AN ABSTRACT OF THE THESIS OF

AN ABSTRACT OF THE THESIS OF

Wen-Shan Chou Chen for the degree of Master of Science in

Nutrition and Food Management presented on May 13, 1992.

Title: Effect of Glycosylated Vitamin B-6 Intake on the Excretion of Vitamin

B-6 in Women

Abstract approved:

Dr. Lorraine T. Miller

The effect of dietary glycosylated vitamin B-6 on the bioavailability of vitamin B-6 was determined in 4 women. A 44-d metabolic-balance diet study was divided into a preliminary 8-d adjustment period followed by two 18-d experimental periods. The subjects were divided into two groups in a crossover design to compare the effect of low- and high-glycosylated vitamin B-6 diets on the bioavailability of vitamin B-6. The total vitamin

B-6 content in the low- and high-glycosylated vitamin B-6 diets was 1.506 mg (8.91 nmol) and 1.897 mg (11.22 jimol), respectively, in which 11 and

22% was the glycosylated form, respectively. Daily 24-h urine specimens were collected by each subject throughout the study; 7- or 8-d fecal collections were made at the end of each experimental period. The four subjects' mean urinary total vitamin B-6 excretion during the low- and high-glycosylated vitamin B-6 periods was 0.76 ± 0.20 and 0.67 ± 0.06

Hmol/24 h, respectively; fecal total vitamin B-6 excretion was 2.98 ± 0.43 and 4.56 ± 0.87 p.mol/24 h, respectively. Expressed as % of total vitamin B-6

intake, the mean urinary total vitamin B-6 excretion was lower during the high-glycosylated vitamin B-6 period (6.0 ± 0.8%) than during the low- glycosylated vitamin B-6 period (8.5 ± 2.4%); in contrast, their mean fecal vitamin B-6 excretion during the high-glycosylated vitamin B-6 period

(40.7 ± 8.2%) was greater than the low-glycosylated vitamin B-6 period (33.6

± 5.4%). In addition, approximately 11% of ingested glycosylated vitamin

B-6 was excreted in urine. These results suggest that dietary glycosylated vitamin B-6 is not completely bioavailable to humans, and the extent of its utilization is not affected by dietary glycosylated vitamin B-6 intake.

Effect of Glycosylated Vitamin B-6 Intake on the Excretion of Vitamin B-6 in Women by

Wen-Shan Chou Chen

A THESIS submitted to

Oregon State University in partial fulfillment of the requirements for the degree of

Master of Science

Completed May 13,1992

Commencement June 1993

APPROVED:

Professor of Nutrition and Food Management in charge of major

Head of Department of Nutrition and Food Management yfin. if IMTTV^-VH*

1

Dean of Graduate bchool

T

Date thesis is presented May 13,1992

Typed by Wen-Shan Chou Chen

TO MY FAMILY

ACKNOWLEDGEMENTS

Many people have helped me to accomplish this degree. I would especially like to thank Dr. Lorraine Miller who gave excellent guidance, constant support and encouragement, not only in my thesis but throughout my graduate studies. Her patience and valuable time spent on this thesis are gratefully appreciated.

My thanks are extended to Dr. Margy Woodburn for her suggestions on my thesis and advice throughout my graduate studies. Her hospitality in inviting me to her house for Thanksgiving is unforgettable.

I also thank my committee members. Dr. Lorraine Miller, Dr. Ann

Messersmith, Dr. James Leklem and Dr. Samuel Vuchinich, for their time and suggestions given in my thesis.

My appreciation is also extended to my fellow graduate students,

Christine Hansen, Xu Wang and Nancy Dunton, for their kind assistance and cooperation in conducting this human diet study and for their friendship throughout my graduate program. Karin Hardin's help in teaching me to do assays and the friendship of Daisy Wan, Ean-Tun Liaw and Jong-Neng Shieh are also appreciated.

Finally, I would like to thank my husband, Yuan-Hsin Chen, and my parents and parents-in-law for their financial and emotional support during the process of completing this M.S. degree.

TABLE OF CONTENTS

INTRODUCTION 1

REVIEW OF LITERATURE 3

Vitamin B-6 3

Biochemical Functions 3

Recommended Dietary Allowance and Actual Dietary Intake 8

Transport and Metabolism 9

The Findings of Glycosylated Forms of Vitamin B-6 11

The Distribution of Glycosylated Forms of Vitamin B-6 in Foods 14

Absorption of Pyridoxine-glucoside 17

The Bioavailability of Vitamin B-6 18

Studies of Glycosylated Vitamin B-6 Bioavailability 19

Animal Studies 19

Human Studies 21

Other Factors Affecting Vitamin B-6 Bioavailability 23

Effect of Fiber 23

Effect of Food Processing 24

MATERIALS AND METHODS 26

Subject Selection 26

Experimental Design and Diets 28

Sample Collection and Preparation 34

Analytical Methods 35

Statistical Analysis 38

RESULTS 39

Subject Profiles 39

Dietary Intake of Total, Glycosylated and Free Vitamin B-6 39

Urinary Excretion of Total, Glycosylated and Free Vitamin B-6 41

Fecal Excretion of Total, Glycosylated and Free Vitamin B-6 46

Total, Glycosylated and Free Vitamin B-6 Excretion in Urine and Feces 46

DISCUSSION 51

SUGGESTIONS FOR FUTURE STUDY 60

SUMMARY AND CONCLUSIONS 61

BIBLIOGRAPHY 62

APPENDICES 70

LIST OF FIGURES

Figure Page

1. Major naturally occurring forms of vitamin B-6 and its metabolite, 4-pyridoxic acid 4

2. Metabolic interconversion of the B-6 vitamers in human liver 5

3. Structure of pyridoxine-£-glucoside 12

4. Structures of pyridoxine-C-glucoside derivatives 13

5. Experimental design and the schedule of sample collection 28

6. Mean daily urinary excretion of total, free and glycosylated vitamin B-6 as a percentage of total vitamin B-6 intake during the adjustment, low- and high-glycosylated dietary periods 44

7. Urinary vitamin B-6 excretion by each subject and group means in response to low- and high-glycosylated vitamin B-6 diets as a percentage of total intake of vitamin B-6 44

8. Urinary % glycosylated vitamin B-6 excretion by each subject and group means in response to low- and high-glycosylated vitamin

B-6 diets 45

9. Mean daily fecal total, free and glycosylated vitamin B-6 excretion as a percentage of total vitamin B-6 intake during the low- and high-glycosylated dietary periods 48 lO.Fecal vitamin B-6 excretion by each subject and group means in response to low- and high-glycosylated vitamin B-6 diets as a percentage of total intake of vitamin B-6 48

11.The amounts of daily total, free and glycosylated vitamin B-6 excreted in both urine and feces from four subjects, expressed as a percentage of total vitamin B-6 intake, during the low- and high-glycosylated dietary periods 50

LIST OF TABLES

Table Page

1. The summary of enzymatic reactions in amino acid metabolism utilizing pyridoxal 5'-phosphate (PLP) 6

2. Percentage of glycosylated vitamin B-6 in plant foods and their processed products 15

3. Description of the subjects 27

4. Adjustment diet 30

5. Experimental diet: low-glycosylated vitamin B-6 diet 32

6. Experimental diet: high-glycosylated vitamin B-6 diet 33

7. Total, glycosylated and free vitamin B-6 in the adjustment, low- and high-glycosylated vitamin B-6 diets 40

8. Total, glycosylated and free vitamin B-6 excreted in urine during the adjustment, low- and high-glycosylated vitamin B-6 dietary periods 42

9. Total, glycosylated and free vitamin B-6 excreted in feces during the low- and high-glycosylated vitamin B-6 dietary periods 47 lO.The amounts of total, glycosylated and free vitamin B-6 excreted in both urine and feces during the low- and high-glycosylated vitamin B-6 dietary periods 49

LIST OF APPENDICES

Appendix Page

1. Initial interview form 70

2. Health/diet history questionnaire 71

3. Initial informed consent form 79

4. Informed consent form 80

5. Daily activity sheet 82

6. Food composites 83

Effect of Glycosylated Vitamin B-6 Intake on the Excretion of Vitamin B-6 in Women

INTRODUCTION

Bioavailability of vitamin B-6 from food is determined by the physiological ability to digest, absorb and utilize this nutrient. In foods, vitamin B-6 is widely distributed and exists in various forms (Orr, 1969;

Gregory, 1980; Kabir, Leklem & Miller, 1983a; Gregory & Leatham, 1990;

Tadera & Orite, 1991). Vitamin B-6jn the form of pyridoxine (PN)- glucoside, a major conjugated form of the vitamin in plant-derived foods, is incompletely utilized by animals (Ink,_Gregory & Sartain, 1986a; Trumbo,

Gregory & Sartain, 1988; Trumbo & Gregory, 1989) as well as humans (Kabir et al., 1983b; Gregory et al., 1991). Glycosylated vitamin B^

1

has been quantified in some plant-derived foods (0-75% of total vitamin B-6), but little or none has been detected in animal-derived foods (Kabir et al., 1983a;

Hardin, Leklem & Miller, 1986; Bills, Leklem & Miller, 1987; Gregory & Ink,

1987; Leklem, 1988). The amount of glycosylated vitamin B-6 in relation to total vitamin B-6 has been suggested as an index of the bioavailability of vitamin B-6 from food (Kabir et al., 1983b). Gregory's research group has observed that purified PN-glucoside exhibits -20-30% bioavailability relative to PN in rats (Ink et al., 1986a; Trumbo et al., 1988; Trumbo &

1

The term glycosylated vitamin B-6 used in this thesis refers to PN- glucoside and other glycosylated forms of vitamin B-6 which can release microbiologically measurable vitamin B-6 by treatment with fi-glucosidase.

Gregory, 1988 & 1989), and 58% in humans (Gregory et al., 1991).

According to the latest available data from a nationwide food consumption survey, the average vitamin B-6 intake by women was 1.16 mg/d (USDA, 1987) and by men was 1.87 mg/d (USDA, 1986). These mean vitamin B-6 intakes were 28% less than the Recommended Dietary

Allowance (NRC, 1989) for women, and 7% for men. Furthermore, food consumption patterns in women from 1977 to 1986 revealed a decreased intake of animal-derived foods and an increased intake of grain products

(USDA, 1986). The low vitamin B-6 intake by free-living women and their change in food habits, with a potential of high glycosylated vitamin B-6 intake, stimulated our interest in understanding the effect of dietary glycosylated vitamin B-6 on the vitamin B-6 status in this group.

Additionally, since most of the previous studies on vitamin B-6 metabolism and bioavailability have been done in males, it is important to understand whether or not gender affects vitamin B-6 metabolism or requirement. In view of these considerations, the objective of the study reported in this thesis was to determine the effect of dietary glycosylated vitamin B-6 on the bioavailability of vitamin B-6 in women. The hypothesis we tested was that the bioavailability of vitamin B-6 from a diet with a relatively high proportion of glycosylated vitamin B-6 would be less than from a diet with less glycosylated vitamin B-6. Using a metabolic balance technique, we measured the intake and excretion of total, glycosylated and free vitamin B-6 by 4 women who were receiving low- and high-glycosylated vitamin B-6 diets.

REVIEW OF LITERATURE

Vitamin B-6

Vitamin B-6 was first identified by Gyorgy (1934) as a preventative of acrodynia (dermatitis) in rats. The essentiality of this nutrient for humans was recognized in the 1950s when certain neurological symptoms in an infant were attributed to a deficiency of vitamin B-6 (Snyderman et al.,

1953).

The term vitamin B-6 is the generic descriptor for all 3-hydroxy-2- methylpyridine derivatives that exhibit vitamin B-6 activity (IUPAC-IUB,

1973). The naturally occurring free forms of vitamin B-6, pyridoxine (PN), pyridoxal (PL) and pyridoxamine (PM), and their respective phosphorylated forms, pyridoxine S'-phosphate (PNP), pyridoxal 5'-phosphate (PLP) and pyridoxamine 5'-phosphate (PMP) are presented in Figure 1. These six forms are interconvertible in mammalian vitamin B-6 metabolism (Figure

2). The chief metabolite of vitamin B-6 excreted in urine is 4-pyridoxic acid

(4-PA) (Figure 1). Vitamin B-6 is labile in near ultraviolet light (Ang, 1979;

Schaltenbrand, Kennedy & Coburn, 1987), but the degree of sensitivity depends on pH. Vitamin B-6 is relatively stable to light in acid solutions, but not in neutral or alkaline solutions.

Biochemical Functions

PLP, a coenzyme form of vitamin B-6, is utilized by over 100 enzymes, most of which are involved in amino acid metabolism

(Sauberlich, 1985). PLP participates in aldolization, decarboxylation, dehydration, racemization, side-chain cleavage and transamination

H,G

3-hydroxy-2-methylpyridme derivatives

R =

-CH

2

OH

-CHO

-CH

2

NH

2

-COOH

5' = -OH

Pyridoxine (PN)

Pyridoxal (PL)

Pyridoxamine (PM)

4-Pyridoxic Acid (4PA)

5' = -OPO,

Pyridoxine Phosphate (PNP)

Pyridoxal Phosphate (PLP)

Pyridoxamine Phosphate (PMP)

FIGURE 1 Major naturally occurring forms of vitamin B-6 and its metabolite, 4-pyridoxic acid.

4-Pyridoxic Acid

■ QJ n <u

Pyridoxine Pyridoxal Pyridoxamine

01

.5 13

Pyridoxine S'-phosphate

Pyridoxine

Phosphate

Oxidase

(FMN)

0)

(A o !8 P-

.S 13

CL,

♦

Pyridoxal S'-phosphate

■

*-

Pyridoxamine

Phosphate

Oxidase

(FMN)

Transaminase

•a g H

01 tn

.5 13

^ (A a.

Pyridoxamine S'-phosphate

FIGURE 2 Metabolic interconversion of the B-6 vitamers in human liver.

(Adapted from Leklem, 1988; Merrill & Henderson, 1990)

On

(Merrill & Burnham, 1990). Examples of these enzymatic reactions utilizing PLP in amino acid metabolism are presented in Table 1. Both PLP and PMP are active as coenzymes for aminotransferases.

TABLE 1

The summary of enzymatic reactions in amino acid metabolism utilizing pyridoxal 5'-phosphate (PLP).

An example of a PLP-dependent enzyme and its chemical reaction is given.

Type of reaction PLP-dependent enzyme Chemical reaction

Aldolization 8-aminolevulinate synthetase

Glycine + succinyl CoA

—> 8-aminolevulinate

Decarboxylation Tryptophan decarboxylase

5-Hydroxytryptophan

-»serotonin + CO2

Dehydration Serine dehydratase Serine

-» pyruvate + NH4

+

Racemization Alanine racemase

Side-chain cleavage Serine transhydroxy- methylase

L-Alanine <-> D-alanine

Serine + tetrahydrofolate

<-> glycine + methylenetetra- hydrofolate + H2O

Transamination Alanine amino- transferase

Alanine + cc-ketoglutarate

<-> pyruvate + glutamate

Glycogen phosphorylase, a key enzyme in carbohydrate metabolism, requires PLP. Muscle glycogen phosphorylase acts as a vitamin B-6 reservoir. Okada, Ishikawa and Watanabe (1991) found that glycogen phosphorylase activity was significantly decreased in gastrocnemius and heart muscles, but not in the liver of vitamin B-6-deficient rats. Decreased enzyme activity in both muscles was due to a decreased amount of enzyme.

Since the in vitro addition of PLP did not increase phosphorylase activity in tissues from vitamin B-6-deficient rats, Okada et al. proposed that glycogen phosphorylase degradation might be related to its lability, or that the synthesis of phosphorylase was dependent on PLP. Black, Guirard and

Snell (1977) reported that high levels of vitamin B-6 intake (70 mg/kg of the diet) resulted in an increased amount of muscle phosphorylase and total muscle vitamin B-6 in rats.

In addition to the coenzyme role of PLP in amino acid and glycogen metabolism, there are other functions of PLP. Gluconeogensis, which is important in maintaining an adequate glucose supply in the body, involves PLP as a coenzyme for alanine and aspartate aminotransferases, which can provide pyruvate and oxaloacetate, respectively, for glucose synthesis. In hormone modulation (Bender, 1989), PLP may function as a terminator of the nuclear actions of steroid hormones by extracting steroid hormone-receptor complexes from nuclear binding. Animal studies, for example, have shown that vitamin B-6 deficiency results in increased and prolonged nuclear accumulation and retention of steroid hormones. The role of vitamin B-6 in immune function may be related to PLP-dependent serine transhydroxy-methylase needed for a normal one-carbon pool for nucleic acid biosynthesis (Miller & Kerkvliet, 1988). In lipid metabolism, a

PLP-dependent enzyme condenses a L-serine and palmitoyl CoA to form 3- dehydrosphinganine, a sphingolipid precursor (McCormick, 1988). The synthesis of neuro-transmitters (e.g., y-aminobutyric acid, histamine, serotonin) requires PLP-dependent enzymes. For instance, PLP-dependent glutamic decarboxylase converts glutamic acid to y-aininobutyric acid, which is essential for preventing convulsive seizures in infants

8

(Henderson, 1985). In the formation of niacin from tryptophan, a PLP- dependent kynureninase catalyzes the conversion of 3-hydroxykynurenine to 3-hydroxyanthranilic add. In erythrocyte function and synthesis of heme, PLP serves as a coenzyme for aminotransferases and 5- aminolevulinate synthetase. The second enzyme catalyzes the condensation of succinyl CoA and glycine to form 8-aminolevulinate, which is a precursor for the biosynthesis of porphyrins in heme.

Additionally, the binding properties of PLP and PL on hemoglobin may be important in treating sickle cell anemia. The binding of PLP to the £-chain of hemoglobin reduces O2 binding affinity, while the binding of PL to the a-chain of hemoglobin increases O2 binding affinity (Reynolds & Natta,

1985).

Recommended Dietary Allowance and Actual Dietary Intake

The Recommended Dietary Allowance (RDA) for vitamin B-6 is according to protein intake: 0.016 mg vitamin B-6 per gram of protein

(NRC, 1989). Based on a protein intake of twice the RDA (e.g., 100 g/d for women, 126 g/d for men), the RDA for women is 1.6 mg/d, and that for men is 2.0 mg/d.

According to data from the Second National Health and Nutrition

Examination Survey (NHANES II), 1976-1980 for 11,658 adults aged 19-74 y, the average of daily vitamin B-6 intake was 1.48 ± 0.01 mg (mean ± SEM) for the total population, and, between the sexes, 1.14 ± 0.01 mg for females and 1.85 ± 0.02 mg for males (Kant & Block, 1990). In this survey, animal products provided 48% and plant products provided 52% of the total vitamin B-6 in the American diet. Beef, alcoholic beverages, potatoes.

ready-to-eat cereals and milk accounted for 34% of total dietary vitamin B-

6.

The U. S. Department of Agriculture (USDA) also conducts nationwide food consumption surveys to assess dietary nutrient intakes.

In 1985, the average vitamin B-6 intake by women 19-50 y was 1.16 mg/d based on a 4-d record (USDA, 1987), and by men 19-50 y was 1.87 mg/d based on a 1-d intake record (USDA, 1986). Additionally, the USDA reported that food consumption patterns in women changed from 1977 to 1986: a shift from whole milk to lowfat/skim milk, decreased intake of animal products

(especially beef consumption from 49 to 28 g/d), and increased consumption of grain products (from 162 to 202 g/d) (USDA, 1986).

Transport and Metabolism

Although other tissues and organs (e.g., erythrocyte, kidney) contribute to vitamin B-6 metabolism, the liver is believed to be the primary organ for the interconversion and metabolism of the vitamin B-6

(Lumeng, Li & Lui, 1985; Merrill & Henserson, 1990). The interconversions among the free and phosphorylated forms of vitamin B-6 in human liver are presented in Figure 2. The free B-6 vitamers (PL, PN and PM) are absorbed forms which are transported into the hepatocytes by diffusion followed by metabolic trapping.

Merrill and Henderson (1990) reviewed the metabolic pathways in the liver which are responsible for converting dietary PN and PM to PL via

PLP. There are several important enzymes involved in these B-6 metabolic pathways. The enzyme for converting PL, PN and PM into their respective

5'-phosphate esters is PL kinase, which is ATP-dependent and is distributed

10 in all mammalian tissues, especially in liver, brain and kidney (Henderson,

1985). Flavin mononucleotide(FMN)-dependent PMP(PNP) oxidase, another important enzyme in the vitamin B-6 metabolism, oxidizes PMP and PNP to PLP (Merrill et al., 1984). In humans, this enzyme is found in liver and erythrocytes (Lumeng et al., 1985). The amount of cytosolic PLP probably plays a regulating role in PLP synthesis by inhibiting PNP/PMP oxidase. This may help avoid PLP accumulation, preventing a wastage of

PLP via PL to 4-PA (Merrill & Henderson, 1990). Evidence strongly suggests that an FAD-dependent aldehyde oxidase catalyzes the conversion of PL to

4-PA in human liver (Merrill et al., 1984). Additionally, aminotransferases interconvert PLP and PMP in mammalian liver. Alkaline phosphatase dephosphorylates phosphorylated B-6 vitamers to their respective free forms. Finally, PLP, PL and 4-PA are three forms of vitamin B-6 released into circulation by the liver. The apparent kinetic properties of these liver enzymes have been reported by Merrill et al. (1984 & 1990). They found that 1) the rates of phosphorylating PN and PL are greater than for dephosphorylating PNP and PLP; 2) the rate of phosphorylating PN and PM is slower than that of converting PNP and PMP to PLP; and 3) the rate of oxidizing PL to 4-PA is rapid and similar to that for phosphorylating PL.

These data provide further understanding of vitamin B-6 metabolic mechanisms in human liver.

The two major forms of vitamin B-6 in blood, PL and PLP, are bound to plasma albumin. Erythrocytes rapidly accumulate PN and PL because vitamin B-6 is metabolized in these cells, and PL and PLP are bound to hemoglobin (Merrill & Burnham, 1990). The pathway of vitamin B-6 metabolism in erythrocytes is similar to that shown in Figure 2 except that

PL is not converted to 4-PA. Since both PL and PLP bind specifically to hemoglobin, erythrocytes also serves as a circulating reservoir of vitamin

B-6 (Lumeng et ah, 1985).

11

The Findings of Glycosylated Forms of Vitamin B-6

Conjugated forms of PN in rice bran were first announced by Scudi

(1942). Subsequently, several other research groups reported evidence of bound (conjugated) vitamin B-6 in plants. For example, Yasumoto et al.

(1976) found some bound forms of vitamin B-6 in cereals and seeds.

Nelson, Burgin and Cerda (1977) reported a bound form of vitamin B-6 in natural orange juice. This vitamin B-6 binder is a heat stable, non-protein molecule of less than 3,500 daltons molecular weight, which binds both PN and PL.

Yasumoto et al. (1977) first identified the chemical structure of a conjugated form of PN from rice bran as S'-CMfi-D-glucopyranosyl) pyridoxine (PIN-fi-G) (Figure 3). Tadera et al. (1979 & 1982) isolated and identified a particulate glucosyltransferase from seedlings of the podded pea, Pisum sativum L. cv. Kinusaya, which catalyzed the synthesis of PN- glucoside from PN and UDP-glucose. Iwami and Yasumoto (1986) purified

6-glucosidase in rice bran to synthesize PN-glucoside from p-nitrophenyl-

6-glucoside and PN. The glucose moiety of PN-glucoside was identified at the 4' and 5' positions of PN ring in this study. Analyzing a variety of foods by high performance liquid chromatography (HPLC), Gregory and Ink

(1987) provided strong evidence that the S'-isomer of PN-glucoside was the predominant glycosylated form of vitamin B-6 in the plant-derived foods.

12

CH2OH

HO

H

3

C—^

1M n

OH H

S'-O-Cfi-D-GlucosylpjoânosyDPyridoxine

FIGURE 3 Structure of pyridoxine-fi-glucoside.

Other PN-glucoside derivatives have been reported, especially by

Tadera's research group. These chemical structures are presented in Figure

4. In the seedlings of podded pea, Pisum sativum L. grown in the presence of PN, Tadera et al. isolated and identified 5

,

-0-[6-0-(3-hydroxy-3-methyl-4- carboxybutanoyl)-fi-D-glucopyranosyl]pyridoxine (HMGPNG) (Tadera et al.,

1983) and 5'-0-(6-0-malonyl-£-D-glucopyranosyl)pyridoxine (malonyl-

PNG) (Tadera et al., 1985). These compounds existed as a low percentage of the total vitamin B-6 in the pea. Later, Tadera, Kaneko and Yagi (1988) found three other PN-glycosides from rice bran, including 5'-0-(fi- cellobiosyl)pyridoxine, 4'-0-(6-D-glucosyl)-5'-0-(fi-cellobiosyl)pyridoxine, and 5'-0-(fi-glucotriosyl)pyridoxine. These three new compounds comprised more than 50% of the PN-glycosides in rice bran. The occurrence of PN oligoglucosides in potatoes has been reported by Addo and Augustin (1988). The significance of these compounds in common foods and their bioavailability were not measured in these studies.

Additionally, Tadera and Orite (1991) isolated and identified the structure of PNX from rice bran as 5'-0-[6-0-((+)-5-hydroxy-dioxindole-3- acetyl)-fi-cellobiosyl] pyridoxine. The PNX fraction in several plant foods

CH

2

OH

-o

CH

2

OH

CH

2

OH

H

OH

CH

2

OH

OH

2

C

\ vT

"/ ^H

"^

O

J O

H /H

\

^ v

OH

»/

V i

H OH H OH

5'-0-((5-Ccllobiosyl)Pyridoxinc

CH

2

OH

CH

2

OH

' -O

HO\?

H

W

H OH H OH H OH

5'-0-(G-Glucotriosyl)Pyridoxine

H OH H OH " "N'

4

,

-0-(G-D-Glucosyl)-5'-0-(g-Ccllobiosyl)[

, yridoxino

HOjC co

2

— H

2 c

OH c

CH

2

OH

.OH

H OH U

3

5'-0-[6-0-(3-Hydroxy-3-Methyl-4-Carboxybutanoyl)- fi-D-Glucopyranosyl)]Pyridoxino

CH

2

OH

C0

2

-H

2

C o

^

CH, »/H co"

H o\?

H

V

H

^"CH, iToH " "N^ ^

5'-0-(6-0-Malonyl-G-D-Glucopyranosyl)Pyridoxinc

H OH H OH

5'-0-[6-0-((+)-5-Hydroxy-Dioxindole-3-Acctyl)-S-Ccllobiosyl)Pyridoxin

FIGURE 4 Structures of pyridoxine-fi-glucoside derivatives.

13

14 has been measured in Tadera et al. (1986). Based on percentage of the total vitamin B-6 content: podded peas contain 21% PNX; defatted soybeans,

27%; rice bran, 38%; and wheat bran, 19%. No detectable amount of PNX was found in cauliflower, spinach, pumpkin, or broad beans. In both studies, they did not determine bioavailability of PNX.

The Distribution of Glycosylated Forms of Vitamin B-6 in Foods

The 5'-isomer of PN-glucoside is the only significant naturally occurring glycosylated form of vitamin B-6 in the plant-derived foods

(Gregory & Ink, 1987). By using fi-glucosidase digestion along with a microbiological assay procedure, our laboratory quantified glycosylated vitamin B-6 in foods (Kabir et al., 1983a; Hardin et al.,1986; Bills et al., 1987).

Animal-derived foods have no detectable amount of glycosylated vitamin

B-6. A list of plant-derived foods and the percentage of total vitamin B-6 as glycosylated vitamin B-6 in above studies is presented in Table 2. The data suggest that glycosylated vitamin B-6 comprises varying proportions of the total vitamin B-6 content in plant foods. Kabir et al. (1983a) investigated the difference in glycosylated vitamin B-6 content between raw and processed foods in broccoli, cauliflower and green beans. These investigators reported that the difference was due to denaturation of fi- glucosidase during processing, decreasing or eliminating the capacity of this enzyme to digest PN-glucoside. The value of % glycosylated vitamin B-6 in a food also varied from one lab worker to another lab worker. Frozen cauliflower, as an example, had 82, 70 and 63% of glycosylated vitamin B-6 measured by Kabir et al., Hardin et al. and Bills et al., respectively. In addition, these variations may be due to other factors, such as food variety.

15

TABLE 2

Percentage of glycosylated vitamin B-6 in plant foods and their processed products

1

Food % Food %

Vegetables

Carrots, canned

Cauliflower, frozen

Broccoli, frozen

Carrots, raw

Cabbage, raw

Sprouts, alfalfa

Potatoes, cooked

Spinach, frozen

Green beans, canned

Peas, frozen

Green beans, raw

Cauliflower, raw

Broccoli, raw

Fruits

Raisins, seedless

Orange juice, cone

Orange juice, fresh

Pineapple, canned

Tomato juice, canned

Apricots, dried

Bananas

75

Grains

Wheat bran

72 ± 9.5

2

Shredded-wheat cereal

59 ± 5.7 Dark rye bread

51 Whole wheat bread

46

42

42

Rice (white), cooked

Whole wheat flour

35

28

15

10

35 ± 7.1

29

3

23

17

14

11

White bread

Corn, frozen

6

6

Fortified wheat-flakes cereal 5

Rice bran 4

5

0

Legumes

Soybeans, cooked

Navy beans, cooked

Peanut butter

65

49 ±3.5 Nuts/Seeds

37 Sunflower seeds, raw

22 Cashews, raw

21

14

10

3

3

Peaches, canned

Walnuts, English

Hazelnuts, raw

Almonds, raw

62

36

13

21

7

4

3

42

18

0 Avocados, fresh

âta from Kabir et al. (1983), Hardin et al. (1986) and Bills et al. (1987) by using C-glucosidase digestion followed by microbiological assay with

Saccharomyces uvarum.

Mean ± SD represents the mean of the % glycosylated vitamin B-6 content from at least three determinations.

3

The average of the % glycosylated vitamin B-6 content comes from two determinations. Other data represent a single determination.

16 maturity, storage time or handling processes.

Using HPLC method, Gregory's research group (Gregory & Ink, 1987;

Gregory & Sartain,1991) reported % PN-glucoside in plant-derived foods: raw carrots, 65-70; fresh orange juice, 69; soy flour, 67; raw green beans, 59; rice bran, 36; raw broccoli, 35; wheat bran, 34; bananas, 6; and oat bran, 0.

Since no studies have directly measured the difference of % glycosylated vitamin B-6 values between these two methods, further investigations will be required.

PN-glucoside has been shown to be absent in both human milk and cow's milk (Kabir et al., 1983a; Gregory & Ink, 1987). On the other hand, other researchers found an apparent occurrence of glycosylated vitamin B-6 in human milk. Reynolds et al. (1986) announced that glycosylated vitamin B-6 accounted for a mean of 15.3% of the total vitamin B-6 in breast-milk collected from 26 Nepalese vegetarian women, who consumed

15.3% of their total dietary vitamin B-6 as glycosylated vitamin B-6. Andon et al. (1989) reported similar glycosylated vitamin B-6 intake (15.4%) by 30

American lactating women, but found significantly less glycosylated vitamin B-6 (2.5% of the total vitamin B-6) in the breast milk. Cho et al.

(1990, abstract) observed 1 and 11% of PN-glucoside of the total vitamin B-6 in the milk of American and Egyptian lactating women, respectively. Total vitamin B-6 of milk collected from Egyptian women was positively correlated with animal protein intake (r = 0.88) and negatively correlated with plant protein intake (r = -0.49) as well as % PN-glucoside intake (r =

-0.59).

17

Absorption of Pyridoxine-glucoside

PN-glucoside has been reported to be absorbed either intact by the intestine (Trumbo & Gregory, 1988) or after hydrolysis by intestinal and/or microbial fi-glucosidase (Trumbo, Banks & Gregory, 1990; Gregory et al.,

1991; Banks & Gregory, 1991). Iwami and Yasumoto (1986), using purified fi-glucosidase from rice bran to measure PN-glucoside absorption in situ in isolated rat jejunal loops, found that PN-glucoside was absorbed across the intestinal wall by simple diffusion in a manner similar to the intestinal uptake of the free forms of vitamin B-6 (Booth & Brain, 1962; Middleton,

1979; Yoshida, Hayashi & Kawasaki, 1981; Middleton, 1985). However, in this study, Iwami and Yasumoto measured the relative disappearance of

PN from the intestinal perfusate and used a purified 4'-isomer of PN- glucoside, rather than the naturally occurring 5'-isomer.

Ink, Gregory and Sartain (1986) detected 85% of dietary PN-glucoside remaining as the glucoside form when investigating the distribution of

[ H]PN-glucoside in rats. They suggested that PN-glucoside was absorbed by the intestinal tract before entering general circulation. This assumption is supported by Hardin et al. (1986, abstract) who determined the absorption of glycosylated vitamin B-6. Two men ingested 350 g of orange juice concentrate, and then their blood was collected at 1,2 and 3 h. In response to the orange juice concentrate, plasma glycosylated vitamin B-6 in 2 men ranged from 16 to 30 nM, which indicated that glycosylated vitamin B-6 was absorbed.

Trumbo, Banks and Gregory (1990) identified and characterized fi- glucosidase which hydrolyzed PN-glucoside in tissues from rats, guinea pigs and humans. They observed that the fi-glucosidase was the most

18 active in the soluble tissue fraction of guinea pig jejunum. Maximum activity of C-glucosidase was at pH 5.5-6.0. Sodium taurocholate acted as an inhibitor of the cytosolic G-glucosidase to hydrolyze PN-glucoside.

According to these characteristics, intestinal fi-glucosidase is a cytosolic enzyme with broad specificity. Additionally, they found that the activity of intestinal fi-glucosidase was approximately six times greater in the human intestinal mucosa than in the rat or guinea pig whole small intestine, and this difference might be related to microbial populations as well as cytosolic fi-glucosidase activity. In this study, Trumbo et al. also examined the effect of intestinal mucosa and the whole intestine on the fi-glucosidase activity.

Activity was similar between these two samples.

Banks and Gregory (1991) evaluated the influence of the intestinal fi- glucosidase activity by chronically feeding rats with either PN-adequate or - deficient diets. They reported that the activity of intestinal fi-glucosidase was not significantly changed by feeding 0, 5,10 or 15 nmol/g of dietary PN- glucoside.

The Bioavailability of Vitamin B-6

Gregory (1988) defined bioavailability as the extent of intestinal absorption and metabolic utilization of a nutrient. Since not all of the vitamin B-6 in food is completely utilizable (Gregory, 1980; Kabir et al.,

1983b; Gregory & Leatham, 1990; Tadera & Orite, 1991), an understanding of the properties and bioavailability of vitamin B-6 compounds in foods becomes important to evaluate adequacy of vitamin B-6 intake. Compared to 100% bioavailability of crystalline PN-HC1 administrated in a formula diet, the bioavailability of vitamin B-6 in an average American diet is 71%

19 based on plasma PLP data, or 79% using urinary total vitamin B-6 data

(Tarr, Tamura & Stokstad, 1981). These data suggest, for example, that the available vitamin B-6 in the average American diet containing 1.52 mg vitamin B-6/d, based on 1985 USD A food consumption surveys (USD A,

1986 & 1987), is approximately 1.14 mg/d.

In addition to glycosylated vitamin B-6, other factors, such as dietary fiber and food processing, affecting vitamin B-6 bioavailability have been indicated (Reynolds, 1988; Gregory, 1990). Bioavailability of PN-glucoside in chemically synthesized form, individual foods or mixed diets has been conducted in animals as well as humans. Studies which determine glycosylated vitamin B-6 bioavailability will be discussed first, and then those which measure other factors or indirectly measure glycosylated vitamin B-6 bioavailability will be discussed later.

Studies of Glycosylated Vitamin B-6 Bioavailability

Animal Studies Tsuji et al. (1977) in an earlier investigation reported that rats utilized PN-glucoside well. They observed that PN- glucoside (chemically synthesized) restored the urinary excretion of xanthurenic acid to a normal level in vitamin B-6-deficient rats. Tsuji et al. suggested that PN-glucoside was converted to free PN by 6-glucosidase either before or after absorption by the small intestine. Gregory's research group conducted a series of studies in rats and found that purified PN- glucoside exhibited very low bioavailability (-20-30%) (Ink et al., 1986a;

Trumbo et al, 1988; Trumbo & Gregory, 1988 & 1989). Trumbo, Gregory and Sartain (1988) analyzed the relationship between the excretion of urinary PN-glucoside and dietary intake of PN-glucoside in a rat depletion-

20 repletion bioassay. Based on slope ratios of dose-response curves, the results showed that the utilization of PN-glucoside relative to PN was 34% from weight gain data, and 10% from plasma PLP concentration data.

Trumbo et al. observed that at various dosage levels a constant 7-9% of the total PN-glucoside intake was excreted in urine within 24 h. These data indicate that the absorption and utilization of PN-glucoside are not affected by level of dietary intake of this compound.

Trumbo and Gregory (1988), investigating the metabolic utilization of PN-glucoside, administrated [

3

H]PN-glucoside and [ C]PN orally or intraperitoneally to vitamin B-6-adequate or -deficient rats. They found that [ H]PN-glucoside was not present in liver and most of the H was excreted in urine by the vitamin B-6-adequate rats but not the vitamin B-6- deficient rats. They estimated that the intestine converted 20% of ingested

PN-glucoside to PN, suggesting that the extent of PN-glucoside hydrolysis was a limiting step in the utilization of this conjugated form in rats. These results are supported by the findings of Ink, Gregory and Sartain (1986a) who recovered 85% of dietary [ H]PN-glucoside as intact PN-glucoside in urine.

In another study, Trumbo and Gregory (1989) investigated the fate of

[

3

H]PN-glucoside and [

14

C]PN administration to lactating rats. Analysis of the isotopic ratio ( H/ C) in the dams' milk and pups' stomach contents showed that 20-25% of the

3

H compared to the

14

C was secreted into the milk. Since no PN-glucoside was found in the milk or in the pups' liver and stomach, Trumbo & Gregory suggested that PN-glucoside was hydrolyzed to PN prior to secretion into milk.

Gilbert and Gregory (1992), administering a single oral dose of 240

21 nmol of [

14

C]PN and either 0, 36 or 72 nmol of unlabeled PN-glucoside to rats, found a significant increase of urinary C and a significant decrease of hepatic C as the unlabeled PN-glucoside dose increased. These data indicated that PN-glucoside varied vitamin B-6 metabolism by impeding the utilization of nonglycosylated vitamin B-6.

Human Studies Leklem et al. (1980) evaluated vitamin B-6 bioavailability among whole wheat bread, white bread and white bread enriched with vitamin B-6 (WB6). They observed that fecal vitamin B-6 excretion significantly increased and urinary 4-PA excretion significantly decreased when whole wheat bread was fed than when the other breads were fed. The urinary and fecal data also indicated that the vitamin B-6 was 5-10% less available from whole wheat bread than from white bread or

WB6.

Kabir, Leklem and Miller (1983b & 1983c) found an inverse relationship between the glycosylated vitamin B-6 content of food and vitamin B-6 bioavailability. This was a 52-d metabolic balance-type study divided into a 10-d adjustment period, followed by three 14-d experimental periods. During the experimental periods, eight young men were fed three diets in which tuna, whole wheat bread or peanut butter supplied approximately half of the total vitamin B-6, and the basal diet supplied the remaining dietary vitamin B-6. Total vitamin B-6 content of the three experimental diets was 1.52-1.56 mg/d. They observed that urinary glycosylated vitamin B-6 excretion increased with an increase of glycosylated vitamin B-6 intake. Based on the excretion of urinary 4-PA, urinary total vitamin B-6 and fecal vitamin B-6 as well as the concentration

22 of plasma PLP, Kabir et al. estimated the average vitamin B-6 bioavailability in whole wheat bread and peanut butter relative to tuna was

75 and 63%, respectively. Additionally, only 4% of the total vitamin B-6 in feces was detected in the glycosylated form when whole wheat bread was fed, but no fecal glycosylated vitamin B-6 was detected during the tuna or peanut butter periods.

Bills (1990, thesis) assessed the bioavailability of vitamin B-6 in 10 plant-derived foods of varying % glycosylated vitamin B-6 content. In a 49- d study, each food was administered as a loading dose equivalent to 0.5 mg of total vitamin B-6. One dose was administrated a day; each dose was separated by two days. Percent bioavailability of vitamin B-6 of each food test dose was determined from a dose response curve constructed from the subjects' urinary excretion of 4-PA following incremental doses (0.2, 0.4 and

0.6 mg) of crystalline PN, which was considered 100% available. Regression analysis showed a significant inverse relationship (R = -0.94; P < 0.01) between the % glycosylated vitamin B-6 and bioavailability of vitamin B-6 from walnuts, bananas, tomato juice, spinach, orange juice and carrots.

However, broccoli and cauliflower had a high positive % bioavailability

(62.2 and 55.9%, respectively), while wheat bran and shredded wheat (a ready-to-eat breakfast cereal product) had low, apparently negative % bioavailability (-45.6 and -22.8%, respectively).

Andon et al. (1989) investigated the vitamin B-6 nutritional status of

30 free-living lactating women, who were not taking a vitamin B-6 supplement. Their mean intake of vitamin B-6 was 1.46 mg of which glycosylated vitamin B-6 comprised 15.4% of the total. Andon et al. reported that dietary glycosylated vitamin B-6 had little effect on these

23 lactating women's plasma PLP concentration, urinary vitamin B-6 and 4-

PA excretion when these women consumed adequate amounts of vitamin

B-6.

Gregory et al. (1991) introduced a stable-isotopic method to examine the bioavailability of PN-glucoside in 5 men. Using labeled urinary 4-PA data, they estimated that the bioavailability of orally administrated PN- glucoside relative to PN was 58%. They detected PN-glucoside in all urine samples after administrating deuterium-labeled PN-glucoside either orally or intravenously. Additionally, utilization of PN-glucoside administrated intravenously was half that given orally. They suggested that fi-glucosidase in the intestinal mucosa and/or microflora might hydrolyze PN-glucoside.

Other Factors Affecting Vitamin B-6 Bioavailability

Effect of Fiber Using a rat jejunal perfusion technique, Nguyen,

Gregory and Cerda (1983) investigated the effect of cellulose, pectin, ligin, fresh carrots and cooked carrots on vitamin B-6 absorption. No inhibitory effect by cellulose, pectin and ligin was observed, but the absorption of B-6 vitamers from carrots was reduced. These researchers suggested that certain naturally occurring fiber components lowered vitamin B-6 bioavailability, while purified or semi-purified dietary fiber had no effect on vitamin B-6 availability.

Kies, Kan and Fox (1984) reported that corn, rice and wheat brans had an adverse effect on vitamin B-6 bioavailability in humans. Twenty grams of com, rice or wheat bran were incorporated into a laboratory-controlled diet containing 1.238 mg of vitamin B-6. Total vitamin B-6 content of corn, rice and whole wheat bran diets was 1.584,1.719 and 1.495 mg vitamin B-6,

24 respectively. The percentage of vitamin B-6 intake excreted as urinary total vitamin B-6 was 6.01, 5.71 and 6.10 in corn, rice and wheat brans, respectively, compared with 9.46 in the control diet. They proposed that the addition of bran increased fiber intake, which might inhibit nutrient absorption and utilization from the gastrointestinal tract.

Shultz and Leklem (1987) compared the vitamin B-6 status of vegetarians and non vegetarians. Vitamin B-6 intake was determined from

3-d dietary records. Plasma PLP concentration as well as urinary 4-PA and vitamin B-6 excretion were lower on the vegetarians than the nonvegetarians, but there were no significant differences. Both groups had similar vitamin B-6 intake, but the vegetarians consumed significantly more fiber than did the nonvegetarians. These investigators concluded that there was no adverse effect of fiber on vitamin B-6 bioavailability with respect to these three measures of vitamin B-6 status between the two groups.

Effect of Food Processing By using the in vitro method to evaluate the effects of food processing on vitamin B-6 bioavailability in lima beans,

Ekanayake and Nelson (1990) reported the vitamin B-6 bioavailability was reduced by 50% during the thermal processing (118

0

C, 30 min) but not during blanching (100 o

C, 5 min). Ink, Gregory and Sartain (1986b), employing a combination of intrinsic and extrinsic labeling in rats, observed that thermal processing of liver and muscle (121

0

C, 45 min) reduced the total [

3

H]vitamin B-6 25 to 30% in these raw tissues. Since rats fed cooked liver or muscle had significantly higher H in the feces than those fed raw liver or muscle, these investigators proposed that it was due

25 to the poor absorption of the thermal degradation products or the reduction of the digestibility of the thermally treated tissue proteins. In another study, Gregory et al. (1986) demonstrated that PLP bound with lysyl residues during thermal processing to form pyridoxallysine which exhibited 50% of vitamin B-6 activity relative to PN in rats (Gregory, 1980).

Tadera et al. (1986) identified 6-hydroxypyridoxine which caused vitamin B-6 losses by PN hydroxylation during processing, storage and cooking of plant foods high in ascorbic acid. This compound exhibited neither vitamin B-6 nor antivitamin B-6 activity in a rat bioassay (Gregory

& Leatham, 1990). Another possible influence on the apparent bioavailability of vitamin B-6 is the synthesis of PN glucosides during the storage. Addo and Augustin (1988) found that during 9 mo of storage % glycosylated vitamin B-6, based on PN-glucoside only, in potatoes increased from 16 to 49%. PN diglucosides also increased significantly.

26

MATERIALS AND METHODS

Subject Selection

Nine apparently healthy women, aged 21-39 y, were recruited by advertisements placed on bulletin boards on the Oregon State University campus. These subjects were selected on the basis of the results of an initial interview (Appendix 1), a health/diet history questionnaire (Appendix 2), and a normal blood chemistry test

2

, which included the measurement of serum cholesterol, electrolytes, enzymes, glucose and protein. The subjects' eligibility for participation in this study included: 1) no medical history of intestinal, renal or metabolic disease; 2) no use of vitamins or other nutritional supplements for at least 3 wk before this study; 3) no food allergies; 4) no alcohol consumption more than 2 oz pure alcohol per week;

5) no smoking for more than 6 mo before this experiment; 6) no use of oral contraceptives or other drugs that alter vitamin B-6 metabolism or affect microbiological assay of this vitamin (e.g., antibiotics); 7) normal menstrual cycle; 8) normal hemoglobin and hematocrit

3

; 9) normal vitamin B-6 status by the measurement of plasma PLP

4

; 10) not involved in strenuous physical activities (e.g., running more than 1 mile/d, bicycling more than 6 miles/d); and 11) willingness to participate the study and eat 3 meals at the laboratory of Nutrition and Food Management Department.

2

Serum was prepared from a fasting blood sample for a chemical screen test done at Good Samaritan Hospital, 3600 Samaritan Drive, Corvallis,

Oregon.

3

Hemoglobin and hematocrit were determined by Jim Ridlington and

Karin Hardin, Nutrition and Food Management Dept., OSU.

4

Plasma PLP was determined by Christine Hansen, Nutrition and Food

Management Dept., OSU.

27

The purpose and design of the study were clearly explained to all subjects. Each subject signed two informed consent forms which had been approved by the Oregon State University Human Subjects Committee on

December 15, 1988: one was signed when she became a potential participant

(Appendix 3); the other was signed after meeting the criteria of the study

(Appendix 4). The description of 9 subjects' age, height, nationality and initial and final weights is listed in Table 3.

TABLE 3

Descrip tion of the subj ects

Subject

No. Race Age y

Height cm kg

Weight

Initial l, 2

Final kg

Group 1

^

2

3

3

4

Mean ± SD

Japanese

Chinese

Indonesian

Caucasian

26 158 45.9

4

46.1

21

39

24

155

148

165

43.6

54.6

55.2

43.5

53.4

55.8

27.5 ±: 7.9 156.5 ±7.0 49.8 ± 5.9 49.7 ± 5.8

Group 2

6

7

8

9

3

10

3

Chinese

Honduran

Caucasian

Caucasian

Chinese

27

24

31

37

30

154

161

175

163

160

47.6.

77.2

54.3

61.0

59.5

46.9

75.9

54.1

60.5

57.9

Mean ± SD 29.8 + 4.9 162.6 + 7.7 59.9 ±11.0 59.1 ± 10.7

Initial and final weights are means for each subject on days 1-5 and 40-

44, respectively, of the study.

No significant difference between the means of initial and final weights for subjects (t test, P > 0.05).

These subjects' urinary and fecal vitamin B-6 data are reported in this thesis.

Based on weight on days 5-9 when her self-weighing became consistent.

28

Experimental Design and Diets

This 44-d study, from April 8 to May 21,1991, was divided into a preliminary 8-d adjustment period followed by two 18-d experimental periods. The subjects were divided into two groups in a crossover design to compare the effect of low- and high-glycosylated vitamin B-6 diets on the vitamin B-6 bioavailability. This experimental design is presented in

Figure 5.

Diet Adjustment Diet

1

Experimental Diet Experimental Diet

Date 4/8 -4/15 4/16-5/3 riftrf

5/4-5/21

Treatment:

Group 1

(Subject 1-4)

Given

0.37 mg Pyridoxine

Oral Supplement Per Day

\\N Low GB6 Diet sNN XNSHieh GB6 Diet^

Group 2

(Subject 6-10)

>OOHigh GB6 DietOOO VSXLOW GB6 DietoN

Food

Composite

Urine

Collection

Fecal Marker

4

Fecal

Collection

Weekly

24 h. Daily

4/24,4/28, 5/2

Two 4-d Composites

5/12,5/16,5/19

5

3-d & 4-d Composites

Figure 5 Experimental design and the schedule of sample collection.

1

Adjustment diet is presented in Table 4.

2

Low GB6 diet (low-glycosylated vitamin B-6 diet) is presented in Table

5.

3

High GB6 diet (high-glycosylated vitamin B-6 diet) is presented in

Table 6.

4

Fecal marker is composed of 50 mg F.D. & C. Blue No. 1 Dye and 200 mg methylcellulose in a gelatin capsule.

5

Subject No. 9 had fecal marker on 5/20 instead of 5/19, so she had two

4-d composites during the second experimental period.

29

The adjustment diet is listed in Table 4, and the experimental diets low in glycosylated vitamin B-6 and high in glycosylated vitamin B-6 are given in Tables 5 and 6, respectively. The total vitamin B-6 content in the adjustment diet was 0.987 mg from food plus 0.37 mg PN supplement; in the low-glycosylated vitamin B-6 diet, 1.506 mg; and in the high- glycosylated vitamin B-6 diet, 1.897 mg. The dietary protein content

5

was

69.3, 84.0 ± 4.3 (mean ± SD, n = 5) and 93.1 ± 4.5 g (n = 6) for the adjustment, low- and high-glycosylated vitamin B-6 diets, respectively. In addition to all known daily required nutrients, adequate energy for the subjects to maintain their body weight was supplied by additional margarine, salad dressing, candy and soft drinks without added natural juice or vitamins.

Chewing gum and table seasonings (e.g., salt, sugar) were also allowed.

Each subject weighed herself before breakfast at the laboratory of Nutrition and Food Management Department and recorded her daily consumption of coffee, tea, soft drinks and candy on a daily activity form (Appendix 5).

To maintain three subjects' constant body weight, a slight diet modification was made from May 5 to May 21. Subjects 3, 7 and 8 were given 35, 40 and 60 g margarine daily, respectively, and whole milk in place of 2% low fat milk. Additionally, 50 and 150 g low protein bread

6

were given to subjects 7 and 8 to supply their daily energy need.

5

The nitrogen content of diets was measured by Ricky Virk, Nutrition and

Food Management Dept., OSU. The total protein values were determined by nitrogen content times appropriate conversion factors.

6

The low protein bread was made with 1 lb "UNIMIX" (Kingsmill Foods

Company Limited, Scarborough, Ontario, Canda), 5 g sugar, 1 3/4 cups warm water and 6 g fast-acting yeast. Subject 7 got an additional 0.1 g protein and 118 kcal from this bread, and subject 8 got an additional 0.3 g protein and 353 kcal.

TABLE 4

Adjustment diet

Food

Breakfast

Shredded wheat

1

Blueberries, frozen

Bread, white enriched

Milk, 2%

Orange juice, frozen reconstituted

Margarine

Pyridoxine supplement

2

Lunch


Cheese, cheddar

Salad

Carrots, raw

Celery, raw

Lettuce, raw

Red cabbage, raw

Salad dressing, French

Apple, fresh

Pears, drained, canned

Gelatin

3

Dinner

Turkey, light meat, cooked

White rice , regular, weight before cooking

Green beans, drained, canned


Cottage cheese, 2% fat

Peaches, drained, canned

Milk, 2%

Graham crackers

Pyridoxine supplement

2

Amount

8

30

40

50

180

160

Variable

25

45

20

30

50

10

Variable

70

100

14

50

45

100

25

40

100

180

40

30

31

TABLE 4 (Continued)

Sunshine Biscuts Inc., Woodbridge, NJ.

2

PN supplement, containing 0.185 mg vitamin B-6 as pyridoxine/10 mL, was prepared by dissolving 0.226 mg PN-HC1 in 0.5% acetic acid. This supplement was prepared in bulk, and each 5-mL portion was dispensed into a vial, sealed, and frozen until use. It was thawed shortly before administering. Two vials were given to each subject at breakfast and at dinner.

3

Gelatin was hydrated in cold water, dissolved in boiling water to 120 mL and diluted with a flavored drink mix (Kool-Aid, Kraft General Foods,

Inc., White Plains, NY). Total volume was 240 mL.

Rice was baked with 120 mL of water in a covered casserole bowl at

350 o

F for 25 min.

32

TABLE 5

Experimental diet: low-glycosylated vitamin B-6 diet

Food Amount

8

Breakfast

Oat cereal

1

, ready-to-eat


Pineapple, drained, canned

Milk, 2%


Margarine

Lunch

Sandwich


Tuna, drained water packed

Egg white, cooked

Dill pickles

Mayonnaise

Salad

Carrots, raw

Celery, raw

Lettuce, raw

Red cabbage, raw


Apple juice, frozen reconstituted

Dinner


White rice , enriched, weight before cooking

Peas, frozen

Pears, drained, canned

Gelatin

3

Milk, 2%

Popcorn, weight after popping

Vanilla cookies

30

37

100

200

200

Variable

75

50

30

10

20

10

40

20

10

Variable

180

100

40

80

100

12

250

15

12

1

Quaker Oat Life, contributed by Quaker Oats Co., Chicago, IL.

Rice was prepared by adding 120 mL water and baking in a covered casserole bowl at 350 o

F for 25 min.

3



4

Nilla Wafers, Nabisco, Inc., East Hanover, NJ.

33

TABLE 6

Experimental diet: high-glycosylated vitamin B-6 diet

Food Amount

8

Breakfast

Oat bran

1

, weight before cooking

Egg white, cooked

Bread, whole wheat

Gelatin

2

Milk, 2%


Margarine

30

45

14

12.5

200

200

Variable

Lunch

Sandwich

Bread, whole wheat

Peanut butter, smooth

Salad

Carrots, raw

Celery, raw

T -ettuce, raw

Red cabbage, raw


Carrots, raw

Peaches, drained, canned

Tomato juice, canned

Dinner


Potato , dehydrated flakes. weight before cooking

Red kidney beans, drained. canned

Gelatin

2

Milk, 2%

Apricots, dried

Vanilla cookies

75

50

20

40

20

10

Variable

30

100

100

40

25

80

12.5

220

30

12

1

Oat bran was prepared by adding 170 mL of water and cooking in the microwave oven for 1.5 min (high power setting).



Potato was prepared by adding 5 g margarine, 135 mL water and a dash of salt, and cooked in the microwave oven on high for 30 s.

Nilla Wafers, Nabisco, Inc., East Hanover, NJ.

34

Sample Collection and Preparation

The schedule for sample collections is listed in Figure 5. A composite of one day's diet (adjustment or each experimental diet) was made weekly throughout this investigation. Composites of animal, plant and milk products of each experimental diet were made separately. Each composite was ground well in a blender, and portions were frozen at -20 o

C until analyzed for total, glycosylated and free vitamin B-6. Food composites did not include mayonnaise, salad dressing, margarine and vitamin B-6-free foods (e.g., candy, coffee, tea, soft drinks).

Throughout the feeding phase of the experiment, the subjects collected 24-h urine specimens daily in plastic bottles containing approximately 5 mL of toluene. During collection and preparation, urine specimens were protected from light and refrigerated as soon as possible to prevent photolysis. The subjects' urine was measured daily. Aliquots of each collection were kept frozen at -20 o

C until analyzed.

The subjects collected their feces in plastic containers during each experimental period. A fecal marker, 50 mg F.D. & C. Brilliant Blue Dye

No. 1 mixed with 200 mg methylcellulose in a gelatin capsule, was given to the subjects at breakfast on 4/24,4/28,5/2 (experimental period 1), 5/12,

5/16 and 5/19 (experimental period 2). Subject 9 received a fecal marker on

5/20 instead of 5/19. The subjects' feces were frozen as soon as possible after collection. Each subject's frozen feces were pooled into 3-d or 4-d composites in a paint bucket and frozen at -20 o

C. After thawing in the refrigerator (4

0

C), each composite was diluted with redistilled water to achieve a desired consistency, degassed in a vacuum desiccator, and mixed thoroughly at room temperature (22-25

0

C) on a paint shaker for 20 min.

35

Portions of each subject's fecal specimens were frozen at -20 o

C for subsequent analysis.

Analytical Methods

For this thesis, the following samples were analyzed: food composites made on 4/11, 5/2 and 5/21; and from subjects 1, 2, 6 and 9, urine collected on 4/13 and 4/15 (adjustment period), 4/27,4/30 and 5/3

(experimental period 1), 5/15, 5/18 and 5/21 (experimental period 2); as well as fecal composites from the two experimental periods. These frozen samples were thawed in the refrigerator (4

0

C) one day before analysis. All samples for the determination of total, glycosylated and free vitamin B-6 assays were prepared in duplicate. All vitamin B-6 analyses were performed at room temperature under yellow fluorescent lights.

In each sample, total vitamin B-6 content was measured in an acid- hydrolyzed sample by microbiological assay with S. uvarum (AOAC, 1990) without the chromatographic step. Food samples were prepared in duplicate by hydrolyzing 2 g of sample at 121

0

C in 200 mL of 0.44 N HCl for

2 h (plant products) or 0.055 N HCl for 5 h (animal products) (Kabir et al.,

1983a). Hydrolyzed fecal samples were prepared by adding 2 g sample to 200 mL of 0.44 N HCl and autoclaving at 121

0

C for 2 h. For hydrolyzed urine,

10 mL of urine were mixed with 50 mL of 0.1 N HCl and heated at 12rc for

30 min (Miller & Edwards, 1981). When the hydrolysates were cool, they were adjusted to pH 4.5 with 6 N KOH. Redistilled water was added to give a final total volume of 100 mL for urine samples, and 250 mL for food and fecal specimens. The diluted hydrolysates were filtered through Whatman

No. 1 filter paper (Whatman Laboratory Products, Clifton, NJ) and diluted

36 further to give 1-2 ng PN equivalents/mL before the microbiological assay.

Glycosylated and free vitamin B-6 were determined by the method of

Kabir, Leklem and Miller (1983a) with a slight modification. Samples for glycosylated and free vitamin B-6 content were prepared without the acid hydrolysis step given above. For determining either glycosylated or free vitamin B-6 in foods or feces, one gram of sample was added to two duplicate beakers (4 beakers total) containing 100 mL of 0.1 M phosphate buffer at pH 5.0, and stirred for 2 h at room temperature (22-25

0

C) to obtain a well-homogenized mixture. Samples for urinary glycosylated or free vitamin B-6 were prepared by mixing 5 mL of urine with 50 mL of 0.1 M phosphate buffer (pH 5.0) in two duplicate beakers. To the mixture in two of the four beakers labelled "glycosylated B-6," 60 units (11 mg) of fi-D- glucosidase (Sigma Chemical Co., St. Louis, MO) were added. No enzyme was added to the two beakers labelled "free B-6." Mixtures in the four beakers for each sample were incubated at 37

0

C for another 2 h in a shaking water bath (GCA/Precision Scientific Co., Chicago, 111) at 40 cycles per min.

Ten milliliters of 1 N HC1 were added to each of the four beakers, and the mixture was steamed for 5 min. Finally, the contents of each beaker were adjusted to pH 4.5 with 6 N KOH, diluted to 100 mL (urinary samples) or

250 mL (food and fecal samples) with redistilled water, mixed well, filtered through Whatman No. 1 filter paper, and diluted further to obtain a final

1-2 ng/mL as PN equivalents before the microbiological assay.

Vitamin B-6 microbiological assay was done by the method of Miller and Edwards (1981). After reading of the percent transmittance values in an Evelyn colorimeter (Rubicon Co., Philadelphia, PA), the data were processed by the Interpolation Unequal Spaced, a calculator program

37

(Hewlett-Packard, Palo Alto, CA), to compute the vitamin B-6 content of each test tube from the standard curve based on a working standard solution of 2 ng PN/mL. The percentage of glycosylated vitamin B-6 in each sample was calculated by the formula (Kabir et al., 1983a): [(G - F)/T] x

100; G = nmol per composite of sample treated with fi-glucosidase, F =

Hmol per composite of sample treated without fi-glucosidase, and T = (imol per composite of sample as determined by microbiological assay after acid hydrolysis. These data were computed by a spreadsheet (Microsoft Excel

Version 2.2, Microsoft, Redmond, WA).

Frozen reconstituted orange juice served as a control throughout the experimental analysis. Preparation of the orange juice control was: one can of frozen orange juice concentrate was thawed at room temperature (22-

25

0

C), mixed well, and centrifuged for 7 min at top speed. The supernatant was decanted into a beaker and the pulp was discarded. Eight-milliliter portions of the orange juice concentrate were placed in vials and frozen at

-20 o

C for later analysis. For each assay, one vial of the orange juice control was used. The preparation of the control sample followed the same procedures as given for plant products except it was stirred with 0.1 M phosphate buffer for 1 h instead of 2 h at room temperature (22-25

0

C) to get a well-mixed sample. For the orange juice control, the means (« = 14) of total, glycosylated and free vitamin B-6, in (ig/100 g, were 197.9 ±11.2 (CV =

5.7%), 88.2 ± 4.0 (CV = 4.5%), and 44.9 ± 3.7 (CV = 8.2%), respectively. The averages of % glycosylated and free vitamin B-6 in this control were 44.7 ±

3.2% (CV = 7.2%) and 22.6 ± 2.3% (CV = 10.2%), respectively.

38

Statistical Analysis

A paired, two tailed t test was applied to compare the 9 subjects' weights at the beginning and end of study. A statistical analysis of variance

(ANOVA) F test was used to analyze the variances for 4 subjects' urinary and fecal data during the low- and high-glycosylated periods. Probability values for both tests equal to or less than 0.05 were regarded as statistically significant.

39

RESULTS

The results of food consumption as well as urinary and fecal excretion of total, glycosylated and free vitamin B-6 in 4 women are reported here. Four subjects were chosen based on the stable creatinine excretion. The remaining subjects' data will be analyzed by another laboratory worker and be reported elsewhere.

Subject Profiles

Nine subjects' nationalities and physiological characteristics are presented in Table 3. These subjects maintained their weights throughout the study. A paired, two-tailed t test indicated that there was no significant difference between the subjects' mean initial (55.4 ± 10.1 kg) and final (54.9

± 9.7 kg) weights (P > 0.05).

Dietary Intake of Total, Glycosylated and Free Vitamin B-6

The amount of total, glycosylated and free vitamin B-6 in the adjustment and two experimental diets is presented in Table 7. The adjustment diet was supplemented daily with 0.37 mg PN, making the subjects' total vitamin B-6 intake 1.357 mg/d. Total vitamin B-6 in the high-glycosylated vitamin B-6 diet (1.897 mg or 11.22 îmol) was 0.391 mg higher than that in the low-glycosylated vitamin B-6 diet (1.506 mg or 8.91

^mol). The calculated vitamin B-6 values from the Food Processor II nutrient database (ESHA Research, Salem, OR) for the adjustment, low- and high-glycosylated vitamin B-6 diets were 0.96,1.37 and 1.43 mg, respectively.

TABLE 7

Diet

Adjustment Diet

3

Total, glycosylated and free vitamin B-6

1 9 in the adjustment, low- and high-glycosylated vitamin B-6 diets '

Total Vitamin B-6 Glycosylated Vitamin B-6 mg mg %

Free Vitamin B-6 mg %

1.357 0.169 12 0.368 27

Low-Glycosylated

Vitamin B-6 Diet 1.506 0.159 11 0.685 45

High-Glycosylated

Vitamin B-6 Diet 1.897 0.421 22 0.669 35

Vitamin B-6 is expressed as milligrams of pyridoxine. Each value was based on the duplicate samples. The data for the low- and high-glycosylated vitamin B-6 diets are based on means of two days' food composites.

Total vitamin B-6 refers to the amount of vitamin B-6 measured by acid hydrolysis following a microbiological assay. Free vitamin B-6 is the amount of free forms of vitamin B-6 measured in a sample that was mixed with 0.1 M phosphate buffer. Glycosylated vitamin B-6 is measured by subtracting free vitamin B-6 sample from the C-glucosidase treated sample. % Glycosylated vitamin B-6 = (Glycosylated Vitamin B-6/Total Vitamin B-

6) X 100. % Free Vitamin B-6 = (Free Vitamin B-6/Total Vitamin B-6) X 100.

Data represent food composite plus a 0.37 mg pyridoxine supplement.

O

41

The % glycosylated vitamin B-6 content of the high-glycosylated vitamin B-6 diet was two times larger than of the low-glycosylated vitamin

B-6 diet (Table 7). In the low-glycosylated vitamin B-6 diet, the mean amounts of total vitamin B-6 in the plant, animal and milk components were 0.639, 0.616 and 0.252 mg/d, respectively; in the high-glycosylated vitamin B-6 diet, 1.431, 0.232 and 0.235 mg/d, respectively. Detailed vitamin B-6 data of the diet composites are given in Appendix 6. The total glycosylated vitamin B-6 content of plant foods in the high-glycosylated vitamin B-6 diet (0.421 mg) was almost three times higher than that in the low-glycosylated vitamin B-6 diet (0.159 mg). Glycosylated vitamin B-6 levels in animal and milk products in both diets were similarly low; these foods contained under 3% glycosylated vitamin B-6.

Urinary Excretion of Total, Glycosylated and Free Vitamin B-6

The effect of the adjustment, as well as the low- and high- glycosylated B-6 diets on each subject's excretion of urinary total, glycosylated and free vitamin B-6 is given in Table 8. The 4 subjects' average urinary total vitamin B-6 excretion was 0.76 ± 0.20 ^mol/24 h when they received the low-glycosylated vitamin B-6 diet, and 0.67 ± 0.06

îmol/24 h when they received the high-glycosylated vitamin B-6 diet.

Urinary total vitamin B-6, expressed as a percentage of the total vitamin B-

6 intake of the adjustment and two experimental diets, is presented in

Figures 6 and 7. When expressed as % excretion of intake, the means of urinary total vitamin B-6 during the low- and high-glycosylated vitamin B-

6 experimental periods were 8.50 ± 2.37 and 6.00 ± 0.75%, respectively.

Three out of the four subjects excreted a higher percentage of total urinary

TABLE 8

Diet and Subject

Adjustment Diet

Subject 1

Subject 2

Subject 6

Subject 9

Mean ± SD

6

Total, glycosylated and free vitamin B-6 excreted in urine during the adjustment, low- and high-glycosylated vitamin B-6 dietary periods '

Total Vitamin B-6

3

/imo//24 h

0.88

0.84

0.60

0.68

0.75 ±0.13

Glycosylated Vitamin B-6

4

Hmol/24 h

0.14

0.13

0.13

0.10

%

16

15

22

15

0.13 ± 0.02 17 ±3.4

Free Vitamin B-6

5 fimol/24 h

0.62

0.59

0.38

0.49

%

71

70

63

72

0.52 ±0.11 69 ± 4.1

Low-Glycosylated

Vitamin B-6 Diet

Subject 1

Subject 2

Subject 6

Subject 9

Mean ± SD

0.72 ± 0.04

0.9010.03

0.48 ±0.05

0.92 ± 0.23

0.76 ± 0.20

0.14 ± 0.02

0.11 ±0.01

0.10 ±0.02

0.07 ±0.03

0.11 ±0.03

19 ±2.0

12 ±1.5

20 ± 2.0

8±2.9

15 ± 5.7

0.54 ± 0.04

0.67 ±0.03

0.35 ± 0.05

0.68 ± 0.18

0.56 ±0.15

75 ± 3.1

74 ± 0.6

72 ± 6.7

74 ± 3.2

74 ±1.3

High-Glycosylated

Vitamin B-6 Diet

Subject 1

Subject 2

Subject 6

Subject 9

Mean ± SD

0.72 ±0.05

0.71 ±0.02

0.62 ±0.03

0.62 ± 0.04

0.67 ±0.06

0.27 ±0.02

0.24 ± 0.05

0.29 ±0.04

0.15 ± 0.02

0.24 ± 0.06

38 ±6.1

34 ±5.3

47 ±5.6

25 ± 4.0

36 ± 9.1

0.50 ± 0.03

0.52 ± 0.02

0.39 ± 0.02

0.43 ± 0.03

0.46 ± 0.06

70 ± 5.2

73 ±1.2

63 ± 0.6

70 ±1.2

69 ± 4.2

TABLE 8 (Continued)

^he data for the adjustment diet are mean of urinary excretion on two days' assays for each subject; for the low- and high-glycosylated vitamin B-6 diets, means are based on three days' assays.

2

Formulas for calculation are given in Table 7.

3

Not significantly different between the low- and high-glycosylated B-6 diets (ANOVA, P > 0.05).

4

Significantly different for both glycosylated and % glycosylated vitamin B-6 values between low- and high- glycosylated B-6 diets (ANOVA, P < 0.05).

5

Not significantly different between the low- and high-glycosylated B-6 diets for both free and % free vitamin

B-6 (ANOVA, P > 0.05).

6

Mean ± SD for 4 subjects.

12

« 10 o o

1

^ Total Vit. B-6

[T] Free Vit. B-6

□

Glycosylated Vit. B-6

Adjustment Low GB6 Diet

Periods

High GB6 Diet

FIGURE 6 Mean daily urinary excretion of total, free and glycosylated vitamin B-6 as a percentage of total vitamin B-6 intake during the adjustment, low- and high-glycosylated (Low/High GB6) dietary periods.

Vertical lines express the standard deviation of the mean for the four subjects. c o

12

X tL) i

10

LGB6

8

+u LGB6 - -

■

HGB6

0

6

> o ^< b£ 2 1-

So

HGB6 CT

''-~-

*.*---

1 2

Experiinental Periods

Subject 1

Subject 2

Mean of Group 1

Subjects

Subject9

Mean of Group 2

FIGURE 7 Urinary vitamin B-6 excretion by each subject and group means in response to low- and high-glycosylated vitamin B-6 (LGB6/HGB6) diets as a percentage of total intake of vitamin B-6. Each subject's point represents the mean of the three days' assays during each experimental period.

44

45 vitamin B-6 on the low-glycosylated B-6 diet than on the high-glycosylated diet (Figure 7).

Individual subject and group means of urinary % glycosylated vitamin B-6 excreted in response to the two experimental dietary periods are given in Figure 8. A higher mean value (36 ±9.1%) was found when the high-glycosylated vitamin B-6 diet was fed; a lower value (15 ± 5.7%) was observed when the low-glycosylated vitamin B-6 diet was fed (Table 8).

This difference was statistically significant (P < 0.05). Averages of urinary glycosylated vitamin B-6 excreted on the low- and high-glycosylated vitamin B-6 diets were 0.11 ± 0.03 and 0.24 ± 0.06 (imol/24 h, respectively

(Table 8).

<0

§

2

UP u

"9 pa

60

50

Q

*

0

"

— — m —

40

>

1

30

; HGB6 a^ "^ ^^ HCBe"""""

^ in I

20

LGB6 if^^"^»^ **+* u

*

■

""»^^^^^Z*^ v x

< ^-.^ ^j LGB6

10

^-« 58

Subject 1

Subject 2

Mean of Group!

Subject 6

Subject 9

Mean of Group 2

D

I.I.

1 2

Experimental Periods

FIGURE 8 Urinary % glycosylated vitamin B-6 excretion by each subject and group means in response to low- and high-glycosylated vitamin B-6

(LGB6/HGB6) diets. Urinary % glycosylated vitamin B-6 calculation based on the formula: Glycosylated Vitamin B-6 in Urine X 100

Total Vitamin B-6 in Urine

Each subject's point represents the mean of the three days' assays during each experimental period.

46

Fecal Excretion of Total, Glycosylated and Free Vitamin B-6

The subjects' excretion of fecal total, glycosylated and free vitamin B-

6 is given in Table 9. Each subject excreted more fecal vitamin B-6 when receiving the high-glycosylated vitamin B-6 diet than when receiving the low-glycosylated vitamin B-6 diet.

Expressed as % of intake, the mean total excretion of fecal vitamin B-

6 was higher during the high-glycosylated vitamin B-6 period than during the low-glycosylated vitamin B-6 period (Figures 9 and 10). Fecal glycosylated vitamin B-6 level accounted for less than 1 % of the dietary vitamin B-6 intake during the two experimental periods. The means for fecal % glycosylated vitamin B-6 were small because no glycosylated vitamin B-6 was detected in the feces of two of the four subjects.

Total, Glycosylated and Free Vitamin B-6 Excreted in Urine and Feces

The four subjects' total urinary and fecal excretion of total, glycosylated and free vitamin B-6 is presented in Table 10. The mean total vitamin B-6 excreted in urine and feces, expressed as |imol/24 h, was 3.74 ±

0.62 during the low-glycosylated B-6 period and 5.23 ± 0.89 during the high- glycosylated vitamin B-6 period. Expressed as % of dietary vitamin B-6 intake, total excretion was 42 ± 7.7 and 47 ± 8.5% on the low- and high- glycosylated B-6 diets, respectively (Figure 11).

Each subject's total urinary and fecal glycosylated vitamin B-6 excretion was lower on the low- than on the high-glycosylated B-6 diet

(Table 10). Expressed as % of intake, the mean glycosylated vitamin B-6 excretion in urine and feces also showed the same tendency (Figure 11): 2 ±

0.9 and 3 ± 1.5% on the low- and high-glycosylated B-6 diets, respectively.

Diet and Subject

Low-Glycosylated

Vitamin B-6 Diet

Subject 1

Subject 2

Subject 6

Subject 9

Mean ± SD

7

Total, glycosylated and free vitamin B-6 excreted in feces during the low- and high-glycosylated vitamin B-6 dietary periods '

Total Vitamin B-6

3

Hmol/24 h

3.08

3.16

2.36

3.32

2.98 ± 0.43

TABLE 9

Glycosylated Vitamin B-6

4 nmol/24 h

0.02

0.14

0.08 n.d.

6

0.06

%

0

4

3 n.d.

1.8

Free Vitamin B-6

5 fxmol/24 h %

2.44

2.14

1.85

2.87

2.33 ± 0.44

79

68

78

86

78 ± 7.4

High-Glycosylated

Vitamin B-6 Diet

Subject 1

Subject 2

Subject 6

Subject 9

Mean ± SD

4.73

4.78

3.33

5.39

4.56 ±0.87 n.d.

0.12

0.28 n.d.

0.10 n.d.

2

8 n.d.

2.5

4.09

3.76

2.56

4.09

3.63 ± 0.73

87

79

77

76

80 ± 5.0

Data represent the mean of two fecal composites, based on nmol/24 h, during each experimental period.


Significantly different between the two diets (ANOVA, P < 0.05).

4

Not significantly different for both glycosylated and % glycosylated vitamin B-6 between the two diets.

Significantly different between the two diets for free vitamin B-6 (ANOVA, P < 0.05); not significantly different for % free vitamin B-6 (ANOVA, P > 0.05).

6 n.d. = not detected

7


48

50 at

S 40 o

30 o

20 "

X

13 10

^ Total Vit. B-6

[v] Free Vit. B-6

□


Low GB6 Diet

Periods

High GB6 Diet

FIGURE 9 Mean daily fecal total, free and glycosylated vitamin B-6 excretion as a percentage of total vitamin B-6 intake during the low- and high-glycosylated (Low/High GB6) dietary periods. Vertical lines represent the standard deviation of the mean for four subjects. c

>-.

X

50

45

40

CO

35

> ^

30 "

la ^5

O Ji

IZ — 25 -

^ 20

HGB6

■

-

■

'a LGB6

Subject 1

Subject2

Mean of Group 1

Subject 6

Suêct9

Mean of Group 2

1 2

Experimental Periods

FIGURE 10 Fecal vitamin B-6 excretion by each subject and group means in response to low- and high-glycosylated vitamin B-6 (LGB6/HGB6) diets as a percentage of total intake of vitamin B-6. Each subject's point represents the mean of two assays during each experimental period.

TABLE 10

The amounts of total, glycosylated and free vitamin B-6 excreted in both urine and feces

during the low- and high-glycosylated vitamin B-6 dietary periods

1,2

Diet and Subject Total Vitamin B-6

3

Glycosylated Vitamin B-6

4

Hmol/24 h Hmol/24 h

Low-Glycosylated

Vitamin B-6 Diet

Subject 1

Subject 2

Subject 6

Subject 9

3.80

4.06

2.84

4.24

0.16

0.25

0.18

0.07

Mean ± SD

6

3.74± 0.62 0.17+ 0.07

Free Vitamin B-6 fj.mol/24 h

2.98

2.81

2.20

3.55

2.89 ± 0.56

5

High-Glycosylated

Vitamin B-6 Diet

Subject 1

Subject 2

Subject 6

Subject 9

Mean ± SD

5.45

5.49

3.95

6.01

5.23 ± 0.89

0.27

0.36

0.57

0.15

0.34± 0.18

4.59

4.28

2.95

4.52

4.09 ± 0.77

âch value represents the amounts of total, glycosylated and free vitamin B-6 excreted in urine and feces during each experimental period.

2


3

Significantly different between the two diets (ANOVA, P < 0.05).

4

Not significantly different between the two diets (ANOVA, P > 0.05).

5

Significantly different between the two diets (ANOVA, P < 0.05).

6


60

»

UH

T3

C nj c

D

50

40

30 c o

•xs

20 w 2

^ *

»*3

^S

10

0

Total Vit. B-6

Free Vit. B-6


Low GB6 Diet

Periods

High GB6 Diet

FIGURE 11 The amounts of daily total, free and glycosylated vitamin B-6 excreted in both urine and feces from four subjects, expressed as a percentage of total vitamin B-6 intake, during the low- and high- glycosylated (Low/High GB6) dietary periods. Vertical lines represent the standard deviation of the mean for four subjects.

50

51

DISCUSSION

Since glycosylated vitamin B-6 is a major determinant of dietary vitamin B-6 bioavailability in humans (Kabir et al., 1983c; Gregory et al.,

1991), understanding of dietary glycosylated vitamin B-6 utilization is important for evaluating nutritional adequacy of diets. The bioavailability of chemically synthesized PN-glucoside (Tsuji et al., 1977; Iwami &

Yasumoto, 1986) and that occurring naturally in foods (Kabir et al., 1983b;

Bills et al., 1987) have been studied in animals as well as humans.

However, information on the amount of this compound in diets affecting human vitamin B-6 status is still unclear.

In the present study, glycosylated vitamin B-6 accounted for 11 and

22% of total vitamin B-6 intake in the low- and high-glycosylated vitamin

B-6 diets, respectively (Table 7). The bioavailability of dietary glycosylated vitamin B-6 was evaluated by measuring urinary and fecal vitamin B-6 excretion of 4 subjects who received these two diets. Bioavailability of dietary vitamin B-6 is influenced by the physiological ability to digest, absorb and utilize this nutrient. A lower concentration of this vitamin and its metabolite in urine and plasma, and, possibly, a higher excretion in feces can indicate a decrease in bioavailability (Leklem et al., 1980; Lindberg et al.,

1983; Kabir et al., 1983c).

The four subjects' mean urinary vitamin B-6 excretion, expressed as

% of total vitamin B-6 intake, was lower during the high-glycosylated vitamin B-6 period (6.0 ± 0.75%) than during the low-glycosylated vitamin

B-6 period (8.5 ± 2.37%) (Figure 6); in contrast, their mean fecal vitamin B-6 excretion during the high-glycosylated vitamin B-6 period (40.7 ± 8.2%) was

52 greater than the low-glycosylated vitamin B-6 period (33.6 ± 5.4%) (Figure

9). We also observed that these four subjects excreted significantly more urinary glycosylated vitamin B-6 when receiving the high-glycosylated vitamin B-6 diet than when receiving the low-glycosylated vitamin B-6 (P

< 0.05) (Table 8). These results suggest that dietary glycosylated vitamin B-6 is not completely bioavailable to humans. Similar results were obtained by

Kabir et al. (1983c), who measured the bioavailability of glycosylated vitamin B-6 in tuna, peanut butter and whole wheat bread. These investigators found that the urinary total vitamin B-6 excretion was significantly lower and fecal total vitamin B-6 excretion was significantly higher when subjects received diets high in glycosylated vitamin B-6 foods

(peanut butter or whole wheat bread) than when they received the one low in glycosylated vitamin B-6 foods (tuna). In the present study, additional information of the subjects' urinary 4-PA excretion, plasma PLP and plasma total vitamin B-6 concentrations, which will be reported elsewhere, will give more conclusive evidence on the bioavailability of dietary glycosylated vitamin B-6. These parameters will provide further evidence on both absorption and utilization of glycosylated vitamin B-6, and also may be useful for interpreting fecal vitamin B-6 excretion as an indicator of bioavailability.

In the present investigation, the four subjects' mean excretion of glycosylated vitamin B-6 in urine, expressed as % of dietary glycosylated vitamin B-6, was 12 and 10% during the low- and high-glycosylated vitamin B-6 dietary periods, respectively (Tables 7 and 8). This percentage ingested glycosylated vitamin B-6 excretion is in agreement with observations reported by Kabir et al. (1983c) and Andon et al. (1989):

53 regardless of differences in the quantity of glycosylated vitamin B-6 ingested, the results of these human studies consistently showed that approximately 11% of this compound is excreted in urine. Gregory et al.

(1991), however, detected higher % urinary PN-glucoside value (35%), which might be related to their experimental design. When vitamin B-6- deficient rats were fed different levels of PN-glucoside, a constant 7-9% of the ingested PN-glucoside was excreted intact in urine (Trumbo et al., 1988).

These results from human and animal studies suggest that the extent of

PN-glucoside utilization is not affected by the level of dietary intake.

Fecal total vitamin B-6 excretion increased significantly during the high-glycosylated vitamin B-6 period (P < 0.05) (Table 9). Regardless of glycosylated vitamin B-6 intake, however, approximately 80% of fecal total vitamin B-6 was excreted as free forms. Additionally, glycosylated vitamin

B-6 was not detected in feces from two subjects; the remaining two subjects excreted less than 3% of fecal total vitamin B-6 in the glycosylated form.

Contributors to the variability of fecal glycosylated vitamin B-6 excretion among the four subjects may include little glycosylated vitamin B-6 normally found in feces, subjects' physiological differences (e.g., gastrointestinal tract functions, types of microflora among these subjects) and experimental error in measuring. These findings are supported by Kabir et al. (1983b), who reported that the majority of fecal vitamin B-6 existed as a free vitamin B-6 form with little or no detectable amount as glycosylated vitamin B-6. Evidence of 6-glucosidase activity by human gut microflora

(Mallett et al., 1989) may explain why little or no glycosylated vitamin B-6 is found in feces.

In the present study, however, it is hard to determine clearly the

54 source of the increased fecal vitamin B-6 during the high-glycosylated vitamin B-6 periods. Increased fecal vitamin B-6 excretion could be due to:

1) decreased absorption of glycosylated vitamin B-6 and other bound forms of vitamin B-6 in the small intestine with subsequent hydrolysis of these compounds in the large intestine; or 2) synthesis of vitamin B-6 by microflora (Samra et al., 1946) in the large intestine.

Diet composition can influence the extent of vitamin B-6 absorption by affecting the access of dietary B-6 compounds to the absorptive surface

(Gregory, 1990) or to the digestive enzymes (Trumbo et al., 1990; Banks &

Gregory, 1991). Dietary fiber has been reported to decrease intestinal absorption of certain nutrients (Kelsay, 1978). According to the calculated values obtained from Food Processor II (ESHA Research, Salem, OR), the dietary fiber content of the high-glycosylated vitamin B-6 diet, 34.8 g, was approximately two times higher than that of the low-glycosylated vitamin

B-6 diet, 17.9 g. For comparison, the recommended fiber intake is 20-30 g with an upper limit of 35 g (Pilch, 1987). Dietary fiber increases wet stool bulk (Slavin, 1987). The weight of the wet stool excretion of the four subjects in this study may reflect the difference in fiber content of the two diets: 189.4 ± 40.8 and 245.5 ± 46.6 g/d during the low- and high-glycosylated vitamin B-6 periods, respectively. Previous studies suggest that certain naturally occurring fiber components may lower vitamin B-6 bioavailability, but purified or semi-purified forms of dietary fiber have no effect (Nguyen et al, 1981 & 1983). The difference may be related to physiological effects of those fibers on nutrient absorption and utilization

(Slavin, 1987). Since a high glycosylated vitamin B-6 diet is also high in fiber, the combined effect of fiber and glycosylated vitamin B-6 on

55 bioavailability of vitamin B-6 in plant-derived foods needs further investigation.

On the other hand, any synthesized vitamin B-6 by the intestinal microflora is not readily absorbed, and it is not a source of vitamin B-6 for the host when vitamin B-6 intake is adequate (Coburn et al., 1989). If the vitamin B-6 synthesized in the colon is available to subjects, plasma PLP concentration and urinary 4-PA excretion will increase when fecal vitamin

B-6 increases. In the present study, data given in Tables 7 and 10 show that a total of 42 and 47% of the ingested vitamin B-6 was excreted into urine and feces during the low- and high-glycosylated vitamin B-6 periods, respectively. Although the results of this present study indicate that vitamin B-6 intake is much greater than excretion, the possibility of utilization vitamin B-6 synthesized by intestinal microflora still can not be eliminated because of lack of information on plasma PLP concentration and urinary 4-PA excretion here.

C-glucosidase, which catalyzes the hydrolysis of various aryl-C-D- glucosides, has been identified in human small intestine (Trumbo et al.,

1990), liver (Daniels et al., 1981) and kidney (Dance et al., 1969), but the comparative activities of this enzyme in human tissues have not been determined. This enzyme activity in guinea pig tissues, as an example, was

2.6 times greater in the jejunum than that in the liver, and there was no detectable activity in the kidney (Trumbo et al., 1990). Therefore, dietary glycosylated vitamin B-6 can be converted to the biologically active PN form through the hydrolysis of this compound by intestinal fi-glucosidase before absorption as well as through the hydrolysis by 6-glucosidase in other tissues following absorption. In humans, the metabolic utilization of

56 orally administrated PN-glucoside was 58% relative to PN, while the utilization of PN-glucoside administrated intravenously was about half that utilized by oral administration (Gregory et al., 1991). This provides evidence that the small intestine is the primary place where dietary glycosylated vitamin B-6 is digested by C-glucosidase to PN in humans. In the present study, although we can not investigate the route of dietary PN- glucoside digestion, glycosylated vitamin B-6 was detected in each of the four subjects' urine samples, which provides evidence of incomplete metabolic utilization of this compound by humans.

The values of total vitamin B-6 determined in food composites of the adjustment, low- and high-glycosylated vitamin B-6 diets were slightly different from calculated data. Mangels et al. (1990), who analyzed self- chosen diets by a microbiological method using S. uvarum as the assay organism, also reported that analyzed vitamin B-6 values were slightly higher than calculated values. The discrepancy between analyzed and calculated values for vitamin B-6 in foods may be due to differences in variety, seasonal variation and vitamin B-6 losses in processing or during storage. Additionally, incomplete food composition data of vitamin B-6 and operational performance by different analysts and laboratories may contribute to these differences.

Interlaboratory variations in vitamin B-6 microbiological assays have been reported by Toepfer and Polansky (1970). They evaluated the results of total and the three free forms of vitamin B-6 contents in 4 foods obtained by 9 collaborators. Although Toepfer and Polansky supplied these collaborators with the same food samples and reagents, and each of the collaborators used the same analytical procedure, the coefficients of

57 variation for total vitamin B-6 values obtained by these collaborators were

17, 20, 21 and 18% in lima beans, whole wheat flour, dried beef liver and enriched bread, respectively. Similar findings were reported by Edwards et al. (1963), who observed the variations of the analyzed data among 13 collaborators were up to 6 times greater on individual foods. Edwards et al. also observed a high degree of variability in results within individual laboratories. In addition, Andon et al. (1989) measuring the vitamin B-6 status indices for 30 lactating women and their infants found a wide variation in vitamin B-6, especially glycosylated vitamin B-6 content. The ranges of dietary, urinary and breast-milk glycosylated vitamin B-6 values were 0.11-4.61 (mean ± SD = 1.33 ± 0.85) îmol/d , 0.01-0.96 (0.15 ± 0.15)

Hmol/d and -71-148 (18 ± 41) nmol/L, respectively. Therefore, inter- and intra-laboratory variation is a factor which should be considered when evaluating the different values in a same assay. Since microbiological assays using the growth of yeast to assess vitamin B-6 values are affected by environmental factors, such as light, temperature and water, a control is highly recommended as an index to evaluate inter-assay reproducibility. In the present study, orange juice concentrate was used to as a control for each assay. The means (n = 14) of orange juice for total, glycosylated, and free vitamin B-6, in îg/lOO g, were 197.9 ± 11.2 (CV = 5.7%), 88.2 ± 4.0 (CV =

4.5%), and 44.9 ± 3.7 (CV = 8.2%), respectively.

As shown in Table 7, the sum of the glycosylated and free vitamin B-

6 accounted for 56% of total vitamin B-6 in food composites. The remaining vitamin B-6, which might be phosphorylated or attached to protein or other compounds in foods, could be hydrolyzed to free vitamin

B-6 by acid hydrolysis at 121

0

C. Since the determination of glycosylated

58 vitamin B-6 contents came from subtraction rather than direct analysis, this might be a source of error in calculating glycosylated vitamin B-6 in food composites (Reynolds, 1990). Additionally, vitamin B-6 in diets may exist in other forms which can not be identified by the present experimental procedures. Tadera and co-workers (Tadera et al., 1983,1985,

1988 & 1991) have identified at least six PN-glucoside derivatives (Figure 4).

PNX, for example, is determined after preliminary treatment with KOH followed by digestion of fi-glucosidase in bran (e.g., rice bran, wheat bran) and legumes (e.g., peas, soybeans) (Tadera et al., 1986). Pyridoxyllysine is a degradation product of vitamin B-6 when PL or PLP binds with proteins during thermal processing (121

0

C) (Gregory, 1980; Gregory et al., 1986).

These compounds may be acid-hydrolyzed to release free vitamin B-6 forms (Gregory & Sartain, 1991), but can not be determined either with phosphate buffer or with fi-glucosidase.

The sum of the glycosylated and free vitamin B-6 was not equivalent to the total vitamin B-6 in either urinary or fecal composites (Tables 8 & 9).

One possible explanation is that some ingested vitamin B-6 may react with food components to synthesize a complex which can not be digested by intestinal mucosa and microflora to release free forms of vitamin B-6. This vitamin B-6 complex can not be identified by our present experimental procedures for the determination of either glycosylated or free vitamin B-6 contents, but can be acid-hydrolyzed to release free forms of vitamin B-6. contributing total vitamin B-6 contents. During the high-glycosylated vitamin B-6 period, three out of four subjects showed the amount of urinary glycosylated and free vitamin B-6 excretion 8% more than that of total vitamin B-6 (Table 8). In addition to experimental error, another

59 possible reason for this might be the synthesis of vitamin B-6 during the 2- h incubation in phosphate buffer.

60

SUGGESTIONS FOR FUTURE STUDY

Urinary and fecal total vitamin B-6 excretion should not be the only criteria used to evaluate glycosylated vitamin B-6 bioavailability. Plasma

PLP and total vitamin B-6 concentration as well as urinary 4-PA excretion should also be considered. These parameters not only give further evidence on absorption and utilization of dietary glycosylated vitamin B-6, but are useful also for interpreting fecal vitamin B-6 excretion data.

Dietary fiber has been suggested as another factor which may lower vitamin B-6 bioavailability (Reynolds, 1988; Gregory, 1990). In the present study, the dietary fiber content of the high-glycosylated vitamin B-6 diet was approximately two times greater than that of the low-glycosylated vitamin B-6 diet. Since a diet high in glycosylated vitamin B-6 is also high in fiber, the combined effect of these two dietary components on vitamin

B-6 bioavailability in plant-derived foods should be further investigated.

Several PN-glucoside derivatives have been identified by Tadera's research group (Tadera et al., 1983,1985,1988 & 1991). Whether these compounds can be measured by the present experimental procedures for glycosylated vitamin B-6 and whether they are metabolized similar to PN- glucoside remain questions. Future research may focus on this area.

61

SUMMARY AND CONCLUSIONS

The objective of this study was to determine the effect of dietary glycosylated vitamin B-6 on the bioavailability of vitamin B-6 in women.

This 44-d diet study was divided into a 8-d adjustment period followed by two 18-d experimental periods in a crossover design. Using a metabolic balance technique, we measured the intake and excretion of total, glycosylated and free vitamin B-6 by 4 women who were receiving low- and high-glycosylated vitamin B-6 diets.

The total vitamin B-6 content in the low- and high-glycosylated vitamin B-6 diets was 1.506 and 1.897 mg, respectively, in which 11 and 22% was the glycosylated form, respectively. Expressed as % of total vitamin B-6 intake, the subjects' mean urinary total vitamin B-6 excretion was lower during the high-glycosylated vitamin B-6 period (6.0 ± 0.8%) than during the low-glycosylated vitamin B-6 period (8.5 ± 2.4%); in contrast, their mean fecal vitamin B-6 excretion during the high-glycosylated vitamin B-6 period (40.7 ± 8.2%) was greater than the low-glycosylated vitamin B-6 period (33.6 ± 5.4%). Although the changes were not significant, each of four subjects responded uniformly. In addition, approximately 11% of ingested glycosylated vitamin B-6 was excreted in urine. These results suggest that dietary glycosylated vitamin B-6 is not completely bioavailable to humans, and the extent of its utilization is not affected by dietary intake.

62

BIBLIOGRAPHY

Addo, C. & Augustin, J. (1988) Changes in the vitamin B-6 content in potatoes during storage. J. Food Sci. 53(3): 749-752.

Andon, M. B., Reynolds, R. D., Moser-Veillon, P. B. & Howard, M. P. (1989)

Dietary intake of total and glycosylated vitamin B-6 and the vitamin B-6 nutritional status of unsupplemented lactating women and their infants.

Am. J. Clin. Nutr. 50: 1050-1058.

Ang, C. Y. W. (1979) Stability of three forms of vitamin B-6 to laboratory light conditions. J. Assoc. Off. Anal. Chem. 62: 1170-1173.

Association of Official Analytical Chemists (1990) Official Methods of

Analysis. 14th ed., pp. 1089-1091, A.O.A.C, Washington, DC.

Banks, M. A. & Gregory, J. F. (1991) Intestinal 6-glucosidase activity is not induced by chronic ingestion of pyridoxine-C-D-glucoside in rats. Nutr. Res.

11:169-175.

Bender, D. A. (1989) Vitamin B-6 requirements and recommendations. Eur.

J. Clin. Nutr. 43: 289-309.

Bills N. D. (1990) In vivo and in vitro determination of the bioavailability of vitamin B-6 from plant foods containing pyridoxine glucoside. Ph.D.

Thesis, Oregon State University.

Bills, N. D., Leklem, J. E. & Miller L. T. (1987) Vitamin B-6 bioavailability in plants foods is inversely correlated with % glycosylated vitamin B-6. Fed.

Proc. 46:1487(abstr).

Black, A. L., Guirard, B. M. & Snell, E. E. (1977) Increased muscle phosphorylase in rats fed high levels of vitamin B-6. J. Nutr. 107: 1962-1968.

Booth, C. C. & Brain, M. C. (1962) The absorption of tritium-labelled pyridoxine hydrochloride in the rat. J. Physiol. 164: 282-294.

Cho, J. Y., Kirksey, A., Galal, O., Bassily, N., Harrison, G. & Jerome, N.

(1990) B-6 vitamers and C-glucoside conjugate levels in milk of American and Egyptian women during 6 months of lactation. FASEB J. 4(3): A668

(abstr).

63

Cobum, S. P., Mahuren, J. D., Wostmann, B. S., Snyder, D. L. & Townsend,

D. W. (1989) Role of intestinal microflora in the metabolism of vitamin B-6 and 4'-deoxypyridoxine examined using germfree guinea pigs and rats. J.

Nutr. 119: 181-188.

Cunnane, S. C, Manku, M. S. & Horrobin, D. F. (1985) Effect of vitamin B-6 deficiency on essential fatty acid metabolism. In: Vitamin B-6: Its role in health and disease. (Reynolds, R. D. & Leklem, J. E. eds.), vol. 13, pp. 447-

451, A. R. Liss, New York, NY.

Dance, N., Price, R. G., Robinson, D. & Stirling, J. L. (1969) fi-Galactosidase,

6-glucosidase and N-acetyl-fi-glucosaminidase in human kidney. Clin.

Chim. Acta. 24: 189-197.

Daniels, L. B., Coyle, P. J., Chiao, Y-B & Glew, R. H. (1981) Purification and characterization of a cytosolic broad specificity fi-glucosidase from human liver. J. Biol. Chem. 256: 13004-13013.

Edwards, M., Benson, E. & Storvick, C. A. (1963) Collaborative study of vitamin B-6 methodology. J. Assoc. Off. Anal. Chem. 46: 396-399.

^-Ekanayake, A. & Nelson, P. (1990) Effect of thermal processing on lima bean vitamin B-6 availability. J. Food Sci. 55: 154-157.

Gilbert, J. A. & Gregory, J. F. (1992) Pyridoxine-5'-fi-D-glucoside affects the metabolic utilization of pyridoxine in rats. J. Nutr. 122: 1029-1035.

Gregory, J. F. (1980) Effects of e-pyridoxyllysine bound to dietary protein on the vitamin B-6 status of rats. J. Nutr. 110: 995-1005.

Gregory, J. F. (1988) Recent developments in methods for the assessment of vitamin bioavailability. Food Tech. 42: 230-238.

Gregory, J. F. (1990) The bioavailability of vitamin B-6. In: Vitamin B-6

(Dakshinamutri, K., ed.), vol. 585, pp. 86-95. Ann. N.Y. Acad. Sci., New

York, NY.

Gregory, J. F. & Ink, S. L. (1987) Identification and quantification of pyridoxine-fi-glucoside as a major form of vitamin B-6 in plant-derived foods. J. Agric. Food Chem. 35: 76-82.

/Gregory, J. F., Ink, S. L. & Sartain, D. B. (1986) Degradation and binding to food proteins of vitamin B-6 compounds during thermal processing. J.

Food Sci. 51:1345-1351.

64

Gregory, J. F. & Leatham, K. (1990) Lack of vitamin B-6 activity of 6- hydroxypyridoxine. J. Food Sci. 55:1143-1146.

Gregory, J. F. & Sartain, D. B. (1991) Improved chromatographic determination of free and glycosylated forms of vitamin B-6 in foods. J.

Agric. Food Chem. 39: 899-905.

Gregory, J. F., Trumbo, P. R., Bailey, L. B., Toth, J. P., Baumgartner, T. G. &

Cerda, J. J. (1991) Bioavailability of pyridoxine-5'-f3-D-glucoside determined in humans by stable-isotopic methods. J. Nutr. 121: 177-186.

Gyorgy P. (1934) Vitamin B-2 and the pellagra-like dermatitis in rats.

Nature 133: 498-499.

Hardin, K., Leklem, J.E. & Miller, L.T. (1986) Glycosylated vitamin B-6(GB6) content of selected foods and plasma levels following ingestion of a food high in GB6. Fed. Proc. 45: 822(abstr).

Henderson, L. M. (1985) Vitamin B-6. In: Present knowledge in nutrition.

(Brown, M. L., ed.), 5th ed. pp. 303-317, The Nutrition Foundation.

Washington, DC.

Ink, S. L., Gregory, J. F. & Sartain, D. B. (1986a) Determination of pyridoxine fi-glucoside bioavailability using intrinsic and extrinsic labeling in the rat. J.

Agric. Food Chem. 34: 857-862.

Ink, S. L., Gregory, J. F. & Sartain, D. B. (1986b) Determination of vitamin B-

6 bioavailability in animal tissues using intrinsic and extrinsic labeling in the rat. J. Agric. Food Chem. 34: 998-1004.

IUPAC-IUB Commission on Biochemical Nomenclature. (1973)

Nomenclature for vitamin B-6 on related compounds. Recommendation.

Eur. J. Biochem. 40: 325-327.

Iwami, K. & Yasumoto, K. (1986) Synthesis of pyridoxine-C-glucoside by rice bran fi-glucosidase and its in situ absorption in rat small intestine. Nutr.

Res. 6: 407-414.

Kabir, H., Leklem, J. E. & Miller, L.T. (1983a) Measurement of glycosylated vitamin B-6 in foods. J. Food. Sci. 48(5): 1422-1425.

Clin. Nutr. 48: 863-867.

Kabir, H., Leklem, J. E. & Miller, L. T. (1983b) Relationship of the glycosylated vitamin B-6 content of foods to vitamin B-6 bioavailability in humans. Nutr. Repts. Intl. 28(4): 709-715.

65

Kabir, H., Leklem, J. E. & Miller, L. T. (1983c) Comparative vitamin B-6 bioavailability from tuna, whole wheat bread and peanut butter in humans. J. Nutr. 113: 2412-2420.

Kant, A.K. & Block, G. (1990) Dietary vitamin B-6 intake and food sources in the US population: NHANES II, 1976-1980. Am. J. Clin. Nutr. 52: 707-716.

Kelsay, J. L. (1978) A review of research on effects of fiber intake on man.

Am. J. Clin. Nutr. 31: 142-159.

Kies, G, Kan, S. & Fox, A. M. (1984) Vitamin B-6 availability from wheat, rice, com brans for humans. Nutr. Repts. Intl. 30(2): 483-491.

Leklem, J. E. (1988) Vitamin B-6 metabolism and function in humans. In:

Current topics in nutrition and disease. (Leklem, J. E. & Reynolds, R. D., eds.), vol. 19, pp. 3-28, A. R. Liss, New York, NY.

Leklem, J. E., Miller, L. T., Perera, A. D. & Peffers, D. E. (1980) Bioavailability of vitamin B-6 from wheat bread in humans. J. Nutr. 110: 1819-1828.

Lindberg, A. S., Leklem, J. E. & Miller, L.T. (1983) The effect of wheat bran on the bioavailability of vitamin B-6 in young men. J. Nutr. 113: 2578-2586.

Lumeng, L., Li, T-K. & Lui, A. (1985) The interorgan transport and metabolism of vitamin B-6. In: Vitamin B-6: Its role in health and disease.

(Reynolds, R. D. & Leklem, J. E. eds.), vol. 13, pp. 35-54, A. R. Liss, New

York, NY.

Mallett, A. K., Bearne, G A. & Rowland, I. R. (1989) The influence of incubation pH on the activity of rat and human gut flora enzymes. J. Appl.

Bacteriol. 66: 433-437.

Mangels, A. R., Moser-Veillon, P. B., Andon, M. B. & Reynold, R. D. (1990)

Vitamin B-6 intakes of lactating women: analyzed vs calculated values. J.

Am. Diet. Assoc. 90: 857-859.

McCormick, D. B. (1988) Vitamin B-6. In: Modern nutrition in health and disease. (Shils, M. E. & Young, V. R. eds.), 7th ed. pp. 376-381, Lea & Febiger,

Philadephia, PA.

Middleton, H. M. (1979) In vivo absorption and phosphorylation of pyridoxine-HCl in rat jejunum. Gastroenterology 76: 43-49.

66

Middleton, H. M. (1985) Uptake of pyridoxine by in vivo perfused segments of rat small intestine: a possible role for intracellular vitamin metabolism.

J. Nutr. 115:1079-1088.

Miller, L. T. & Edwards, M. (1981) Microbiological assay of vitamin B-6 in blood and urine. In: Methods of Vitamin B-6 Nutrition (Leklem, J. E. &

Reynolds, R. D., eds.), pp. 45-55, Plenum, New York, NY.

Merrill, A. H. & Burnham, F. S. (1990) Vitamin B-6. In: Present knowledge in nutrition. (Brown, M. L., ed.), 6th ed. pp. 155-162, International Life

Sciences Institute-Nutrition Foundation. Washington, DC.

Merrill, A. H. & Henderson, J. M. (1990) Vitamin B-6 metabolism by human liver. In: Vitamin B-6 (Dakshinamutri, K., ed.), vol. 585, pp. 110-

117, Ann. N. Y. Acad. Sci., New York, NY.

Merrill, A. H., Henderson, J. M., Wang, E., McDonald, B. W. & Millikan,

W. J. (1984) Metabolism of vitamin B-6 by human liver. J. Nutr. 114: 1664-

1674.

Miller, L. T. & Kerkvliet, N. I. (1988) Vitamin B-6 in immunology. In:

Current topics in nutrition and disease. (Leklem, J. E. & Reynolds, R. D., eds.), vol. 19, pp. 133-146, A. R. Liss, New York, NY.

National Research Council (1989) Vitamin B-6. In: Recommended Dietary

Allowances. 10th ed., pp. 142-150, National Academy Press, Washington,

DC.

Nelson, E. W., Burgin, C. W. & Cerda, J. J. (1977) Characterization of food binding of vitamin B-6 in orange juice. J. Nutr. 107: 2128-2134.

Nguyen, L. B., Gregory, J. F. & Cerda, J. J. (1983) Effect of dietary fiber on absorption of B-6 vitamers in a rat jejunal perfusion study. P. S. E. B. M.

173: 569-573.

Okada, M., Ishikawa, K. & Watanabe, K. (1991) Effect of vitamin B-6 deficiency on glycogen metabolism in the skeletal muscle, heart, and liver of rats. J. Nutr. Sci. Vitaminol. 37: 349-357.

Pilch, S. M. (1987) Physiological effects and health consequences of dietary fiber. Bethesda, Md: Life Sciences Research Office, Federation of American

Societies for Experimental Biology, pp. 149-157.

^Reynolds, R. D. (1988) Bioavailability of vitamin B-6 from plant foods. Am.

J. Clin. Nutr. 48: 863-867.

67

Reynolds, R. D. (1990) Determination of dietary vitamin B-6 intake: is it accurate? Am. J. Clin. Nutr. 90: 799-801.

Reynolds, R. D., Acharya, S., Leklem, J. E. & Moser, P. B. (1986) Effects of low maternal dietary intake of calcium, selenium and vitamin B-6 upon breast milk composition in Nepal. In: Human lactation 2. Maternal and environmental factors. (Hamosh, M. & Goldman, A.S., eds.), pp. 205-213,

Plenum Press, New York, NY.

Reynolds, R. D. & Natta, C. L. (1985) Vitamin B-6 and sickle cell anemia. In:

Vitamin B-6: Its role in health and disease. (Reynolds, R. D. & Leklem, J. E. eds.), vol. 13, pp. 301-316, A. R. Liss, New York, NY.

Samra, P. S., Snell, E. E. & Elvehjem, C. A. (1946) The vitamin B-6 group.

VIII. Biological assay of pyridoxal, pyridoxamine and pyridoxine. J. Biol.

Chem. 165: 55-63.

Sauberlich, H. E. (1985) Interaction of vitamin B-6 with other nutrients. In:

Vitamin B-6: Its role in health and disease. (Reynolds, R. D. & Leklem, J. E. eds.), vol. 13, pp. 193-217, A. R. Liss, New York, NY.

Schaltenbrand, W. E., Kennedy, M. S. & Coburn, S. P. (1987) Low- ultraviolet "white" fluorescent lamps fail to protect pyridoxal phosphate from photolysis. Clin. Chem. 33: 631-632.

Scudi, J. V. (1942) Conjugated pyridoxine in rice bran concentrates. J. Biol.

Chem. 145: 637-639.

Shultz, T. D. & Leklem. J. E. (1987) Vitamin B-6 status and bioavailability in vegetarian women. Am. J. Clin. Nutr. 46: 647-651.

Slavin, J. L. (1987) Dietary fiber: classification, chemical analyses, and food sources. J. Am. Diet. Assoc. 87:1164-1171.

Snyderman, S. E., Holt, L. E., Carretero, R. & Jacobs, K. (1953) Pyridoxine deficiency in the human infant. Am. J. Clin. Nutr. 1: 200-207.

Tadera, K., Arima, M., Yoshino, S., Yagi, F. & Kobayashi, A. (1986)

Conversion of pyridoxine into 6-hydroxypyridoxine by food components, especially ascorbic acid. J. Nutr. Sci. Vitaminol. 32: 267-277.

Tadera, K., Kaneko, T. & Yagi, F. (1986) Evidence for the occurrence and distribution of a new type of vitamin B-6 conjugate in plant foods. Agric.

Biol. Chem. 50(11): 2933-2934.

68

Tadera, K., Kaneko, T. & Yagi, F. (1988) Isolation and structural elucidation of three new pyridoxine-glycosides in rice bran. J. Nutr. Sci. Vitaminol. 34:

167-177.

Tadera, K., Mori, E., Yagi, F., Kobayashi, A., Imada, K. & Imabeppu, M.

(1985) Isolation and structure of a minor metabolite of pyridoxine in seedlings of Pisum sativum L. J. Nutr. Sci. Vitaminol. 31: 403-406.

Tadera, K., Nagano, K., Yagi, F., Kobayashi, A., Imada, K., and Tanaka, S.

(1983) Isolation and structure of a new metabolite of pyridoxine in seedlings of Pisum sativum L. Agric. Biol. Chem. 47: 1357-1359.

Tadera, K., Nakamura, M. & Kobayashi, A. (1978) Biosynthesis of 5'-0-(fi-D- glucopyranosyl)pyridoxine in germinating rice seeds. Vitamins 52: 17-22.

Tadera, K., Nakamura, M., Yagi, F. & Kobayashi, A. (1979) A particulate glucosylaltransferase catalyzing the formation of 5

,

-0-(6-D- glucopyranosyl)pyridoxine from pyridoxine: the occurrence in the seeding of Pisum Sativum L. J. Nutr. Sci. Vitaminol. 25: 347-350.

Tadera, K. & Orite, K. (1991) Isolation and structure of a new vitamin B-6 conjugate in rice bran. J. Food Sci. 56(1): 268-269.

Tarr, J. B., Tamura, T. & Stokstad, E. L. R. (1981) Availability of vitamin B-6 and panothenate in an average American diet in man. Am. J. Clin. Nutr.

34:1328-1337.

Toepfer, E. W. & Polansky, M. M. (1970) Microbiological assay of vitamin B-

6 and its components. J. Assoc. Off. Anal. Chem. 53: 546-550.

Trumbo, P. R., Banks, M. A. & Gregory, J. F. (1990) Hydrolysis of pyridoxine-

S'-G-D-glucoside by a broad-specificity fi-glucosidase from mammalian tissues. P. S. E. B. M. 195: 240-246.

Trumbo, P. R. & Gregory, J. F. (1988) Metabolic utilization of pyridoxine-6- glucoside in rats: influence of vitamin B-6 status and route of administration. J. Nutr. 118: 1336-1342.

Trumbo, P. R. & Gregory, J. F. (1989) The fate of dietary pyridoxine-6- glucoside in the lactating rat. J. Nutr. 119: 36-39.

Trumbo, P. R., Gregory, J. F. & Sartain, D. B. (1988) Incomplete utilization of pyridoxine-fi-glucoside as vitamin B-6 in the rat. J. Nutr. 118: 170-175.

69

Tsuji, H., Okada, J., Iwami, K. & Yasumoto, K. (1977) Availability as vitamin B-6 and small intestinal absorption of pyridoxine-6-D-glucoside in rats. Vitamins (Kyoto) 51:153-159.

USDA (U. S. Department of Agriculture) (1986) Nationwide Food

Consumption Survey. Continuing Survey of Food Intakes by Individuals:

Men 19-50 Years, 1 Day, 1985. Report No. 85-3. Nutrition Monitoring

Division, Human Nutrition Information Service. U. S. Department of

Agriculture, Hyattsville, Md. 94 pp.



Women 19-50 Years and Their Children 1-5 Years, 1 Day, 1986. Report No.

86-1. Nutrition Monitoring Division, Human Nutrition Information

Service. U. S. Department of Agriculture, Hyattsville, Md. 98 pp.



Women 19-50 Years and Their Children 1-5 Years, 4 Day, 1985. Report No.

85-4. Nutrition Monitoring Division, Human Nutrition Information

Service. U. S. Department of Agriculture, Hyattsville, Md. 182 pp.

Yasumoto, K., Iwami, K., Tsuji, H., Okada, J. & Mitsuda, H. (1976) Bound forms of vitamin B-6 in cereals and seeds. Bitamin 50: 327-333.

Yasumoto, K., Tsuji, H., Iwami, K. & Mitsuda, H. (1977) Isolation from rice bran of a bound form of vitamin B-6 and its identification as 5'-0-(fi-D- glucopyranosyDpyridoxine. Agric. Biol. Chem. 41: 1061-1067.

Yoshida, S., Hayashi, K. & Kawasaki, T. (1981) Pyridoxine transport in brush border membrane vesicles of guinea pig jejunum. J. Nutr. Sci. Vitaminol.

27: 311-317.

APPENDICES

APPENDIX 1

Initial interview form

Name Age _

Address Phone (home)

Phone (work) . hours:

Height Present weight Length of time you have maintained present weight

OC user: yes no

Vitamin user: yes no If yes, how long? Willing to stop? _

Activity level: Do you have a current fitness program? yes no

If yes, list normal week's activities and intensity:

How long have you maintained this level of fitness?

70

Do you have any food preferences or foods you will not eat? Please list:

Are you currently on any medications? yes no If yes, please list:

Will you be able to be on campus the entire 44 days (April 8 - May 22)? yes no

Any other helpful information on the person:

Would this person be a good subject?

Appointment scheduled: Date Time With whom?

APPENDIX 2

Health/diet history questionnaire

HEALTH / DIET HISTORY-Aug 1990 version

CONFIDENTIAL

Dr. Jim Leklem Project Name

Dept. Nutrition & Food Management

Oregon State University Project Dates

Code #: Today's Date:

Age: Date of Birth: Place of Birth:.

Sex: M / F Predominant Place of Residence:

Present Employment:

Race (circle one): a. American Indian b. Black c. Caucasian d. Hispanic e. Chinese f. Japanese g. Polynesian/Pacific Islander h. Other Asian (specify) i. Other (specify)

Marital Status (circle one): a. Single b. Married c. Divorced/Separated d. Widowed

How many people live in your household?

Do you have any children? Yes No If yes, give ages

Females : MENSTRUAL and REPRODUCTIVE HISTORY

When did your last menstrual period begin?

Do you have regular menstrual periods? Yes No

How long is your menstrual cycle?.

Do you have problems with your menstruation? Yes No

If yes, please explain:

Are you pregnant? _ Yes _No Breast Feeding? _ Yes _No

Have you ever been pregnant? Yes No

If yes, how many times?

How many children have you carried?

Please check if you have had any of the following complications of pregnancy:

1. hyperemesis gravidarum (morning sickness)

2. pre-eclampsia or eclampsia (toxemia)

3. high blood pressure

4. severe edema (swelling of your legs and feet)

5. numbness and tingling in your hands, wrists, or arms

6. gestational diabetes

7. premature birth(s) (please indicate gestational age of infant(s))

8. kidney or bladder infections

9. premature rupture of membranes

10. small for dates infant (less than 5 lbs or 2500 g at term)

71

HEIGHT / WEIGHT: Height (ft. & in.) Present weight:.

Most ever weighed ; What year _

Length of time you have maintained current weight

DIETARY HISTORY

Dieting: Are you currently on a special diet? Yes No

If yes, for what purpose? (please check as many as apply):

1. weight loss

2. weight gain

3. control serum lipids

4. diabetes

5. kidney failure

6. ulcers

7. diverticulitis

8. allergies

9. heart trouble

10. high blood pressure

11. pregnancy

12. breast feeding

13. other (please specify):

If you are on a diet, was it prescribed by a doctor, dietitian, or nurse? Yes No

If you are on a diet, what kind is it? (please check as many as apply):

1. low fat

2. low protein

3. high protein

4. low salt

5. low carbohydrate

6. low sugar

7. low calorie

8. low cholesterol

9. high calorie

10. a bland diet

11. other (please specify):

If you are currently on a diet, for how long have you been on this diet?.

If dieting, is your dieting associated with any commercial weight loss program?

Yes No If yes, please specify what program:

72

Are you a vegetarian? Yes No If yes, circle the type of vegetarian diet you follow: a. ovo-lacvto b. ovo c. lacto d. vegan

Do you take vitamins? (circle one): a. yes, daily b. yes, frequently (3 to 6 times/wk) c. often (once or twice/wk) d. occasionally (less than once/wk) e. never

If yes, what type, how much, and for how long have you taken them?

Type Amount per day How lono have vou taken

Do you take any other nutritional supplements (such as iron, calcium, other minerals, amino acids, fiber, supplement drinks [such as Ensure], etc)? Yes No

Type Amount Per day How long have vou taken

Please list all foods which you refuse to eat, can not eat, or prefer not to eat:

73

Please list those foods and beverages that you eat/drink almost every day:

HABITS: A. Smoking:

1) Do you currently smoke? Yes 2_ No

If yes, please check below what you do smoke, and how much per day:

Cigarettes Packs per day

Cigars Number per day

Pipe Pipe Loads per day

At what age did you start smoking?

2) If you do not currently smoke, did you ever smoke? Yes No

If yes, at what age did you start?

If yes, when did you quit?

Was this the only time you have quit? Yes No

If you quit, please check below what you did smoke, and how much per day:

Cigarettes Packs per day


Pipe Pipe Loads per day.

3) Does anyone else in your household smoke? Yes No

If yes, please list type and how much per day:

Cigarettes Packs _per day


Pipe Pipe Loads per day.

B. Alcohol:

1) Do you drink alcoholic beverages? Yes No

If yes, how many times do you drink per month?

If yes, what do you drink and how many drinks do you consume each time you drink?

Beer Number of drinks at one time

Wine Number of drinks at one time

Liquor Number of drinks at one time

Other Number of drinks at one time

C. Caffeine:

1) Do you drink beverages containing caffeine? Yes No

If yes, which of the following beverages do you drink, and how much?

Coffee Number of cups per day

Tea Number of cups per day.

Soda Number of 12 oz servings per day

2) Do you drink any decaffeinated or caffeine-free beverages? Yes No

If yes, which of the following beverages do you drink, and how much?

Coffee Number of cups per day

Tea Number of cups per day

Soda Number of 12 oz servings per day

D. Diet Soda Pop and other Sugarless Beverages

1) Do you drink any beverages containing artificial sweetners? Yes No

If yes, what do you drink and how many drinks (ounces, servings) per day?

74

EXERCISE LEVEL: Are you currently involved in a regular exercise program?

Yes No If yes, describe:

TYP9 Of Exercise # Minutes (continuous) Distance covered or reoititions # davs/wk

75

Do you monitor your heart rate during exercise? Yes No

If yes, what heart rate do you try to maintain while exercising?.

If you do not have a regular fitness program, what types of exercise would you get in a typical week?

MEDICAL HISTORY:

Have you ever had a glucose tolerance test? Yes No If yes, please explain when, the reason, and the results:

Have you ever had a stress electrocardiogram? Yes No If yes, please explain when, the reason, and the results:

Have you ever had any health risk screening tests, such as serum cholesterol, blood glucose, or blood pressure? Yes No If yes, please explain what tests you had, and what were the results and recommendations you received:

MEDICAL HISTORY (Check any condition for which you have been diagnosed and give AGE at diagnosis):

Diagnosis Age at Diagnosis

1. acquired immunodeficiency syndrome (AIDS)

2. diabetes

3. hypoglycemia.

4. hypothyroidism_

5. hyperthyroidism,

6. goiter

7. osteoporosis.

8. hepatitis

9. cirrhosis

10. kidney stones.

,11. nephritis

12. cystitis

13. high blood pressure.

14. angina

15. ulcer

16. pancreatitis

17. ulcerative colitis

18. recurring gastritis

19. allergies/hayfever

20. hypoadrenalism (Addison's disease).

21. spastic colon/diverticulitis

22. carpal tunnel syndrome

23. rheumatoid arthritis

24. systemic lupus erythematosus

25. mental depression requiring regular medication

26. asthma

27. insomnia requiring frequent medication

28. emphysema.

29. heart problems (specify) _

. 30. cancer (specify type)

. 31. chronic infection (specify).

32. tuberculosis

33. chronic headache or other pain (specify)

34. hereditary condition (specify)

35. premenstrual syndrome

36. other condition (specify)

Comments:

76

Are you currently suffering from any.cold, flu, or allergy symptoms? Yes No

If yes, please specify:

Do any of your first-degree relatives (mother, father, brother, sister, son, daughter) have any of the following conditions? Yes No If yes, indicate which condition and his/her relationship to you:

1. diabetes

2. heart disease before age 60

3. cancer before age 60

4. high blood pressure before age 60

5. allergies

Have you ever had a nerve conduction/muscle stimulation study? Yes No

If yes, when, for what reason, and what were the results?

Have you ever had any other special diagnostic tests (such as special X-ray studies or a CAT-scan) Yes No If yes, please specify:

77

SURGICAL HISTORY (Please specify any type of surgery you have had and the date and age when it occurred):

Operation Age or Year

MEDICATION HISTORY (Check any which you take on a regular basis and when and how often): Medication " Taking Currently? How often?

1. sleeping tablets

2. aspirin.

, 3. cold medications.

. 4. barbiturates

, 5. tranquilizers

6. diuretics

. 7. blood pressure tablets.

8. antibiotics

9. thyroid hormones.

10. oral contraceptives.

11. insulin s

12. oral hypoglycemics.

13. corticosteroids

14. estrogens (female hormones).

15. isoniazid

16. pain medications _

17. muscle relaxants.

18. theophylline

19. antiarrhythmatics.

20. ulcer medications.

21 antacids

22. digoxin

23. antidepressants.

24. seizure medications

25. other medications (please specify):

How long did you fast prior to having your blood drawn? more than 12 hrs

8-12 hrs less than 8 hrs

COMMENTS:

Checked by Date.

78

APPENDIX 3

Initial informed consent form

Department of Nutrition and Food Management

Oregon State University

Informed Consent

I am interested in being considered as a possible participant in a human metabolic study on the interrelationship between dietary protein and vitamin B-6. To determine my eligibility for participating in this investigation, I understand that I will be asked to do the following:

1. Answer a questionnaire.

2. Come to 107 MLM on between 7 and 8 am without having eaten or drunk anything except water since 7 pm the night before. This includes vitamins or any other nutritional supplements. A registered medical technologist will draw 20 mL of blood from a vein in my forearm.

I also understand:

1. That I will receive no direct benefits except information on my vitamin B-6 status and results of the chemistry screen (glucose, electrolytes, enzymes, etc.).

2. That I may receive a slight bruise at the site of blood drawing. A sterile, disposable needle will be used to draw blood into evacuated- tubes.

3. Any information obtained from rae will be confidential. The only persons who have access to information obtained fro- me are the two principal investigators. All information will be filed in one of the investigator's office.

4. That I am not comnitred to participate in any metabolic study even if I meet all of the criteria.

5. Persons at risk for hepatitis B or HTLV III (com-only called AIDS) should not participate in this preliminary investigation (screening) since the donation of both and blood and urine are required to determine my eligibility.

All of my questions have been answered to my satisfaction. Should I have any further questions I can contact either Dr. Jim Leklem or Dr.

Lorraine Miller between 8 and 5 weekdays at 737-3561.

Name

Date

Co-Principal Investigator_

79

80

APPENDIX 4

Informed consent form

Department of Nutrition and Food Management

Oregon State University

Informed Consent

The purpose of this investigation is to compare the bioavailability of vitamin B-6 from a diet high in glycosylated vitamin B-6 to a diet low in glycosylated vitamin B-6 in women.

Glycosylated vitamin B-6 is a form of vitamin B-6 in plants.

I have received a thorough explanation of this study. I understand the following:

1. I will not have taken any vitamin or any other nutritional supplement for at least three weeks before the beginning of this project.

2. This is a 44-day study. The study ends the morning after the last day of the study.

3. I will consume only those foods and beverages that are served to me or permitted.

4. During this study I will not engage in any strenuous physical activity such as running more than one mile per day or bicycling more than 6 miles per day.

5. I will keep a record of my daily activities which I will turn in each day.

6. I will collect complete 24-hour urine specimens in the containers that are provided for me. If I accidentally lose some urine I will immediately report this to a member of the research staff.

7. During the last 8 days of each of the two experimental periods, I will collect all of my feces in the containers provided for me. If I accidentally lose a specimen I will immediately report this to a member of the research staff.

I will be administered a fecal marker (FDC Blue No. 1) at regular intervals during the last 8 days of each experimental period.

8. At regular intervals, a total of 9 times, a registered medical technologist will draw 20 mL (equivalent to less than two tablespoons) of blood from my forearm. On the last day of this investigation the medical techologist will withdraw an additional 10 mL for a chem screen which will be done at Good Samaritan Hospital. On the days that blood is drawn, between 7 and 8 a.m., I am not to eat or drink anything except water since 7 p.m. the night before. I understand that this procedure may cause a slight bruise.

9. All information obtained from me will be confidential.

My data will be identified by a code number. The only persons who will have access to may data are the two principal investigators, the medical technologist and the graduate students who are assisting in this research.

10. My participation in this research is voluntary and I can withdraw at any time without loss of benefits.

11. At the end of this investigation I will teceive in a lump sum $4.50 for each day that I participated.

12. The co-principal investigators reserve the right to remove me from this project if I do not adhere to the requirements of this research. Examples include not showing up for meals, not adhering strictly to the diet, failure to report losses of urine or feces to a member of the research staff. Urine specimens will be assayed daily to determine completeness of collection and adherence to the diet.

13. I will incur no medical risks from participating in this research. I will receive some benefits: a nutritially balanced diet furnished free of charge for 44 days; $4.50 for each day I participated; and results of my vitamin B-6 status and other laboratory analyses will be available to me if I request.

14. Persons who are at increased risk for hepatitis B or

HTLV III (commonly called AIDS) should not participate in this investigation since they will be requested to give their blood and excreta.

15. This study will be conducted from April 8 to May 22

(morning).

All of my questions have been answered to my satisfaction.

If I have any questions I will call either Dr. Lorraine T. Miller at 737-0970 or Dr. James E. Leklem at 737-0969.

Name

Present address_

Date

Co-Principal Investigator_

81

APPENDIX 5

Daily activity sheet

Diet Study

Dept. of Nutrition and Food Management Name

Oregon State University Date

DAILY ACTIVITY SHEET

1. Record all activity for the previous day and length spent at each.

Activity Length of Time Time of Day *

(fraction of hours)

Sleep

Sitting

Walking

Physical work

Other activities

Other sports or activities,

(indicate type)

* M - morning; A - afternoon; E - evening; L - late night/early morning

2. Record all "free" foods in exact amounts used. Indicate type also used, decaf, etc.

Coffee (cups)

82

Tea (cups)

Diet Pop

3. How do you feel today? Excellent

Good

Fair

Poor

4. Any medications? (i.e., aspirin, etc.)

5. Other unusual events, exams, injuries, etc.

6. Did you turn your urine bottles in and pick up clean ones?

7. Did you weigh yourself today? Your weight today.

8. Other comments.

Diet/Composite

Adjustment Diet

Plant Foods:

Breakfast & Dinner

Plant Foods: Lunch

Animal Foos

Milk Products

Total

Total Vitamin B-6 mg (fimol)

0.364 (2.15)

0.176 (1.04)

0.254(1.50)

0.193 (1.14)

0.987 (5.83)

Low-Glycosylated

Vitamin B-6 Diet

5/2/91

Plant Foods

Animal Foods

Milk, 2%

Total

5/21/91

Plant Foods

Animal Foods

Milk. 2%

Total

0.602 (3.56)

0.595 (3.52)

0.249(1.48)

1.446(8.56)

0.675 (3.99)

0.636 (3.76)

0.254(1.50)

1.565(9.25)

APPENDIX 6

Food Composites

1,

2

Glycsoylated Vitamin B-6 mg (jdmol) %

0.107(0.64)

0.046 (0.28)

0.002 (0.01)

0.014 (0.09)

0.169 (1.02)

0.147 (0.86)

0.009 (0.05)

0.011 (0.06)

0.167 (0.97)

0.141 (0.84)

0.004 (0.02)

0.005 (0.03)

0.150 (0.89)

29

26

1

7

17

24

2

4

12

21

1

2

10

Free Vitamin B-6 mg (fimol) %

0.122 (0.72)

0.075 (0.44)

0.049 (0.29)

0.122 (0.72)

0.368 (2.17)

34

43

19

63

37

0.324(1.92)

0.180(1.07)

0.164 (0.97)

0.668 (3.96)

0.326 (1.93)

0.196(1.16)

0.179(1.06)

0.701 (4.15)

54

30

66

46

48

31

70

45

00

APPENDIX 6 (Continued)

Diet/Composite

High-Glycosylated

Vitamin B-6 Diet

5/2/91

Plant Foods

Animal Foods

Milk, 2%

Total

Total Vitamin B-6 mg (nmol)

1.515(8.96)

0.220(1.30)

0.233(1.38)

1.968(11.64)

Glycsoylated Vitamin B-6 mg (nmol) %

0.398 (2.35)

0.006 (0.04)

0.009 (0.06)

0.413 (2.45)

5/21/91

Plant Foods

Animal Foods

Milk, 2%

Total

1.346(7.96)

0.243 (1.44)

0.237(1.40)

1.826(10.80)

0.417 (2.47)

0.007 (0.04)

0.005 (0.03)

0.429 (2.54)

1

Values are based on the duplicate samples for each composite.


31

3

2

23

26

3

4

21

Free Vitamin B-6 mg (fimol) %

0.422 (2.50) 28

0.046 (0.27) 21

0.154 (0.91) 66

0.622 (3.68) 32

0.500 (2.96) 37

0.049 (0.29) 20

0.167 (0.99) 70

0.716 (4.24) 39

2

AN ABSTRACT OF THE THESIS OF

Related documents

Products

Support

AN ABSTRACT OF THE THESIS OF

Related documents

Add this document to collection(s)

Add this document to saved

Suggest us how to improve StudyLib