietary supplements. In contrast, the majority of vitamin B12 in the diet of omnivores came from animal products, defined as meat, fish, seafood, da iry products, and eggs, followed by dietary supplements (89). Dunn-Emke et al. (2005) assessed dietar y intake of vitamin B12 among vegans with prostate cancer following a well-planned, low-fat diet. Subjects were given specific dietary guidelines for this diet along with a soy protein powde r that contained isoflavones (potential inhib itor of cancer growth). After 6 months, 3-day food records were obtained. The mean vitamin B12 intake for these subjects was 3.7 g/day, with 81% of the vitamin B12 intake from supplemental soy protein and 19% from fortified plant foods (90). Leblanc et al. (2000) determined vita min B12 intake among lactoovovegetarians, lactovegetarians, and vegetarians following a macrobiotic diet. Participants recorded dietary intake using a 5-day food record. Th e nutrient software used to analyze the food records contained additional f oods found in a vegetarian diet. Only 80% of these foods

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30 contained data on vitamin B12 content. Afte r analysis, it was determined that the women who followed a macrobiotic diet had a signifi cantly lower (P<0.05) dietary vitamin B12 intake compared to the women in the other vegetarian groups. There were no differences in vitamin B12 intake among vegetarian men. The median intakes of vitamin B12 for all three groups (men and women) were belo w the RDA for vitamin B12 (range of 0-1.73 g/day) (11). Vitamin B12 Status in the United States Based on data from NHANES III (1994-1998) mean serum vitamin B12 was 382 pmol/L for individuals 4 years and older (91). Younger males and females (4-5 years) had the highest median serum vitamin B12 c oncentration for all ag e groups. Men and women 70 years and over, had th e lowest median serum vitami n B12 concentrations (i.e., 286 pmol/L and 322 pmol/L, respectively). Based on ethnicity, non-Hispanic black Americans had the highest median concen tration (419 pmol/L) followed by Mexican Americans (368 pmol/L). In contrast to non-Hispanic white Americans had the lowest median concentration (329 pmol/L) (91). Data from the NHANES III (1998-94) indicat ed that the prevalence of Americans with a serum vitamin B12 concentration le ss than 148 pmol/L incr eased with age. Thirteen percent of individuals 70 years and ol der, 12% of individuals 60 to 69 years, 9% of individuals 50 to 59 years, 8% of individua ls 40 to 49 years, and 2% of individuals 30 to 39 had a serum vitamin B12 concentrati on less than 148 pmol/L. Serum vitamin B12 concentration less than 74 pmol/L was found in 1% of the total population over 4 years of age (91). Vitamin B12 deficiency related to prot ein-bound vitamin B12 malabsorption in the elderly is estimated between 10 to 30% ( 45). Carmel et al. (2002) conducted a study

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31 evaluating the prevalence of elevated homocy steine concentration in young Asian Indians in the United States. They found that a larg e percentage of this population had elevated plasma homocysteine concentration compared to healthy controls. Fifty-nine percent of men and 23.8% of women had low vitamin B12 status as defined by a vitamin B12 concentration <180 pmol/L. They concluded that elevated homocysteine concentration was possibly caused by low dietary vitamin B12 intake (92). Refsum et al. (2001) also studied an Asian Indian popul ation and found that 47% had vitamin B12 deficiency, and 77% had hyperhomocysteinemia regardless of whether they followed a vegetarian or non-vegetarian diet (93). Vegetarianism Definitions of Vegetarianism and Prevalence in the United States There are many definitions of the term vegetarian. Vegetarians may exclude all animal products (vegan); exclude meat and egg products, but include dairy (lactovegetarian); or exclude meat products but include dairy and eggs (lactoovovegetarian). Some vegetarians (pes co-vegetarian) may re strict beef, poultry, and pork consumption, but consume fish, dairy, and eggs. In 1979, the USDA reported that 1.7% of Americans were vegetarians (94). A 2003 national Harris Inter active survey reported that ap proximately 2.5% of Americans describe themselves as vegetarians (84, 95). According to data obtained from the CSFII 1994-1996, 1998, more individuals in the 20 to 29 age group define themselves as vegetarian. The age group with the lowest number of people defining themselves as vegetarians was the 60 to 69 year old group ( 84). There are many factors which motivate individuals to become vegetarian such as h ealth, ethical, or religious attitudes (96).

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32 Assessment of Dietary Adequa cy of Vegetarian Diets Haddad et al. (1999) used a 24-hour recall and 4-day food records to determine the dietary adequacy of vegetarian diets among 45 healthy adults. In this small study, vegans had lower total protein, fat, saturated fat, monounsaturated fat, cholesterol, and higher dietary fiber intake (87). The European Prospective Investigation into Cancer and Nutrition-Oxford (EPIC-Oxford) study, which included approximately 66,000 meateaters and vegetarians, including vegans, in the United Kingdom conf irmed the results of Haddad et al. (1999) in a much larger study. This study used 7-day food records and a FFQ developed for the United States Nurses ’ Health study to determine differences among dietary intake of meat-consumers, fi sh-consumers, vegetarians, and vegans. Subjects were categorized base d on their answers to questions regarding meat (i.e., beef, poultry, and pork), fish, dairy or egg inta ke. The differences between the meatconsumers and the vegans were more pronounced than between the meat-consumers and vegetarians. The meat-consumers reported highe r mean calorie, protein, fat, saturated fat, and vitamin B12 intake, and lower carbohydrate, fiber, folate, and iron intake than the vegetarians and vegans. The intake of v itamin B6 was relatively similar across groups (<10% difference). Serum vitamin B12 concen tration was not determined in this study (97). The American Dietetic Association and Diet itians of Canada have recognized the possible limitations of a vegeta rian diet, but have taken th e position that a carefully planned vegetarian diet can provide the necessary needed nutrients (98). To better determine the adequacy of vegetarian diets, mo re data are needed rela ted to the intake of macronutrients (i.e., carbohydrate, protein, and fat) and micronutrients (i.e., vitamin B12, folate, vitamin B6, zinc, and iron).

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33 Assessment of Beef Consumption and Health There is an increasing trend for American s to exclude or limit beef intake from their diets for health reasons, as review ed by White and Frank (1994) (99). The Food Marketing Institute in 2000 reported that 68 % of American consumers felt their diet could be healthier. In doing so, 68% percen t were increasing their fruit and vegetable intake, 22% were decreasing b eef consumption, and 9% were increasing their intake of chicken and turkey (100). In recent years, beef consumption has been linked to cardiovascular disease (17, 101, 102). The literature regarding beef intake and various markers of cardiovascular disease are conflicting. In 1991, data from the Coronary Artery Risk Development in Young Adults (CARDIA) study were used to de termine relationships between meat (i.e., red meat and poultry) consumption and lipid profiles. Frequency of meat consumption was assessed using a FFQ. The data suggested that those who ate meat less than one time per week had lower total chol esterol and low-density lipopr otein (LDL) concentrations compared to those who consumed meat more than one time per week (102). Nicklas et al. (1995) analyzed 24-hour diet ary recalls in 504 young adults. Those whose meat consumption fell at the 75th percentile or higher did not m eet the Dietary Guidelines for Americans for total fat, saturated fat, and choles terol. In contrast to the Slattery et al. (1991) study, even though th e percent of calor ies from fat was higher than the recommended amount, there were no significant differences in blood li pid profiles in any of the meat consumption quartiles (103). Fr aser (1999) used a FFQ to assess dietary intake and mortality in approximately 34,000 non-Hispanic white Ca lifornia Seventh-Day Adventists. This FFQ included 51 different f ood types. The participants were categorized into three groups; vegetarian (i.e., no meat, poultry, and/or fi sh), semivegetarian (i.e.,

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34 poultry and fish < 1 time per week), a nd nonvegetarian (beef, poultry, and fish consumers). Males who consumed red meat mo re frequently than 3 times per week had a relative risk of 2.5 for devel oping fatal ischemic heart disease. Those who consumed red meat more than 1 time per week had a rela tive risk of 1.86 for developing colon cancer (101). The following studies promote lean beef intake by providing supporting data relative to the role of fat content in beef and other meats on lipid profiles. The O’Dea study in 1990 confirmed that high frequency of beef consumption was not associated with poor health status, but beef fat was. Te n participants ate a c ontrolled diet of verylow fat lean beef (i.e., 9% en ergy from fat) for 3 weeks. Du ring these 3 weeks, total LDL and cholesterol significantly de creased from baseline. Beef drippings were added to the diet during weeks 4 and 5, increasing total fat intake to 20% of total calories at week 4 and 30% at week 5. Total LDL and cholestero l significantly increase d by week 5. It was concluded that lean beef could be incorporat ed into diets instead of reducing overall beef intake (104). Beauchesne-Rondeau et al. (2003) compared the effect s of incorporating lean beef, poultry, and fish on lipid profiles in hypercholesterolemic patients. These patients recorded 3-day food diaries before each 26-day treatment diet. All three treatments were associated with a reduction in total cholesterol and LDL concentration. It was concluded that regardless of the protei n source, decreased saturated fat in the diet could decrease cardiovascular di sease risk (105). Snetselaar et al. (2004) used three 24hour recalls to assess dietar y intake and compared that to serum blood lipid data in adolescents. Subjects were randomized to a lean beef diet or a lean poultry and fish diet. No differences were determined in total cholesterol concentration and saturated fat intake

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35 between the two diets (106). However, a dolescents on the lean poultry and fish group were found to have significantly lower ir on status after 3 months on this diet. Assessment of Dietary Adequa cy of Beef Consumption Beef is the most commonly consumed m eat product in the American diet. Per capita, Americans consume approximately 65 pounds of beef per year, followed by 55 pounds of poultry, and 50 pounds of turkey (1 7). Combined data from the Nationwide Food Consumption Survey (NFCS) 1977-78 a nd the CFSII 1989-91 and 1994-95 indicate that individuals eating separate cuts of beef have decrease d, but beef consumption from meat mixtures has increased (107). The fat co mposition of beef has been reported to be 50% monounsaturated fatty acids, 46% satu rated fatty acids, and 4% polyunsaturated fatty acids. Compared to othe r protein sources, beef provi des the highest concentration of zinc and iron per 3 oz serving. Beef also is rich in protein, vitamin B12, and choline (17). The current 2005 Dietary Guidelines for Amer icans continue to maintain that beef is important in a well-rounded diet as long as leaner cuts of meat and sensible portion sizes are consumed (108). Research Rational and Potent ial Application of Findings Total dietary vitamin B12 intake has been assessed among meat-consumers and vegetarians. The majority of these studies assessing vitamin B12 intake have focused on vegetarian intakes using meat-consumers as th e relative control group. These studies are focused on measuring the outcomes related to a vegetarian lifestyle. It has been confirmed that the more restrictive the diet the lower the dietary vitamin B12 intake and a lower serum vitamin B12 concentration. Beef a highly concentrated source of vitamin B12, is often restricted in the diet of meat-c onsumers and vegetarians for health reasons. Restriction of beef could resu lt in lower dietary vitamin B12 intake and lower status,

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36 which may have multiple adverse health consequences such as neurological abnormalities, impaired fetal development, and risk for cardiovascular disease. Therefore, it is important to assess the effect of beef consumption on total dietary intake of vitamin B12, which has not been the focus of previous investigations. The present study categorized subjects based on frequency of beef intake. This type of categorization has been done only in studies assessing the association between beef consumption and cardiovascular disease, bu t not how beef intake is associated with vitamin B12 intake and status. In additi on, the present study compared the vitamin B12 contribution from dietary sources of v itamin B12 consumed by different beefconsumption groups. In this comparison, the contribution of vitamin B12 from specific animal sources, including beef, were estimat ed. Yoshino et al. ( 2005) recently published estimates of vitamin B12 intake and the pr oportion of vitamin B12 contributed from various food sources, excludi ng vitamin B12 from fortif ied sources, among Japanese adults over a 25 year period using an FFQ (109). Unlike the study by Yoshino et al. (2005) the FFQ used in the current study has been modified to contain all vitamin B12 sources including current fo rtified soy-based sources that vegetarians commonly consume. The assessment methods and nutrient databases used in previous studies to calculate dietary vitamin B12 intake were not completely tailored for vegetarian foods. There are many vitamin B12-fortified products available for vegetarians consumers. Manufacturers such as Worthington, Loma Linda, Morning Star Farms, and Boca produce a large number of vitamin B12 fortif ied soy products such as soy burgers, soy crumbles, soy bacon, soy chicken patties, a nd vegetarian cold cuts. Unlike previous

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37 studies, the present study uses a database that had been modified to assess the contribution of these fortified produc ts to dietary vitamin B12 intake. The present study is the first to strati fy subjects based on their frequency of consumption of beef products and to compare the vitamin B12 intake and food sources to vitamin B12 concentration among the beef cons umption groups. This study also is the first to use a modified diet ary history questionnaire specifically for vitamin B12containing foods and use a nutrient database where 100% of foods have information on vitamin B12 content. The data from this study provides valuable information regardi ng frequency of beef consumption and beef’s role as a contributor to total dietar y vitamin B12 intake in the diet. The data from this study will assi st consumers in making educated decisions regarding the frequency of cons umption of beef related to th e potential impact on vitamin B12 status. Data from this study provides new informati on to nutrition educators and dietetic practitioners regarding the relati onship between consumption of foods within specific food groups and the impact on vitamin B12 status. This new information can be incorporated into educational materials fo r consumers and practitioners related to optimizing vitamin B12 status through dietary means.

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38 CHAPTER 3 MATERIALS AND METHODS Study Design and Methods Overview Healthy adult male and female volunteers (n = ~1,000) were recruited by flyers, newspaper, and radio advertisements for 1 y ear to participate in the study. Prospective subjects were screened by phone using a screening questionnaire (Appendix A) to determine if they met the following inclusi on criteria: (a) age 18 to 49 years, (b) nonsmoking, (c) no use of prescription medications (birth control was allowed), (d) low alcohol intake (< one drink per day), (e) no history of chronic diseases, (f) non-pregnant, (g) non-lactating, (h) no use of vitamin B12 supplements within the last 6 months; and (i) no major dietary changes within the last 3 ye ars. An attempt was made to screen out consumers of highly-fortified cereal as well. Eligible males and females (n = 388) were selected to participate in the study. Subjects were scheduled to have fast ing blood samples drawn and to receive detailed instructions regarding the comple tion of the DHQ (Appendix B). Following the collection of 70 milliliters (mL) of blood that was processed for multiple analyses including plasma vitamin B12, subjects were provided with a comprehensive instruction lasting 15 to 20 minutes on the procedures to complete the DHQ (Appendix C). Subjects took the DHQ forms with them to complete at home and mail back to the primary investigator within 2 we eks of instruction. Total dietary vitamin B12 intake, in additi on to other nutrients, and plasma vitamin B12 concentration were assessed. Subjects we re compensated $50 for their participation

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39 in the study. The University of Florida, Inst itutional Review Board approved this protocol and all subjects signed approved informed consent forms. Diet History Questionnaire Overview The contribution of beef and beef-containi ng foods to total vitamin B12 intake was assessed by the NCI DHQ. This study was a co llaborative effort betw een investigators at the University of Florida and the University of North Carolina, Chapel Hill (UNC). The original DHQ from NCI was modified with added questions for beef containing foods and vitamin B12-fortified foods. The Diet*Cal c Analysis program, also provided by NCI, was used to analyze the questionnaire res ponses. This program is a free software program that can be downloaded from the NCI website ( www.riskfactor.cancer.gov ). The nutrient database within the software is from the USDA Survey Nutrient Database and NDS-R, University of Minnesota. Th e Diet*Calc Analysis program nutrient database was last updated in August 2004. Modifications Made to the Paper Versio n of the Diet History Questionnaire The DHQ from NCI was modified in such a way that vitamin B12-containing foods could be isolated in the data analysis. A comprehensive review of the original DHQ was conducted to determine if vitamin B12-contai ning foods were missing from the DHQ. Fifteen questions (including embedded questi ons) were added and twenty-six questions were modified to distinguish between vita min B12-containing food items and other food items. For example, an original DHQ questi on may have referred to the frequency of lasagna consumption. The DHQ was modified to determine the frequency of consumption of lasagna made with beef, lasa gna made with meat other than beef, and lasagna made without meat. Seven questions were changed to reflect beef containing

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40 foods. For example, the original DHQ asked a bout cold cuts. Questions like these were modified to be more specific, and in this example, asked only about beef containing cold cuts. Eight questions were added to reflect soy substitute products. These questions referred to commercial products available to consumers that are possibly fortified with vitamin B12. The ten supplement questions fr om the original DHQ were deleted since this study population did not consume any supplements. Modifications Made to Diet*Calc Analysis Software Since modifications were made to th e paper version of the DHQ, corresponding modifications needed to be made to the Di et*Calc Analysis software. The software changes were imperative so the DHQ would be compatible with the Diet*Calc Analysis software. The modifications to the Diet*Calc Analysis software were completed by staff in the Nutrition Epidemiology Core at UNC. The Questionnaire Data Dictionary (*qdd) file within the Diet*Calc Analysis program was modified to be compatible with the changes made to the DHQ. The *qdd file c ontained coding information for interpreting scanned DHQs. Once the *qdd file was update d allowing the program to correctly interpret new questions, the Nutrient and F ood Group database within the program also was modified. The nutrient content of vitamin B12-containing foods that were added to the DHQ were extensively reviewed using the USDA National Nutrient Database for Standard Reference (4) and ma nufacturer websites for specific product information. For example, if the old question asked about co ld cuts and the new revised question was regarding only beef cold cu ts, then the Nutrient and F ood Group database was updated to only reflect nutrients that were provided from beef cold cuts.

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41 Data Generation When the paper version of the DHQ wa s scanned (procedure for scanning discussed later), food items or questions were linked to a predetermined Food Identification Number (FIN). Every FIN ha d defined nutrients for gender and serving size. FINs were found in the Diet*Calc Nu trient and Food Group database. Diet*Calc used the FIN associated with each response to assess the nutrient intake of the individual. When Diet*Calc identified a FIN from the AS CII text file, it used the database to calculate an individual’s nut rient intake. This Nutrient and Food Group database was converted into a Microsoft Excel spreadsh eet with all the FI Ns and corresponding nutrient values per serv ing size and gender. This spread sheet was used to identify the beef and beef-containing foods assessed in this study. Diet*Calc produced three data files availabl e for analysis. The Details.txt file was an expanded version of the data calculated from the ASCII text file. This file allowed the investigator to compare FINs to individual nu trient intake and daily frequency of intake. The second file was a Results.txt file, whic h was a condensed version of the Detail.txt file. This file provided co mbined daily nutrient intake values for each individual regardless of the food sources. The third f ile was a Report.txt file, which provided a summary document of daily nutrient intake fo r each individual. This file was not used for data analysis, but for individual feedback when a participant indicated they wanted their dietary intake results from the DHQ. Diet History Questionnaire Instruction Pretest Prior to beginning the study, a pretest of the DHQ instruction and scanning was conducted with a group of 20 Master of Science and Doctoral students in addition to five vegetarian and vegan consumers. This pretes t was done to ensure that the instructions

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42 and handouts provided to the subjects fo r completing the DHQ were clear and understandable. Those who partic ipated in the pretest were instructed using the same script that would be used for the study populat ion. They were expected to follow these instructions and complete the DHQ as if they were a participant in the study. This group was given an additional survey asking vari ous questions regardi ng length of time to complete the DHQ, if any frequently consum ed foods were missing from the DHQ, and if they had additional comments that might allow for the DHQ instruction to be better understood (Appendix D). Comments from this pretest group were reviewed and incorporated into the DHQ and into the DHQ instructions. Human Subjects Procedures Qualified subjects reported to the Fo od Science and Human Nutrition Building during their scheduled appoint ment following an overnight fast. The subjects were required to be fasting for 8 hours prior to having their blood drawn. The study was explained to the subjects, and if they agreed to continue, they were asked to sign the approved informed consent form. Subjects w ho granted consent were assigned a subject identification number. A phlebotomist drew 70 mL of blood for analysis. During the blood draw, subjects were asked again if they were taking a v itamin B12-containing supplement. Subjects received a snack after their blood was draw n prior to receiving the DHQ instructions. Diet History Questionnaire Instructions After completion of the blood draw, each participant was given a 20-minute group instruction reviewing the pr ocedures to follow when completing the questionnaire. During this time, the instructions were read aloud and explained in detail using examples of questions and scenarios. Subjects were ag ain asked if they consumed a vitamin B12-

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43 containing supplement 6 months pr ior to the study. If a subjec t indicated that they did, specific information regarding supplement use was recorded. To ensure that portion sizes were reported accurately, subjects were taught how to use food labels to determine portion sizes, and instructed on how to use measuring cups and spoons for visual representation of a porti on size. Subjects also were given additional information that aided in completion of th e DHQ. This information, which included a schematic drawing of the proper procedure to mark or change an answer, and handouts on “Caffeinated vs. Non-caffeinated Beve rages,” “Fortified Cereals,” and “Seasonal Fruits and Vegetables,” were reviewed dur ing the group instruction (Appendix E). The purpose of these handouts was to ensure th at the DHQ would be s canned correctly, and that subjects would be better prepared to consistently answ er the questions presented in the DHQ. Subjects also were given two samp le questions reviewing the procedure to average their intake over 12 months. These ex amples were based on usual dietary intake and referred to specific questions in the DHQ. Subjects were given the opportunity to ask further questions if anything was unclear. Sample Collection and Processing Processing of Plasma fo r Vitamin B12 Analysis Blood samples were drawn into Vacutain er tubes (Vacutainer Blood Collection Set; Becton Dickinson, Vacutainer System s; Franklin Lakes, NJ) containing K3 ethylenediaminetraacetic acid (EDTA) as an anticoagulant and immediately placed on ice. Following centrifugation at 2000 x g for 30 minutes at 4C, aliquot s were stored at -20C until plasma vitamin B12 analysis.

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44 Processing the Diet History Questionnaire Subjects were encouraged to return thei r DHQ within 2 weeks. If they did not return the DHQ within 2 weeks, they were contacted at the phone number or email address provided during the initial screeni ng. All returned DHQs were reviewed for completeness. The purpose of the review was to ensure that the DHQ had been completed and that no question had a missing response. To prevent bias, marked responses to questions were not reviewed. Questions fo r which a response was missing were noted, and the subject was contacted to obtain the mi ssing information. If the participant was phoned, the questions for which responses were missing were read aloud to the participant and their re sponse was recorded on the DHQ. If the participant was contacted via email, then the question and answer responses were typed. The DHQ was sent to Optimal Solutions Corporation (OSC), Lynbrook, New York, in groups of 100 to be scanned electronicall y. Once the scanning process was completed for each group of 100, OSC mailed the DHQs and el ectronic data in the form of an ASCII text file to UNC. The ASCII text file wa s uploaded at UNC into the Diet*Calc Analysis program modified for this version of the DHQ. Analytical Methods Formation of Dietary Groups (Objective 1) Subjects who consumed meat were grouped based on their frequency of consumption of beef (e.g., steak, roast, hambur ger, etc) as reported from the DHQ results (Table 3-1). Table 3-1. Daily and weekly beef intake frequency Daily Frequency Weekly FrequencyDiet Group 0.286 – 1.0 2.0 – 7.0 Frequent Beef 0.0001 – 0.285 0.0007 – 1.995 Seldom Beef <0.0001 <0.0007 Never Beef

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45 If the daily frequency of consumption of th ese foods was greater than or equal to a daily frequency of 0.286 (equivalent to > 1 time a week), the subjects were grouped in the frequent beef group. If the daily frequenc y was greater than 0 .0001 but less than 0.286 (equivalent to 1 time a week), subjects were gr ouped into the seldom beef group. Individuals who never consume beef foods but who consumed other types of meat including seafood were placed in the never beef group. Subjects were categorized as vegetarians only if their fre quency of consumption of all meat products (i.e., beef, poultry, pork, mixed dishes, and seafood) was zero. Identification of Food Groups from the Diet History Questionnaire (Objective 2) All foods included in the DHQ were categor ized into specific food groups (i.e., beef, pork/other meat, poultry, dairy, eggs, s eafood, cereal, soy, meal replacement, mixed dishes with meat type unknown, non-dairy fats, beans, rice/pasta, soups/sauces, breads/cracker/cakes/pies, nut s/seeds, vegetables, fruit, syrup/honey/gelatin/candy, and beverages). Dietary vitamin B12 intake was estimated from the following food groups: (1) beef, (2) pork, (3) poultry, (4) dairy, (5) eggs, (6) seafood, (7) soy, (8) cerea l, (9) meal replacement, and (10) mixed dishes w ith meat type unknown. The remaining food groups were grouped together as (11) “other ”. Table 3.2 indicates the types of foods found within each food group. Analysis of Additional Nutrients Total calorie, carbohydrate, fat, saturated fa t, and protein intake were determined using information reported from the DHQ. Tota l dietary intake of other micronutrients (folate and vitamin B6) relevant to vitami n B12 metabolism also was evaluated. Key minerals (e.g., iron and zinc) known to be c oncentrated in beef, but possibly limited in the diet of vegetarians, also were analyzed.

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46 Table 3-2. Foods categorized within each food group Food Group Types of Food Included Beef Steak, roast, hamburger, ground beef, beef hotdog, beef sausage, beef cold cuts, mixed dishes made with beef Poultry All cuts of chicken and turkey (breast, wing, etc), chicken and turkey cold cuts, ground chicken and turkey Pork and other meats Ham, pork (all cuts ), bacon, ham cold cuts, veal, venison, lamb, shortribs, liver Dairy Milk, cheese, yogurt, cr eam, pudding, ice cream, butter Eggs and egg mixtures Eggs, mayonnaise Seafood* Fish Shellfish Tuna, fried and not fried fish Oysters, clams, muscles, shrimp, crab, lobster Cereals** Highly-fortified Low-fortified All-Bran, Multigrain Cheerios, Complete, Just Right Fruit and Nut, KASHI Heart-to-Heart, Mueslix, Product 19, Smart Start, Special K, Total (all types) Oatmeal, grits, all other types of cereals not listed above Soy Tofu, soymilk, soy substitute products (Morningstar Farms, Worthington/Loma Linda), egg substitutes Meal replacement Power bars, Nutrigrain bars, Balance bar, Zone bar Mixed dishes with meat type unknown Pasta with meat/fish sauce, Mexican food, lasagna, chili or pizza with meat other than beef Other Non-dairy fats, beans, rice/pasta, soups/sauces, breads/cracker/cakes/pies, nuts/ seeds, vegetables, fruit, syrup/honey/gelatin/candy, and beverages Seafood was analyzed as one food group a nd as separate food groups of fish and shellfish. **Cereal was analyzed as one food group and as separate food groups of highly-fortified and low-fortified cereals. Hi ghly-fortified cereals contained >25% of the Daily Value for vitamin B12. Low-fortified cereals containe d <25% of the Daily Value for vitamin B12. Plasma Vitamin B12 Concentration (Objective 3) The plasma vitamin B12 concentration of all subjects was determined using MP Biomedicals, Inc SimulTRAC-S B12/Folate Radioassay kit (Orangeburg, NY). This is a competitive protein binding assay using cobalt-57 (57Co). Dithiothreitol (DTT) solution was added to the vitamin B12/folate tracer (57Co, 125I, borate buffer with human serum albumin, dextran, potassium cyanide, dye and preservative). This mixture was added to

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47 the plasma samples and boiled for 15 mi nutes. The boiling process inactivates endogenous binding proteins and co nverts all cobalamin forms to cyanocobalamin. Once cooled, purified IF was added to the mi xture and incubated for 1 hour. During incubation, endogenous cyanocobalamin and 57Co compete for binding sites to IF. The mixture was centrifuged. Bound cobalamin (lab eled and unlabelled) accumulated at the bottom in a pellet, while unbound cobalamin was in the supernant. The radioactivity of the pellet was measured using a gamma counter. Vitamin B12 concentration was inversely related to the measured radioact ivity. During the assay, samples were covered with aluminum foil to decrease exposure to light. A plasma vitamin B12 concentration a bove 221 pmol/L was considered normal. Plasma vitamin B12 concentrations betw een 148 and 221 pmol/L were considered marginally deficient, and a concentration below 148 pmol/L was considered deficient (66). Statistical Methods The statistical analysis was performed us ing SAS, version 9.1, SAS Institute Inc. Cary, NC, USA. An initial analysis was c onducted to determine whether the four dietary groups differed significantly with respect to the following demographic variables: gender, age, body mass index (BMI), ethnicity, marital status, employment status, student status, and educational level. For th e continuous demographic variab les, a one-way analysis of variance (ANOVA) was used to determine whether the mean responses differed among the dietary groups. A chi-squa re test was used to determ ine whether the proportion of responses in each of the categories differed among groups when considering categorical response variables. Significant differences in gender, age, BMI, marital status, and student status were found among the four dietary groups. BMI a nd gender were highly

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48 correlated. As a consequence, after adju sting for gender, mean BMI did not differ significantly among dietary groups. Similarly age was highly correlated with student and marital status. After adjusting for age, neith er student status nor marital status differed significantly among dietary groups. Therefor e, only gender and age were used as covariates in the analysis. For each of the dependent variables, a one-way ANOVA was conducted with age and gender as the covariates and allowing for differences in variances among the four dietary groups. Plasma vita min B12 concentration was log transformed for normality. Reported plasma concentra tions are back-log transformed. Pearson’s correlation was used to determine the rela tionship between vitamin B12 intake and concentration.

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49 CHAPTER 4 RESULTS Demographic Characteristic s of the Study Population Subjects Three hundred eighty-eight subjects were en rolled in the study. After enrollment, 62 of the subjects were excluded due to admission during the study interview that a vitamin B12-containing supplement had been taken within the past 6 months. Six subjects were excluded because they failed to return their completed DHQ. Data from 18 subjects were not included because the to tal calorie intake re ported from the DHQ was less than 600 calories or greater than 5,000 calories, which are deemed to be implausible. Therefore, 302 (137 males, 165 females) eligib le subjects were included and the meat consumers in this group were further categori zed into three groups based on frequency of beef consumption. The dietary beef intake gr oups were as follows: (1) frequent beef consumers (n = 97), (2) seldom beef consumer s (n = 42), and (3) never beef consumers (n = 42). Those who excluded all meat produc ts (i.e., beef, poultry, pork, mixed dishes containing meat, and seafood) were grouped into a fourth dietary group referred to as the vegetarian group (n = 121). The vegetarian group consisted of l actoovovegetarians (n = 66), lactovegetarians (n = 47), and vegans (n = 8). Demographic Characteristics The demographic characteristics of the st udy population are presented in Table 4-1. The mean age and BMI (kg/m2) (mean SD) for all the participants were 25 14 years and 24 9, respectively. The mean education level was 15 5, which is equivalent to

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50 the junior level at a university. Gender a nd age were significantly different (p<0.0001); as were martial status (p = 0.02), student status (p = 0.001), and BMI (p = 0.03) among the four groups. Table 4-1. Demographic charac teristics of dietary groups1,5 Demographic Variable Frequent Beef (n = 97) Seldom Beef (n = 42) Never Beef (n = 42) Vegetarian (n = 121) p-value Age2 23.2 6.1 23.7 5.1 26.0 7.2 28.1 8.8 <0.0001 BMI2,3 24.5 5.4 24.0 3.7 22.7 2.9 23.0 4.3 0.0333 Education level2 14.6 2.1 15.1 2.7 15.2 1.7 15.3 2.2 0.1336 Gender (%)4 <0.0001 Female 34 69 79 55 Male 66 31 21 45 Ethnicity (%)4 0.1039 Asian 10 14 12 24 White 60 64 74 56 African American 8 5 2 5 Hispanic 19 10 7 9 Other 2 7 5 5 Marital status (%)4 0.0177 Single 89 88 86 76 Married 11 12 14 24 Student status (%)4 0.0007 Full-time 79 69 60 55 Part-time 4 10 12 3 Not a student 16 21 29 41 Employed (%)4 0.2484 Yes 56 60 74 63 No 44 40 26 37 1Means expressed as mean standard deviation (SD). 2One-way ANOVA was used for statis tical comparisons between groups. 3Body Mass Index (BMI) calculated as kg/m2. 4Chi-square test was used for statistical comparisons between groups. 5Due to rounding, percentages will not always sum to 100. Dietary Vitamin B12 Intake (Objectives 1 and 2) Total vitamin B12 intake (mean SD) am ong the four dietary groups is presented in Table 4-2. Vitamin B12 inta ke is expressed in this tabl e as total vitamin B12 intake ( g/day) consumed and as g/1000 calories consumed. Unad justed for calories, total vitamin B12 intake in the seldom beef group was lower (p = 0.001) than the frequent beef group. Vitamin B12 intake in the never beef group and vege tarian group also were lower (p<0.0001) than the frequent beef group. The unadjusted vitamin B12 intake of the never

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51 beef group was lower (p = 0.05) than the se ldom beef group. The vitamin B12 intake unadjusted for calories in the vegetarian group was lower (p<0.0001) than the seldom beef group and lower (p = 0.02) than the never beef group. After adjusting for calorie intake ( g/l000 kcals), the never beef and vegetarian groups had lower (p < 0.0001) vitamin B12 inta kes than the frequent beef and seldom beef groups. There were no differences (p = 0.27) in vitamin B12 intake ( g/l000 kcals) among the frequent beef and seldom beef groups. The vitamin B12 intake of the vegetarian and never beef intake groups did not differ (p = 0.60). Table 4-2. Daily total dietar y intake of vitamin B121 Frequent Beef (n = 97) Seldom Beef (n = 42) Never Beef (n = 42) Vegetarian (n = 121) Vitamin B12 (g) 8.2 4.0 5.9 3.52 4.5 2.72, 3 3.3 2.82,3,4 Vitamin B12 (g/1000 kcals) 3.3 1.4 3.7 2.2 2.0 1.42,3 1.9 1.52,3 1Means expressed as mean SD. On e-way ANOVA was used for statis tical comparisons between groups adjusted for age and gender. 2Significantly lower than frequent beef (p<0.05). 3Significantly lower than seldom beef (p<0.05). 4Significantly lower than never beef (p<0.05). The total vitamin B12 intake (g) among the four dietary groups was compared to the Estimated Average Requirement (EAR) as illustrated in Figure 4-1. The frequent beef group consumed 4.1 times more vitamin B12 than the EAR. The seldom beef group consumed 3 times more vitamin B12 than the EAR. The never beef group consumed 2.3 times more than the EAR, and the vegeta rian group consumed 1.7 times more vitamin B12 than the EAR (Figure 4-1).

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52 Frequent Beef Seldom Beef Never Beef Vegetarian 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 EAR 2.0 g/dDietary GroupMean Total Vitamin B12 Intake ( g/d) Figure 4-1. Mean vitamin B12 intake comp ared to recommended intakes. EAR (2.0 g/day) is based on recommendation fo r non-pregnant, non-lactating healthy adults. The daily mean vitamin B12 intake ( g/1000 kcals) contributed by various food sources is presented in Table 4-3. Beef, Poultry, Pork, Seafood, and Mixed Foods Among meat consumers (i.e., frequent beef seldom beef, and never beef groups), vitamin B12 from beef in the seldom beef group was lower (p<0.0001) than vitamin B12 from beef in the frequent beef group. Vitami n B12 from poultry in the never beef group was lower (p = 0.004) than vitamin B12 from poultry in the frequent beef group and lower (p = 0.03) than the vitamin B12 from poultry in the seldom beef group. There were no differences (p = 0.90) in vitamin B12 fr om poultry among the frequent beef and seldom beef groups. There were no differen ces in vitamin B12 intake from pork or seafood, including shellfish and fish, among the four dietary groups. Vitamin B12 from mixed foods that contained meat (type unde terminable) in the never beef group was

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53 lower (p<0.0001) than the vitamin B12 intake from mixed foods in the frequent beef group and the seldom beef group. There were no differences (p = 0.18) in vitamin B12 intake from mixed foods among the frequent beef and seldom beef group. Dairy and Eggs There were no differences in vitamin B12 intake from dairy products among all dietary groups. Vitamin B12 intake from e ggs in the vegetarian group was lower (p = 0.001) than the frequent beef, lower (p = 0.001) than the seldom beef, and lower (p = 0.0004) than the never beef groups. Cereal Vitamin B12 intake from all types of cer eal was lower (p = 0.01) in the frequent beef and lower (p = 0.003) in the never beef group compared to the seldom beef group. The vitamin B12 intake from cereal (all types) in the never beef gr oup also was lower (p = 0.03) than that in the vegetarian group. There were no differences in vitamin B12 intake from all types of cereal among the fre quent beef group and ve getarians (p = 0.13); frequent beef group and never beef group (p = 0.10); and seldom beef group and vegetarians (p = 0.14). Cereal was further se parated to determine differences in mean vitamin B12 intake from highly-fortified cer eals and low-fortified cereals. Vitamin B12 intake from highly-fortified cereals in the frequent beef and never beef group was lower (p<0.01) than the seldom beef group. Vitamin B12 intake from highly-fortified cereals in the never beef group also was lower (p = 0.02) than the vitamin B12 intake from highlyfortified cereals in the vege tarian group. There were no diffe rences in mean vitamin B12 intakes from low-fortifie d cereal across all groups.

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54 Fortified Soy Products, Meal Replacements, and Other Sources Vitamin B12 intake from fortified soy pr oducts in the frequent beef and seldom beef groups was lower (p<0.0001) than observed in the vegetarian group. Vitamin B12 intake from fortified soy products in the fr equent beef group also was lower (p<0.0001) than the never beef group. Vitamin B12 inta ke from fortified soy products in the never beef group was lower (p = 0.04) than the ve getarian group. No differences were found for vitamin B12 intake from meal replacement sources among the dietary groups. Vitamin B12 intake from other sources in the frequent beef group was lower (p = 0.03) than the vitamin B12 intake from other s ources in the never b eef group and (p = 0.001) the vegetarian group. Naturally-Occurring and Fortified Sources Vitamin B12 intakes from food sources in which vitamin B12 occurs naturally was compared with vitamin B12 intake from for tified foods. Naturally -occurring vitamin B12 intake in the never beef group was lower (p< 0.0001) than the frequent beef group and (p = 0.01) the seldom beef group. The vege tarian group had lower (p<0.0001) vitamin B12 intake from naturally-occurring sources th an the frequent beef, seldom beef, or never beef groups. The frequent beef group had a lower (p = 0.0004) intake of vitamin B12 from fortified foods than the seldom b eef group, never beef group (p = 0.03), and vegetarian groups (p<0.0001). Vitamin B12 from fortified f oods in the never beef group was lower (p = 0.04) than the vegetarian group.

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55 Table 4-3. Daily mean vitamin B12 contribution by dietary sources ( g/1000 kcals)1 Dietary Source Frequent Beef (n = 97) Seldom Beef (n = 42) Never Beef (n = 42) Vegetarian (n = 121) Beef 0.94 0.5 0.16 0.22 --Poultry 0.07 0.1 0.07 0.1 0.03 0.12,3 -Pork 0.10 0.1 0.14 0.6 0.05 0.3 -Seafood 1.00 1.2 1.24 1.5 0.83 1.0 -Shellfish 0.78 1.1 0.86 1.1 0.58 0.7 -Fish 0.21 0.3 0.34 0.4 0.27 0.3 -Mixed 0.08 0.1 0.06 0.1 0.01 0.002,3 -Dairy 0.53 0.3 0.51 0.4 0.43 0.3 0.51 0.4 Eggs 0.07 0.1 0.10 0.1 0.10 0.1 0.03 0.12,3,4 Cereal 0.25 0.53 0.75 1.2 0.15 0.43,5 0.44 1.1 High-fortified cereal 0.21 0.53 0.71 1.2 0.11 0.43,5 0.41 1.1 Low-fortified cereal 0.03 0.00 0.02 0.1 0.03 0.1 0.04 0.1 Soy products 0.03 0.14,5 0.12 0.35 0.24 0.35 0.39 0.6 Meal replacement 0.08 0.2 0.10 0.3 0. 06 0.1 0.13 0.3 Other 0.15 0.14,5 0.18 0.1 0.20 0.1 0.22 0.2 Naturally-occurring products 2.93 1.3 2.40 1.9 1.49 1.12,3 0.70 0.62,3,4 Fortified products 0.41 0.63,4,5 1.20 1.4 0.75 1.05 1.17 1.5 1Means expressed as mean SD. On e-way ANOVA was used for statis tical comparisons between groups adjusted for age and gender. 2Significantly lower than frequent beef group (p<0.05). 3Significantly lower than seldom beef group (p<0.05). 4Significantly lower than never beef group (p<0.05). 5Significantly lower than vegetarian group (p<0.05). The proportion of vitamin B12 intake provide d by different dietary sources to total vitamin B12 intake (g/1000 kcals) is illust rated in Figure 4-2. In the frequent beef group, the largest contributors to vitamin B 12 intake were seafood (30%), beef (28.5%), and dairy products (16%). Among the seldom beef group, the largest contributors to vitamin B12 intake were seafood (33.5%), cere al (20%), and dairy (13.8%). Sources in the never beef group that cont ributed the largest proportion of vitamin B12 were seafood (41%), dairy (21.3%), and soy products (12%). Dairy (27%), cereal (23.4%), and soy products (20.7%) were the la rgest contributors to vitamin B12 intake among the vegetarians (Figure 4-2).

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56 0%20%40%60%80%100% Vegetarian Never Beef Seldom Beef Frequent Beef Beef Seafood Dairy Cereal Poultry Pork Eggs Soy products Mixed Meal Replacement Other Figure 4-2. Percent of total vitamin B12 intake from f ood sources. Proportions were determined based on adjusted mean intakes of vitamin B12 from each food source. 0%20%40%60%80%100% Vegetarian Never Beef Seldom Beef Frequent Beef Highly-Fortified cereal Low-Fortified cereal Figure 4-3. Percent of total v itamin B12 intake from cereal sources. Proportions were determined based on adjusted mean intakes of vitamin B12 from each cereal source. The proportion of vitamin B12 provided by sub-groupings of foods within a category is shown in Figures 4.3 and 4.4. Highly -fortified cereals provided the majority of vitamin B12 derived from cereal sources in all dietary intake groups (Figure 4-3). Vitamin B12 derived from a subgroup of seafood (i.e., shellfish and fish) revealed that

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57 shellfish provided a larger proportion of vitamin B12 among the groups who consumed seafood (Figure 4-4). 0%20%40%60%80%100% Vegetarian Never Beef Seldom Beef Frequent Beef Shellfish Fish Figure 4-4. Percent of total v itamin B12 intake from shellfish and fish. Proportions were determined based on adjusted mean intakes of vitamin B12 from each seafood source. The proportion of vitamin B12 intake provided from natura lly-occurring or fortified foods is illustrate d in Figure 4.5. When the v itamin B12 content of foods consumed was categorized as naturally-occurr ing or fortified, it wa s found that naturallyoccurring sources of vitamin B12 provided a larger proportion of this nutrient compared to fortified products in the fr equent beef, seldom beef, and never beef groups. In the vegetarian group, fortified products provide d a larger proportion of the vitamin B12 content of the diet (Figure 4-5).

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58 0%20%40%60%80%100% Vegetarian Never Beef Seldom Beef Frequent Beef Naturally-occurring Fortified Figure 4-5. Percent of total v itamin B12 intake from naturally-occurring and fortified sources of vitamin B12. Proportions were determined based on adjusted mean intakes of vitamin B12 from naturally-occurring or fortified food sources. Dietary Intake of Macronut rients and Micronutrients Table 4-4 presents the energy and macr onutrient intakes (mean SD) among the four dietary groups reported fr om the DHQ. To control for differences in energy intake, protein, carbohydrate, fat, and saturated fa t were standardized and expressed as g/1000 kcals consumed. Energy Intake The energy or calorie intake of the seldom beef, never beef, and vegetarian groups was lower (P<0.0001) than the calorie intake of the frequent beef group. There were no differences in energy intake among the seldom beef, never beef, and vegetarian groups. Protein Intake The protein intake of the seldom beef, ne ver beef, and vegetarian groups also was lower (p<0.05) than the protein intake of th e frequent beef group. Th e protein intake of the vegetarian group was lower (P<0.0001) than th e protein intake of the seldom beef and never beef group.

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59 Carbohydrate Intake The carbohydrate intake of the frequent beef group was lower (p<0.0001) than the carbohydrate intake of the seldom beef, ne ver beef, and vegetarian groups. The carbohydrate intake of the seldom beef and never beef groups was lower (p<0.05) than the carbohydrate intake of the vegetarian group. Fat Intake Fat intake was lower (p = 0.01) in the se ldom beef group and lower (p = 0.0001) in the never beef group compared to the frequent beef group. Fat intake in the vegetarian group also was lower (p<0.0001) than the frequent beef group. There were no differences in fat intake among the seldom beef, never beef, and vegetarian groups. Saturated Fat Saturated fat intake in the seldom beef never beef, and vegetarian groups was lower (P<0.0001) than the intake of saturated fat in the frequent beef group. There were no differences in saturated fat intake among th e seldom beef, never beef, and vegetarian groups. Table 4-4. Daily mean dietary intake of macronutrients (per 1000 kcals consumed)1 Nutrient Frequent Beef (n = 97) Seldom Beef (n = 42) Never Beef (n = 42) Vegetarian (n = 121) Energy (kcals) 2489 1026 1729 6502 1856 7312 1862 809 2 Protein (g) 41 9 39 72 37 92 33 92,3,4 Carbohydrate (g) 112 213,4,5 129 195 132 155 140 21 Fat (g) 40 7 37 82 36 52 36 82 Saturated Fat (g) 12 3 9 32 9 32 10 32 1Means expressed as mean SD. On e-way ANOVA was used for statis tical comparisons between groups adjusted for age and gender. 2Significantly lower than frequent beef (p<0.05). 3Significantly lower than seldom beef (p<0.05). 4Significantly lower than never beef (p<0.05). 5Significantly lower than vegetarian (p<0.05).

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60 Table 4-5 presents the daily intakes of micronutrients (mean SD) (i.e., folate and vitamin B6) relevant to vitamin B12 metabolism and key minerals known to be concentrated in beef, but possi bly limited in the diet of vege tarians (i.e., iron and zinc). To control for differences in energy intake among the four dietary groups these nutrients also were standardized and expressed as g/1000 kcals consumed. Folate Intake The frequent beef group had a lower (p<0.0001) folate intake than the seldom beef, never beef, and vegetarian groups. There were no differences in folate intake among the seldom beef, never beef and vegetarian groups. Vitamin B6 Intake The frequent beef group had a lower (p = 0.0001) intake of vitamin B6 than the seldom beef, never beef (p = 0.03), the ve getarian groups (p = 0.001). There were no differences in vitamin B6 intake among the seldom beef, never b eef, and vegetarian groups. Iron Intake The frequent beef group had a lower (p< 0.0001) intake of iron than the seldom beef, never beef, and vegetarian groups. Th e seldom beef and the never beef groups had a lower intake of iron (p = 0.003; p = 0.001. respectively) co mpared to the vegetarian group. There were no differences in iron intake between the seldom beef and never beef groups (p = 0.72). Zinc Intake The zinc intake of the never beef group was lower than the zinc intake of the frequent beef group (p = 0.001), seldom beef group (p = 0.002), and vegetarian groups (p = 0.02).

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61 Table 4-5. Daily mean dietary intake of micronutrients (per 1000 kcals consumed)1 Nutrient per 1000 kcals Frequent Beef (n = 97) Seldom Beef (n = 42) Never Beef (n = 42) Vegetarian (n = 121) Folate (g) 180 483,4,5 271 85 273 93 287 107 Vitamin B6 (mg) 1.0 0.33,4,5 1.3 0.4 1.2 0.4 1.2 0.6 Iron (mg) 7.4 1.93,4,5 10.4 3.85 10.1 3.85 13.1 7.6 Zinc (mg) 5.6 1.8 6.1 2.7 4.5 1.52,3,5 5.4 2.6 1Means expressed as mean SD. On e-way ANOVA was used for statis tical comparisons between groups adjusted for age and gender. 2Significantly lower than frequent beef (p<0.05). 3Significantly lower than seldom beef (p<0.05). 4Significantly lower than never beef (p<0.05). 5Significantly lower than vegetarian (p<0.05). Plasma Vitamin B12 Concentration (Objective 3) Plasma vitamin B12 concentrations (mean SD) for the four groups are presented in Table 4-6. There were no differences in the plasma vitamin B12 concentration among the dietary groups (p = 0.70). As illustrated in Figure 4-6, mean plasma vitamin B12 concentration was above 221 pmol/L, the limit set as normal, for all dietary groups. Table 4-6. Plasma vitamin B12 concentration1 Frequent Beef (n = 97) Seldom Beef (n = 42) Never Beef (n = 42) Vegetarian (n = 121) Plasma vitamin B12 (pmol/L) 280 114 289 114 279 139 263 143 1Means expressed as mean SD. On e-way ANOVA was used on log-transformed means for statistical comparisons between groups adjusted for age and gender. Plasma concentrations are based on back-log transformed means. Vitamin B12 Status Vitamin B12 status was based on plasma v itamin B12 concentration and defined as normal (>221 pmol/L), marginally deficien t (148-221 pmol/L), or deficient (<148 pmol/L). The percent of indivi duals who were considered no rmal, marginally deficient, and deficient is shown in Figure 4-7. Vitamin B12 status was normal in 74% of the frequent beef consumers, 74% of the seldom beef consumers, 69% of the never beef

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62 consumers, and 61% of the vegetarians. Vita min B12 status was marginally deficient in 20% of the frequent beef consumers, 21% of the seldom beef, 19% of the never beef consumers, and 22% of the vegetarians. Vita min B12 status was deficient in 6% of the frequent beef consumers, 5% of the seldom beef consumers, 12% of the never beef consumers, and 17% of the vegetarians. Ther e were no differences in the percentage of individuals who were normal, marginally defi cient, or deficient among the four groups (p = 0.18). The percentage of indi viduals who were deficient based on plasma vitamin B12 concentration (<148 pmol/L) was approximate ly two-fold higher in the non-beef consumers [never beef (12%); and vegetari ans (17%)] compared to beef consumers [frequent beef (6%); and seldom (5%)]. Frequent Beef Seldom Beef Never Beef Vegetarian 0 50 100 150 200 250 300 Normal (221 pmol/L)Dietary GroupPlasma Vitamin B12 Concentration (pmol/L) Figure 4-6. Plasma vitamin B12 concentration (pmol/L). Normal vitamin B12 status was defined as a plasma vitamin B12 c oncentration greater than 221 pmol/L.

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63 Frequent Beef Seldom Beef Never Beef Vegetarian 0 10 20 30 40 50 60 70 80Deficient Marginally Deficient Normal Percent of Individuals Figure 4-7. Vitamin B12 status among dietary groups. Stat istical comparisons were determined using a Chi square test. Vitamin B12 status was defined as follows: (1) deficient (<148 pmol/L); (2) marginally deficient (148-221 pmol/L); and (3) normal (>221 pmol/L). The mean plasma vitamin B12 concentration for the non-vegetarian and vegetarian groups are presented in Table 4-7. The mean plasma vitamin B12 concentration (mean SD) was lower (p = 0.01) in the vegetarian group than the non-vegetarian group. Table 4-7. Plasma vitamin B12 concentra tion among non-vegetarians and vegetarians1 Non-Vegetarians (n = 181) Vegetarians (n = 121) Plasma vitamin B12 (pmol/L) 313 124 280 1462 1Means expressed as mean SD. On e-way ANOVA was used for statistical comparisons controlled for BMI. 2Significantly lower than non -vegetarians (P = 0.01) The percent of individuals w ho were deficient in the nonvegetarian and vegetarian groups are presented in Figure 48. The percent of individuals who were deficient in the non-vegetarian group (7%) was lower (p = 0.01) th an the percent of individuals who were deficient in the vegetarian group (17%).

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64 Non-Vegetarians Vegetarians 0 2 4 6 8 10 12 14 16 18 20Percent of Deficient Individuals Figure 4-8. Percent of indivi duals deficient among non-vegetarians and vegetarians. Statistical comparisons were determined using a Chi Square test (p = 0.01). Vitamin B12 deficiency was defined as <148 pmol/L. Vitamin B12 Intake and Status The association between total vitamin B12 intake and plasma vitamin B12 concentrations for all individuals was assesse d. Dietary vitamin B12 intake was weakly but significantly correlated with plasma vitamin B12 c oncentration (r = 0.23, p<0.0001).

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65 CHAPTER 5 DISCUSSION AND CONCLUSION The focus of this study was to assess vitami n B12 intake and stat us in healthy men and women who consumed beef at variable frequencies and to estimate the relative contribution of specific foods to vitamin B 12 intake. Previous vitamin B12 intake assessment has primarily focused on the adequacy of vitamin B12 intake in individuals who consume vegetarian diets compared to that of individuals who consume nonvegetarian diets. Beef, a highly concentrated source of vitamin B12 and often restricted in the diets of meat-consumers and vegetarian s for health reasons, is hypothesized to be an important contributor of dietary vitamin B12 intake among meat-consumers. Prior to the current study, the role of beef in contri buting to vitamin B12 intake had not been explored. Consumption of beef at least one time a w eek or more resulted in higher dietary vitamin B12 intake than that observed in th e other dietary intake groups. Exclusion of beef from the diet resulted in lower vitami n B12 intake compared to the vitamin B12 intake of beef consumers. These data indica te that frequent beef consumption plays an important role in providing vitamin B12 in th e diet of meat consumers. Miller et al. (1991) assessed frequency of intake of othe r vitamin B12 containing foods such as dairy, eggs, and seafood among vegeta rians in relation to serum vitamin B12 concentration. They did not, however, assess the relationship be tween frequency of intake of these foods and dietary intake of vitamin B12. Since this study only involved vegetarians, frequency of beef consumption and the relation to vi tamin B12 intake and status could not be

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66 assessed. Until this current study, the effect of beef intake frequency on total dietary intake of vitamin B12 had not been addressed. Previous investigations have reported that mean daily vitamin B12 intake among meat-consumers was consistently above 2.0 g /day (84-86, 97). National data from NHANES 1999-2000 indicate that Americans of all age groups are consuming well above the EAR (2.0 g/day) for vitamin B12 (32). In the current study, daily vitamin B12 intake for all meat consumption groups exceeded the EAR. Data from the present study indicated that as beef intake frequency increased so did the intake of vitamin B12 as illustrated by the fact that the vitamin B12 intake among the frequent beef consumers was 4.1 times above the EAR compared to 2.3 times the EAR in the individuals who never consumed beef, but included all other animal products. Analysis of the CSFII 1995 data for men and women indicated that the top three foods contributing to the vitamin B12 intake we re mixed foods, milk and milk drinks, and beef (20). Beef contributes approximately 2.2 g/serving of vitamin B12. Therefore, when categorizing subjects based on their freque ncy of beef intake, as done in the current study, one might assume that beef would pr ovide the largest proportion of vitamin B12 among the beef consumers. Unexpectedly, this was not found among all beef consumption groups. Beef contributed 28.5% among the frequent beef consumers, but only 4% among the seldom beef consumers. In all three meat consumption groups (frequent, seldom, and never) seafood and dair y consistently contributed a substantial percentage of dietary vitami n B12 in the diet. Seafood cont ributed at least 30% (range 30-41%) of the total dietary vitamin B12 inta ke and dairy provided no less than 13.8% (range 13.8-21.3%) of the tota l dietary vitamin B12 intake among these groups.

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67 Differences in data from the current study a nd the CSFII 1995 data may be attributed to differences in the categorization of food groups In the current study, the type of meat found in the mixed foods determined the ca tegory in which it was placed. For this reason, an independent category for mixed foods was not created in the current study. In the current study, beef intake among th e frequent beef consumers was the only meat (compared to poultry and pork) that provided a substantial amount of vitamin B12 in the diet. In contrast, in the seldom beef group, beef did not contribute a large proportion to dietary vitamin B12 intake. Yosh ino et al. (2005) i nvestigated dietary sources of vitamin B12 among Japanese adults irrespective of diet t ype. It was reported that seafood, meats, and dairy provided a larger proportion of vitamin B12 in the diet than other sources of vitamin B12. Larss on and Johansson (2005) separated their study population into vegetarians and non-vegetarian s in a similar manner as was done in the current study. All animal products were combined together when assessing the contribution of vitamin B12 from dietary sour ces by these investigator s (89). Therefore, the contribution of dietary vitamin B12 intake from various animal sources could not be assessed. In the current study, sp ecific food groups that provided vitamin B12 to the diet were characterized to a greater extent than was done in previous investigations. For example, the contribution of vitamin B12 from different types of seafood (i.e., shellfish and fish) was determined, as well as, the contribution of vitamin B12 from highlyfortified cereal versus low-fo rtified cereals. In addition, the overall vitamin B12 intake was characterized as naturally-containing s ources or fortified food sources. Among meat consumers, the vitamin B12 provided by seaf ood was predominately from shellfish; the

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68 vitamin B12 from cereal was from highly-fo rtified cereals ; and a larger proportion of vitamin B12 intake of meat consumers wa s from naturally-occ urring food sources. In comparison to beef consumers, vegetari ans had a lower intake of vitamin B12. These findings are consistent with previous investigations (85, 86). Even with lower intakes of vitamin B12 among the vegetarian s, the intake of vitamin B12 among the vegetarian group was 1.7 times above the EAR in the current study. The addition of so many fortified products in the marketplace that are acceptable to vegetarians has made it possible for individuals who fo llow a vegetarian diet pattern to consume enough vitamin B12 to maintain an adequate intake. Seve ral studies that have assessed vitamin B12 intake of vegetarian and vegan populations ha ve reported that dietary intake of vitamin B12 may have been low because the nutrient database used to assess intake did not contain foods frequently consumed by vegeta rians or because the nutrient database was not complete, in that information regarding vitamin B12 content of all consumed foods was not included (10, 11). To address this re search need, the FFQ used to assess dietary intake of vitamin B12 in the current study wa s modified to contain vitamin B12-fortified foods that are commonly consumed by vegetari ans. Vegetarians get a large proportion of their vitamin B12 from dairy, highly-fortifie d cereals, and fortified soy products, which likely explains why low dietary vitamin B12 intake in the vegetarian group was not observed in the current study. In addition to a low vitamin B12 intake, th e intake of calories, protein, fat, and saturated fat were lower in vegetarians co mpared to frequent beef-consumers. The vegetarians also had higher intakes of carbohydr ates, folate, vitamin B6, and iron than the

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69 frequent beef-consumers. This nutrient intake pattern was similar to what was reported in the EPIC-Oxford study by Da vey et al. (2003). Beef is a rich source of iron and zinc, ther efore, it has been assumed that increased beef consumption would be associated with a higher intake of iron and zinc. This was not always true in our study populat ion. Iron was actually lower in all of the meat consuming groups compared to the vegetarian group. This may occur because a higher proportion of the vitamin B12 intake among of vegetarians was from highly-fortified cereals, products that are also enriched with iron. Zinc inta ke did not differ among the vegetarian and the frequent or seldom beef gr oups. The group that never consumed beef but consumed other animal products had a lower zinc intake than the frequent beef, seldom beef, and vegetarian groups. These findings suggest that in meat consumers, beef may play an important role in maintaining ad equate dietary zinc intake. Mean plasma vitamin B12 concentrati on in our study popula tion did not differ among the dietary groups. This is surprisi ng since previous investigations support the conclusion that vegetarians, especially vega ns, have a lower vitamin B12 concentration compared to non-vegetarians (10, 87, 88). It may be possible that our vegetarian population consumed enough vitamin B12 from an imal and fortified foods to maintain a normal plasma vitamin B12 concentration. NHANES III (1994-1998) indicated that the mean vitamin B12 concentration of all Amer icans was 382 pmol/L. In the current study, the mean vitamin B12 concentration of each dietary intake group was lower than that observed in this national survey. The m eans of each group did not exceed 290 pmol/L, however, this concentration is still considered clinically normal. It is important to note that conclusions regarding stat us can not be based on mean values alone since means can

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70 be affected by outliers. It is more appropria te to use the percentage of individuals with concentrations below the accepted normal range to assess vitamin B12 status. In the current study, no significant difference was found among the percent of individuals who were normal, marginally defici ent, or deficient. Although, it is striking that 17% of vegetarians and 12% of never beef consumer s were deficient compared to 6% of the frequent beef and 5% of the seldom beef groups. A strength of this study is that supplement users were excluded from participation making it possible to assess intake of vitami n B12 from only dietary sources. This study also used a modified FFQ that containe d a comprehensive numb er of vitamin B12containing foods and vitamin B12-fortif ied food products commonly consumed by vegetarians. The addition of these foods a llowed for better dietary assessment of vitamin B12 intake. Another strength of this study was that it was possi ble to directly compare the frequency of beef consumption to dietar y vitamin B12 intake. No other study has grouped subjects in this manner, therefore prev ious investigations have been unable to determine the effect of beef consum ption on dietary vitamin B12 intake. Differences within the vegetarian group w ith respect to the various subgroups of vegetarian food patterns may be considered a weakness of the study. For example, when comparing the contribution of vitamin B12 intake from eggs, the results of the vegetarian group may be different if the lactoovove getarian, lactovegetarian, and vegan subcategories classifications were eval uated separately. Since vegans and lactovegetarians do not consume eggs, this ma y be one reason why the vegetarian group as a whole had a lower vitamin B12 intake from eggs compared to the meat-consumers.

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71 The small sample size of the meat-consuming dietary groups is another notable weakness. This may reflect why no differe nces were found for plasma vitamin B12 concentration. Due to the limitations and curren t scrutiny of the reliability of the plasma vitamin B12 assay, this method of assessment may not truly reflect vitamin B12 status. Other vitamin B12 status indica tors that are more reflective of functional status are being measured by other investigators involved in the current research study and will be reported separately. Due to a small sample size among the fre quent beef group, seldom beef group, and never beef group, these groups were pooled to evaluate the relative difference between non-vegetarian and vegetarian consumers regardless of beef intake. These comparisons were performed as part of a separate analysis. The mean plasma vitamin B12 concentration of the non-vegetarian group was higher than the vegetarian group, this was not found with the previous four dietary gr oups. Also, the percent of individuals who were deficient in the non-vegetarian group were lower than the vegetarians, which again, were not observed with the four dietary groups. These data i ndicate that the small sample size of the beef intake subcategories may e xplain why no differences were detected in vitamin B12 status among the four dietary groups. To conclude, this study used a modified FFQ for vitamin B12 food sources to compare vitamin B12 intake among beef consum ers and vegetarians. The hypothesis that high dietary intake frequency of beef would be associated wi th a greater intake of total dietary vitamin B12 was supported by the data in this study. Increased frequency of beef intake was not associated with a higher mean plasma vitamin B12 concentration. However, the percentage of individuals who were deficient am ong non-vegetarians was

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72 lower than vegetarians. Beef did not provide a larger proportion of vitamin B12 intake among all meat consumers. In the frequent beef group, seafood provided a comparable proportion of total vitamin B12 intake as b eef. Overall, seafood and dairy provided a substantial proportion of vitamin B12 among meat consumers. Dietetic practitioners and nutrition educators can use these data to promote beef, seafood, and dairy consumption for optimizing vitamin B12 status through diet ary means. Future studies should evaluate the impact of frequency of intake of seaf ood and dairy food sources on total vitamin B12 intake.

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73 APPENDIX A SUBJECT DATA COLLECTION FORM Introduction I am calling in regard to your interest in our nutrition study; do you have a few minutes right now? This is a UF Nutrition department study a nd involves coming in one morning for about 1 hour for a fasting blood sample, we take a bout 1 ounces of blood, and you only need to fast 8 hours. We will give you a breakfast sn ack right afterward, and then give a brief explanation of a food frequency questionnair e you will be taking home. You will be asked to mail it back in the provided envel ope, and once we receive the questionnaire you would get paid the $50. I just have to ask you some questions to s ee if you are eligible for our study and to get background information, OK? How old are you? 18-49 Do you smoke? no Are you pregnant or breastfeeding ? no Do you take any prescription medications other than oral contraceptives ? no If not within the age range or if they answer yes to any question: I am very sorry, but you do not meet our exclusion criteria, but thank you for your interest. Now I just have a few questions about your diet to see what specific category of our study you would fit in to. Please answer as best you can, estimates are ok and consider all instances of when you might eat the items I will ask about, even if only occasionally. Do you take a multi-vitamin, complex, red star nutritional ye ast, or any other supplement or additive ever? If they take a multivitamin, B complex, red star nutritional yeast, complete the session through all diet info but do not record. C onclude 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.

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74 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. Concl ude 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 appointme nt 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 below. Do you eat breakfast cereals? o Yes o No Name/Brand Quantity Frequency Are you a vegan, vegetarian or meat eater? Vegan – this means you eat NO animal de rived foods intentionally (if they eat small amt like in cake then OK) Vegetarian – this means you eat NO b eef, chicken, turkey, pork, or fish How often do you eat … Never Rarely (<1 x/mo) Occasionally (1-4 x/mo) Frequently (2-4 x/wk) Always (5-7 x/wk) Beef Chicken Turkey Pork Fish Eggs Cheese Cow’s Milk Yogurt Other Dairy

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75 Do you follow a restricted diet such as o No red meat o Lactose-free o Kosher o Weight loss o Weight gain o Low salt o Low fat o Low cholesterol o Low carbohydrate o Hypoallergenic (If so) How long have you consumed this type of diet? ________________________________________________________________________ Have you made any major dietary ch anges within the last 3 years? o No o Yes; How long ago did you make cha nges and what changes did you make? ____________________________________________________________ NO YES Do you consume alcoholic beverages? How often/quantity

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76 Health Information I am going to ask you a few questions ab out your health to determine if you are eligible for our study. I will be recording this information, but it will be kept confidential and is this ok with you? _______ Height: Weight: Have you do you currently have any of the following? NO YES Alcoholism Anemia Blood clots Bronchitis Cystic Fibrosis Dermatitis Diabetes Eating disorders/Chronic nausea or vomiting Food allergy Gall bladder disease GI problems/ Lactose intolerance Gout Migraines Hemorrhoids Hepatitis/Liver disease Heart disease/High cholestero l/High blood pressure HIV Kidney disease Neurological disorder Obesity Seizures/Stroke Thyroid problem Tumors/Cancer Ulcers Other Have you been hospitalized 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 acce ss to medical information on you? o Yes o No

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77 Demographic Information What is your birth date? _______/_______/_________ Month Day Year How would you describe your race or ethnic background? o White o Black or African American o American Indian or Alaska Native o Hispanic or Latino o Asian o Native Hawaiian or Other Pacific Islander o Other _________________________________________________ What is the highest level of school or training that you have completed? [Circle only one response] Grade school 01 02 03 04 05 06 07 08 High school 09 10 11 12 Technical school or college 13 14 15 16 Graduate or professional 17 18 19 20+ Don’t know X Marital status? o Single/never married o Married o Separated o Divorced o Widowed Are you a full-time or part-t ime student? Are you employed? o Full time o Yes o Part time o No o Not a student o Student employee

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78 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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79 APPENDIX B DIET HISTORY QUESTIONNAIRE

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119 APPENDIX C SCRIPT FOR DIET HISTORY QUE STIONNAIRE INSTRUCTIONS Diet History Questionnaire: Thank you for participating in our study. We are going to spend about 15 minutes reviewing the Diet History Qu estionnaire, going over the inst ructions and answering any questions that you might have. I would like to explain what is in your packet. You have been given a copy of each of the informed consents that you signed this morning. These are for you to review when you get home. Th ey are your copies and do not need to be sent back. You have also been giving a copy of the Diet History Questionnaire. Everyone’s is exactly the same, there are no diffe rences and they were printed at the same time. They are all white in color and doubl e sided. Before filling out the questionnaire make sure you answer the 4 questions on the front. There is also a “bar code” or “subject id” label on the front. (Show participants where this is located.) This has already been filled in for you. It is the same number you received today. So, when you mail it back to us we only know that subject #250 has turned theirs in, but we do not know the name. Lastly, you have been given a return envel ope. Once you complete the questionnaire, you will mail it to us using the envelope. The only thing we need back in your packet is the questionnaire. You can keep everything else. The envelope has been pre-addressed to us and we have already paid for it. Do not put any postage on the envelope. The purpose of the Diet History Questionnaire is to give us an idea of the type of foods you have been eating over the past year. This is a very accurate questionnaire and has been tested against other quest ionnaires that have been used in research studies. As long as the person completing the que stionnaire is honest and accura te, the information that we learn from this questionnaire is very true to their actual food intake. The information will tell us a lot about your nutriti onal status. If you are intereste d, sometime in the future we can share this information with you. The questionnaire will only ask you about food items. It will not ask about any thing additional that you might take, such as vita mins, minerals, multivitamin, protein powder, or even nutritional yeast. If you do take anything additional, even once a month, that is fine, we just need to know about it. We record the information and when we get the results from the questionnaire, we add that info rmation to the final results to give us an overall idea of your total intake. (If they take a supplement/nut rition yeast have them record it on colored paper and put into file. If they do not remember, give them the paper to take home and return with the questionnaire. Record their subject number a nd put a note in their file (on colored paper) that says, “Subject will return supplement paper with questionnair e.” If they take

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120 nutritional yeast make sure you distinguish whether it is made by RED STAR or if it is from bulk (like Ward’s). Red Star adds v itamins/minerals especia lly B12, bulk does not.) We have given you two sets of instructions. It is important to r ead both instructions before you begin. The General Instructions ar e located on the first page of the Diet History Questionnaire and the A dditional Instructions are incl uded in your envelope. The General Instructions combined with the A dditional Instructions will guide you while completing the questionnaire. Next, we will review the Additional Instructions (Read each instruction out-loud) Instruction #1: When answering each question, think about your diet over the past year and NOT the past few weeks. We would like to get an idea of the type of food you have eaten for the past year. Therefore, it is important that you focus on your average intake over the past year and not you intake over the past few week s. Sometimes we tend to eat the same thing day-to-day or even w eek-to-week. If we were to fill out the questionnaire based on one day or one week, it wouldn’t be an accurate representation of your overall food intake. Instruction #2: Several questions refer you to additional handouts that have been provided in your packet. Please be sure to use the handouts when you get to these questions. We have provided you a chart the gives you the question number and the name of the handout to use when answering these que stions. There is also a reminder next to these questions on the Diet History Qu estionnaire reminding you to refer to the handouts. These are what the three handouts look like. (Show participants the three handouts) Explain Seasonal Handout When you reach the questions that ask you how often you eat a fruit or vegetable when it is “in season”. You want to first look at the handout called Seasonal Fruits and Vegetables. This handout gives you a list of wh en these fruits and vegetables are in season. So, as an example let’s look at ques tion #19a on page 6. This question asks you, how often you eat fresh peaches, nectarines, or plums when in season. If you look at the Seasonal Fruit and Vegetable handout, it states that peaches are in season from June to September, nectarines are in season from July to September, and plums are in season from June to October. Therefore, you want to answer question 19a based on how often you eat these fruits within those months. Question 19b asks you how often you eat these fruits the rest of the year. So, you woul d think about how ofte n you eat these fruits

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121 during the months that they are not considered “in season” Please use our handout as a guide for when fruits are in season. Pleas e do not use other materials or your own knowledge about when fruits and vegetables are in season. As a reminder, if a question like question #19a, asks you about multiple food items, you want to make sure you think of each time you eat those foods individually and add those times together. For example, I eat a fres h peach once a week, a nectarine once a week, and a plum twice a week, when in season. Theref ore, I eat these fruits a total of 4 times p/wk. I would mark 3-4 times per w eek as my answer for question #19a. Does anyone have any questions about how to use the handouts? Instruction #3: If you have any questions while comple ting the questionnai re, please contact Amanda Brown at 352-392-1991 ext 246. Please leave a voice message with your name, contact number, and best time or way to reach you. Next, we will review the General Instructions located on the first page of the Diet History Questionnaire. Instruction #1: Answer each question as well as you can. Estimate if you are not sure. A guess is better than leaving a blank. For some questions, it may be helpful if you measure out how much you usually eat or drink of a certain food or beverage by using measuring cups and spoons. You may also want to review food labels of the foods you eat because we do not want you to under-estimate or over-estimate the amount of a certain food you are eating. For example, I occasionally have a bottle of ora nge juice for breakfast. Since one bottle is not provided as an answer choice on the Di et History Questionnair e, referring to the label on the orange juice bottle will help me determine the amount of cups or ounces that I am drinking. When I look at the juic e bottle, I find that each time I drink the entire juice bottle; I am drinking 15 fl. oz (roughly 2 cups ) of orange juice. Therefore, I can mark 15 fluid ounces or 2 cups as my answer. Instruction #2 Use only a black ballpoint pen. Do not use a pencil or felt-tip pen. Do not fold, staple, or tear the pages. (Hold up an example of a black ballpoint pen) We have provided you with a black ball-point pen that you can use to fill out the questionnaire. Please do not fold the questionnaire when you send it back to us. The envelope that is in your packet is large enough that you can slip the questionn aire right into th e envelope and you do not have to fold it. As a reminder, you only have to send the Diet History Questionnaire back. You can keep the rest of the material that is in your packet.

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122 Instruction #3 & 4 When selecting an answer, put an X in the box next to your answer. If you make any changes, cross out the incorrect answer and put an X in the box next to the correct answer. Also draw a circle around the correct answer. Please look at the handout en titled “Directions on How to Change your Answer Choice”. The question asks, “Over the past 12 months, how ofte n did you drink tomato or vegetable juice?” Over the past 12 months, you decided that you drink vegetable juice or tomato juice less than one time per mont h. You select “a” as your first answer, but then you remember you actually drink it much more frequently. So, the next step would be to cross out answer “a” and to ma rk an “X” in your new answer. Then you would want to circle your new answer, so it is clear which is your final answer. Instruction #5 If you mark NEVER, NO, or DON’T KNOW for a question, please follow any arrows or instructions that di rect you to the next question. Open your questionnaire to page 13 questi on #48g. This question asks “how often do you add cheese or cheese sauce to your pot atoes”. Notice that you have the option to select “Never”. If you never add cheese or cheese sauce to y our potatoes you would mark an “X” in the “never” box. Please noti ce that next to the “never” choice it also states the question number you should go to next In this example, next to the “never” choice it says go to question 49. Does everyone see this? (Pause) There is also an arrow that directs you to the next page. It is important to be aware of the proper question to go to. The arrows will direct you, but make sure you go to the question that is stated next to the “n ever” choice. In this example, you are directed to question 49. If you follow the arrow and turn th e page, it seems that you should answer question 48h. Although, this is wrong. You want to go to question 49. (Pause) Does anyone have any questions about marking never and how to know what question to answer next? Additional Information to Discuss: I want to go through some additional ques tions to make sure you understand how to answer these questions. Each question asks you how often you eat a sp ecific food and the amount you eat. Please approach each question t houghtfully. It is very important that we get the most accurate information about your diet. Open the questionnaire to page 2 question #1. This question asks you, “How often do you drink tomato juice or vegetable juice?” Think about how many times a day or a week you drink tomato or vegetable juice and then mark the appropriate answer. If you drink tomato or vegetable juice you would continue to the second part of the question: “1a”. This question asks about the serving amount each time you drank tomato or vegeta ble juice. Once completing question “1a”

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123 you can continue to question 2, and so forth. If you never drink tomato or vegetable juice, select never and follow the directions to the next appropriate questi on. In this example, you would go to question number 2. Remember when answering these questions, we want you to think about your diet over the past year and NOT the past few weeks. I want to go over a few examples, which you may find helpful when completing the questionna ire. We have provided you with written examples of what I am about to cover. 1. "Please look at page 16 question #57. Th is question asks, “How often did you eat pancakes, waffles, or French toast?” I go to IHOP for breakfast on the first Sunday of every month of the year. I always order one medium size Belgian waffle. I never make waffles, pancakes, or French toast at home. I only eat waffles at IHOP. Since there are 12 mont hs in a year, and I go on the first Sunday of each month, that is 12 times a year. 12 months divided into 12 times, gives you an average of 1 time per month. Which makes sense because there is only 1 first Sunday of every month, so I would only eat waffles 1 time per month. If you look at question #57, I would mark an "X" for 1 time per month. For question #57a, I would answer 1 to 3 medium pieces." Here is another example: 2. Please look at page 31 question #107. This question asks, “How often did you eat ice cream or ice cream bars, (including lo w-fat or fat-free)?” I only eat ice cream during the summer months of June, July, a nd August. During that time, I eat 1 cup of ice cream twice a week. That means I eat ice cream 8 times during each of those months. So that adds up to 24 times. Since I do not eat ice cream during the other months, I can say that I only have ice cream 24 times a year. If I remember that there are 12 months in a year that means, on average, I only have ice cream twice a month. If you look at question #107 on page 31, I would mark an “X” for 2-3 times a month. For question #107a, I would mark to 1 cups or 1 to 2 scoops. Do you have any questions about these two examples? (Pause) All instructions have been written down, so please re-read each dire ction before starti ng the Diet History Questionnaire. If you have any questions please let me know. Remember the questionnaire you have been given in your packet has questions on th e front and back of each page. Please make sure you answer each question as directed by the arrows. Again, it is very important you take your time filling out th e questionnaire and be as thorough as possible. The information we gather is very important. Y ou have my number if you have any questions while filling out the questionnaire. Once you return the questionnaire we can process your payment. We will not begin the proces sing of your payment until we receive your Diet History Questionnaire. You have two week s to complete this questionnaire. Please

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124 return only the Diet History Questionnaire by ______________________. If you cannot return the questionnaire within 2 weeks, pl ease contact Amanda Brown to find a date by which you can return it. We prefer to have to questionnaire returned at some point rather than never having it returned at all. This completes the session for today. (Make sure you collect their clipboard, consent form, and payment form. Restoc k clipboards when needed)

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125 APPENDIX D DIRECTIONS AND SURVEY FOR DIET HISTORY QUESTIONNAIRE PRETEST Thank you for agreeing to complete the Diet History Questionnaire. Please follow the General Instructions located on the first pa ge of the questionnai re and the Additional Instructions attached to th e front of the Diet History Questionnaire. The General Instructions combined with the Additional Instructions wi ll guide you while completing the questionnaire. Upon completion of the que stionnaire, please answer the following questions. Be specific with your responses. Please record the time at which you started and completed the questionnaire. Time started: Time completed: If you have any questions while comp leting the questionnaire, please email Amanda Brown at abrown14@ufl.edu or phone at 352-392-1991 ext 246 Please leave a voice message with your name, contact number, and best time or way to reach you. Please return the questionnair e by Tuesday, September 28, 2004. Thank you in advance! For bonus credit please print name : ________________________________ Your response will be kept confidential

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126 Please answer the following questions after completing the Diet History Questionnaire. 1. How long did it take for you to comple te the Diet Hist ory Questionnaire? 2. How clear were the General In structions for the Diet Hi story Questionna ire? Please explain. 3. Were there any questions on the Diet Hist ory Questionnaire you did not understand? If yes, please list the question number and explain why you did not understand the question. 4. Were there any questions on the Diet Hist ory Questionnaire you could not answer? If yes, please list the question number a nd explain why it was difficult to answer. 5. Were there any questions on the Diet Hi story Questionnaire for which the response you would have given was not an option? If yes, please provide the question number, the response you would have given, and w hy you would have given that response.

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127 6. Is there any food item you eat frequently that was not listed on the Diet History Questionnaire? 7. Please provide additional comments in the space provided below.

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128 APPENDIX E ADDITIONAL INSTRUCTIONS PACKET Please read these instructions prior to begi nning the Diet History Questionnaire. These additional instructions combin ed with the “General Instru ctions” (found on the first page of the Diet History Questi onnaire) will guide you while completing the questionnaire. ADDITIONAL INSTRUCTIONS 1. When answering each question, think about your diet over the past year and NOT the past few weeks. 2. Several questions refer you to additional handouts that have been provided in your packet. Please be sure to use thes e handouts when you get to these questions. If you have any questions while comple ting the questionnaire, please contact Amanda Brown at 352-392-1991 ext 246 Please leave a voice message with your name, contact number, a nd best time or way to reach you. Question Number Handout 8e Caffeinated versus Non-caffeinated Beverages 13b Fortified Cereal 19a, 21a, 22a, 23a, 24a, 25a, 34a, 42a Seasonal Fruits and Vegetables

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129 Remember when answering these qu estions, we want you to think about your diet over the past year and NOT the past few weeks. Here are a few examples, that you may find helpful when completing the questionnaire. 1. Please look at question #57 on page 16. I go to IHOP for breakfast on the first Sunday of every month of the year. I always order one medium size Belgian waffle. I never make waffles, pancakes, or French toast at home. I only eat waffles at IHOP. Since there are 12 months in a year, I eat 12 waffles a year or 1 waffle a month. If you look at question #57, I would mark an "X" for 1 time per month. For question #57a, I would answer 1 to 3 medium pieces. 2. Please look at question #107 on page 31. I only eat ice cream during the summer months of June, July, and August. During th at time, I eat 1 cup of ice cream twice a week. That means I eat ice cream 8 times during each of those months. So that adds up to 24 times. Since I do not eat ice cream during the other months, I can say that I only have ice cream 24 times a year. If I remember that there are 12 months in a year that means, on average, I only have ice cream twice a month. If you look at question #107 on page 31, I would mark an “X” for 2-3 times a month. For question #107a, I would mark to 1 cups or 1 to 2 scoops.

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130 Directions on How to Change Your Answer Choice Over the past 12 months how often did you drink tomato juice or vegetable juice ? a. 1 time per month or less b. 2-3 times per month c. 1-2 times per week d. 3-4 times per week e. 5-6 times per week a. 1 time per month or less b. 2-3 times per month c. 1-2 times per week d. 3-4 times per week e. 5-6 times per week a. 1 time per month or less b. 2-3 times per month c. 1-2 times per week d. 3-4 times per week e. 5-6 times per week You select “a” as your first answer, but then you change your mind. Step #1 Cross out answer “a” Step #2 Mark an “X” in the box of your new choice and circle that answer too

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131 Please use this list for Question 8e Caffeinated Non-caffeinated A & W Creme Soda 7-Up Barq’s Root Beer A & W Root Beer Canada Dry Cola Diet 7-Up Coffee Diet A & W Root Beer Diet A & W Creme Soda Diet Sprite Diet Coke with Lemon Minute Maid Orange Diet Cherry Coke Sprite Cherry Coke Cherry Pepsi Coke Diet Coke Diet Barq’s Root Beer Diet Dr. Pepper Diet Mountain Dew Diet Pepsi Diet RC Cola Diet Shasta Cola Diet Snapple Flavored teas Diet Sunkist Orange Dr. Pepper Hot Chocolate Mix Jolt Lipton Brisk Mellow Yellow Mountain Dew Mountain Dew Code Red Nestea Sweetened & Unsweetened Iced Teas Pepsi Pepsi One Pepsi Twist RC Cola Shasta Cola Snapple Flavored teas Sunkist Orange Surge Tea: Iced, Brewed, Instant, Green

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132 Please use this list for Question 13b All-Bran – Buds All-Bran – Extra Fiber All-Bran – Original Cheerios – Multigrain Complete Oat Bran Flakes Complete Wheat Bran Flakes Just Right Fruit and Nut KASHI Heart to Heart Kellogg’s Low Fat Granola no raisins Kellogg’s Low Fat Granola with raisins Mueslix Product 19 Smart Start Smart Start Soy Protein Special K Total – Original Total Whole Grain Total Brown Sugar and Oat Total Corn Flakes Total Raisin Bran

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133 Please use this list for Questions 19a, 21a, 22a, 23a, 24a, 25a, 34a, 42a Fruit or Vegetable Months When In Season Cantaloupe March to July Corn August to June Grapefruit September to June Honeydew Melon June to October Nectarines July to September Oranges October to June Peaches June to September Plums June to October Strawberries October to June Tangelos November to February Tomatoes September to June

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134 LIST OF REFERENCES 1. Banerjee RV, Frasca V, Ballou DP, Matthews RG. Participation of cob(I)alamin in the reaction catalyzed by meth ionine synthase from Esch erichia coli: a steady-state and rapid reaction kinetic anal ysis. Biochemistry 1990;29:11101-9. 2. Beck W. Cobalamin (Vitamin B12). 3rd ed. In: Rucker RB, Suttie JW, McCormick DB, Machlin LJ, eds. Handbook of Vitamins. New York, NY: Marcel Dekker, Inc., 2001:463-512. 3. Seetharam B, Alpers DH. Absorption and transport of cobalamin (vitamin B12). Annu Rev Nutr 1982;2:343-69. 4. US Department of Agriculture Agricu ltural Research Services. USDA National Nutrient Database for Standard Refere nce Release 17. Version current 29 August 2005. Internet: http://www.nal.usda.gov/ fnic/foodcomp/Data/SR17/sr17.html (accessed 15 May 2005). 5. Herrmann W, Geisel J. Vegetarian lifesty le and monitoring of vitamin B-12 status. Clin Chim Acta 2002;326:47-59. 6. Mezzano D, Kosiel K, Martinez C, Cuevas A, Panes O, Aranda E, Strobel P, Perez DD, Pereira J, Rozowski J, Leighton F. Cardiovascular Risk Factors in Vegetarians: Normalization of Hyperh omocysteinemia with Vitamin B12 and Reduction of Platelet Aggregation with n-3 Fatty Acids. Thrombosis Research 2000;100:153-160. 7. Pittock SJ, Payne TA, Harper CM. Reversible myelopathy in a 34-year-old man with vitamin B12 deficiency. Mayo Clin Proc 2002;77:291-4. 8. Groenen PMW, van Rooij IALM, Peer PGM, Gooskens RH, Zielhuis GA, Steegers-Theunissen RPM. Marginal maternal vitamin B12 status increases the risk of offspring with spina bifida. Am J Obstet Gynecol 2004;191:11-17. 9. Miller DR, Specker BL, Ho ML, Norman EJ Vitamin B-12 status in a macrobiotic community. Am J Clin Nutr 1991;53:524-9. 10. Rauma AL, Torronen R, Hanninen O, Mykka nen H. Vitamin B-12 status of longterm adherents of a strict uncooked vegan diet ("living food diet ") is compromised. J Nutr 1995;125:2511-5.

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142 102. Slattery ML, Jacobs DR, Jr., Hilner JE, Caan BJ, Van Horn L, Bragg C, Manolio TA, Kushi LH, Liu KA. Meat consumption a nd its associations with other diet and health factors in young adults: the CARD IA study. Am J Clin Nutr 1991;54:930-5. 103. Nicklas TA, Farris RP, Myers L, Berens on GS. Impact of meat consumption on nutritional quality and cardiovascular ri sk factors in young adults: the Bogalusa Heart Study. J Am Diet Assoc 1995;95:887-92. 104. O'Dea K, Traianedes K, Chisholm K, Leyde n H, Sinclair AJ. Cholesterol-lowering effect of a low-fat diet containing lean beef is reversed by the addition of beef fat. Am J Clin Nutr 1990;52:491-4. 105. Beauchesne-Rondeau E, Gascon A, Berger on J, Jacques H. Plasma lipids and lipoproteins in hypercholester olemic men fed a lipid-lowering diet containing lean beef, lean fish, or poultry. Am J Clin Nutr 2003;77:587-93. 106. Snetselaar L, Stumbo P, Chenard C, Ahrens L, Smith K, Zimmerman B. Adolescents eating diets rich in either lean beef or lean poultry and fish reduced fat and saturated fat intake and those eating beef maintained serum ferritin status. J Am Diet Assoc 2004;104:424-8. 107. Enns C, Goldman J, Cook A. Trends in Food and Nutrient Intakes by Adults: NFCS 1977-78, CSFII 1989-91, and CSFII 1994-95. Family Economics and Nutrition Review 1997;10:2-15. 108. Food and Nutrition Information Center. US Department of Agriculture. Dietary Guidelines for Americans 2005. Version current 8 December 2005. Internet: http://www.nal.usda.gov/fnic/dga/ (accessed 15 January 2006). 109. Yoshino K, Inagawa M, Oshima M, Yakota K, Umesawa M, Endo M, Yamagishi K, Tanigawa T, Sato S, Shimamoto T, Iso H. Trends in dietary intake of folate, vitamins B6, and B12 among Japanese adults in two rural communities from 1974 through 2001. J Epidemiol 2005;15:29-37.

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143 BIOGRAPHICAL SKETCH Amanda was born in Herndon, Virginia. She received her undergraduate degree in human nutrition, foods, and exercise from Vi rginia Tech in Blacksburg, Virginia, in 2001. After graduation she was accepted into The National Institute s of Health (NIH) Dietetic Internship in Bethes da, Maryland. In 2003, she passed the Registration Exam for Dietitians and practiced as a Registered Diet itian before enrolling in graduate school at the University of Florida. As a Registered Dietitian she worked with low-income women, infants, and children teaching basic nutriti on messages. In 2005, she was awarded the Presidential Recognition Award at the Universi ty of Florida. In 2006, Amanda received the North American College and Teachers of Agriculture (NACTA ) Graduate Student Teaching Award. Her hobbies include playing soccer at a competitive level and singing in an a cappella group. Upon completion of her graduate degree, she plans to return to practice as a Registered Dietitian.


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Permanent Link: http://ufdc.ufl.edu/UFE0014280/00001

Material Information

Title: Relationship between Beef Consumption and Vitamin B12 Intake and Status in Healthy Men and Women
Physical Description: Mixed Material
Copyright Date: 2008

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RELATIONSHIP BETWEEN BEEF CONSUMPTION AND VITAMIN Bl2 INTAKE
AND STATUS OF HEALTHY MEN AND WOMEN














By

AMANDA L. BROWN


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA


2006


































Copyright 2006

by

Amanda L. Brown
















ACKNOWLEDGMENTS

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

Gail P.A. Kauwell, PhD, and Anna-Maria Siega-Riz, PhD. I especially would like to

thank my committee chair Lynn B. Bailey, PhD. Her continuous guidance and

encouragement were amazing. She is a remarkable mentor and role model.

I would like to thank Dave Maneval for his direction while working in the

laboratory. I also would like to thank Karla Shelnutt, PhD, Kristina von-Castel-Roberts,

and Claire Edgemon. All of these women were amazing support systems and were always

willing to listen.

In addition, I would like to thank my family. They truly are my foundation. They

are remarkable listeners and always provide the best advice. I also would like to thank

Danny Wittmann. Everyday he reminded me of my hard work and of my

accomplishments. He always told me how proud he was of me and what a good j ob I

was doing. I would not have been able to complete this program without his support.





















TABLE OF CONTENTS


page

ACKNOWLEDGMENT S ............ ..... ___ .............. iii...


LI ST OF T ABLE S ............_ ..... ..__ .............. vii..


LIST OF FIGURES ............_...... ._ ..............viii...


LIST OF ABBREVIATIONS ............. ...... ._ .............. ix....


AB STRAC T ................ .............. xi


CHAPTER


1 INTRODUCTION ................. ...............1.......... ......


Hypotheses............... ...............
Specify c Obj ectives ................ ...............2............ ....

2 LITERATURE REVIEW .............. ...............3.....


Vitamin B 12 ................. ...............3................
Chem istry .............. ...............3.....
Dietary Sources .............. ...............4.....
Absorption ................ ...............6.................
Transport............... ...............7
Storage ................. ...............7.................
Excreti on ................. ...............8.................
Biochemical Functions .............. ...............8.....
Dietary Reference Intakes .............. ...............10....
Vitamin Bl2 Defieiency ................. ...............11................
Etiology ................. ...............11.......... .....
Clinical symptom s.............. .. ....... ...............1
Health related risks of a vitamin B l2 deficiency ................. ................ ...15
Vitamin Bl 2 Status Assessment ................. ...............20...............
Serum Vitamin B l2 Concentration .............. ...............20....

Methylmalonic Acid Concentration .............. ...............20....
Holotranscobalamin Concentration ........._.._.. ....._.. ......_.._............2
Serum Homocysteine Concentration............... .............2
Megaloblastic Anemia............... ...............22.












Dietary Intake Assessment in Adults .............. ...............22....
Twenty-four hour recall method .............. ...............22....
M ultiple-day food record .............. ...............24....
Food frequency questionnaire .............. ...............25....
Assessment of vitamin Bl2 intake .......__ ......... ___ .........__ ......26
Vitamin B l2 Status in the United States .............. ...............30....

Vegetari ani sm ........ .. .. ..... ..__.........__ ..........._ .........3
Definitions of Vegetarianism and Prevalence in the United States....................3 1
Assessment of Dietary Adequacy of Vegetarian Diets .........__ ..... ............. ..32
Assessment of Beef Consumption and Health. ................ .......___ .........__ ..33
Assessment of Dietary Adequacy of Beef Consumption ............__.. ........._......3 5
Research Rational and Potential Application of Findings............. ..__.........__ ....3 5

3 MATERIALS AND METHODS .............. ...............38....


Study Design and Methods Overview ....__ ......_____ .......___ ...........3
Diet History Questionnaire ............_ ..... ..__ ...............39...
O verview ............... .. ......... ...... .... .. .................3
Modiaications Made to the Paper Version of the Diet History Questionnaire ....39
Modiaications Made to Diet*Calc Analysis Software............. ..._.........__ ...40
Data Generation................ .. ...... ...............4
Diet History Questionnaire Instruction Pretest ................. ......___ ........._._...41
Human Subj ects Procedures ............_...... .__ ...............42...
Diet History Questionnaire Instructions .............. ...............42....
Sample Collection and Processing.................. ..... ..........4
Processing of Plasma for Vitamin Bl2 Analysis ............_ .... ...__...........43
Processing the Diet History Questionnaire ....._.__._ ..... ... ._ ........._......44
Analytical Methods............... ...............44
Formation of Dietary Groups ..................... ...... ................4
Identification of Food Groups from the Diet History Questionnaire ..................45
Analysis of Additional Nutrients ................. ...............45................
Plasma Vitamin B l2 Concentration .............. ...............46....
Statistical Methods............... ...............47


4 RE SULT S .............. ...............49....


Demographic Characteristics of the Study Population............... ...............4
Subj ects ........._..... .. ..._ ._ ...............49....
Demographic Characteristics............... ............4
Dietary Vitamin Bl2 Intake..................... .. ...............5
Beef, Poultry, Pork, Seafood, and Mixed Foods .....__.___ ..... .._. .. ............52
Dairy and Eggs .............. ...............53....
C ereal ............... ... ....... ........ ........ ..... ... ...........53
Fortified Soy Products, Meal Replacements, and Other Sources ................... .....54
Naturally-Occurring and Fortified Sources .......................__ ...............54
Dietary Intake of Macronutrients and Micronutrients ................. ........___..........5 8
Energy Intake............... ...............58.












Protein Intake............... ...............58.
Carbohydrate Intake .............. ...............59....
Fat Intake ........._.__....... .__. ...............59....
Saturated Fat............... ...............59..
F ol ate Intake .............. ...............60....
Vitamin B6 Intake .............. ...............60....
Iron Intake .............. ...............60....
Zinc Intake .........._.... ........__ ...............60....
Plasma Vitamin Bl2 Concentration .............. ...............61....
Vitamin Bl 2 Status. ........ ................. .........._. .......6
Vitamin Bl2 Intake and Status ............_._ ...............64......._ ...


5 DISCUSSION AND CONCLUSION .............. ...............65....


APPENDIX


A SUBJECT DATA COLLECTION FORM ................. ...............73.......... ...


B DIET HISTORY QUESTIONNAIRE............... .............7


C SCRIPT FOR DIET HISTORY QUESTIONNAIRE INSTRUCTIONS ................119


D DIRECTIONS AND SURVEY FOR DIET HISTORY QUESTIONNAIRE
PRETEST .............. ...............125....


E ADDITIONAL INSTRUCTIONS PACKET ................ .............................128


LIST OF REFERENCES ................. ...............134................


BIOGRAPHICAL SKETCH ................. ...............143......... ......


















LIST OF TABLES


Table pg

2-1 Dietary vitamin Bl 2 sources ........... ........... ...............6 ....

3-1 Daily and weekly beef intake frequency ........._.. ......... ......_. ...........44

3-2 Foods categorized within each food group .............. ...............46....

4-1 Demographic characteristics of dietary groups .........._..._.. ........_........._......50

4-2 Daily total dietary intake of vitamin B12 ....._.._.. .... ..._._.. ... ...._........5

4-3 Daily mean vitamin Bl2 contribution by dietary sources..........._.._.. ........._.._....55

4-4 Daily mean dietary intake of macronutrients .............. ...............59....

4-5 Daily mean dietary intake of micronutrients ................. ......__ ........._._ ...61

4-6 Plasma vitamin Bl2 concentration............... .............6

4-7 Plasma vitamin Bl2 concentration among non-vegetarians and vegetarians ..........63



















LIST OF FIGURES


Figure pg

2-1 Structure of cobalamin .............. ...............4.....

2-2 Vitamin Bl2 function in the remethylation pathway of homocysteine to
m ethionine. .............. ...............9.....

2-3 Role of cobalamin in the formation of succinyl-CoA ................. ......................10

4-1 Mean vitamin B l2 intake compared to recommended intakes. ............. ..... ...........52

4-2 Percent of total vitamin B l2 intake from food sources ................. .....................56

4-3 Percent of total vitamin B l2 intake from cereal sources ................. ................ ...56

4-4 Percent of total vitamin B l2 intake from shellfish and fish ................. ................. 57

4-5 Percent of total vitamin B l2 intake from naturally-occurring and fortified
sources of vitamin Bl2 2................ ...............58...............

4-6 Plasma vitamin Bl2 concentration ................. ...............62........... ..

4-7 Vitamin Bl2 status among dietary groups .............. ...............63....

4-8 Percent of individuals deficient among non-vegetarians and vegetarians. ........._....64
















LIST OF ABBREVIATIONS


Abbreviation

AMPM
ANOVA
BMI
CARDIA
CNS
CSFII
DHQ
DNA
DTT
EAR
EDTA
EPIC-Oxford

FFQ
FIN

IF
kcal
kg

LDL
m
MCV
mg
mL
MMA
MS
NCI
NDS-R
NFCS
NHANES
nmol
NTD
OSC
oz
pmol
RDA


automated-multiple-pass method
analysis of variance
body mass index
coronary artery risk development in young adults
central nervous system
Continuing Survey of Food Intakes by Individuals
Diet History Questionnaire
deoxyribonucleic acid
dithiothreitol
Estimated Average Requirement
ethylenediaminetraacetic acid
European prospective investigation into cancer and nutrition-
Oxford
food frequency questionnaire
food identification number
gram
intrinsic factor
kilocalories
kilogram
liter
low-density lipoprotein
meter
mean corpuscular volume
milligram
milliliter
methylmalonic acid
methionine synthase
National Cancer Institute
Nutrient Data System for Research
Nationwide Food Consumption Survey
National Health and Nutrition Examination Survey
nanomole
neural tube defects
Optimal Solutions Corporation
ounce
picomole
Recommended Dietary Allowance


Meaning









SAH s-adeno syl hom ocy stei ne
SAM s-adeno syl methi oni ne
SD standard deviation
TC transcobalamin
THF tetrahydrofolate
UL Tolerable Upper Intake Level
UNC University of North Carolina, Chapel Hill
USDA United States Department of Agriculture
wk week
Clg microgram
Cpmol micromole
*qdd Questionnaire Data Dictionary
125I iodine-125
5Co cobalt-57
















Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

RELATIONSHIP BETWEEN BEEF CONSUMPTION AND VITMAIN Bl2 INTAKE
AND STATUS OF HEALTHY MEN AND WOMEN

By

Amanda L. Brown

May 2006

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

Vitamin B l2 is an essential nutrient required in the diet to ensure normal cell

division and nervous system function. Dietary sources of naturally-occurring vitamin

Bl2 are limited to those of animal origin; therefore diets restricting animal products are

likely to be vitamin Bl2 deficient. Beef, a highly concentrated source of vitamin Bl2

that is often restricted in the diet of both meat-consumers and vegetarians for health

reasons, is hypothesized to be a maj or contributor to dietary vitamin Bl2 intake among

meat-consumers. It is important to assess the effect of beef consumption on total dietary

intake of vitamin Bl2 and plasma vitamin Bl2 concentration, which is the focus of this

investigation.

Vitamin B l2 intake and plasma concentration were assessed among frequent beef

consumers (beef intake >1 time/wk) (n=97), seldom beef consumers (beef intake I 1

time/wk) (n=42), and never beef consumers (n=42), and compared to a vegetarian group

(n=121). The dietary vitamin Bl2 intake (mean + SD Gig/1000 kcal/d) of the never beef









group (2.0 & 1.4) and vegetarian group (1.9 & 1.5) was both lower (p < 0.0001) than the

frequent beef group (3.3 A 1.4) and the seldom beef group (3.7 & 2.2). In the frequent

beef group, the largest contributors to vitamin B l2 intake were seafood (30%), beef

(28.5%), and dairy products (16%). Among the seldom beef group, the largest

contributors to vitamin Bl2 intake were seafood (33.5%), cereal (20%), and dairy

(13.8%). Sources in the never beef group that contributed the largest proportion of

vitamin Bl2 were seafood (41%), dairy (21.3%), and soy products (12%). There were no

differences (p = 0.70) in mean plasma vitamin Bl2 concentration (range 263 to 289

pmol/L) among the four dietary groups. The percentage of individuals who were

deficient based on plasma vitamin Bl2 concentration (<148 pmol/L) was approximately

two-fold higher in the non-beef consumers [never beef (12%); and vegetarians (17%)]

compared to beef consumers [frequent beef (6%); and seldom (5%)].

The present study was the first to stratify subj ects based on their frequency of

consumption of beef products and to compare the dietary vitamin Bl2 intake and food

sources to vitamin Bl2 concentration among beef consumption groups. This study also

was the first to use a dietary history questionnaire modified specifically for vitamin Bl2-

containing foods and to use a nutrient database where 100% of foods have information on

vitamin Bl2 content. These data indicate that consumption of beef at least one time per

week plays an important role in providing adequate amounts of vitamin Bl2 in the diet of

meat consumers. In addition to beef, seafood and dairy products were maj or contributors

to dietary vitamin Bl2 intake. Dietetics practitioners and nutrition educators can use

these data to promote beef, seafood, and dairy consumption for optimizing vitamin Bl2

status through dietary means.















CHAPTER 1
INTTRODUCTION

Vitamin Bl2, a water-soluble vitamin, plays an important role in the remethylation

of homocysteine to methionine (1) and the formation of succinyl CoA, a Kreb's cycle

intermediate, from L-methylmalonyl CoA (2). Vitamin B l2 is an essential nutrient that is

naturally present only in dietary sources of animal origin. Foods that are naturally rich

sources of vitamin Bl2 are seafood, organ meats (especially liver), beef, poultry, pork,

dairy products (e.g., milk, cheese, yogurt), and eggs (especially egg yolk) (3). Compared

to other meat products (e.g., poultry, pork, and some types of Eish), beef has the highest

concentration of vitamin Bl2 per 3 ounce (oz) serving. Dairy products and eggs have

smaller amounts of vitamin Bl2 per serving than meat products (4).

Consumption of a vegetarian diet, which restricts animal sources, may limit dietary

intake of vitamin Bl12 and impair vitamin Bl2 status (5). Vitamin Bl12 deficiency has

been related to elevated homocysteine concentration, a risk factor for cardiovascular

disease, impaired fetal development, and neurological abnormalities (6-8).

Dietary intake of vitamin Bl2 has been assessed in meat-consumers and

vegetarians by using a number of different assessment methodologies including 24-hour

food recalls, multiple-day food records, and food frequency questionnaires (FFQ).

Dietary vitamin Bl2 intake has been reported to be consistently lower in vegetarians than

meat-consumers when the vegetarian group does not consume supplements or vitamin

Bl12-fortified products (9-11). The impact of frequent beef consumption on total dietary









vitamin Bl2 intake and plasma vitamin Bl2 concentration relative to other food sources

among meat-consumers has not been previously investigated.

Hypotheses

1. High frequency of beef intake is associated with a greater intake of dietary vitamin
Bl2 and higher plasma vitamin Bl2 concentration compared to other food sources.

2. Among meat eaters, beef and beef-containing foods are the largest contributors to
dietary vitamin Bl2 intake relative to other food sources.

Specific Objectives

The overall goal of this study was to assess vitamin Bl2 intake and status in healthy

individuals who consumed beef more frequently compared to those who consumed

vitamin Bl2 from other food sources. The obj ectives of this research study were as

follows:

1. To determine the differences in mean dietary vitamin Bl2 intake [micrograms (Clg)
per 1000 kilocalories (kcals)] among all dietary groups.

2. To characterize the daily contribution of dietary vitamin Bl2 intake from beef
sources (Clg/1000 kcals) relative to the total dietary vitamin Bl2 intake from all
food sources and other types of animal-based foods.

3. To assess the relationship between dietary vitamin Bl2 intake and plasma vitamin
Bl2 concentration within each dietary group.















CHAPTER 2
LITERATURE REVIEW

Vitamin B12

Chemistry

The chemical properties of vitamin Bl2, also known as cobalamin, have been

previously reviewed (12). Cobalamin, a water-soluble vitamin has a molecular mass of

1,580 daltons. Cobalamin is a general term for a group of corrinoids that are cobalt-

containing compounds (12). The structure of cobalamin consists of a macrocyclic corrin

ring, a 5,6-dimethylbenzimidazole nucleotide, and an aminopropanol group (Figure 2-1).

The macrocyclic corrin ring consists of four reduced pyrrole rings linked to one cobalt

atom at the center. Attached to the central cobalt atom is a lower (alpha) ligand 5,6-

dimethylbenzimidazole nucleotide (13). Numerous upper (beta) ligands such as -CN, -

OH, -H20, -NO2, 5'-deoxyadenosyl, and -CH3 can be covalently bound to the central

cobalt atom. Addition of a ligand creates various forms of cobalamin, such as

cyanocobalamin, hydroxocobalamin, aquocobalamin, nitritocobalamin,

adenosylcobalamin, and methylcobalamin, respectively (12).

Methylcobalamin and adenosylcobalamin are active coenzymes and can be derived

from other cobalamin forms (2). Cyanocobalamin is the commercial form produced by

industry (12). In tissue, a "coenzyme synthetase" system converts cyanocobalamin and

hydroxocobalamin to adenosylcobalamin. In this reaction, ATP provides a 5'-deoxy-5'-

adenosyl moiety that is moved to the cobalamin. Hydroxocobalamin can be methylated in

the cytosol to form methylcobalamin (2).









H2N O
H2N ~O Beta Ligand
O H30 32
H2( H
Corrin Ring H3NHC X OI

H3C` C~O2
\NN
O CH3

NH2H3C CH, = CH3


H3 \INH OONH

Alpha Lig a n dO O O NH CH3


H 3H
HO


Figure 2-1. Structure of cobalamin. Adapted from Martens et al. (2002), p. 276 (12).

During exposure to light, methylcobalamin, adenosylcobalamin, and

cyanocobalamin are unstable. Light disrupts the carbon-cobalt bond and decreases

enzyme activity (12). Cyanocobalamin is stable in aqueous solutions, low pH, and high

temperatures (14).

Dietary Sources

Cobalamin, which will be referred to subsequently as vitamin Bl2, is an essential

nutrient that is naturally present only in dietary sources of animal origin (3). Only certain

microorganisms (members of Archea and some eubacteria) can synthesize vitamin B l2

as reviewed by Raux et al. (2000) (15). Animal sources rich in vitamin B l2 have

obtained the vitamin from these microorganisms. Sources rich in vitamin Bl2 are seafood

(e.g., fish and shellfish), organ meats (especially liver), beef, poultry, pork, dairy products

(e.g., milk, cheese, yogurt), and eggs (especially egg yolk) (16). It may be possible to









acquire vitamin Bl2 from plant sources if they are contaminated with vitamin Bl2

producing-microorgani sms (12).

Beef has the potential to provide a large amount of dietary vitamin Bl2 since one 3

oz serving contains approximately 2.2 Cpg of vitamin B l2 (4), almost providing the

Recommended Dietary Allowance (RDA) for vitamin B l2 (RDA = 2.4 Cpg; non-pregnant,

non-lactating adults). Compared to all other meat products (e.g., poultry, pork, and some

types of fish), beef has the highest concentration of vitamin B l2 per 3 oz serving (Table

2-1) (4). Dairy products and eggs have smaller amounts of vitamin B l2 per serving

compared to meat. Seafood provides a higher amount of vitamin Bl2 in one 3 oz serving

than beef. Specifically, one 3 oz serving of shellfish (e.g., clams, mussels, and oysters)

provides approximately 20 Cpg per serving of vitamin B l2 (4). Although, according to a

report by the United States Department of Commerce, seafood, including shellfish, is

consumed in much smaller amounts per year (per capital 16.6 pounds) compared to beef

(per capital 65 pounds) (17-19).

Adenosylcobalamin and hydroxocobalamin are the predominant vitamin Bl2 forms

found in meat, fish, poultry, pork, and eggs (16). Methylcobalamin and

hydroxocobalamin are the predominant forms of vitamin Bl2 found in dairy products

(16). The form of vitamin Bl2 in commercial supplements and fortified foods such as

breakfast cereals, soy products, and meal replacement formulas is predominately

cyanocobalamin (20).










Table 2-1. Dietary vitamin Bl2 sources
Vitamin Bl2
Content (Clg) Vitamin Bl2 Content
Dietary Source per 100 grams (g) per serving (Clg) Serving Size
Beef 2.50 2.2 3 ounce
Chicken 0.34 0.29 3 ounce
Turkey 0.34 0.29 3 ounce
Pork 0.75 0.64 3 ounce
Milk 0.45-0.53 1.1-1.3 1 cup
Cheese 0.35-1.55 0.10-0.44 1 ounce
Yogurt 0.47 1.07 8 ounce
Egg 1.42 0.64 1 large
Seafood
Shellfish
Clams, mussels, oysters 24-49 20-42 3 ounce
Shrimp, lobster 1.4-3.1 1.2-2.6 3 ounce
Fish
Cod, tuna, salmon 1.05-2.8 0.89-2.38 3 ounce
Adapted from United States Department of Agriculture, National Nutrient Database for Standard
Reference, Release 17 (2005) (4).
Absorption

The mechanism of dietary vitamin Bl2 absorption has been reviewed previously

(21). In the stomach, dietary vitamin Bl2 bound to protein is disassociated by pepsin and

hydrochloric acid. The acid environment increases the vitamin B l2 binding affinity for R

proteins (i.e., cobalophilins or haptocorrins), which are secreted by salivary glands and

the gastric mucosa (3, 21). These R proteins prevent denaturation of the vitamin from

chemicals produced in the stomach. Intrinsic factor (IF), a glycoprotein secreted by the

parietal cells of the stomach in response to stimulation by food, is released but does not

bind to free vitamin B l2 in the stomach (21). The vitamin B l2-R protein complex and IF

then travel to the small intestine. Vitamin Bl2 is released from the R protein complex in

the duodenum by pancreatic proteases. These proteases hydrolyze the R proteins thereby

releasing vitamin B l2 (21). The alkaline environment enhances the binding of vitamin

Bl2 to IF, and the IF-vitamin Bl2 complex then travels to the ileum for receptor-

mediated uptake (21). The intestinal cells of the ileum have receptors for vitamin Bl2









called cubilins. It is unclear whether both IF and vitamin Bl2 are absorbed during this

process or if only vitamin Bl2 is absorbed. It is known that this is a calcium dependent

process (14). Vitamin Bl2 is absorbed throughout the ileum, but the greatest amount is in

the distal third. Most absorption of vitamin Bl2 is receptor-mediated, but approximately

1% is absorbed by passive diffusion (22).

Transport

Once vitamin B l2 is internalized into the enterocyte it can take 3 to 4 hours before

the vitamin is in circulation (2). Vitamin Bl2 is bound in the plasma to two proteins:

haptocorrin and transcobalamin (TC) (23). Haptocorrin accounts for 80% of all vitamin

Bl2 bound in circulation, while TC accounts for 20% of all vitamin Bl2 bound in

circulation. Transcobalamin carries vitamin Bl2 in a one-to-one ratio (24). Tissue cells

have receptors only for TC, making it the maj or transport protein for cellular uptake.

Once vitamin Bl2 crosses the cell membrane of peripheral tissues, vitamin Bl2 is

released from TC into the cytosol (3).

Storage

Unlike most water-soluble vitamins, vitamin B l2 can be stored in the body for a

considerable length of time due to enterohepatic circulation via continuous bile secretion

(25). The liver is the main storage tissue followed by the muscles accounting for 60%

and 30% of total body stores, respectively. Vitamin B l2 also can be stored in small

amounts in other tissues such as bone, kidneys, heart, muscle, brain, and spleen (26).

Approximately 2 to 5 milligrams (mg) of vitamin B l2 can be stored in the body (2)

predominately in the form of adenosylcobalamin and methylcobalamin (26).









Excretion

Vitamin B l2 is excreted primarily through feces originating primarily from

unabsorbed bile (27). Excess vitamin Bl2 losses through urine occur when the serum

vitamin Bl2 concentration surpasses the binding ability of the transport proteins in the

blood (22). There is an inverse relationship between fecal and urinary losses and storage.

If storage of vitamin Bl12 decreases, then excretion of vitamin Bl12 will decrease and

more will be conserved. Approximately, 0. 1% to 0.2% of stored vitamin B l2 is lost each

day (28).

Biochemical Functions

Vitamin Bl2 plays an important role in two enzymatic processes. The first is a

methylcobalamin dependent pathway that remethylates homocysteine to methionine

(Figure 2-2). This reaction occurs in the cytoplasm of the cell. Cobalamin bound to the

enzyme methionine synthase (MS) acts as an acceptor of a methyl group donated from 5-

methyltetrahydrofolate (5-methyl THF). Methionine synthase acts as a catalyst during

this remethylation reaction by removing the methyl group from 5-methyl THF and adding

it to cob(I)alamin, forming methylcobalamin and regenerating THF. Methylcobalamin

donates the methyl group to homocysteine, resulting in remethylation of homocysteine to

methionine (1). Methionine can further be converted to S-adenosylmethionine (SAM).

SAM functions to provide methyl groups for over 100 methylation reactions including

deoxyribonucleic acid (DNA), protein, and phospholipid synthesis (29). In a vitamin B l2

deficiency, the human body lacks sufficient amounts of cobalamin to accept the methyl

group from 5-methyl THF. Folate is trapped in the 5-methyl THF form preventing the

regeneration of THF. Since THF is required to produce 5,10 methylene THF used for

DNA synthesis, a vitamin Bl2 deficiency leads to decreased DNA synthesis and









impaired cell division (30). Homocysteine cannot be remethylated to methionine,

resulting in a build-up of homocysteine in the blood (5).

SDNA Synthesis DNA Methylation



T i: THF- Methionine


SAM




I5-CH,-THF~ Homocysteine


Figure 2-2. Vitamin Bl2 function in the remethylation pathway of homocysteine to
methionine. Adapted from Scott (1999), p. 442 (21). THF = tetrahydrofolate;
5,10-CH3-THF = 5, 10 methylenetetrahydrofolate; 5-CH3-THF = 5-
methyltetrahydrofolate; MS = methionine synthase; SAM = s-
adenosylmethionine; SAH = s-adenosylhomocy steine.

The second vitamin Bl2-dependent reaction is an adenosylcobalamin dependent

pathway, in which L-methylmalonyl CoA is converted to succinyl CoA (Figure 2-3).

This reaction occurs in the mitochondria. The oxidation of methionine, threonine,

isoleucine, and odd chain fatty acids results in the formation of propionyl CoA.

Propionyl CoA is then converted to D-methylmalonyl CoA by a biotin, ATP, and

magnesium dependent enzyme, propionyl CoA carboxylase. Methylmalonyl CoA

racemase converts D-methylmalonyl CoA to L-methylmalonyl CoA. Methylmalonyl

CoA mutase is adenosylcobalamin dependent and requires two adenosylcobalamin

molecules to transform L-methylmalonyl CoA to succinyl CoA (2). Succinyl CoA is a

key Kreb's cycle intermediate. In a deficiency of adenosylcobalamin, methylmalonyl










CoA builds up, is hydrolyzed to methylmalonic acid (MMA), and MMA ultimately

accumulates in the blood (12).

Isoleucine, Valine, Odd-chain
Methionine, Threonine Fatty Acids




Propionyl-CoA

Propionyl-CoA arboylas
(D)-Methylmalonyl-CoA


(L)-Methylmalonyl-CoA

Methylmalonyl-CoA-utas
Succinyl-CoA

Citric Acid
Cycle

Figure 2-3. Role of cobalamin in the formation of succinyl-CoA. Adapted from Horster
and Hoffmann (2004), p. 1073 (31).

Dietary Reference Intakes

The RDA for vitamin Bl2 is based on the amount of vitamin Bl12 required for

maintenance of normal hematological status and serum vitamin Bl2 concentration

considering estimates of intake and turnover (20). The RDA for male and female adults

is 2.4 Gig/day (non-pregnant, non-lactating) (20). During pregnancy and lactation, the

vitamin Bl2 RDA increases to 2.6 and 2.8 Gig/day, respectively (20). Adults greater than

51 years of age frequently malabsorb food-bound vitamin Bl2, therefore, it is

recommended that their vitamin Bl2 RDA (2.4 Gig/day) be derived from consumption of

foods fortified with vitamin B l2 or from vitamin B l2 supplements (20).









Dietary data from the National Health and Nutrition Examination Survey

(NHANES) 1999-2000 indicated that Americans of all age groups are consuming well

above the RDA for vitamin Bl2. The mean vitamin Bl2 intake for males and females

aged 20 to 39 years was 6.1 and 4.0 Gig/day, respectively, and the intakes for ages 40 to

59 years was 6. 1 and 4. 1 Gig/day, respectively. Individuals over 60 years of age had a

slightly lower vitamin Bl2 intake, although the intake still surpassed the RDA. In this age

group, reported intake of vitamin Bl2 for males was 5.3 Gig/day and for females was 3.9

Gig/day (32). Data from the Continuing Survey of Food Intakes by Individuals (CSFII)

1995 was used to estimate the percent that specific dietary sources contributed to vitamin

Bl2 intake. For men, the sources that contributed the greatest percentage of dietary

vitamin Bl2 were mixed foods (18.5%), beef (15%), and milk and milk drinks (10%).

For women, the greatest percentage of total dietary vitamin B l2 intake was from mixed

foods (16.4%), milk and milk drinks (14.6%), and beef (12%) (20).

To date, no adverse effects of large doses of vitamin Bl2 have been reported from

food or supplements. No adverse or toxic effects have been reported in studies of patients

who receive very large doses of vitamin B l2 for the treatment of pernicious anemia (33).

There is no Tolerable Upper Intake Level (UL) established for vitamin Bl2 (20).

Vitamin B12 Deficiency

Etiology

In addition to dietary inadequacy of vitamin Bl2, there are numerous other causes

contributing to a vitamin B l2 deficiency including pernicious anemia, food-bound

malabsorption, pancreatic insufficiency, jejunal bacterial overgrowth, tropical sprue, or

gastric or ileal resection (22). Drugs such as nitrous oxide, metformin, and stomach acid

blockers can also impair vitamin Bl2 status (34-36). Only vitamin Bl2 deficiency









associated with pernicious anemia, food-bound malabsorption, and dietary deficiency

will be discussed in the following sections.

Pernicious anemia. Pernicious anemia is an autoimmune disease that occurs when

the body produces auto-antibodies against the parietal cells. This causes loss of parietal

cells and a subsequent decrease in IF. Other auto-antibodies can bind to IF and block the

vitamin Bl2 binding site. Lack of IF due to the absence or reduction of parietal cell mass

or blockage of the IF-Bl2 binding site results in vitamin Bl2 malabsorption (21). The

exact number of Americans with pernicious anemia is unknown. Although, based on

studies of elderly it has been estimated that 1.9% of the population over the age of 65

years has pernicious anemia (37).

Historically, a Shilling's test was the primary test used to diagnose pernicious

anemia. This test involves giving an individual a radioactive oral dose and a flushing

dose of crystalline cobalamin and measuring the amount of radioactive cobalamin

excreted in the urine over time (3 8). This test is not used as widely by clinicians as it was

in the past (39). Alternative methods for diagnosing pernicious anemia include testing an

individual's serum for IF antibodies or testing for parietal cell antibodies (3 8).

In 1926, Minot and Murphy used raw liver to cure pernicious anemia in humans.

Vitamin B l2 was eventually isolated and termed the "anti-pernicious anemia factor"

(40). Individuals with pernicious anemia can be initially treated with weekly

intramuscular inj sections of 1,000 Cpg of cyanocobalamin for up to 2 months, preceded by

monthly inj sections for life (29). Kuzminski et al. (1998) reported that 2,000 Cpg per day of

oral cobalamin may be an effective alternative to intramuscular inj sections (41).









Food-bound malabsorption. Vitamin Bl2 absorption requires secretion of

stomach acid to disassociate the vitamin from any bound components. With inadequate

amounts of gastric acid, vitamin Bl2 will not be released from the protein moiety

resulting in decreased vitamin absorption and increased excretion (21). Vitamin Bl2

consumed in a crystalline form is not bound, so regardless of stomach acid secretion,

crystalline vitamin Bl2 will be absorbed normally (42). Drugs such as antacids and

proton-pump inhibitors may contribute to food-bound malabsorption of vitamin Bl12 by

decreasing gastric acid production (34).

To determine if vitamin Bl12 deficiency is due to food-bound malabsorption,

research laboratories use a protein-bound cobalamin absorption test (43). In this

technique, radiolabelled vitamin Bl2 is mixed with powdered egg yolk and made into an

omelet. The omelet is consumed by the patient and 1.5 hours later the patient is given an

unlabelled dose of hydroxocobalamin. Urinary excretion of vitamin Bl2 is then

measured (43).

Individuals over the age of 51 are at risk for food-bound vitamin Bl2

malabsorption due to the age-related decrease in stomach acid or achlorhydria (42, 44).

As a part of the RDA recommendations, individuals older than 5 1 years of age are

advised to obtain vitamin Bl2 from fortified foods sources or from vitamin Bl2

supplements (20). These sources are not protein bound, therefore these sources can be

absorbed through the normal mechanism (45).

Dietary deficiency. Those who restrict their intake of vitamin Bl2 containing

foods, such as vegetarians or vegans, are at risk for vitamin Bl2 deficiency, unless they

consume vitamin Bl2 from fortified foods or supplements (5). Since vitamin Bl2 can be









stored in the body, a decrease in dietary intake may not affect vitamin Bl2 concentration

initially (9). Those who restrict their intake of vitamin B l2 containing foods (naturally

occurring, fortified, or supplements) for longer durations are at higher risk for a vitamin

Bl2 deficiency due to depletion of body stores (9, 10).

Clinical symptoms

Megaloblastic anemia. Vitamin Bl2 deficiency occurs in stages as previously

reviewed (5, 38). Vitamin Bl2 deficiency begins with a reduction in serum concentration

followed by decreased cellular vitamin B l2 concentration. Metabolic abnormalities occur

next as indicated by increased homocysteine and MMA concentrations and decreased

DNA synthesis (5, 38). The last stage is indicated by the presence of megaloblastic

anemia (5). Megaloblastic anemia results in large abnormally shaped erythrocytes and

elevated mean corpuscular volume (MCV). Similar to a folate deficiency, leukocytes

become enlarged and hypersegmentation of the nuclei occurs. Treatment with vitamin

Bl2 can reverse changes in the erythrocytes and leukocytes (2). Presence of

megaloblastic anemia may indicate a possible deficiency of vitamin Bl2, although it is

not a strong diagnostic tool since a folate deficiency results in the same type of anemia

(3 8).

Neurological disorders. According to a review by the Institute of Medicine

(1998), neurological abnormalities occur in 75 to 90% of individuals with vitamin Bl2

deficiency. They generally appear long after the deficiency has occurred making it a poor

assessment tool for vitamin Bl2 status (20). Neurological abnormalities are caused by

demyelination of the central nervous system (CNS). The myelin sheath and spinal cord

near the brainstem are affected first. Degradation of the CNS can occur before a vitamin

Bl2 deficiency is recognized in an individual. Lindenbaum et al. (1988) found that 28%









of individuals with neurological abnormalities presented with normal hematological

indices. Tingling and numbness in extremities, memory loss, disorientation, and dementia

are common neurological signs of a deficiency (46). These symptoms are not specific for

a vitamin Bl2 deficiency since they also can be present among individuals who have

other neurological conditions. Therefore, an individual can not be diagnosed with a

vitamin Bl2 deficiency based only on presentation of neurological symptoms (3 8).

Health related risks of a vitamin B12 deficiency

As a person restricts more animal food sources, the risk of inadequate intake of

vitamin Bl2 increases (5). Since vegetarians restrict various animal food sources,

thereby restricting naturally-occurring food sources of vitamin Bl2, they are at risk for a

defei ency. This is especially the case if vitamin Bl12-fortified foods or supplements are

not consumed. Data continue to support a relationship between vegetarian diets and

increased risk for birth defects, neurological abnormalities, and elevated homocysteine

concentration related to a vitamin Bl2 deficiency (6-8).

Birth defects. Women of childbearing age who consume a vitamin Bl2 deficient

diet are at an increased risk for impaired fetal development if they are pregnant. Folic

acid is generally the vitamin associated with birth defects, specifically neural tube defects

(NTD) such as spina bifida. There is, however, strong evidence supporting a role for low

vitamin Bl2 status as an NTD risk factor, which may be related to the interdependent role

of folic acid and vitamin Bl2 in one carbon metabolism (8, 47-49). Kirke et al. (1993)

compared maternal folate and vitamin Bl2 concentrations before the birth of a child.

After delivery, these women were grouped based on presence of an NTD in the child.

Both plasma folate and vitamin Bl2 concentrations were lower in women who had a

child with an NTD compared to those who did not give birth to a child with an NTD.









Low vitamin Bl2 status was considered an independent risk factor for NTD (49).

Groenen et al. (2004) investigated maternal vitamin Bl2 status and the risk for spina

bifida in offspring. In this study, serum vitamin B l2 concentrations of mothers and their

children who had spina bifida were compared to those of control mothers. The mothers

with children who had spina bifida had a mean serum vitamin Bl2 concentration that was

21% lower [<185 picomoles per liter (pmol/L)] than that of the control mothers and was

associated with a three and a half-fold increase in spina bifida risk (8).

van Rooij et al. (2003) evaluated vitamin Bl2 status of mothers and infants with

nonsyndromic orofacial clefts. Mothers who had decreased serum vitamin Bl2

concentration (<185 pmol/L) had a three-fold increased risk of having offspring with a

nonsyndromic orofacial cleft. There were no differences between median serum vitamin

Bl2 concentrations in the affected versus the control infants (48). Afman et al. (2001)

investigated the association between vitamin B l2 binding by TC and the risk of NTD.

They found that mothers with children who are affected with an NTD had a significantly

higher mean homocysteine concentration and lower TC and vitamin Bl2 concentrations

than mothers with normal children. They hypothesized that decreased binding of vitamin

Bl2 by transcobalamin, which resulted in a vitamin Bl2 deficiency as indicated by

increased homocysteine concentration, was associated with increased risk for birth

defects. They concluded that vitamin Bl2 supplementation may be needed along with

folic acid supplementation in women of childbearing age (47).

Neurological abnormalities. Neurological abnormalities can occur in individuals

with a vitamin B l2 deficiency (20). Depending on the time at which a neurological

disorder persists, some can be reversed. Pittock et al. (2002) discussed a case-report of a









34-year old man with reversible myelopathy who suffered from pernicious anemia. He

was treated with intramuscular inj sections of cyanocobalamin every two weeks. After one

month of treatment, all clinical symptoms had completely reversed to normal (7).

Louwman et al. (2000) investigated the effect of marginal cobalamin status on

adolescent cognitive functioning when following a macrobiotic diet. Data from 28

adolescents on vegan diets and 24 adolescents on non-vegetarian diets indicated that

adolescents on macrobiotic diets performed worse on psychological tests than adolescents

on non-vegetarian diets (50).

There are numerous published studies that show neurological abnormalities in

infants who are exclusively breast-fed from mothers following a vegetarian or vegan diet

and who have poor maternal vitamin Bl12 status (5 1-5 5). The first report (Jadhav et al.

1962) included six cases occurring in India. None of the infants had absorption problems,

but all had megaloblastic anemia attributed to poor vitamin B l2 intake due to low

maternal vitamin B l2 breast milk concentrations. Half of the mothers had inadequate

vitamin B l2 breast milk concentrations because of their restriction of animal products,

the other half suffered from vitamin Bl2 malabsorption. All infants were born without

complications, but began to display abnormal skin pigmentation, apathy, anemia,

involuntary movements, and developmental regression around the ages of 7 to 12 months.

Upon diagnosis, they were treated with oral cobalamin and the symptoms were reversed

(56).

Elevated homocysteine concentration. As discussed previously, with inadequate

amounts of vitamin Bl12, homocysteine cannot be remethylated and homocysteine can

build up in the blood (5). There are many proposed mechanisms by which an elevated









homocysteine concentration may increase the risk for cardiovascular disease, as reviewed

by Ueland et al. (2000) (57). One hypothesis is that homocysteine may increase

endothelial permeability by decreasing tumor necrosis factor and increasing thrombin

formation, which may exacerbate the development of atherosclerotic plaque and

contribute to the progression of cardiovascular disease (58).

Since vegetarians limit or exclude animal products, which are the only naturally-

occurring sources of dietary vitamin B l2, they are at an increased risk for a vitamin B l2

deficiency and hyperhomocysteinemia (59). Data from NHANES III showed that two-

thirds of United States men and women with elevated homocysteine concentrations had

low serum vitamin Bl2 concentrations (60). Gao et al. (2003) used an FFQ to investigate

dietary patterns and serum homocysteine concentration in a Chinese population.

Individuals were categorized into three dietary groups based on greater intake of calories

from: (1) milk and fruit, (2) red meat, and (3) unbleached, refined cereals (rice and flour

products). Forty percent of subj ects in the refined cereals group had a high homocysteine

concentration and 67% had a low serum vitamin Bl2 concentration. This group was four

to Hyve times more likely than individuals in the milk and fruit group or red meat group to

have these hematological factors. They concluded that dietary patterns where the

maj ority of calories were from low vitamin Bl12 sources contributed to the increase in

homocysteine concentration in Chinese adults (61).

Obeid et al. (2002) measured homocysteine and MMA concentrations among

individuals following various types of vegetarian diets. It was shown that vegans,

followed by the combined lacto/lactoovovegetarian group, had the highest median

homocysteine concentration, lowest median serum vitamin Bl2 concentration, and









highest median MMA concentration (62). A report by Herrmann et al. (2003) further

supported these findings of an association between vitamin B l2 intake and increased

homocysteine concentration in vegetarians. They reported that 67% of vegans, 38% of

vegetarians, and 16% of omnivores had elevated homocysteine concentrations. These

researchers concluded that the vitamin Bl2 status of vegetarians and vegans could be

described as marginal to severely deficient compared to a more normal vitamin Bl2

concentration in the omnivore control group. These data indicated that decreased serum

vitamin Bl2 concentration was directly correlated with elevated homocysteinemia in

vegetarians (63). Kraj covivova-Kudlackova et al. (2000) measured homocysteine

concentration in vegetarians and reported that 53% of vegans, 29% of vegetarians, and

5% of omnivores had hyperhomocysteinemia. Serum vitamin Bl2 concentrations were

significantly lower in 28% of vegetarians, 78% vegans, and none of the omnivores (64).

Research findings support the conclusion that vitamin B l2 supplements can correct

hyperhomocysteinemia. Mezzano et al. (2000) studied vegetarians with poor serum

vitamin Bl2 status. After one intramuscular inj section of cyanocobalamin (10,000 Cpg),

serum vitamin Bl2 concentration increased significantly above normal from 110 & 46

pmol/L to 393 A 151 pmol/L) and mean total homocysteine concentration decreased

significantly [from 12.4 & 4.7 micromoles per liter (Cpmol/L) to 7.9 & 31 Cpmol/L)] (6).

Recently, Bor et al. (2006) reported that a daily intake of at least 6 Cpg of vitamin B l2

(from food or supplements) reduced blood homocysteine, MMA, and vitamin Bl2

concentrations in postmenopausal women (65).









Vitamin B12 Status Assessment

Serum Vitamin B12 Concentration

Serum vitamin Bl2 is generally the first diagnostic test performed in a clinical

setting on patients suspected of having a vitamin Bl2 deficiency (2). Established

standards that have been used in both clinical and research settings are defined as

follows: (a) deficient (<148 pmol/L); (b) marginally deficient (148-221 pmol/L); and (c)

normal (>221 pmol/L) (66); although depending on the method of analysis, the normal

range can vary from laboratory to laboratory. Ranges of vitamin Bl2 values using

radioassay methods are found to be higher and less accurate compared to microbiological

assays, which are generally lower and more accurate (2). The use of serum vitamin Bl2

concentration as an indicator of vitamin Bl2 status is currently under much scrutiny

because of the potential for false normals (2). Lindenbaum et al. (1990) found that MMA

concentration predicted a vitamin B l2 deficiency two times more than serum vitamin

Bl2 concentration, and five percent of individuals who were diagnosed with a clinical

vitamin Bl2 deficiency had a normal serum vitamin Bl2 concentration (67). In contrast,

Bolann et al. (2000) reported that serum vitamin Bl2 concentration was the best indicator

of vitamin Bl2 status and should be used first in determining if the symptoms

experienced by an individual are vitamin Bl2 related (68). Currently other metabolites of

vitamin B l2 metabolism such as MMA, are considered a better determinate of vitamin

Bl2 status. Holo-TC is under investigation as a potentially more sensitive vitamin Bl2

status assessment indicator.

Methylmalonic Acid Concentration

Serum MMA concentration is considered a functional measure of intracellular

vitamin Bl2 status (20) Vitamin Bl2 is required to convert L-methylmalonyl CoA to









succinyl CoA. In the event of a vitamin Bl2 deficiency, this pathway is disrupted due to

decreased enzyme activity. Consequently, the concentration of MMA increases in the

blood and in response to a vitamin Bl2 deficiency in the urine (12). The normal serum

concentration range for MMA is 73 to 271 nanomoles per liter (nmol/L) (2). Urinary

MMA may be an earlier status indicator than other status indicators since MMA can

become elevated before serum vitamin B l2 concentration drops below 148 pmol/L (2).

Renal insufficiency is associated with an increase in the concentration of MMA

independent of vitamin Bl12 deficiency (3 8). MMA concentration also can be elevated in

the elderly regardless of vitamin Bl2 status (69). The photometric methods used to

determine serum MMA concentration are very costly. For this reason serum MMA

concentration is not used routinely as a clinical diagnostic test (2). Gultepe et al. (2003)

developed a modified procedure for measuring urinary MMA concentration that is more

sensitive than the older photometric method (70).

Holotranscobalamin Concentration

Serum holo-TC is a biologically active complex of vitamin Bl2 and

transcobalamin. Since serum vitamin Bl2 concentration may not always be a reliable

indicator of vitamin B l2 status, holo-TC is currently being investigated as a more

sensitive test for vitamin B l2 assessment (71). Our research group is currently

investigating the influence of the polymorphism transcobalamin 776C G (TC

776C G) on the concentration of serum holo-TC, and the reliability of holo-TC as an

indicator of vitamin B l2 status. von Castel-Dunwoody et al. (2005) reported an inverse

relationship between plasma homocysteine and serum holo-TC, and plasma vitamin Bl2

concentrations. These findings correlated to previous studies investigating the

relationship between homocysteine and holo-TC concentrations. Holo-TC concentration









may be helpful in characterizing vitamin Bl2 status in marginally defieient individuals

(72).

Serum Homocysteine Concentration

Vitamin Bl2 and folate are required for the remethylation of homocysteine to

methionine (1), and vitamin B6 is required for the transulfuration of homocysteine to

cystathionine (12). Serum homocysteine concentration may provide an indication of

intracellular vitamin Bl2 status (20). Serum homocysteine concentration below 12

Cpmol/L is considered normal (60). In a vitamin Bl2 deficiency, serum homocysteine

concentration can be elevated. Since there are three vitamins (e.g., vitamin Bl2, folate,

and vitamin B6) that play a role in converting homocysteine to other compounds in the

body, homocysteine concentration is not a specific determinant for vitamin Bl2

deficiency (73).

Megaloblastic Anemia

Megaloblastic anemia is diagnosed when hematocrit and hemoglobin are low, but

MCV is high. Vitamin Bl2 and folate deficiencies, among other disorders, can cause this

type of anemia. Therefore, megaloblastic anemia is not a definitive status indicator of a

vitamin Bl2 deficiency. If folic acid is given to treat the condition, the anemia will

reverse to normal, even if the underlying cause is due to vitamin Bl2 deficiency.

However, the neurological complications of a deficiency will not resolve and can result in

further damage (3 8).

Dietary Intake Assessment in Adults

Twenty-four hour recall method

The twenty-four hour recall method of dietary intake assessment can be

administered in-person or over the phone and can be used for large epidemiological









studies. This method is inexpensive, requires no literacy, and has a high response rate.

Twenty-four hour recalls rely heavily on memory and individuals may omit food items or

even report food items not consumed that day (74). The three-pass method is generally

used to obtain complete dietary recalls (75). In this method, an individual will simply

start listing all the foods they ate without interruption. After the individual has given a

brief list of the foods they ate, the interviewer asks specific questions regarding each food

item eaten, portion size, and the meal time. For example, if the person said they had a

piece of bread, the interviewer should ask, the type of bread (e.g., multi-grain, white, etc),

additional products added, the amount, and time of day consumed. Next, the interviewer

repeats the foods that were reportedly consumed at that meal or snack, to see if any

additional foods were omitted. The interviewer should address snacks between meals,

beverages, and food consumed during the night. Individuals generally think of their main

meals and disregard daytime or evening snacks. Food models and measuring cups can be

used with the individual to help them correctly identify their portion size (75).

The United States Department of Agriculture (USDA) recently revised their

method from three-steps to five-steps for interviewing during a 24-hour dietary recall.

They have developed a very comprehensive automated-multiple-pass method (AMPM)

(75). It was developed to keep the attention of individuals during the interview and to

correct for under-reporting (75). This five-step method has more passes and increased

opportunities to add forgotten foods. The computerized method uses a database with

predetermined questions, possible follow-up questions, and instructions for the

interviewer. The AMPM contains 2,400 questions and 21,000 response options (75, 76).

The AMPM is directly linked to the USDA Food and Nutrient Database for Dietary









Studies for nutrient analysis. Moshfegh et al. (2001) pilot tested this method in 700

individuals and found a higher average daily caloric intake and more foods reported than

the CSFII (1996) that used a three-step method (77).

Multiple-day food record

The multiple-day food record involves instruction of an individual to write

everything they ate on a food record that is provided (78). A food record can be self-

administered or administered by an interviewer. If self-administered, instructions should

be provided with the diet record. Example instructions would include information on

food description, preparation methods, brand names, and ingredients in mixed foods (78).

Kolar et al. (2005) provided a 12-page serving size booklet with pictures to illustrate and

quantify serving sizes during their study. Records are reviewed for completeness and

entered into a nutrient database program such as Nutrient Data System for Research

(NDS-R), University of Minnesota (78). Since they require review and manual

correction, multiple-day diet records are generally discouraged for large epidemiological

studies (78). Dietary records do not rely on memory, but can be tedious for the individual

completing the record (79). The reliability of the nutrient data from the food record

increases with the numbers of days recorded (80). Craig et al. (2000) measured the effect

of atypical days, as described by participants, on nutrient intake estimates in 1,090

women. Those who reported an atypical day as less than normal or higher than normal

had lower intakes or higher intakes, respectively, of all nutrients. They concluded that

atypical days in an individual's diet can affect estimated nutrient intakes in research

studies that use the multiple-day food record (80).









Food frequency questionnaire

A FFQ is used to determine usual dietary intake (81). This type of questionnaire is

commonly used for large epidemiological studies (81). A FFQ can be self-administered

and is less expensive than other dietary assessment methods (82). Twenty-four hour

recall methods and multiple-day food records are more detailed than a FFQ. A FFQ uses

general food groupings, such as high-fiber cereal, whereas a 24-hour recall method or

multiple-day food record provides the specific brand name of cereal that was consumed.

A FFQ has preset questions and portion sizes to which an individual responds. These

types of questionnaires are developed to be scanned by a computer and uploaded into a

software package (81). There generally is no option for the individual to add a personal

response not listed on the questionnaire. Food frequency questionnaires need to be

validated before using them for a large epidemiological study to ensure that the

questionnaire is capturing true dietary intake (82).

The National Cancer Institute (NCI) has validated a FFQ referred to as the Diet

History Questionnaire (DHQ) (81). Frequency of intake and portion size for 124

individual food items is captured in this FFQ. Forty-four of these questions have

embedded questions that ask about seasonal intake, type of fats added, and whether the

food item was low-fat or fat free (81). The original DHQ also includes ten questions

regarding dietary supplements, and four summary questions (81). Portion size choices

were determined from analyses of data from the CSFII (1994-1996). Based on responses

from 10,019 adults during this study, portion size choices were changed in the

questionnaire. Instead of using portion sizes such as small, medium, and large, this

questionnaire provides portion sizes specific to the food item such as "less than 1 cup,"

"1-2 cups," and "more than 2 cups". Also, based on data from the CSFII (1994-1996),









5,261 individual foods were categorized into 170 food groups (83). These food groups

are the basis of the 124 food items used in the DHQ (81). Nutrient estimations are

calculated for each food item listed in the DHQ by multiplying the daily frequency by a

nutrient value specific for the gender of the individual and the portion size selected.

The NCI DHQ was validated in 2001 against repeated 24-hour recalls and

compared to the Block and Willett FFQ (81). The Block FFQ is an eight-page

questionnaire that covers 106 food items. Portion size choices are described as small,

medium, or large (81). The questionnaire has thirteen dietary supplement questions,

eight questions regarding use of added fats, five summary questions, and six questions

regarding restaurant eating. The Willett FFQ is a four page questionnaire with 126 food

items. The questionnaire is unique in that it does not have a separate portion size

question. Individuals select one response in which the frequency and portion size are

combined together in one response. The questionnaire also includes ten dietary

supplement questions and ten questions about added fats (81). The validation study for

the DHQ consisted of 1,301 men and women who completed four telephone 24-hour

recalls during one session. The subjects were then randomized to receive the DHQ and

Block FFQ or the DHQ and Willett FFQ. Compared to the 24-hour recall method, it was

found that the DHQ was more accurate in determining nutrient intake than either the

Block or Willett FFQs (81).

Assessment of vitamin B12 intake

The assessment of vitamin Bl2 intake among vegetarians and non-vegetarians has

generally been assessed along with other nutrients to provide an overall profile of dietary

adequacy of a vegetarian diet in contrast to a non-vegetarian diet. Studies where only









vitamin B l2 intake has been assessed (generally in relationship to serum concentration

status) have largely been conducted with vegans compared to non-vegetarians.

Haddad and Tanzman (2003) used CSFII 1994-1996 data to compare the dietary

intake of various nutrients including vitamin Bl2 among self-defined vegetarians and

non-vegetarians in the United States. Dietary data was collected using two 24-hour

dietary recalls. Men and women ages 20 years and above who reported consuming no

meat had a vitamin Bl2 intake lower than those who consumed meat (84). Two separate

studies by Barr and Broughton (2000) and Janelle and Barr (1995) reported findings in

Canadian women that were similar to those reported in a United States study. Both

studies compared nutrient intake, including vitamin Bl2, among vegetarian and non-

vegetarian women in Canada. The study by Barr and Broughton (2000) assessed dietary

intake using the 24-hour recall method, and the study conducted by Janelle and Barr

(1995) assessed dietary intake using 3-day food frequency questionnaires. Among

women, vitamin B l2 intakes of the vegetarians were lower than non-vegetarians (85, 86).

Haddad et al. (1999) also assessed vitamin Bl2 intake and status of vegans and

non-vegetarians. To measure dietary intake, a 24-hour recall and 4-day food records

were used. Vitamin Bl2 intake among the female vegans was lower than the vitamin

Bl2 intake among the female non-vegetarians. No differences in vitamin Bl2 intake

were reported among the male vegan and non-vegetarian groups. Serum vitamin B l2

concentration (male and female combined) did not differ among the vegan and non-

vegetarian groups (87).

Miller et al. (1991) assessed vitamin Bl2 intake in relation to serum vitamin Bl2

status among individuals who consumed a macrobiotic diet but sometimes included dairy,










eggs, and seafood in their diet. Dietary intake was obtained although the specific

assessment tool used (i.e., food record, FFQ, or dietary recall) was not indicated. To

determine dietary vitamin Bl2 intake, the frequency of intake was multiplied by the

vitamin Bl2 content of the foods. This number was then added to a frequency score that

was based on how often specific foods (e.g., dairy, eggs, seafood) were consumed. This

study compared the frequency intake of dairy, eggs, and seafood in relation to serum

vitamin Bl2 concentration. Those who consumed dairy products I 1 time/week or > 1

time/week had a significantly higher serum vitamin Bl2 concentration than those who

never consumed dairy products. No differences were found in frequency of egg or

seafood intake on serum vitamin B l2 concentration between intake groups. Serum

vitamin Bl2 concentration was then compared to tertiles of vitamin Bl2 intake score.

Vitamin Bl2 intake scores of 0, 0.01 to 0. 15, and 0. 16 to 1.7 were defined as low,

medium, and high, respectively. Adults who had a low vitamin B l2 intake score had

significantly lower serum vitamin Bl2 concentration than those with a score of medium

or high. (9).

Rauma et al. (1995) used 5-day food records to determine vitamin Bl2 intake

among vegans who followed a strict uncooked diet and non-vegetarians. The mean

vitamin Bl2 intake from the vegan group was significantly lower than the non-vegetarian

group (1.8 versus 6.2 Cpg/day, respectively). Although, the authors stated that dietary

assessment of vitamin Bl2 of the vegan group was underreported because their database

did not contain data for the foods consumed on the "uncooked diet". Serum vitamin Bl2

concentration was correlated (r = 0.63; p < 0.01) with dietary vitamin Bl2 intake among









the veg~ans. Contribution of total vitamin B l2 from various food sources consumed were

not determined in this study (10).

Larsson and Johansson (2002) compared dietary intake of various nutrients

including vitamin Bl2 among 16 to 20 year old vegans and omnivores in Sweden. In this

study, participants were interviewed for 1 to 2-hours, 1 to 2 weeks apart to determine

dietary intake. Dietary vitamin Bl2 intakes for female and male vegans (0.0 + 0.1, 0.11

0.03, respectively) were significantly lower (p<0.0001) than female and male omnivores

(5.0 & 2.5, 5.9 & 1.5, respectively) (88). Larsson and Johansson (2005) published

additional data comparing dietary sources of vitamin Bl2, in addition to other nutrients.

Among the vegans, 100% of vitamin B l2 intake was from dietary supplements. In

contrast, the maj ority of vitamin Bl2 in the diet of omnivores came from animal

products, defined as meat, Eish, seafood, dairy products, and eggs, followed by dietary

supplements (89).

Dunn-Emke et al. (2005) assessed dietary intake of vitamin Bl2 among vegans

with prostate cancer following a well-planned, low-fat diet. Subjects were given specific

dietary guidelines for this diet along with a soy protein powder that contained isoflavones

(potential inhibitor of cancer growth). After 6 months, 3-day food records were obtained.

The mean vitamin Bl2 intake for these subj ects was 3.7 Cpg/day, with 81% of the vitamin

Bl2 intake from supplemental soy protein and 19% from fortified plant foods (90).

Leblanc et al. (2000) determined vitamin B l2 intake among lactoovovegetari ans,

lactovegetarians, and vegetarians following a macrobiotic diet. Participants recorded

dietary intake using a 5-day food record. The nutrient software used to analyze the food

records contained additional foods found in a vegetarian diet. Only 80% of these foods









contained data on vitamin Bl2 content. After analysis, it was determined that the women

who followed a macrobiotic diet had a significantly lower (P<0.05) dietary vitamin B l2

intake compared to the women in the other vegetarian groups. There were no differences

in vitamin B l2 intake among vegetarian men. The median intakes of vitamin B l2 for all

three groups (men and women) were below the RDA for vitamin Bl2 (range of 0-1.73

Cpg/day) (11).

Vitamin B12 Status in the United States

Based on data from NHANES III (1994-1998), mean serum vitamin Bl2 was 382

pmol/L for individuals 4 years and older (91). Younger males and females (4-5 years)

had the highest median serum vitamin Bl2 concentration for all age groups. Men and

women 70 years and over, had the lowest median serum vitamin B l2 concentrations (i.e.,

286 pmol/L and 322 pmol/L, respectively). Based on ethnicity, non-Hispanic black

Americans had the highest median concentration (419 pmol/L) followed by Mexican

Americans (368 pmol/L). In contrast to non-Hispanic white Americans had the lowest

median concentration (329 pmol/L) (91).

Data from the NHANES III (1998-94) indicated that the prevalence of Americans

with a serum vitamin Bl2 concentration less than 148 pmol/L increased with age.

Thirteen percent of individuals 70 years and older, 12% of individuals 60 to 69 years, 9%

of individuals 50 to 59 years, 8% of individuals 40 to 49 years, and 2% of individuals 30

to 39 had a serum vitamin Bl2 concentration less than 148 pmol/L. Serum vitamin Bl2

concentration less than 74 pmol/L was found in 1% of the total population over 4 years of

age (91).

Vitamin Bl2 deficiency related to protein-bound vitamin Bl2 malabsorption in the

elderly is estimated between 10 to 30% (45). Carmel et al. (2002) conducted a study









evaluating the prevalence of elevated homocysteine concentration in young Asian Indians

in the United States. They found that a large percentage of this population had elevated

plasma homocysteine concentration compared to healthy controls. Fifty-nine percent of

men and 23.8% of women had low vitamin Bl2 status as defined by a vitamin Bl12

concentration <180 pmol/L. They concluded that elevated homocysteine concentration

was possibly caused by low dietary vitamin Bl2 intake (92). Refsum et al. (2001) also

studied an Asian Indian population and found that 47% had vitamin Bl2 deficiency, and

77% had hyperhomocysteinemia regardless of whether they followed a vegetarian or

non-vegetarian diet (93).

Vegetarianism

Definitions of Vegetarianism and Prevalence in the United States

There are many definitions of the term vegetarian. Vegetarians may exclude all

animal products (vegan); exclude meat and egg products, but include dairy

(lactovegetarian); or exclude meat products but include dairy and eggs

(lactoovovegetarian). Some vegetarians (pesco-vegetarian) may restrict beef, poultry,

and pork consumption, but consume fish, dairy, and eggs.

In 1979, the USDA reported that 1.7% of Americans were vegetarians (94). A 2003

national Harris Interactive survey reported that approximately 2.5% of Americans

describe themselves as vegetarians (84, 95). According to data obtained from the CSFII

1994-1996, 1998, more individuals in the 20 to 29 age group define themselves as

vegetarian. The age group with the lowest number of people defining themselves as

vegetarians was the 60 to 69 year old group (84). There are many factors which motivate

individuals to become vegetarian such as health, ethical, or religious attitudes (96).









Assessment of Dietary Adequacy of Vegetarian Diets

Haddad et al. (1999) used a 24-hour recall and 4-day food records to determine the

dietary adequacy of vegetarian diets among 45 healthy adults. In this small study, vegans

had lower total protein, fat, saturated fat, monounsaturated fat, cholesterol, and higher

dietary fiber intake (87). The European Prospective Investigation into Cancer and

Nutrition-Oxford (EPIC-Oxford) study, which included approximately 66,000 meat-

eaters and vegetarians, including vegans, in the United Kingdom confirmed the results of

Haddad et al. (1999) in a much larger study. This study used 7-day food records and a

FFQ developed for the United States Nurses' Health study to determine differences

among dietary intake of meat-consumers, Eish-consumers, vegetarians, and vegans.

Subj ects were categorized based on their answers to questions regarding meat (i.e., beef,

poultry, and pork), Eish, dairy or egg intake. The differences between the meat-

consumers and the vegans were more pronounced than between the meat-consumers and

vegetarians. The meat-consumers reported higher mean calorie, protein, fat, saturated fat,

and vitamin Bl2 intake, and lower carbohydrate, fiber, folate, and iron intake than the

vegetarians and vegans. The intake of vitamin B6 was relatively similar across groups

(<10% difference). Serum vitamin Bl2 concentration was not determined in this study

(97).

The American Dietetic Association and Dietitians of Canada have recognized the

possible limitations of a vegetarian diet, but have taken the position that a carefully

planned vegetarian diet can provide the necessary needed nutrients (98). To better

determine the adequacy of vegetarian diets, more data are needed related to the intake of

macronutrients (i.e., carbohydrate, protein, and fat) and micronutrients (i.e., vitamin Bl2,

folate, vitamin B6, zinc, and iron).









Assessment of Beef Consumption and Health

There is an increasing trend for Americans to exclude or limit beef intake from

their diets for health reasons, as reviewed by White and Frank (1994) (99). The Food

Marketing Institute in 2000 reported that 68% of American consumers felt their diet

could be healthier. In doing so, 68% percent were increasing their fruit and vegetable

intake, 22% were decreasing beef consumption, and 9% were increasing their intake of

chicken and turkey (100).

In recent years, beef consumption has been linked to cardiovascular disease (17,

101, 102). The literature regarding beef intake and various markers of cardiovascular

disease are conflicting. In 1991, data from the Coronary Artery Risk Development in

Young Adults (CARDIA) study were used to determine relationships between meat (i.e.,

red meat and poultry) consumption and lipid profiles. Frequency of meat consumption

was assessed using a FFQ. The data suggested that those who ate meat less than one time

per week had lower total cholesterol and low-density lipoprotein (LDL) concentrations

compared to those who consumed meat more than one time per week (102). Nicklas et al.

(1995) analyzed 24-hour dietary recalls in 504 young adults. Those whose meat

consumption fell at the 75th percentile or higher did not meet the Dietary Guidelines for

Americans for total fat, saturated fat, and cholesterol. In contrast to the Slattery et al.

(1991) study, even though the percent of calories from fat was higher than the

recommended amount, there were no significant differences in blood lipid profiles in any

of the meat consumption quartiles (103). Fraser (1999) used a FFQ to assess dietary

intake and mortality in approximately 34,000 non-Hispanic white California Seventh-Day

Adventists. This FFQ included 51 different food types. The participants were categorized

into three groups; vegetarian (i.e., no meat, poultry, and/or fish), semivegetarian (i.e.,










poultry and fish < 1 time per week), and nonvegetarian (beef, poultry, and fish

consumers). Males who consumed red meat more frequently than 3 times per week had a

relative risk of 2.5 for developing fatal ischemic heart disease. Those who consumed red

meat more than 1 time per week had a relative risk of 1.86 for developing colon cancer

(101).

The following studies promote lean beef intake by providing supporting data

relative to the role of fat content in beef and other meats on lipid profiles. The O'Dea

study in 1990 confirmed that high frequency of beef consumption was not associated

with poor health status, but beef fat was. Ten participants ate a controlled diet of very-

low fat lean beef (i.e., 9% energy from fat) for 3 weeks. During these 3 weeks, total LDL

and cholesterol significantly decreased from baseline. Beef drippings were added to the

diet during weeks 4 and 5, increasing total fat intake to 20% of total calories at week 4

and 30% at week 5. Total LDL and cholesterol significantly increased by week 5. It was

concluded that lean beef could be incorporated into diets instead of reducing overall beef

intake (104). Beauchesne-Rondeau et al. (2003) compared the effects of incorporating

lean beef, poultry, and fish on lipid profiles in hypercholesterolemic patients. These

patients recorded 3-day food diaries before each 26-day treatment diet. All three

treatments were associated with a reduction in total cholesterol and LDL concentration.

It was concluded that regardless of the protein source, decreased saturated fat in the diet

could decrease cardiovascular disease risk (105). Snetselaar et al. (2004) used three 24-

hour recalls to assess dietary intake and compared that to serum blood lipid data in

adolescents. Subj ects were randomized to a lean beef diet or a lean poultry and fish diet.

No differences were determined in total cholesterol concentration and saturated fat intake









between the two diets (106). However, adolescents on the lean poultry and fish group

were found to have significantly lower iron status after 3 months on this diet.

Assessment of Dietary Adequacy of Beef Consumption

Beef is the most commonly consumed meat product in the American diet. Per

capital, Americans consume approximately 65 pounds of beef per year, followed by 55

pounds of poultry, and 50 pounds of turkey (17). Combined data from the Nationwide

Food Consumption Survey (NFCS) 1977-78 and the CFSII 1989-91 and 1994-95 indicate

that individuals eating separate cuts of beef have decreased, but beef consumption from

meat mixtures has increased (107). The fat composition of beef has been reported to be

50% monounsaturated fatty acids, 46% saturated fatty acids, and 4% polyunsaturated

fatty acids. Compared to other protein sources, beef provides the highest concentration

of zinc and iron per 3 oz serving. Beef also is rich in protein, vitamin B l2, and choline

(17). The current 2005 Dietary Guidelines for Americans continue to maintain that beef is

important in a well-rounded diet as long as leaner cuts of meat and sensible portion sizes

are consumed (108).

Research Rational and Potential Application of Findings

Total dietary vitamin Bl2 intake has been assessed among meat-consumers and

vegetarians. The majority of these studies assessing vitamin Bl2 intake have focused on

vegetarian intakes using meat-consumers as the relative control group. These studies are

focused on measuring the outcomes related to a vegetarian lifestyle. It has been

confirmed that the more restrictive the diet the lower the dietary vitamin Bl2 intake and a

lower serum vitamin Bl2 concentration. Beef, a highly concentrated source of vitamin

Bl2, is often restricted in the diet of meat-consumers and vegetarians for health reasons.

Restriction of beef could result in lower dietary vitamin Bl2 intake and lower status,









which may have multiple adverse health consequences such as neurological

abnormalities, impaired fetal development, and risk for cardiovascular disease.

Therefore, it is important to assess the effect of beef consumption on total dietary intake

of vitamin B l2, which has not been the focus of previous investigations.

The present study categorized subjects based on frequency of beef intake. This

type of categorization has been done only in studies assessing the association between

beef consumption and cardiovascular disease, but not how beef intake is associated with

vitamin Bl2 intake and status. In addition, the present study compared the vitamin Bl2

contribution from dietary sources of vitamin Bl2 consumed by different beef-

consumption groups. In this comparison, the contribution of vitamin Bl2 from specific

animal sources, including beef, were estimated. Yoshino et al. (2005) recently published

estimates of vitamin Bl2 intake and the proportion of vitamin Bl2 contributed from

various food sources, excluding vitamin Bl2 from fortified sources, among Japanese

adults over a 25 year period using an FFQ (109). Unlike the study by Yoshino et al.

(2005) the FFQ used in the current study has been modified to contain all vitamin Bl2

sources including current fortified soy-based sources that vegetarians commonly

consume. The assessment methods and nutrient databases used in previous studies to

calculate dietary vitamin Bl2 intake were not completely tailored for vegetarian foods.

There are many vitamin Bl2-fortified products available for vegetarians consumers.

Manufacturers such as Worthington, Loma Linda, Morning Star Farms, and Boca

produce a large number of vitamin B l2 fortified soy products such as soy burgers, soy

crumbles, soy bacon, soy chicken patties, and vegetarian cold cuts. Unlike previous









studies, the present study uses a database that had been modified to assess the

contribution of these fortified products to dietary vitamin B l2 intake.

The present study is the first to stratify subj ects based on their frequency of

consumption of beef products and to compare the vitamin B l2 intake and food sources to

vitamin Bl2 concentration among the beef consumption groups. This study also is the

first to use a modified dietary history questionnaire specifically for vitamin Bl2-

containing foods and use a nutrient database where 100% of foods have information on

vitamin B l2 content.

The data from this study provides valuable information regarding frequency of beef

consumption and beef s role as a contributor to total dietary vitamin Bl2 intake in the

diet. The data from this study will assist consumers in making educated decisions

regarding the frequency of consumption of beef related to the potential impact on vitamin

Bl2 status. Data from this study provides new information to nutrition educators and

dietetic practitioners regarding the relationship between consumption of foods within

specific food groups and the impact on vitamin B l2 status. This new information can be

incorporated into educational materials for consumers and practitioners related to

optimizing vitamin Bl2 status through dietary means.















CHAPTER 3
MATERIALS AND METHODS

Study Design and Methods Overview

Healthy adult male and female volunteers (n = ~1,000) were recruited by flyers,

newspaper, and radio advertisements for 1 year to participate in the study. Prospective

subj ects were screened by phone using a screening questionnaire (Appendix A) to

determine if they met the following inclusion criteria: (a) age 18 to 49 years, (b) non-

smoking, (c) no use of prescription medications (birth control was allowed), (d) low

alcohol intake (< one drink per day), (e) no history of chronic diseases, (f) non-pregnant,

(g) non-lactating, (h) no use of vitamin B l2 supplements within the last 6 months; and (i)

no maj or dietary changes within the last 3 years. An attempt was made to screen out

consumers of highly-fortified cereal as well. Eligible males and females (n = 388) were

selected to participate in the study.

Subj ects were scheduled to have fasting blood samples drawn and to receive

detailed instructions regarding the completion of the DHQ (Appendix B). Following the

collection of 70 milliliters (mL) of blood that was processed for multiple analyses

including plasma vitamin Bl2, subj ects were provided with a comprehensive instruction

lasting 15 to 20 minutes on the procedures to complete the DHQ (Appendix C). Subj ects

took the DHQ forms with them to complete at home and mail back to the primary

investigator within 2 weeks of instruction.

Total dietary vitamin B l2 intake, in addition to other nutrients, and plasma vitamin

Bl2 concentration were assessed. Subjects were compensated $50 for their participation









in the study. The University of Florida, Institutional Review Board approved this protocol

and all subj ects signed approved informed consent forms.

Diet History Questionnaire

Overview

The contribution of beef and beef-containing foods to total vitamin B l2 intake was

assessed by the NCI DHQ. This study was a collaborative effort between investigators at

the University of Florida and the University of North Carolina, Chapel Hill (UNC). The

original DHQ from NCI was modified with added questions for beef containing foods

and vitamin Bl2-fortified foods. The Diet*Calc Analysis program, also provided by NCI,

was used to analyze the questionnaire responses. This program is a free software

program that can be downloaded from the NCI website (wnw. riskfactor. cancer.gov).

The nutrient database within the software is from the USDA Survey Nutrient Database

and NDS-R, University of Minnesota. The Diet*Calc Analysis program nutrient

database was last updated in August 2004.

Modifications Made to the Paper Version of the Diet History Questionnaire

The DHQ from NCI was modified in such a way that vitamin B l2-containing foods

could be isolated in the data analysis. A comprehensive review of the original DHQ was

conducted to determine if vitamin Bl12-containing foods were missing from the DHQ.

Fifteen questions (including embedded questions) were added and twenty-six questions

were modified to distinguish between vitamin Bl2-containing food items and other food

items. For example, an original DHQ question may have referred to the frequency of

lasagna consumption. The DHQ was modified to determine the frequency of

consumption of lasagna made with beef, lasagna made with meat other than beef, and

lasagna made without meat. Seven questions were changed to reflect beef containing









foods. For example, the original DHQ asked about cold cuts. Questions like these were

modified to be more specific, and in this example, asked only about beef containing cold

cuts. Eight questions were added to reflect soy substitute products. These questions

referred to commercial products available to consumers that are possibly fortified with

vitamin Bl2. The ten supplement questions from the original DHQ were deleted since

this study population did not consume any supplements.

Modifications Made to Diet*Calc Analysis Software

Since modifications were made to the paper version of the DHQ, corresponding

modifications needed to be made to the Diet*Calc Analysis software. The software

changes were imperative so the DHQ would be compatible with the Diet*Calc Analysis

software. The modifications to the Diet*Calc Analysis software were completed by staff

in the Nutrition Epidemiology Core at UNC. The Questionnaire Data Dictionary (*qdd)

file within the Diet*Calc Analysis program was modified to be compatible with the

changes made to the DHQ. The *qdd file contained coding information for interpreting

scanned DHQs. Once the *qdd file was updated allowing the program to correctly

interpret new questions, the Nutrient and Food Group database within the program also

was modified. The nutrient content of vitamin Bl12-containing foods that were added to

the DHQ were extensively reviewed using the USDA National Nutrient Database for

Standard Reference (4) and manufacturer websites for specific product information. For

example, if the old question asked about cold cuts and the new revised question was

regarding only beef cold cuts, then the Nutrient and Food Group database was updated to

only reflect nutrients that were provided from beef cold cuts.









Data Generation

When the paper version of the DHQ was scanned (procedure for scanning

discussed later), food items or questions were linked to a predetermined Food

Identification Number (FIN). Every FIN had defined nutrients for gender and serving

size. FINs were found in the Diet*Calc Nutrient and Food Group database. Diet*Calc

used the FIN associated with each response to assess the nutrient intake of the individual.

When Diet*Calc identified a FIN from the ASCII text file, it used the database to

calculate an individual's nutrient intake. This Nutrient and Food Group database was

converted into a Microsoft Excel spreadsheet with all the FINs and corresponding

nutrient values per serving size and gender. This spreadsheet was used to identify the

beef and beef-containing foods assessed in this study.

Diet*Calc produced three data files available for analysis. The Details.txt file was

an expanded version of the data calculated from the ASCII text file. This file allowed the

investigator to compare FINs to individual nutrient intake and daily frequency of intake.

The second file was a Results.txt file, which was a condensed version of the Detail.txt

file. This file provided combined daily nutrient intake values for each individual

regardless of the food sources. The third file was a Report.txt file, which provided a

summary document of daily nutrient intake for each individual. This file was not used

for data analysis, but for individual feedback when a participant indicated they wanted

their dietary intake results from the DHQ.

Diet History Questionnaire Instruction Pretest

Prior to beginning the study, a pretest of the DHQ instruction and scanning was

conducted with a group of 20 Master of Science and Doctoral students in addition to five

vegetarian and vegan consumers. This pretest was done to ensure that the instructions









and handouts provided to the subj ects for completing the DHQ were clear and

understandable. Those who participated in the pretest were instructed using the same

script that would be used for the study population. They were expected to follow these

instructions and complete the DHQ as if they were a participant in the study. This group

was given an additional survey asking various questions regarding length of time to

complete the DHQ, if any frequently consumed foods were missing from the DHQ, and if

they had additional comments that might allow for the DHQ instruction to be better

understood (Appendix D). Comments from this pretest group were reviewed and

incorporated into the DHQ and into the DHQ instructions.

Human Subjects Procedures

Qualified subj ects reported to the Food Science and Human Nutrition Building

during their scheduled appointment following an overnight fast. The subj ects were

required to be fasting for 8 hours prior to having their blood drawn. The study was

explained to the subj ects, and if they agreed to continue, they were asked to sign the

approved informed consent form. Subj ects who granted consent were assigned a subj ect

identification number.

A phlebotomist drew 70 mL of blood for analysis. During the blood draw, subj ects

were asked again if they were taking a vitamin Bl12-containing supplement. Subj ects

received a snack after their blood was drawn prior to receiving the DHQ instructions.

Diet History Questionnaire Instructions

After completion of the blood draw, each participant was given a 20-minute group

instruction reviewing the procedures to follow when completing the questionnaire.

During this time, the instructions were read aloud and explained in detail using examples

of questions and scenarios. Subj ects were again asked if they consumed a vitamin Bl12-









containing supplement 6 months prior to the study. If a subj ect indicated that they did,

specific information regarding supplement use was recorded.

To ensure that portion sizes were reported accurately, subj ects were taught how to

use food labels to determine portion sizes, and instructed on how to use measuring cups

and spoons for visual representation of a portion size. Subj ects also were given additional

information that aided in completion of the DHQ. This information, which included a

schematic drawing of the proper procedure to mark or change an answer, and handouts

on "Caffeinated vs. Non-caffeinated Beverages," "Fortified Cereals," and "Seasonal

Fruits and Vegetables," were reviewed during the group instruction (Appendix E). The

purpose of these handouts was to ensure that the DHQ would be scanned correctly, and

that subj ects would be better prepared to consistently answer the questions presented in

the DHQ. Subj ects also were given two sample questions reviewing the procedure to

average their intake over 12 months. These examples were based on usual dietary intake

and referred to specific questions in the DHQ. Subj ects were given the opportunity to

ask further questions if anything was unclear.

Sample Collection and Processing

Processing of Plasma for Vitamin B12 Analysis

Blood samples were drawn into Vacutainer@ tubes (Vacutainer@ Blood Collection

Set; Becton Dickinson, Vacutainer@ Systems; Franklin Lakes, NJ) containing K3

ethylenediaminetraacetic acid (EDTA) as an anticoagulant and immediately placed on

ice. Following centrifugation at 2000 x g for 30 minutes at 4oC, aliquots were stored at

-20oC until plasma vitamin Bl2 analysis.









Processing the Diet History Questionnaire

Subj ects were encouraged to return their DHQ within 2 weeks. If they did not

return the DHQ within 2 weeks, they were contacted at the phone number or email

address provided during the initial screening. All returned DHQs were reviewed for

completeness. The purpose of the review was to ensure that the DHQ had been completed

and that no question had a missing response. To prevent bias, marked responses to

questions were not reviewed. Questions for which a response was missing were noted,

and the subj ect was contacted to obtain the missing information. If the participant was

phoned, the questions for which responses were missing were read aloud to the

participant and their response was recorded on the DHQ. If the participant was contacted

via email, then the question and answer responses were typed.

The DHQ was sent to Optimal Solutions Corporation (OSC), Lynbrook, New York,

in groups of 100 to be scanned electronically. Once the scanning process was completed

for each group of 100, OSC mailed the DHQs and electronic data in the form of an ASCII

text file to UNC. The ASCII text file was uploaded at UNC into the Diet*Calc Analysis

program modified for this version of the DHQ.

Analytical Methods

Formation of Dietary Groups (Objective 1)

Subj ects who consumed meat were grouped based on their frequency of

consumption of beef (e.g., steak, roast, hamburger, etc) as reported from the DHQ results

(Table 3-1).

Table 3-1. Daily and weekly beef intake frequency
Daily Frequency Weekly Frequency Diet Group
0.286 1.0 2.0 7.0 Frequent Beef
0.0001 0.285 0.0007 1.995 Seldom Beef
<0.0001 <0.0007 Never Beef









If the daily frequency of consumption of these foods was greater than or equal to a

daily frequency of 0.286 (equivalent to > 1 time a week), the subj ects were grouped in the

frequent beef group. If the daily frequency was greater than 0.0001 but less than 0.286

(equivalent to I 1 time a week), subj ects were grouped into the seldom beef group.

Individuals who never consume beef foods but who consumed other types of meat

including seafood were placed in the never beef group. Subj ects were categorized as

vegetarians only if their frequency of consumption of all meat products (i.e., beef,

poultry, pork, mixed dishes, and seafood) was zero.

Identification of Food Groups from the Diet History Questionnaire (Objective 2)

All foods included in the DHQ were categorized into specific food groups (i.e.,

beef, pork/other meat, poultry, dairy, eggs, seafood, cereal, soy, meal replacement, mixed

dishes with meat type unknown, non-dairy fats, beans, rice/pasta, soups/sauces,

breads/cracker/cakes/pies, nuts/seeds, vegetables, fruit, syrup/honey/gelatin/candy, and

beverages). Dietary vitamin B l2 intake was estimated from the following food groups:

(1) beef, (2) pork, (3) poultry, (4) dairy, (5) eggs, (6) seafood, (7) soy, (8) cereal, (9) meal

replacement, and (10) mixed dishes with meat type unknown. The remaining food

groups were grouped together as (1 1) "other". Table 3.2 indicates the types of foods

found within each food group.

Analysis of Additional Nutrients

Total calorie, carbohydrate, fat, saturated fat, and protein intake were determined

using information reported from the DHQ. Total dietary intake of other micronutrients

(folate and vitamin B6) relevant to vitamin Bl2 metabolism also was evaluated. Key

minerals (e.g., iron and zinc) known to be concentrated in beef, but possibly limited in

the diet of vegetarians, also were analyzed.









Table 3-2. Foods categorized within each food grou
Food Group Types of Food Included
Beef Steak, roast, hamburger, ground beef, beef hotdog, beef
sasge, beef cold cuts, mixed dishes made with beef
Poultry All cuts of chicken and turkey (breast, wing, etc), chicken
and turkey cold cuts, ground chicken and turkey
Pork and other meats Ham, pork (all cuts), bacon, ham cold cuts, veal, venison,
lamb, shortribs, liver
Dairy Milk, cheese, yogurt, cream, pudding, ice cream, butter
Eggs and engg mixtures Eggs, mayonnaise
Seafood*
Fish Tuna, fried and not fried fish

Shellfish Oysters, clams, muscles, shrimp, crab, lobster
Cereals**
Highly-fortified All-Bran, Multigrain Cheerios, Complete, Just Right Fruit
and Nut, KASHI Heart-to-Heart, Mueslix, Product 19,
Smart Start, Special K, Total (all types)

Low-fortified Oatmeal, grits, all other types of cereals not listed above
Soy Tofu, soymilk, soy substitute products (Morningstar Farms,
Worthington/Loma Linda), egg substitutes
Meal replacement Power bars, Nutri-grain bars, Balance bar, Zone bar
Mixed dishes with meat Pasta with meat/fish sauce, Mexican food, lasagna, chili or
tyeunknown p izza with meat other than beef
Other Non-dairy fats, beans, rice/pasta, soups/sauces,
breads/cracker/cakes/pies, nuts/seeds, vegetables, fruit,
syrup/honey/gelatin/candy, and beverages
* Seafood was analyzed as one food group and as separate food groups of fish and
shellfish.
**Cereal was analyzed as one food group and as separate food groups of highly-fortified
and low-fortified cereals. Highly-fortified cereals contained >25% of the Daily Value for
vitamin Bl2. Low-fortified cereals contained <25% of the Daily Value for vitamin Bl12.

Plasma Vitamin B12 Concentration (Objective 3)

The plasma vitamin Bl12 concentration of all subj ects was determined using MP

Biomedicals, Inc SimulTRAC-S Bl2/Folate Radioassay kit (Orangeburg, NY). This is a

competitive protein binding assay using cobalt-57 ( 7Co). Dithiothreitol (DTT) solution

was added to the vitamin B l2/folate tracer ( 7Co, 125I, borate buffer with human serum

albumin, dextran, potassium cyanide, dye and preservative). This mixture was added to










the plasma samples and boiled for 15 minutes. The boiling process inactivates

endogenous binding proteins and converts all cobalamin forms to cyanocobalamin. Once

cooled, purified IF was added to the mixture and incubated for 1 hour. During

incubation, endogenous cyanocobalamin and 57Co compete for binding sites to IF. The

mixture was centrifuged. Bound cobalamin (labeled and unlabelled) accumulated at the

bottom in a pellet, while unbound cobalamin was in the supernant. The radioactivity of

the pellet was measured using a gamma counter. Vitamin Bl2 concentration was

inversely related to the measured radioactivity. During the assay, samples were covered

with aluminum foil to decrease exposure to light.

A plasma vitamin Bl2 concentration above 221 pmol/L was considered normal.

Plasma vitamin Bl2 concentrations between 148 and 221 pmol/L were considered

marginally defieient, and a concentration below 148 pmol/L was considered defieient

(66).

Statistical Methods

The statistical analysis was performed using SAS, version 9.1, SAS Institute Inc.

Cary, NC, USA. An initial analysis was conducted to determine whether the four dietary

groups differed significantly with respect to the following demographic variables: gender,

age, body mass index (BMI), ethnicity, marital status, employment status, student status,

and educational level. For the continuous demographic variables, a one-way analysis of

variance (ANOVA) was used to determine whether the mean responses differed among

the dietary groups. A chi-square test was used to determine whether the proportion of

responses in each of the categories differed among groups when considering categorical

response variables. Significant differences in gender, age, BMI, marital status, and

student status were found among the four dietary groups. BMI and gender were highly










correlated. As a consequence, after adjusting for gender, mean BMI did not differ

significantly among dietary groups. Similarly age was highly correlated with student and

marital status. After adjusting for age, neither student status nor marital status differed

significantly among dietary groups. Therefore, only gender and age were used as

covariates in the analysis. For each of the dependent variables, a one-way ANOVA was

conducted with age and gender as the covariates and allowing for differences in variances

among the four dietary groups. Plasma vitamin Bl2 concentration was log transformed

for normality. Reported plasma concentrations are back-log transformed. Pearson's

correlation was used to determine the relationship between vitamin Bl2 intake and

concentration.















CHAPTER 4
RESULTS

Demographic Characteristics of the Study Population

Subj ects

Three hundred eighty-eight subjects were enrolled in the study. After enrollment,

62 of the subj ects were excluded due to admission during the study interview that a

vitamin Bl2-containing supplement had been taken within the past 6 months. Six

subj ects were excluded because they failed to return their completed DHQ. Data from 18

subj ects were not included because the total calorie intake reported from the DHQ was

less than 600 calories or greater than 5,000 calories, which are deemed to be implausible.

Therefore, 302 (137 males, 165 females) eligible subjects were included and the meat

consumers in this group were further categorized into three groups based on frequency of

beef consumption. The dietary beef intake groups were as follows: (1) frequent beef

consumers (n = 97), (2) seldom beef consumers (n = 42), and (3) never beef consumers (n

= 42). Those who excluded all meat products (i.e., beef, poultry, pork, mixed dishes

containing meat, and seafood) were grouped into a fourth dietary group referred to as the

vegetarian group (n = 121). The vegetarian group consisted of lactoovovegetarians (n =

66), lactovegetarians (n = 47), and vegans (n = 8).

Demographic Characteristics

The demographic characteristics of the study population are presented in Table 4-1.

The mean age and BMI (kg/m2) (mean & SD) for all the participants were 25 A 14 years

and 24 & 9, respectively. The mean education level was 15 & 5, which is equivalent to







50


the junior level at a university. Gender and age were significantly different (p<0.0001);

as were martial status (p = 0.02), student status (p = 0.001), and BMI (p = 0.03) among

the four groups.

Table 4-1. Demographic characteristics of dietary groups"
Demographic Frequent Beef Seldom Beef Never Beef Vegetarian p-value
Variable (n = 97) (n = 42) (n = 42) (n = 121)
Age2 23.2 & 6.1 23.7 & 5.1 26.0 & 7.2 28.1 & 8.8 <0.0001
BMI2,3 24.5 & 5.4 24.0 & 3.7 22.7 & 2.9 23.0 & 4.3 0.0333
Education level 14.6 & 2.1 15.1 & 2.7 15.2 & 1.7 15.3 & 2.2 0.1336


55
45

24
56
5


Gender (%/)4
Female
Male
Ethnicity (%)4
Asian
White
African
American
Hispanic
Other
Marital status (%)4
Single
Married
Student status (%)4
Full-time
Part-time
Not a student
Employed (%)4


<0.0001


0.1039


0.0177


0.0007



0.2484


Yes 56 60 74 63
No 44 40 26 37
Leans expressed as mean a standard deviation (SD).
20ne-way ANOVA was used for statistical comparisons between groups.
3Body Mass Index (BMI) calculated as kg/m2.
4Chi-square test was used for statistical comparisons between groups.
5Due to rounding, percentages will not always sum to 100.

Dietary Vitamin B12 Intake (Objectives 1 and 2)

Total vitamin B l2 intake (mean + SD) among the four dietary groups is presented

in Table 4-2. Vitamin Bl2 intake is expressed in this table as total vitamin Bl2 intake

(Cpg/day) consumed and as Cpg/1000 calories consumed. Unadjusted for calories, total

vitamin Bl2 intake in the seldom beef group was lower (p = 0.001) than the frequent beef

group. Vitamin Bl2 intake in the never beef group and vegetarian group also were lower

(p<0.0001) than the frequent beef group. The unadjusted vitamin Bl2 intake of the never









beef group was lower (p = 0.05) than the seldom beef group. The vitamin B l2 intake

unadjusted for calories in the vegetarian group was lower (p<0.0001) than the seldom

beef group and lower (p = 0.02) than the never beef group.

After adjusting for calorie intake (Cpg/1000 kcals), the never beef and vegetarian

groups had lower (p < 0.0001) vitamin Bl2 intakes than the frequent beef and seldom

beef groups. There were no differences (p = 0.27) in vitamin Bl2 intake (Cpg/1000 kcals)

among the frequent beef and seldom beef groups. The vitamin B l2 intake of the

vegetarian and never beef intake groups did not differ (p = 0.60).

Table 4-2. Daily total dietary intake of vitamin Bl21
Frequent Beef Seldom Beef Never Beef Vegetarian
(n = 97) (n = 42) (n = 42) (n = 121)
Vitamin B l2 (Clg) 8.2 f 4.0 5.9 f 3.52 4.5 f 2.72, 3 3.3 f 2.82,3,4
Vitamin B l2 (Clg/1000 kcals) 3.3 f 1.4 3.7 f 2.2 2.0 f 1.42,3 1.9 & 1.52,3
'Means expressed as mean + SD. One-way ANOVA was used for statistical comparisons between groups
adjusted for age and gender.
2Significantly lower than frequent beef (p<0.05).
3Significantly lower than seldom beef (p<0.05).
4Significantly lower than never beef (p<0.05).

The total vitamin Bl2 intake (Clg) among the four dietary groups was compared to

the Estimated Average Requirement (EAR) as illustrated in Figure 4-1. The frequent

beef group consumed 4. 1 times more vitamin Bl2 than the EAR. The seldom beef group

consumed 3 times more vitamin Bl2 than the EAR. The never beef group consumed 2.3

times more than the EAR, and the vegetarian group consumed 1.7 times more vitamin

Bl2 than the EAR (Figure 4-1).













1E 7.0

d 6.5






> 3.0

S2.0 1I EAR 2.0 tpg/d

S1.0


Frequent Beef Seldom Beef Never Beef Vegetarian

Dietary Group

Figure 4-1. Mean vitamin B l2 intake compared to recommended intakes. EAR (2.0
Cpg/day) is based on recommendation for non-pregnant, non-lactating healthy
adults .

The daily mean vitamin Bl2 intake (Cpg/1000 kcals) contributed by various food

sources is presented in Table 4-3.

Beef, Poultry, Pork, Seafood, and Mixed Foods

Among meat consumers (i.e., frequent beef, seldom beef, and never beef groups),

vitamin Bl2 from beef in the seldom beef group was lower (p<0.0001) than vitamin Bl2

from beef in the frequent beef group. Vitamin Bl2 from poultry in the never beef group

was lower (p = 0.004) than vitamin B l2 from poultry in the frequent beef group and

lower (p = 0.03) than the vitamin B l2 from poultry in the seldom beef group. There were

no differences (p = 0.90) in vitamin B l2 from poultry among the frequent beef and

seldom beef groups. There were no differences in vitamin Bl2 intake from pork or

seafood, including shellfish and fish, among the four dietary groups. Vitamin Bl2 from

mixed foods that contained meat (type undeterminable) in the never beef group was









lower (p<0.0001) than the vitamin Bl2 intake from mixed foods in the frequent beef

group and the seldom beef group. There were no differences (p = 0. 18) in vitamin Bl2

intake from mixed foods among the frequent beef and seldom beef group.

Dairy and Eggs

There were no differences in vitamin Bl2 intake from dairy products among all

dietary groups. Vitamin B l2 intake from eggs in the vegetarian group was lower (p =

0.001) than the frequent beef, lower (p = 0.001) than the seldom beef, and lower (p =

0.0004) than the never beef groups.

Cereal

Vitamin Bl2 intake from all types of cereal was lower (p = 0.01) in the frequent

beef and lower (p = 0.003) in the never beef group compared to the seldom beef group.

The vitamin B l2 intake from cereal (all types) in the never beef group also was lower (p

= 0.03) than that in the vegetarian group. There were no differences in vitamin Bl2

intake from all types of cereal among the frequent beef group and vegetarians (p = 0. 13);

frequent beef group and never beef group (p = 0. 10); and seldom beef group and

vegetarians (p = 0.14). Cereal was further separated to determine differences in mean

vitamin Bl2 intake from highly-fortified cereals and low-fortified cereals. Vitamin Bl2

intake from highly-fortified cereals in the frequent beef and never beef group was lower

(p<0.01) than the seldom beef group. Vitamin Bl2 intake from highly-fortified cereals in

the never beef group also was lower (p = 0.02) than the vitamin Bl2 intake from highly-

fortified cereals in the vegetarian group. There were no differences in mean vitamin Bl2

intakes from low-fortified cereal across all groups.









Fortified Soy Products, Meal Replacements, and Other Sources

Vitamin B l2 intake from fortified soy products in the frequent beef and seldom

beef groups was lower (p<0.0001) than observed in the vegetarian group. Vitamin Bl2

intake from fortified soy products in the frequent beef group also was lower (p<0.0001)

than the never beef group. Vitamin Bl2 intake from fortified soy products in the never

beef group was lower (p = 0.04) than the vegetarian group. No differences were found

for vitamin B l2 intake from meal replacement sources among the dietary groups.

Vitamin B l2 intake from other sources in the frequent beef group was lower (p = 0.03)

than the vitamin Bl2 intake from other sources in the never beef group and (p = 0.001)

the vegetarian group.

Naturally-Occurring and Fortified Sources

Vitamin Bl2 intakes from food sources in which vitamin Bl2 occurs naturally was

compared with vitamin B l2 intake from fortified foods. Naturally-occurring vitamin B l2

intake in the never beef group was lower (p<0.0001) than the frequent beef group and

(p = 0.01) the seldom beef group. The vegetarian group had lower (p<0.0001) vitamin

Bl2 intake from naturally-occurring sources than the frequent beef, seldom beef, or never

beef groups. The frequent beef group had a lower (p = 0.0004) intake of vitamin Bl2

from fortified foods than the seldom beef group, never beef group (p = 0.03), and

vegetarian groups (p<0.0001). Vitamin Bl2 from fortified foods in the never beef group

was lower (p = 0.04) than the vegetarian group.






































































vegetarians (Figure 4-2).


Table 4-3. Daily mean vitamin B l2 contribution by dietary sources (yg/1000 kcals)'


Beef
Poultry
Pork
Seafood
Shellfish
Fish
Mixed
Dairy
Eggs
Cereal
High-fortified
cereal
Low-fortified
cereal
Soy products
Meal replacement
Other
Naturally-occurring
products
Fortified products


Dietary Source


Frequent Beef
(n = 97)
0.94 & 0.5
0.07 & 0.1
0.10 + 0.1
1.00 & 1.2
0.78 & 1.1
0.21 & 0.3
0.08 & 0.1
0.53 & 0.3
0.07 & 0.1
0.25 & 0.53
0.21 & 0.53

0.03 & 0.00

0.03 f 0.14,5
0.08 & 0.2
0.15 f 0.14,5
2.93 A 1.3

0.41 f 0.63,4,5


Seldom Beef
(n = 42)
0.16 & 0.22
0.07 & 0.1
0.14 & 0.6
1.24 & 1.5
0.86 & 1.1
0.34 & 0.4
0.06 & 0.1
0.51 & 0.4
0.10 + 0.1
0.75 A 1.2
0.71 + 1.2

0.02 & 0.1

0.12 & 0.3"
0.10 + 0.3
0.18 & 0.1
2.40 & 1.9

1.20 & 1.4


Never Beef
(n = 42)

0.03 0.12,3
0.05 0.3
0.83 1.0
0.58 0.7
0.27 0.3
0.01 0.002,3
0.43 0.3
0.10 0.1
0.15 0.43,5
0.11 0.43,5

0.03 ~0.1

0.24 0.3"
0.06 0.1
0.20 0.1
1.49 1.12,3

0.75 1.0'


Vegetarian
(n = 12 1)








0.51~ 0.4
0.03 0.12,3,4
0.44 1.1
0.41 1.1

0.04 0.1

0.39 0.6
0.13 0.3
0.22 0.2
0.70 0.62,3,4

1.17 1.5


1Means expressed as mean + SD. One-way ANOVA was used for statistical comparisons between groups
adjusted for age and gender.
2Significantly lower than frequent beef group (p<0.05).
3Significantly lower than seldom beef group (p<0.05).
4Significantly lower than never beef group (p<0.05).
5Significantly lower than vegetarian group (p<0.05).

The proportion of vitamin B l2 intake provided by different dietary sources to total

vitamin Bl2 intake (Clg/1000 kcals) is illustrated in Figure 4-2. In the frequent beef


group, the largest contributors to vitamin Bl2 intake were seafood (30%), beef (28.5%/),

and dairy products (16%). Among the seldom beef group, the largest contributors to


vitamin Bl12 intake were seafood (3 3.5%), cereal (20%), and dairy (13.8%). Sources in


the never beef group that contributed the largest proportion of vitamin Bl2 were seafood


(41%), dairy (21.3%), and soy products (12%). Dairy (27%), cereal (23.4%), and soy


products (20.7%) were the largest contributors to vitamin B l2 intake among the











O Beef
a Seafood
g Dairy
9 Cereal
M Poultry
B Pork
aEggs
B Soy products
E Mixed
a Meal Replacement
B Other


Frequent Beef


Seldom Beef


Never Beef



Vegetarian


0% 20% 40% 60% 80% 100%

Figure 4-2. Percent of total vitamin B l2 intake from food sources. Proportions were
determined based on adjusted mean intakes of vitamin Bl2 from each food
source.


Frequent Beef


Seldom Beef


Never Beef


Vegetarian


:


o Highly-Fortified cereal
aLow-Fortified cereal


0% 20% 40% 60% 80% 100%

Figure 4-3. Percent of total vitamin Bl2 intake from cereal sources. Proportions were
determined based on adjusted mean intakes of vitamin B l2 from each cereal
source.

The proportion of vitamin B l2 provided by sub-groupings of foods within a

category is shown in Figures 4.3 and 4.4. Highly-fortified cereals provided the maj ority

of vitamin B l2 derived from cereal sources in all dietary intake groups (Figure 4-3).

Vitamin Bl2 derived from a subgroup of seafood (i.e., shellfish and fish) revealed that
















:


shellfish provided a larger proportion of vitamin Bl2 among the groups who consumed

seafood (Figure 4-4).


Frequent Beef


Seldom Beef


Never Beef


Vegetarian


SShellfish


0% 20% 40% 60% 80% 100%

Figure 4-4. Percent of total vitamin Bl2 intake from shellfish and fish. Proportions were
determined based on adjusted mean intakes of vitamin Bl2 from each seafood
source.

The proportion of vitamin B l2 intake provided from naturally-occurring or

fortified foods is illustrated in Figure 4.5. When the vitamin Bl2 content of foods

consumed was categorized as naturally-occurring or fortified, it was found that naturally-

occurring sources of vitamin Bl2 provided a larger proportion of this nutrient compared

to fortified products in the frequent beef, seldom beef, and never beef groups. In the

vegetarian group, fortified products provided a larger proportion of the vitamin Bl2

content of the diet (Figure 4-5).

























0% 20% 40% 60% 80% 100%


Figure 4-5. Percent of total vitamin Bl2 intake from naturally-occurring and fortified
sources of vitamin Bl2. Proportions were determined based on adjusted mean
intakes of vitamin B l2 from naturally-occurring or fortified food sources.

Dietary Intake of Macronutrients and Micronutrients

Table 4-4 presents the energy and macronutrient intakes (mean + SD) among the

four dietary groups reported from the DHQ. To control for differences in energy intake,

protein, carbohydrate, fat, and saturated fat were standardized and expressed as Cpg/1000

kcals consumed.

Energy Intake

The energy or calorie intake of the seldom beef, never beef, and vegetarian groups

was lower (P<0.0001) than the calorie intake of the frequent beef group. There were no

differences in energy intake among the seldom beef, never beef, and vegetarian groups.

Protein Intake

The protein intake of the seldom beef, never beef, and vegetarian groups also was

lower (p<0.05) than the protein intake of the frequent beef group. The protein intake of

the vegetarian group was lower (P<0.0001) than the protein intake of the seldom beef and

never beef group.





:


Frequent Beef


Seldom Beef


Never Beef


Vegetari an


a Naturally-occurring
| nFortified










Carbohydrate Intake

The carbohydrate intake of the frequent beef group was lower (p<0.0001) than the

carbohydrate intake of the seldom beef, never beef, and vegetarian groups. The

carbohydrate intake of the seldom beef and never beef groups was lower (p<0.05) than

the carbohydrate intake of the vegetarian group.

Fat Intake

Fat intake was lower (p = 0.01) in the seldom beef group and lower (p = 0.0001) in

the never beef group compared to the frequent beef group. Fat intake in the vegetarian

group also was lower (p<0.0001) than the frequent beef group. There were no

differences in fat intake among the seldom beef, never beef, and vegetarian groups.

Saturated Fat

Saturated fat intake in the seldom beef, never beef, and vegetarian groups was

lower (P<0.0001) than the intake of saturated fat in the frequent beef group. There were

no differences in saturated fat intake among the seldom beef, never beef, and vegetarian

groups.

Table 4-4. Daily mean dietary intake of macronutrients (per 1000 kcals consumed)'
Nutrient Frequent Beef Seldom Beef Never Beef Vegetarian
(n = 97) (n = 42) (n = 42) (n = 12 1)
Energy (kcals) 2489 & 1026 1729 & 6502 1856 & 7312 1862 & 809 2
Protein (g) 41 f 9 39 f 72 37 f 92 33 f 92,3,4
Carbohydrate (g) 112 & 213,4,5 129 195 132 & 155 140 & 21
Fat (g) 40 & 7 37 & 82 36 & 52 36 & 82
Saturated Fat (g) 12 f 3 9 f 32 9 f 32 10 f 32
'Means expressed as mean + SD. One-way ANOVA was used for statistical comparisons between groups
adjusted for age and gender.
2Significantly lower than frequent beef (p<0.05).
3Significantly lower than seldom beef (p<0.05).
4Significantly lower than never beef (p<0.05).
5Significantly lower than vegetarian (p<0.05).










Table 4-5 presents the daily intakes of micronutrients (mean + SD) (i.e., folate and

vitamin B6) relevant to vitamin Bl2 metabolism and key minerals known to be

concentrated in beef, but possibly limited in the diet of vegetarians (i.e., iron and zinc).

To control for differences in energy intake among the four dietary groups these nutrients

also were standardized and expressed as Cpg/1000 kcals consumed.

Folate Intake

The frequent beef group had a lower (p<0.0001) folate intake than the seldom beef,

never beef, and vegetarian groups. There were no differences in folate intake among the

seldom beef, never beef, and vegetarian groups.

Vitamin B6 Intake

The frequent beef group had a lower (p = 0.0001) intake of vitamin B6 than the

seldom beef, never beef (p = 0.03), the vegetarian groups (p = 0.001). There were no

differences in vitamin B6 intake among the seldom beef, never beef, and vegetarian

groups.

Iron Intake

The frequent beef group had a lower (p<0.0001) intake of iron than the seldom

beef, never beef, and vegetarian groups. The seldom beef and the never beef groups had

a lower intake of iron (p = 0.003; p = 0.001. respectively) compared to the vegetarian

group. There were no differences in iron intake between the seldom beef and never beef

groups (p = 0.72).

Zinc Intake

The zinc intake of the never beef group was lower than the zinc intake of the

frequent beef group (p = 0.001), seldom beef group (p = 0.002), and vegetarian groups

(p = 0.02).










Table 4-5. Daily mean dietary intake of micronutrients (per 1000 kcals consumed)'
Nutrient Frequent Beef Seldom Beef Never Beef Vegetarian
per 1000 kcals (n = 97) (n = 42) (n = 42) (n = 12 1)
Folate (pLg) 180 & 483,4,5 271 f 85 273 & 93 287 & 107
Vitamin B6 (mg) 1.0 + 0.33,4,5 1.3 0.4 1.2 & 0.4 1.2 & 0.6
Iron (mg) 7.4 & 1.93,4,5 10.4 3.85 10.1 &3.85 13.1 & 7.6
Zinc (mg) 5.6 f 1.8 6.1 f 2.7 4.5 f 1.52,3,5 5.4 f 2.6
'Means expressed as mean + SD. One-way ANOVA was used for statistical comparisons between groups
adjusted for age and gender.
2Significantly lower than frequent beef (p<0.05).
3Significantly lower than seldom beef (p<0.05).
4Significantly lower than never beef (p<0.05).
5Significantly lower than vegetarian (p<0.05).

Plasma Vitamin B12 Concentration (Objective 3)

Plasma vitamin Bl2 concentrations (mean + SD) for the four groups are presented

in Table 4-6. There were no differences in the plasma vitamin Bl2 concentration among

the dietary groups (p = 0.70). As illustrated in Figure 4-6, mean plasma vitamin Bl2

concentration was above 221 pmol/L, the limit set as normal, for all dietary groups.

Table 4-6. Plasma vitamin Bl2 concentrations

Frequent Beef Seldom Beef Never Beef Vegetarian
(n = 97) (n = 42) (n = 42) (n = 12 1)

Plasma vitamin
280 + 114 289 1 114 279 1 139 263 1 143
B12 (pmol/L)

'Means expressed as mean + SD. One-way ANOVA was used on log-transformed means for statistical
comparisons between groups adjusted for age and gender. Plasma concentrations are based on back-log
transformed means.

Vitamin B12 Status

Vitamin Bl2 status was based on plasma vitamin Bl2 concentration and defined as

normal (>221 pmol/L), marginally defieient (148-221 pmol/L), or deficient (<148

pmol/L). The percent of individuals who were considered normal, marginally defieient,

and defieient is shown in Figure 4-7. Vitamin Bl2 status was normal in 74% of the

frequent beef consumers, 74% of the seldom beef consumers, 69% of the never beef









consumers, and 61% of the vegetarians. Vitamin Bl2 status was marginally deficient in

20% of the frequent beef consumers, 21% of the seldom beef, 19% of the never beef

consumers, and 22% of the vegetarians. Vitamin Bl2 status was deficient in 6% of the

frequent beef consumers, 5% of the seldom beef consumers, 12% of the never beef

consumers, and 17% of the vegetarians. There were no differences in the percentage of

individuals who were normal, marginally deficient, or deficient among the four groups (p

= 0. 18). The percentage of individuals who were deficient based on plasma vitamin Bl2

concentration (<148 pmol/L) was approximately two-fold higher in the non-beef

consumers [never beef (12%); and vegetarians (17%)] compared to beef consumers

[frequent beef (6%); and seldom (5%)].






I I I I Normal
E 200-(221 pmol/L)










Frequent Beef Seldom Beef Never Beef Vegetarian
Dietary Group

Figure 4-6. Plasma vitamin Bl2 concentration (pmol/L). Normal vitamin Bl2 status was
defined as a plasma vitamin B l2 concentration greater than 221 pmol/L.















e 3 I EDeficient
3 E# lMarginally Deficient
3 H ENormal






Frequent Beef Seldom Beef Never Beef Vegetarian

Figure 4-7. Vitamin B l2 status among dietary groups. Statistical comparisons were
determined using a Chi square test. Vitamin Bl2 status was defined as
follows: (1) deficient (<148 pmol/L); (2) marginally defieient (148-221
pmol/L); and (3) normal (>221 pmol/L).

The mean plasma vitamin B l2 concentration for the non-vegetarian and vegetarian

groups are presented in Table 4-7. The mean plasma vitamin Bl2 concentration (mean +

SD) was lower (p = 0.01) in the vegetarian group than the non-vegetarian group.

Table 4-7. Plasma vitamin Bl2 concentration among non-vegetarians and vegetarians

Non-Vegetarians Vegetarians
(n = 181) (n = 121)

Plasma vitamin B12 (pmol/L) 313 A 124 280 & 1462


IMeans expressed as mean + SD. One-way ANOVA was used for statistical comparisons controlled for
BMI.
2Significantly lower than non-vegetarians (P = 0.01)

The percent of individuals who were defieient in the non-vegetarian and vegetarian

groups are presented in Figure 4-8. The percent of individuals who were defieient in the

non-vegetarian group (7%) was lower (p = 0.01) than the percent of individuals who were

defieient in the vegetarian group (17%).






64










20









Non-Vegetarians Vegetarians


Figure 4-8. Percent of individuals deficient among non-vegetarians and vegetarians.
Statistical comparisons were determined using a Chi Square test (p = 0.01).
Vitamin B l2 deficiency was defined as <148 pmol/L.

Vitamin B12 Intake and Status

The association between total vitamin Bl2 intake and plasma vitamin Bl2

concentrations for all individuals was assessed. Dietary vitamin Bl2 intake was weakly

but significantly correlated with plasma vitamin Bl2 concentration (r = 0.23, p<0.0001).















CHAPTER 5
DISCUSSION AND CONCLUSION

The focus of this study was to assess vitamin B l2 intake and status in healthy men

and women who consumed beef at variable frequencies and to estimate the relative

contribution of specific foods to vitamin Bl2 intake. Previous vitamin Bl2 intake

assessment has primarily focused on the adequacy of vitamin B l2 intake in individuals

who consume vegetarian diets compared to that of individuals who consume non-

vegetarian diets. Beef, a highly concentrated source of vitamin B l2 and often restricted

in the diets of meat-consumers and vegetarians for health reasons, is hypothesized to be

an important contributor of dietary vitamin Bl2 intake among meat-consumers. Prior to

the current study, the role of beef in contributing to vitamin B l2 intake had not been

explored.

Consumption of beef at least one time a week or more resulted in higher dietary

vitamin Bl2 intake than that observed in the other dietary intake groups. Exclusion of

beef from the diet resulted in lower vitamin Bl2 intake compared to the vitamin Bl2

intake of beef consumers. These data indicate that frequent beef consumption plays an

important role in providing vitamin Bl2 in the diet of meat consumers. Miller et al.

(1991) assessed frequency of intake of other vitamin B l2 containing foods such as dairy,

eggs, and seafood among vegetarians in relation to serum vitamin Bl2 concentration.

They did not, however, assess the relationship between frequency of intake of these foods

and dietary intake of vitamin B l2. Since this study only involved vegetarians, frequency

of beef consumption and the relation to vitamin B l2 intake and status could not be









assessed. Until this current study, the effect of beef intake frequency on total dietary

intake of vitamin Bl2 had not been addressed.

Previous investigations have reported that mean daily vitamin B l2 intake among

meat-consumers was consistently above 2.0 Gig/day (84-86, 97). National data from

NHANES 1999-2000 indicate that Americans of all age groups are consuming well

above the EAR (2.0 Gig/day) for vitamin Bl2 (32). In the current study, daily vitamin

Bl2 intake for all meat consumption groups exceeded the EAR. Data from the present

study indicated that as beef intake frequency increased so did the intake of vitamin Bl2

as illustrated by the fact that the vitamin Bl2 intake among the frequent beef consumers

was 4.1 times above the EAR compared to 2.3 times the EAR in the individuals who

never consumed beef, but included all other animal products.

Analysis of the CSFII 1995 data for men and women indicated that the top three

foods contributing to the vitamin Bl2 intake were mixed foods, milk and milk drinks, and

beef (20). Beef contributes approximately 2.2 Gig/serving of vitamin B l2. Therefore,

when categorizing subj ects based on their frequency of beef intake, as done in the current

study, one might assume that beef would provide the largest proportion of vitamin B l2

among the beef consumers. Unexpectedly, this was not found among all beef

consumption groups. Beef contributed 28.5% among the frequent beef consumers, but

only 4% among the seldom beef consumers. In all three meat consumption groups

(frequent, seldom, and never) seafood and dairy consistently contributed a substantial

percentage of dietary vitamin Bl2 in the diet. Seafood contributed at least 30% (range

30-41%) of the total dietary vitamin B l2 intake and dairy provided no less than 13.8%

(range 13.8-21.3%) of the total dietary vitamin Bl2 intake among these groups.









Differences in data from the current study and the CSFII 1995 data may be attributed to

differences in the categorization of food groups. In the current study, the type of meat

found in the mixed foods determined the category in which it was placed. For this

reason, an independent category for mixed foods was not created in the current study.

In the current study, beef intake among the frequent beef consumers was the only

meat (compared to poultry and pork) that provided a substantial amount of vitamin Bl2

in the diet. In contrast, in the seldom beef group, beef did not contribute a large

proportion to dietary vitamin Bl2 intake. Yoshino et al. (2005) investigated dietary

sources of vitamin Bl2 among Japanese adults irrespective of diet type. It was reported

that seafood, meats, and dairy provided a larger proportion of vitamin Bl2 in the diet

than other sources of vitamin Bl2. Larsson and Johansson (2005) separated their study

population into vegetarians and non-vegetarians in a similar manner as was done in the

current study. All animal products were combined together when assessing the

contribution of vitamin B l2 from dietary sources by these investigators (89). Therefore,

the contribution of dietary vitamin B l2 intake from various animal sources could not be

assessed. In the current study, specific food groups that provided vitamin Bl2 to the diet

were characterized to a greater extent than was done in previous investigations. For

example, the contribution of vitamin Bl2 from different types of seafood (i.e., shellfish

and fish) was determined, as well as, the contribution of vitamin Bl2 from highly-

fortified cereal versus low-fortified cereals. In addition, the overall vitamin Bl2 intake

was characterized as naturally-containing sources or fortified food sources. Among meat

consumers, the vitamin Bl2 provided by seafood was predominately from shellfish; the









vitamin B l2 from cereal was from highly-fortified cereals; and a larger proportion of

vitamin Bl2 intake of meat consumers was from naturally-occurring food sources.

In comparison to beef consumers, vegetarians had a lower intake of vitamin Bl2.

These Eindings are consistent with previous investigations (85, 86). Even with lower

intakes of vitamin Bl2 among the vegetarians, the intake of vitamin Bl2 among the

vegetarian group was 1.7 times above the EAR in the current study. The addition of so

many fortified products in the marketplace that are acceptable to vegetarians has made it

possible for individuals who follow a vegetarian diet pattern to consume enough vitamin

Bl2 to maintain an adequate intake. Several studies that have assessed vitamin Bl2

intake of vegetarian and vegan populations have reported that dietary intake of vitamin

Bl2 may have been low because the nutrient database used to assess intake did not

contain foods frequently consumed by vegetarians or because the nutrient database was

not complete, in that information regarding vitamin B l2 content of all consumed foods

was not included (10, 11). To address this research need, the FFQ used to assess dietary

intake of vitamin Bl2 in the current study was modified to contain vitamin Bl12-fortified

foods that are commonly consumed by vegetarians. Vegetarians get a large proportion of

their vitamin Bl2 from dairy, highly-fortified cereals, and fortified soy products, which

likely explains why low dietary vitamin Bl2 intake in the vegetarian group was not

observed in the current study.

In addition to a low vitamin Bl2 intake, the intake of calories, protein, fat, and

saturated fat were lower in vegetarians compared to frequent beef-consumers. The

vegetarians also had higher intakes of carbohydrates, folate, vitamin B6, and iron than the










frequent beef-consumers. This nutrient intake pattern was similar to what was reported in

the EPIC-Oxford study by Davey et al. (2003).

Beef is a rich source of iron and zinc, therefore, it has been assumed that increased

beef consumption would be associated with a higher intake of iron and zinc. This was not

always true in our study population. Iron was actually lower in all of the meat consuming

groups compared to the vegetarian group. This may occur because a higher proportion of

the vitamin Bl2 intake among of vegetarians was from highly-fortified cereals, products

that are also enriched with iron. Zinc intake did not differ among the vegetarian and the

frequent or seldom beef groups. The group that never consumed beef but consumed

other animal products had a lower zinc intake than the frequent beef, seldom beef, and

vegetarian groups. These findings suggest that in meat consumers, beef may play an

important role in maintaining adequate dietary zinc intake.

Mean plasma vitamin Bl2 concentration in our study population did not differ

among the dietary groups. This is surprising since previous investigations support the

conclusion that vegetarians, especially vegans, have a lower vitamin Bl2 concentration

compared to non-vegetarians (10, 87, 88). It may be possible that our vegetarian

population consumed enough vitamin Bl2 from animal and fortified foods to maintain a

normal plasma vitamin B l2 concentration. NHANES III (1994-1998) indicated that the

mean vitamin Bl2 concentration of all Americans was 382 pmol/L. In the current study,

the mean vitamin B l2 concentration of each dietary intake group was lower than that

observed in this national survey. The means of each group did not exceed 290 pmol/L,

however, this concentration is still considered clinically normal. It is important to note

that conclusions regarding status can not be based on mean values alone since means can









be affected by outliers. It is more appropriate to use the percentage of individuals with

concentrations below the accepted normal range to assess vitamin B l2 status. In the

current study, no significant difference was found among the percent of individuals who

were normal, marginally deficient, or deficient. Although, it is striking that 17% of

vegetarians and 12% of never beef consumers were deficient compared to 6% of the

frequent beef and 5% of the seldom beef groups.

A strength of this study is that supplement users were excluded from participation

making it possible to assess intake of vitamin Bl2 from only dietary sources. This study

also used a modified FFQ that contained a comprehensive number of vitamin Bl12-

containing foods and vitamin B l2-fortified food products commonly consumed by

vegetarians. The addition of these foods allowed for better dietary assessment of vitamin

Bl2 intake. Another strength of this study was that it was possible to directly compare the

frequency of beef consumption to dietary vitamin Bl2 intake. No other study has

grouped subj ects in this manner, therefore previous investigations have been unable to

determine the effect of beef consumption on dietary vitamin Bl2 intake.

Differences within the vegetarian group with respect to the various subgroups of

vegetarian food patterns may be considered a weakness of the study. For example, when

comparing the contribution of vitamin B l2 intake from eggs, the results of the vegetarian

group may be different if the lactoovovegetarian, lactovegetarian, and vegan

subcategories classifications were evaluated separately. Since vegans and

lactovegetarians do not consume eggs, this may be one reason why the vegetarian group

as a whole had a lower vitamin Bl2 intake from eggs compared to the meat-consumers.









The small sample size of the meat-consuming dietary groups is another notable

weakness. This may reflect why no differences were found for plasma vitamin Bl2

concentration. Due to the limitations and current scrutiny of the reliability of the plasma

vitamin Bl2 assay, this method of assessment may not truly reflect vitamin Bl2 status.

Other vitamin Bl2 status indicators that are more reflective of functional status are being

measured by other investigators involved in the current research study and will be

reported separately.

Due to a small sample size among the frequent beef group, seldom beef group, and

never beef group, these groups were pooled to evaluate the relative difference between

non-vegetarian and vegetarian consumers regardless of beef intake. These comparisons

were performed as part of a separate analysis. The mean plasma vitamin Bl2

concentration of the non-vegetarian group was higher than the vegetarian group, this was

not found with the previous four dietary groups. Also, the percent of individuals who

were deficient in the non-vegetarian group were lower than the vegetarians, which again,

were not observed with the four dietary groups. These data indicate that the small sample

size of the beef intake subcategories may explain why no differences were detected in

vitamin Bl2 status among the four dietary groups.

To conclude, this study used a modified FFQ for vitamin Bl2 food sources to

compare vitamin Bl2 intake among beef consumers and vegetarians. The hypothesis that

high dietary intake frequency of beef would be associated with a greater intake of total

dietary vitamin B l2 was supported by the data in this study. Increased frequency of beef

intake was not associated with a higher mean plasma vitamin Bl2 concentration.

However, the percentage of individuals who were deficient among non-vegetarians was










lower than vegetarians. Beef did not provide a larger proportion of vitamin B l2 intake

among all meat consumers. In the frequent beef group, seafood provided a comparable

proportion of total vitamin Bl2 intake as beef. Overall, seafood and dairy provided a

substantial proportion of vitamin Bl2 among meat consumers. Dietetic practitioners and

nutrition educators can use these data to promote beef, seafood, and dairy consumption

for optimizing vitamin Bl2 status through dietary means. Future studies should evaluate

the impact of frequency of intake of seafood and dairy food sources on total vitamin Bl2

intake.















APPENDIX A
SUBJECT DATA COLLECTION FORM

Introduction

I am calling in regard to your interest in our nutrition study; do you have a few minutes
right now?

This is a UF Nutrition department study and involves coming in one morning for about 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? 18-49
Do you smoke? no
Are you pregnant or breastfeeding? no
Do you take any prescription medications other than oral contraceptives? no

If not n ithrin the age range or if they answer yes to any question:
I am very sorry, but you do not meet our exclusion criteria, but thank you for your
interest.

Now I just have a few questions about your diet to see what specific category of our
study you would fit in to. Please answer as best you can, estimates are ok and
consider all instances of when you might eat the items I will ask about, even if only
occasionally.

Do you take a multi-vitamin, complex, red star nutritional yeast, or any other
supplement or additive ever?

If they takettttt~~~~~~~tttttt a naultivitanttt~~~~tttttin,~t B complex, red star nutritional yeast, complete the session
through all diet info but do not record. Conchede by confirming their 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 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.


If the interviewee fulfills all selection criteria continue with the questionnaire, record info
on moderate/non-fortified cereal consumption below.

Do you eat breakfast cereals?
o Yes
o No

Name/Brand Quantity Frequenc





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 Occasionally Frequently Always
(<1 x/mo) (1-4 x/mo) (2-4 x/wk) (5-7 x/wk)
Beef
Chicken
Turkey
Pork
Fish

Cheese
Cow's Milk

Other Dir



















(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/quantit


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










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: Wei ht:

Have you do you currently have any of the following? NO YES
Alcoholism
Anemia
Blood clots
Bronchiti s
Cystic Fibrosis
Dermatiti s
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 pessure
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






















































Are you a full-time or part-time student?
o Full time
o Part time
a Not a student


Are you employed?
0 Yes
0 No
o Student employee


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
one response]


or training that you have completed? [Circle only


Grade school
High school
Technical school or college
Graduate or professional
Don't know X


04
12
16
20+


05 06 07 08


Marital status?


Single/never married
Married
Separated
Divorced
Widowed













Name

M F Last First Middle
Address


Street Apt. #



City Zip, code
Phone


Day Evening. Cell
E-mail


Name of person and phone number to call in case of an emergency if you are invited
to participate in this study:





If we need to contact you, and can not reach you where/with who can a message be
left?


Contact Information


How did you hear about our study?














































GENERAL INSTRUCTIONS
* Answer each question as best you can. Estimate if you are not sure. A guess is
better than leaving a blank.

* Use only a black ball-point pen. Do not use a pencil or felt-tip pen. Do not fold,
staple, or tear the pages.

* Put an Xin the box next to your answer.

* If you make any changes, cross out the incorrect answer and put an X in the box
next to the correct answer. Also draw a circle around the correct answer.

* If you mark NEVER, NO, or DON'T KNOW for a question, please follow any
arrows or instructions that direct you to the next question.

BEFORE TURNING TH E PAG E, PLEASE COMPLETE THE FOLLOWInNG QU ESTIONS.


Today's date:


In what month were
you born?



030 Mar
040 Apr
asO May
060 Jun
070 Jul
080 Aug
090 Sep
100 oct
110 Nov
120 Dec


In what year were
you born?


Are you male or
female?


MONTH


030 Mar
040 Apr
asO May
060 Jun
070O Jul
080 Aug
090 Sep
100 oct
110 Nov
120 Dec


DAY


o1 0

02 02
03 03
04
Os
OS
07
Og
09


YEAR

0 2004
O 2005
O 2oon


APPENDIX B
DIET HISTORY QUESTIONNAIRE



NATIONAL INSTITUTES OF HEALTH


Diet History Questionnaire


02 02
03 03
04 04
05 05
06 06
07 07
08 08



SBAR CODE LABEL OR SUBJECT ID
HERE

|_I_I_I_I__I_I_

















1. Over the past12 months, how often did you drink
tomato juice or vegetable juice?

rC0 NEVER (GO TO QUESTION 2)


] 1 time per day
O 2-3 times per day
O 4-5 times per day
.] 6 or more tirnes per day


1 time per month or less
2-3 times per month
1-2 times per week
3-4 times per week
5-6 times per week


O 1 time per day
O 2-3 tmesper day
O 4 times per day
OBB 6 rrore tim~s per day


1 time per month or less
2-3 times per month
1-2 tirrs per week
3- tires per week
5-6 times per week


la. Each tirne you drank tomato juice or
vegetable juice, how rnuch did you usually
drink?

0 Less than% ?cup (6 ounces)
3 M 10 1 Y cups (6 to 10 ounces)
.] Mcre Itan 1% cups (10 ounces)

Ov~er the oast 1Z morns,~ how often did you drink
orange juice?

SO NEVER (GO TO QUESTION 3)


4 Each Tirne you~ dranre fruil drinks how much
an~d you~ u~sually dn~nk?

O Less than 1 cup (8 ounces)
O 1 to 2 cups (8 to 16 ounces)
O1 More than 2 cups (16 ounces)

4 How often where your fruit drinks diet or
sugar-free drinks?

O Almost never or never
O About % of the time
01 About %/ of the time
O About M~ of the time
D Almost always or always

5. How often did you drink milk, including lactose-
free milk (but NOT reallk suba~sllinlesl as a
beveragle (NOT in coffee. NOT in cereal)?
.Pleasel include chocolate milk, flavored milk like
Ovaltine or Quick, and hot chocolate.)

r- O NEVER (GO TO QUESTION 6)


1 time per month or less
2-3 times per month
1-2 times per week
3-4 times per week
5-6 times per week


2.Each time you dranke orange juice, how
much did you usu~ally drini?

CI Less than ?b at~ (C calncel
02: o1/ c ps (6 to 10 ri e)


3. Over the past12 months, how often did you drink
other 100% fruit juice or '100%6 fruit juice
mixtures (such as apple, grape, pineapple,
grapefruit or -Il sers I?

- O NEVER (GO TO Q~UESTIONI 4)


1 time per month or less
2-3 times per month
1-2 times per week
3-4 times per week
5-8 tirres per week


1 time per month or less
2-3 times per month
1-2 times per week
3-4 times per week
5-6 tires per week


3 1 time per day
.] 2-3 times per day
3 46 times per day
3 6 or more times per day


Sa. Each time you drank milk as a beverage,
how much did you usually drinkI

O Less than 1 cup (8 ounces)
O 1 to 11' .ups 16 ro 12 ounces)
0 More than 1% oups (12 ounces)



Please continue on next pagle.


3a. Each time you drank other fruit juice or fruit
juice mixtures, how much did you usually
drink?

O Less than %,' cup (6 ounces)
O % to 1% cups (6 to 12 ounces)
O1 More than 1r GuPs (12 OunceS)


2'


Over the past 12 months...

4 How often did you drink other fruit drinks (such
as cranberry cocktail, HI-C, lemonade, or Kool-
Aid, diet or regular)',

p-0 NEVER (GO TO QUESTION 5)


O 1 time per day
O 2-3 times per day
O 4-5 times per day
O 6 or more tires per day


O 1 tirne per day
O] 2-3times per day
0 4--5times per day
O 6or morre timKs per day




















5b3. What kind of milk dldyou usually drink?

O] Whole milk (including lacrose-free variety)

O 1 1 mlarn e~rCluel~ng 13ctose-11ee vj..eh*
O] susem enter cr wI- 13r m..u r~~Cema(J ng planes
free variety)

6. How often did you drink a milk substitute such
as say or rice milk as a beverage (NOT in
Cot.r-,~ IlnOT in cereal:~ !Plr aae include milk
substitute used in make other ~Xrflmf eerage suh
chocolate "milk', Ovralline, Quickr and hot
chocolate.)

O NEVER (GO TO QUESTION 7)

O] 1tim per monthor less OC 1 time pe~rday
O 2--3times per moth O 2--3 imesper day
OI 1--2 times per w~eek O] 4-5res per day
O] 5-4times p~her Cee De r moretires
O] 5-6 tmes per week per day

6a. Each time you drank a rnik substituteas a
beverage, how r;ucth didr r(ou uS mally danllk-

O7 Less than 1 cup (8 ounces)
l] 1 10 1%A cups (8 to 12 wounes)
O] More? tan 1'X cups (12 ounces)

6b. Wvhat kdindof milk substitute did you usually
drink?

O Soy milk (8'" Continent)
O Soy milk (Whiltewave Silk)

O Sc. 0 Ik tetlher crans)
O3 Rice milk


Over the past 12 months...

'. How often did you drink real replacement,
energy, or high-protein beverages such as
Instant Breakfast, Ensure, Shmf~ast. A a nr~Eoge,
Boost or others?

- O NEVER (GO TO QUESTION 8)

OI 1 time per month orless O] 1 ime per day
OI 2--3 times per month O] 2--3 imes per day
O] 1-2 times per week O] 4--5 times per day
OI 3-4 times per wlek OI 6or more tunes per day
O 5-e times per week


Ta. Each time you drank meal replacement,
energy, or high protein beverages, how
much did you usually drink?

O] Less than 1 cup (8 ounces)
0 1 to lva cups (8 ta i2 ounces)
O More than 1% cups (12 ounces)

8. Over the past 12 months, u0.0 ?ou drink? soft
drinks, soda, or pop?

-0NO (GO TO QLJES ION 9)

O] YES


8a. How often did you drink soft drinks, seda,
or pop IN THE SUMMER?

C3 NNEVE


1 time per day
2-3 times per day
4-5j tmes per day
6 or more times
per day


1 time per day
2-3 times per day
4-5 times per day
6 or more time-s
pe~rday


80. Each time you drank soft drinks, soda, or
pop, how much :jl:j ,onl usa:IIl, drink,

O7 Less than 12 ounces or less than 1 carl or bottle
OI 12 to Is ounces or 1 can or bottle
O3 More than 16 ounces or more than 1 can or bottle

8d. How often were these soft drinks, soda, or
pop diet or sugar-free?

A 1m~lm1 never nrerO
^ bonur: ;r ll e I~nn
A b.our 3. of Ihe I.me~
Abotlr I 31 the ImP
] I3 L3 *3)- 5I~ Ofr Jln-iy


O 1 time per mnhorm les IS O
OI 2-3 tim~s per month ]
0 1-2 times paBer wek
O1 30timgs per week
O] 5 times per wee


8b H-ow often did you drink soft drinks, soda,
or pop DURING THE REST OF THE YEAR?

O NEVER


O3 1 time per month or less O
O] 2-3 tlmesper month ]
O 1-2 tim~s per weekB O
OM 3-timesper week
O 5-6 times per week



















8e. How often were these soft drinks. soda, or
Pop caireine-free, ISee Iera of caffeine and
caffeine-free sodes provided by the
researcher,)

OI Almost nCeve or never
O About % of the? time
OI About % of the time
O3 About % of he! time
O] Almost alwsys or always

9, Over the past 12 months, did you drinkt beer?

-O NO (GO TO QUESTION 10)

OI YES


Sa ow often did you drink beer INI THE
SUMMER?

O3 NEVER

O 1time per month or lss O 1timeper day
O 2-3 times prrnonth O ^-5n wa~rs pr1!
O 1-2times pe~r ek O7 -> emes er :20,
O7 3-4times per week De cr more times
OI 5-6 times perumek per day

9b. How often did you drink beer DURING THE
REST OF THE YEAR?

O NEVER

O 1time permonth or lss O] 1 me perday
] 2-3 times per month O 2-3 homes per day
O 1-2 imes perrgl wee O -5 Omes perday
O 3-4 tmes permkl De Bor more tlnes
O 5-6 emes per veek per day

90. Each time you drank beer, how much did you
usually drnnkO

[7 Les Ihain a 12-oune can or bottle
0 1 loi 3 12cunce cans or bottles
O More than 31I2-ounce cans or bottles


Over the past 12 months...

10. How often did you drinkt wine or wine coolers?

O NEVER (GO TO QUESTION 11)

0 1 time per month or lss O 1 time per day
I 2-3 tlmes per month ] 2-3 timesper day
1 1-2 times per week U 4-6 times p~r day
] 3-4tlmes perweekt De cr moretlmes
OI 5- times per week perday

10a. Each time~ you drank wine or wine coolers,
how much did you usually drink?

O Less than 5 ounces or leas than 1 glass
3 l) r12 cu :n:.es c~r l i: 2 glarses
SMore than 1; OulnCeb or more thRn L glasses5

11 How often did you drinks liquor or mixed drinks?

-O NEVER (GO TO QUESTION 12)

OI 1 timeper moth orless O 1time per dy
OI 2-3 times permonth O 2-3 timesper day
I 1-2 times perwpeek n 4-5 tmesper day
O] 3-4 timrs per week 0 6or moretimes
OM ~l-times per week per day

11a. Each time you drank liquor or mixed drinks,
how much did you usually drink*?

_] LPss inan 1 5hot or hq4uer
3 I I:; 35r,::rs nf liquor
Oj More than a shots of liquor

12. 0.(3ie Tre cnst 12 rinanres did you eat oatmeal,
grits, or other cooked cereal?

O NO (GO TO QUESTION 13)

["] YES


12.How often did you eat oatmeal, grits, or
other cooked cereal IN THE WINTER:,

OJ NEVER

O1 1-s ames per winter 2 times per week
C] 7-11 omes per er r I 3-4 times perweek
[ 1 time per month |] S timesper week
O7 2- times permonth 0 1 timeper day
O 1 time per week O 2 or moretim~es
per day



















12b How often did you eat oatmeal, grits, or
other cooked cereal DURING THE REST
OF: THE YEAR?

O3 NEVER


13c How often was the cold cereal you ate? any
other type of cold cereal (such as Corn
F~jines, PIce ahSpie Frostfed = lakTS Fruit
Loops, Capn Crunrch, nr rnersl'o

] AlmCFi newerI L' never
3 abour t f 11;9 I~lnp
.] ;tbout :: 3f the time
3 irowl I of the time~
O] Almost always or alw~ys

13d. W~as milk or a milk substitute added to your
cold cereal?

-O NO (Go To ouESTION 14l)

CO YEs
13e. What kind of milk or milk substitute wars
usually added?

II Whnle milt 1100:~1.I"Cing -lcae-tre 3ar~ety)
O 1% Ilal mi~lk Irl.nluding.( lact::se rrs ar.@,4ty

I 6r j- con ~;t.; c
3 o ll a~lpvave Silk)





13f Each time milk or a milk substitute was
added to your cold cereal, how mU~h was
usually added?


S O ::r~s er 1 c upD

14 How oilsol de jou~! ear applesauce~

""' O NEVER (GO'O QUESTIOhi lS)


O3 1-6 times peryear
OI 7-11 times peryear
O1 1 time per month
O] 2-3 tlmespermonth
OI 1 time per welek


2 times per week
9- tirres per week
Er6 times per week e
1 turne per day
2or rare hmes
per day


12c. Each time you ate oatmeal, grits, or other
cooked cereal, how much did you usually
eat?

O Less than% lcup
U % to 1% cups
O IMorethan 1% cups

13. How often did you eat cold cereal?

O- C NEve (Go To ouEsrION 14)


1-6 mes per year
7--11 times per year
1 time per month
2-3 times per month
1 time per week


13a Each time you ate cold cereal, how rnuch
did you usually eat?

O Less than 1 cup
01lto2%cups
O] More than 2Yr cupa

13b, How offen was the cold cereal you ate a
fortified cereal? (See list of fortified
cereals provided by/ the researcher )

OI Almost never or never
O About % of the time
OI About of the time
| | About %/ of the time
OI Almost always or always


14 times per year
7-11 lirnes per year
1 time? per month
2-3 times per month
1 time per welek


2times per wegek
3-4 times per weekb
5-6 times per week
1 time? pe~r day
2 or more! times per day


Please continue on next page.


O 2 times per week
I] 3-4 times per wesek
13 5-8 times per week
O3 1 tm per day
O 2 ormore tim~ses pr ay



















14a. Each time you ate applesauce, how much


OI Less than Yz eup
0%iX o cup
J M~ore than 1 cuP

15. How often did you eat apples?

I O NEVER (GO TO QUESTION 1S)

OI 1-e emers p~r yer OI 2 times per wreek
O1 7--11 times per year OM times per week
O7 1 time per month O 5-6times per w~eek
O] 2-3times permonth O 1 time per day
07 1 time per week ] 2or more times per day

15a. Each time you ate apples, howv many~ dld you
usually eat?

O7 Less than 1 apple
31 apple
O] More than 1 apple

116. How often did you eat pears (fresh, canned, or
froze nP

O NEVER (GO TO QUESTION 17)

O] 1-6 times peryear 1 2 times per wesk
O] 7-11 times per year O 3-4 times per week
OI 1 time per month U 5-8 times per week
OI 2-3 times per month O ime per day
O] 1 time per wpee O 2or mor timess per day

16a. Each time you ate pears, how many did you
usually eat?

OI Less than 1 pear
0 1pear
]3 More than 1 pear

17. How often did you eat bananas?



O 1-e tmes per year C] 2 times per week
] 7-1 times per yeart OI 5- times perweek
O 2E-3times permonth O 1 time per day
O] 1 turne per Week O 2 or more tunes pr daY


Over the past 12 months...

17a. Each time you ate bananas, how many did
you usually eat?

[7 Lesrs thiin 1 banana

0 More Ianar banana

18 How often did you eat dried fruit, such as prunes
orraisins (not including dried apricots)?

O NEVER (GO TO QUESTION 19)

[I 1-6 tmes per year 0 times per we
II 7-11 res per year I 3-4 times per week
O 1 eme per month OR E-tlmes perweekl
O] 2-3 times permonth C] 1tlme perday
OI 1 time per week OI 2 rmore bres per day


18a. Each time you ate dried fruit, how much did
you usually eat (not including dried apricots)?

U Less. rthan I lablespocr~s
70 2r eep:0
O More thrain S tablespoons

9, Over the past 12 months, did you ~eat peaches,
nectarines, or plums?

- O] NO (GO TO QUESTION 20)
OI YES


19a. How often did you eat fresh peaches,
nectarines, or plurns WHEN IN SEASON?
(See list for description of "in season" )

[I NEVER

O1 1-e times per season O 2 times per week
] 7-11 tmes per season OI 3-4 tim-s per week
3 1time per mnth O7 $-times per week
3 2--3 tmes per rnonth O3 1 fime perday
O] 1time per ~eek O7 2or more times
per day

19b. How often did you eat peaches, nectarinels,
or plums (fresh, canned, or frozen) DURING
THE REST OF THE YEAR?

O NEVER

O] 1-6 times per year 0I 2 times per weekt
O] 7-11 times per year O 3- times per weepk
OI Irn~ Fr meretr O 5 times per week
OI i-: na-uw for **:**In 0 1 tim per day
O 1 t ne perweek 0] 2or moreImess
per day




















190. Each time you ate peaches, nectarines, or
plums, how muclih Jlra ,oe a s salI, eaPi

OI Less than 1 fruit or less than %Xcup
0]1 to 2fruilts orYXto ~CUP
J Mlore than 2 fruits or more than Y1 cup

20. How, often did you eat grapes?

[- O NEVER (GO TO QUESTION 21)


C] 2tlmes pe~r wee
03 3-4 ties per wek
C] 5- trrs per week
0] 1 time per day
03 2or more times per day


1-6 times pert ytar
7-~11 times per year
1 time per flanth
2-3 time~ per month
1 time~ per week


20a. Each time you ate grapes, how much did
you usually eat

OI Less than % cup or less than 10 grapes
0 % to 1 cup or 10 to 30 grapes
3 More than 1 cup or more than 30 grapes

!1. Cser the pasI 1; mon~lhs, did you eat
cantaloupe?

-- O] NO (GO TO QUESTION 22)

O7 YES


21a H-ow often did you eat fresh cantaloupe
WYHEN IN SEASON? (See list for description
of "in Seasoff.)

[3 NEVER


5 1-8 times per season
3 7-11tmes per season
D 1tim e per month
I 2-3 tmes per month
D time per week


2 tires per vegek
M4 times per week
5-B times per week
1 time per day
2 or moem times
per day


22b. How often did you eat fresh or frozen
melon, other than cantaloupe (sU~ch as
watermelon or honek-ewI DURING THE
REST OF THE YEAR?

] NEVER


1-6 times per season
7-11 times per season
1 time per month
2-3 ties per onth


2 times per wneek

1 trnes per seeK
- rarne ere tmek

p ca s


2 turneS per weegk
3- tunes per week
5--6 times pr weekL

20r rare times
per day


21b. H-ow oftendid you eat fresh or frozen
cantaloupe DURING THIE REST OF
THE YEAR?

O NEVER


22c Each time you ate melon other than
cantaloupe, how rnuch diclyou usually eat7

C] Less than % cup or 1 small wedge
O] Mto 2 cups or 1 medilum wedge
[7 More than 2 cups or 1 large wedge


1-6 turnes per ye~ar
7-11 times per year
1 time per mnth
2-3 times per month
1 time per week rl


2 times per week
3--4 times per weekh
5-6 times per we~ek
1 time? per day
2 or more times
per day


Over the past 12 months...

210. Each time you ate cantaloupe, how much
did you usually eat?

[7 _esrs thiin Y4 melon or less than % cup
Ot, Irl31; jlrtoloUP
[7 Mcre man~ % melon or more than 1 cup

22, Over the sast 12 months, did you eat melon,
other than cantaloupe (such as watermelon or




O YES


228. ow often did you eat fresh melon, other
than cantaloupe Isuch; ap wstermelon or
nor~ewen]* WHEN IN SEASON7 (SEIE liSt for
description of "in season .)

0 NEVER


O 1-e t es per year 0 ]
C] 7-11 times per year II
[7 2-tlmen per mn th O
0 1 time per week O



















23. Ower Ihe pasI 1, rnonits, 113 ,ol, en
strawberries'





23a How often did you eat fresh strawberries
WYHEN IN SEASON' I (See InsI ror dtesonption of
"in season .)

OIC NE c oo~ro 4I]NVER


07 2times per wteek
On 3tirnes per weekl
On 5-times per wee~k
[I 1 time per ay
O 2 or mcrre ames
per day


1-6times per season
7--11 times per season
1 time per month
2--3tlmes peFr month
1 time per week


2times per week
3-4 timers per week
5-6 tirnes per week c
1 time per day
2 or more times
per day


24c, Each time you ate oranges, tangerines, or
tangelos, how many did you usually eat?

U Less than 1 fruit
O fruit
0 Mdore than 1 fruit

25. Over tree cast 12 months, did you eat grapefruit?

-O NO (GO TO QUESTION 26)
YES


25~a Ho fren did ,nou ER fresh grapefruit
WHENI IN SEASON', ISee lst lor aesolpl~on
of "in season .)

O7 NEVER


23b. How often did you eat fresh or frozen
strawberries DURING THE REST OF THE
YEARR

O] NEV ER

OI 1-Gtimes per year OI 2 times per week
C 7-11 times per year O 3-4times per week
O 1 time permonth O] 5-6times per Reek
O 2-3times per month O17 time per day
0J 1 time per week O] 2or mor times
per day

23c. Eia:l 1teri youI ale- strawberries, how rnuch
did you usually eat?

O7 Less that'. a cp ori les E n 3 berries
[7 '.. I:1 cup~ r .. Ic ? be~res
O More rhan~ -'.:ur> or more than 8 berries

24. Over the past 12 months, did you eat oranges,
tangerines, or tangelos >

-0No (Oo To ouESTION 25)
YES


24a. How oft en did you eat fresh oranges, tangerines
or tangelos WHEN IN SEASON? (Se~e list for
(12b:rilan(" of "in season''.)

OI NEVER

O] 1-etimes per season 1 2tirnes per week
O] 7-11 times perseason O 3-4 times per woeek
O 1 time per month O7 5-times per wele
O] 2-3timesper month 01 Itim per day
O 1 Itime per week 7 20r moreemes
per day


O1 1-6 tmes per season
3 7-11 times per season
S 1time per month
3 2--3 tmes per month
O 1 ime per week


O 2 times per weerk
O SA times per ~eek
O M -itimes per wee
CI 1 time per day
O 2 or more times
per day


Over the past 12 months...

24tt How often did you eat oranges, tangerines,
or tangelos (fresh or canned) DURING: THE
RES OF THE YEAR?

II NEVER


01 1--6 times per year
O 7--11 times per year
OI 1 time per month
O 2-3 timets per month
O3 1 time per werek


25b. How 3rien aid~ you ear grapefruit rlresno~r
conned)l DURING THE REST OF THE YEAR?

O3 NEVER

O 31-6 tmes per~ yea 02tlmes per wee!k
OI 7-11 times per year O 3-4 terrs per week
O 1 time: per nonth II sa times per week
O 2-3 timses em onth O] 1time per day
O] 1 time per week C] 2or rare tmes
per day

25c Each time you ate grapefruit, how much did
you USUally eiat?

[7 LIs~s r+,jn ngrapefruit

O MOre inan H grapefrmit



















26. How often did you eat other kinds of fruit such
as pineapple, mangoes, blueberries, or others',

O NEVERW (GO TO QUESTION 27)

O7 1-G emes pr year O 2 times per wee
OI 7-11 timersper yer O 3-4 tunes per week
07 1time per month O 5-6 tims cprer eek
C] 2--3tims per month O ime per day
[I 1 time p~e~rweek OI 2 ormore tmes per day

26a. Each time you ate other kinds of fruit, how
much did you usually eat?

3 Less than '/ cup
3%~ to %cup
3 Uorethan % cup

27. How often did you eat COOKED greens (such as
spinach, turnip, collard, mustard, chard, or k~ale)?

C O NEVER (GO TO QUESTION 28)

O] 1-6times peryear O 2 timts per wek
O] 7-11 times per year O] 3-4tires per week
OI 1 time permonth OI 5-6 tmes pwer ek
OI2-3times permonth O 1time per day
O] 1 time per week O] 2 ormore times per aY

27a. _arl~ Ilme you a~r COOKED greens, how


O Less than tS cup

3 UOeithaun l cup

28. How often did you eat RAW greens (such as
spmnach, turnip, collard, mustard, chard, or k~ale)?
(We will ask about lettuce later)

O NEVER (GO TO QUESTION 29)

O 1-e ornes er year ] 2 times per week


C] 2--3 tues permonth 1 time perday
OI 1 time per gelek O 2 or more times per day

28a, E~ach time you ate RAW greens, how much
did you usually eat?

O Leas than Ye cup

SAot ethcan1 cup


Over the past 12 months...

29. How often did you eat coleslaw?

O NEVER (GO TO QUESTION 30)

OI 1-e times per yar 0 2 tim~s pe~r we
O 7-1times per year 3--4 times per week
O timne permonth O 5j--6 tmes perweek
C] 2-3times permonth 1 Itlmeper day
O 1 time per weeke [I 2 omorE mers pe~rday

29a. Each time you ate coleslaw, how much did
you usually eat?

CH roe 7.cn:;ap
O More I~~n *A canR

:0How often did you eat sauerkraut or cabbage
(other than coleslaw)?

O NEVER (GO TO QUESTION 31)

] 1-6 times per year O] 2 tlmes per week
II 7-11 times tperyear [I] '-r times per wheek
0 1 tim per month O] 5~--time per week
O 2--3timesper month O 1 time per day
O] 1 time perrrweek [I 2 ormore tmes perday

30a. Each time you ate sauerkraut or cabbage,
how much did you usually eat?



S O rescr dtan 1 cup
31. How offen did you eat carrots (fresh, canned, or
f rozen)?

0 NEVER (GO TO QUESTION 32)

O] 1-6 timesper year C] 2 -times per mekl

O 2-3 times per month [I time perday
O] 1 time per week ] 2 omore bres per day

31a. Each time you ate carrots, how much did
you usually eat7

O7 Lrss rhan ** cup or less than 2 baby canrots
El :. in :' .lu ::r 2 to 5 baby calrrts
[7 twice dhan : cup or more than 5 baby carrots



















32. How often did you eat string beans or green
beans (fresh, canned, or frozen)?

-O NEVER (GO TO QUESTION 33)

O] l-e eimes pe~yr ye3 2 times per w~eek
O7 7-11 times per yer O] 3-timrs per wele
OI 1 tame per month O7 5--6 tmes per week
0I 2-3times permonth O7 time per day
OI 1 time perr wtee OI 2 ormore times per day

32a. Each time you ate: string beans or green
beans, how much did you usually eat?

O] Less than Wj cup
Os]' to 1 cup
O More than 1 cup

-1.How often did you eat peas (fresh, canned, of
frozen)?

O NEVER (GO TO QUESTION 34)

0I 1-6 imes per year OI 2 times per week
O 7-11 times per yer O 0 times per week
O] 1tim per month O] 5-8times per week
] 2-s times pr m month O time perday
O7 1 time pe~rweeke O 2 or more times per day

33a. Each time you ate peas. *x:-w mru::t candc rou
usually eat?

O1 Less than M cup
]% int % cup
O] More than%3/ cup

:4.Over the past 12 months, did you eat corn?

O NO (GO TO QUESTION 35)

YES

34a, How oft en did you eat fresh corn WVHEN IN
SEASON? (See list for description of in
season" )

O] NEVER

] 1-6 tlmrs per season O] 2 tmes per week
O3 'f-11 (res per seas~n O 3--4 timpes peree
O] 1trme per moth On t-Bimes per week
O] 2-3 times pe~rmonth O 1 time per daY
O] time per week O] 2or more ores
perrday


Over the past 12 months...

34tt How often did you eat corn (fresh, canned, or
frozen) DURING THE REST OF THE YEAR?

C3 NEVER

O 1-e times per yar O 2tirres prlerek
[7 7-11 times per year I -4 times per week
O 1ti~p~me pr oth r] 5-6tim~s per week
O 2-3 times per mnth 3 Itim per day
0 1 timrnper weekt 7 2or mlor times
per day

34c. Each time you ate corn, how much did you
usually eat?

O7 Lwe rhan 1 euar er less than M cup
Uc1 a. or; I 1 1 cup
O M~::re II an 1 ear or more than 1 cup


35. Over the past 12 months, how often did you eat
broccoli (fresh or frozen)?

0 NEVER (OO TO QUESTION 36)

C] 1-6 times per yer O3 times per mgek
II 7-11 tirres per year OI 5- timrs per week
0 1tim per month O 5- times per week
O] 2-3 tlmes pr nmonh O] 1tlnm per lay
O] 1 time per week C1 2or more bres per day

35a. Each time you ate broccoli, how much did
you usually eat?

El Less rn % Cup
U More than 1 cup

:i.How often did you eat caulifower or Brussels
sprouts :rert~r~or frozen)?

NEVER (OO TO QUESTION 37)

O 1-6 times per year O 2 times per week
O 7-11 times pr year 0 3S-4times per week
O 1timre per month O] 5-times per wek
OI 2- times per month C] 1timreper day
O 1 time perrrweek O 2 ormore mes perday

36a. Each time you ate caulAlower or Brussels
sprouts, how much did you usually eat?

O L'ErtjnVanLcp
C1erellan :c up