The Bioavailability of Vitamin B, Recent Findings" JESSE F. GREGORY 111 Food Science and Human Nutrition Department University of Florida Gainesville, Florida 3261 1-0163
INTRODUCTION The nutritional status of a person, with respect to vitamin B,, is a function of the quantity of the vitamin ingested and its bioavailability. Although various definitions of bioavailability have been proposed, the concept of bioavailability is best used in the broadest sense in reference to the extent of intestinal absorption and metabolic utilization of all dietary forms of the vitamin. This paper is a review of recent findings in the study of vitamin B, bioavailability. Emphasis will be directed toward research techniques for the study of vitamin B, bioavailability and various factors known to influence the bioavailability of the vitamin. Much of the discussion will be focused on the development and application of in vivo isotopic methods and recent studies concerning the nutritional properties of glycosylated forms of vitamin B, found in plant-derived foods. Additional information concerning the bioavailability of vitamin B, may be found in several reviews.'-5 In spite of recent improvements in analytical methods, there is a great need for more precise data concerning the quantity and forms of vitamin B, present in the diet. Even more fragmentary is present knowledge of the bioavailability of the vitamin, which is not sufficient to permit an accurate a priori assessment of dietary adequacy. Variability in digestive function among individuals may also influence the extent or rate of absorption of vitamin B, from foods, which would further complicate attempts to tabulate the bioavailability of nutrients. As a result, the development of data bases concerning the bioavailability of vitamin B, in various foods does not appear feasible a t this time.
EXPERIMENTAL PROCEDURES Bioassay Methods The concentration of biologically available vitamin B, in foods is generally determined by bioassays using the rat or chick. Traditional bioassay procedures have been limited by the potential for inaccuracy caused by the influence of dietary components on the production of vitamin B, by intestinal microorganisms. Animal bioassays are 'The author gratefully acknowledges support of this research by Grant No. 83-CRCR-1-1240 from the Competitive Research Grants Office, United States Department of Agriculture, and Grant No. DK37481 from the National Institutes of Health. 86
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subject to variable accuracy when applied to the measurement of available vitamin B, in foods. Both the rat and the chick are sensitive to influences of diet composition, especially with respect to fermentable carbohydrate, on the production of vitamin B, by the intestinal microflora!.' Large differences in the concentration of fecal vitamin B, have been detected in rats fed diets containing various dried foods as the source of vitamin B,.8 Recycling of this fecal vitamin B, via coprophagy or direct intestinal absorption could bias quantitative bioassays, even in procedures in which coprophagy is prevented.' Bioassays of available vitamin B, in individual foods or mixed diets also have been performed using human subjects. These procedures are lengthy, limited in precision, and often require relatively large quantities of the vitamin to elicit a measurable Recent applications of radioisotopic and stable-isotopic methods have provided an important alternative for the study of vitamin B, bioavailability. Attempts are frequently made to calculate the vitamin B, balance (ingested versus urinary and fecal excretion) of B, vitamers and 4'-pyridoxic acid (4PA) in bioassays employing human subjects. In such experiments, the use of data concerning fecal vitamin B, has little validity. Fecal material contains microbiologically synthesized B, vitamers in addition to unabsorbed vitamin B, from dietary sources. The microbial contribution to fecal vitamin B, would depend on the influence of dietary components on the number and types of intestinal microorganisms. Isotopic Methods
Significant advances have been made recently in the development of procedures, particularly isotopic, for the assessment of vitamin B, bi~availability.~ Advantages in the use of isotopic methods include: (a) the ability to determine specifically the fate of the administered labeled compound, (b) the ability to examine the kinetics of turnover and metabolism of the labeled compound, and (c) lack of interference by vitamin B, produced by intestinal microflora. A major question to be addressed in the design of isotopic methods in bioavailability studies is how to administer the labeled c o m p o ~ n d ( s ) Extrinsic .~ enrichment involves direct addition of the labeled compound to the diet or tested food. This approach requires the assumption that the labeled compound behaves in an analogous manner to that naturally present in the food. Alternatively, intrinsic enrichment involves introduction of the labeled compound into a plant or animal tissue by in vivo administration of the compound. In this manner the labeled compound undergoes metabolism and cellular distribution analogous to the endogenous forms of the vitamin. Radioisotopes Radioisotopic methods offer a powerful alternative to bioassays for the study of vitamin B, bioavailability using animal models. Typically a single test meal containing the labeled compound(s) is administered. By analysis of excreta and tissues for total radioactivity and the distribution of labeled B, vitamers, the extent of absorption, and metabolism of the test compounds can be determined. In studies involving the metabolism of intraperitoneally injected with ['4C]pyridoxine
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(PN), only 3% of the isotope was found in feces and intestinal contents over 24 hours po~t-dose.'~ These findings indicate that, when using an oral dose of labeled vitamin B,, most of the radioactivity in feces and intestinal contents would represent unabsorbed dietary vitamin B,, and would be a direct measurement of the extent of absorption of the test compound. Fecal isotopic excretion following oral administration of labeled P N represents approximately 10-17% of the dose over 24 hours when administered to rats in typical diets.'"'' The simultaneous administration of two isotopically labeled forms of the vitamin, for example, labeled with 'H and I4C,permits direct comparisons of the bioavailability of various forms of vitamin B, as well as determination of the effect of the route of
Stable Isotopes
The application of stable-isotopic methods to the study of vitamin B, bioavailability permits the use of isotopically labeled compounds in human subjects without the potential hazards of radioisotopes. Additional advantages of stable isotopic studies have been discussed in recent Deuterium-labeled forms of vitamin B, may be readily synthesized by methods developed by Coburn et aL2' These labeling procedures permit the synthesis of all forms of vitamin B, in bideutero (d2) form, with two deuterium atoms a t the 5'-methylene position, or in pentadeutero (d5) form, with two deuterium atoms at the 5'-position and three a t the 2'-methyl position. Quantitative analysis in stable isotopic studies involves measurement of the molar ratio of various labeled and unlabeled species of vitamin B, or 4PA in urine or other biological materials usually by gas chromatography-mass spectrometry (GCMS). Hachey et a1.*' developed procedures for G C M S (positive-ion electron-impact mode) which have been applied to assess the in vivo kinetics and pool sizes of vitamin B, by analysis of deuterium labeling of urinary 4PA in humans.23 Alternatively, G C M S analysis in the negative-ion chemicalionization mode provides greater ~ e n s i t i v i t yBecause .~~ vitamin B, compounds can be synthesized in d2 and d5 form, dual-label studies can be performed with experimental design analogous to those performed in animals with 'H and I4C for the evaluation of bioavailability and in vivo kinetics.
FACTORS AFFECTING THE BIOAVAILABILITY OF VITAMIN B, Most evidence indicates that the intestinal absorption of vitamin B, occurs by passive diffusion of vitamin B, compounds in nonphosphorylated form.25Diet composition could influence the extent of absorption of the vitamin by affecting the access of dietary B, compounds to the absorptive surface, either by binding or entrapment phenomena or by retarding diffusion through effects on the environment of the brush-border membrane. Whether food components (e.g. dietary fiber) retard the absorption of vitamin B, through such mechanisms is unclear. Tarr et al. determined the net bioavailability of vitamin B, in a mixed American diet using a long-term bioassay method." They observed a mean bioavailability of approximately 75% relative to P N in a formula diet. The factors responsible for this incomplete bioavailabil-
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ity were not determined. The study of Tarr et al. was important because it indicated that, although incomplete, the overall bioavailability of vitamin B, in a typical diet is reasonably high. The factors responsible for the incomplete bioavailability have not been determined. Dietary Fiber The influence of various forms of dietary fiber ozf vitamin B, bioavailability has been examined in animals and humans. N o physical interaction occurs in vitro between B, vitamers and purified neutral and anionic polysaccharides.26 Purified forms of dietary fiber have little or no effect on the bioavailability of P N in bioassays using rats and chicks and little effect in intestinal perfusion s t ~ d i e s .The ~ , ~results ~ of studies with human subjects also indicate little or no adverse effect of dietary fiber.‘z328
OH
FIGURE 1. Structural formula of 5’-O-(&D-glucopyranosyl)-pyridoxine, the primary form of glycosylated vitamin B, in foods.
Glycosylated Forms of Vitamin B, Although evidence of “bound” forms of vitamin B, had been reported for many
year^,'^-^' the first isolation and structural identification of a glycosylated B, compound was reported in 1977. In these studies, Yasumoto et al. isolated a compound from rice bran, which was identified as 5’-0-(P-D-glucopyranosyl)-pyridoxine(PN-glucoside; FIG. This compound was also found in germinating rice,33 peas,34 and alfalfa sprouts.35Incubation of germinating alfalfa seeds with P N was found to be a convenient method of biological synthesis of PN-glucoside in unlabeled, radiolabeled, or deuterium-labeled form. PN-glucoside appears to be formed in plants by enzymatic tran~glycosylation.~~,~~~~~ The glucose moiety of PN-glucosides has been detected a t the 5’ and 4‘ positions of the P N ring in various plants and in vitro enzymatic reaction^.^^.".^^ The presence of the 5’-isomer as the primary naturally occurring form of PN-glucoside suggests that the transglycosylation reaction exhibits specificity for this position in most plant tissue^.^' The existence of several derivatives of PN-glucoside has been reported. Tadera et al. isolated and identified forms of 5‘-O-(P-~-glucopyranosyl)-pyridoxine in which the C-6 position of the glucosyl moiety was esterified to either malonic acid or 3-hydroxy-
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3-methyl-glutaric acid (HMG).40s41 These compounds were found to be minor components of the total vitamin B, in pea seedlings grown in the presence of 1 m M PN, with observed molar ratios of PN: PN-glucoside : HMG-PN-glucoside : malonyl-PNglucoside of 7 : 30 : 7 : 1. Three additional PN-glycosides have been reported in rice bran, including 5’-0-(~-cellobiosyl)-pyridoxine,4‘-0-(@-D-glucosyl)-5’-~ -(@-cellobiosy1)-pyridoxine, and 5’-0-(P-~ellotriosyl)-pyridoxine.~~ The significance of these compounds as components of the total vitamin B, in foods is unclear; however, PN-5’glucoside has been the only sibnificant glycosylated form of vitamin B, detected in chromatographic studies of a variety of foods to date.35Tadera et nl. have reported the existence of an unidentified minor class of vitamin B, conjugate termed “B6X” that yields a response in microbiological assays only after sequential alkaline treatment and hydrolysis with @ - g l u c ~ s i d a s ePresumably, .~~ B6X comprises the esterified forms of PN-glucoside, in which the esters must be removed by alkaline hydrolysis to render the glycoside susceptible to enzymatic hydrolysis. The quantitative analysis of glycosylated forms of vitamin B, in foods can be performed by microbiological or HPLC methods.44A microbiological method devised by Kabir et al. involves measurement of microbiologically active vitamin B, before and after P-glucosidase treatment of an aqueous extract of the food sample.45This method may underestimate the concentration of glycosylated vitamin B, during analysis of raw samples that contain endogenous P-glucosidase activity. Alternatively, HPLC with fluorometric detection permits either the direct quantification of PN-glucoside in intact form or its measurement following enzymatic hydr~lysis?~ P N has been found to be the sole glycosylated form of vitamin B, in foods analyzed to date. Although PN-glucoside comprises 25-75% of the total vitamin B, in many plant-derived food^:^,^^ its bioavailability for humans has not been determined conclusively. PN-glucoside has been found to undergo intestinal absorption in intact form in the rat in ~ i t r o ”and . ~ ~in vivo.I6 Tsuji et al. reported that chemically synthesized PN-glucoside was biologically active as vitamin B, in a rat bioassay?6 In contrast, a rat bioassay by Trumbo et nl. indicated that PN-glucoside isolated from alfalfa sprouts exhibited only 10-30% bioavailability relative to PN.47 Studies conducted using radiolabeled PN-glucoside in the rat have shown that, while PN-glucoside is effectively absorbed, it undergoes little metabolic utilization as vitamin B, and is rapidly excreted in the ~ r i n e . ’ ~The . ’ ~extent of absorption of [3H]PN-glucoside from diets containing intrinsically enriched alfalfa sprouts was slightly less than that of purified [3H]PNglucoside,’6 which suggests an inhibitory effect of plant tissues. Initial studies using bioassays with human subjects indicated that the percentage of glycosylated vitamin B, in several foods was inversely related to the bioavailability of the vitamin.48However, further studies indicated that this correlation was inconsistent when additional foods were Stable-isotopic techniques have been employed to evaluate the bioavailability of purified deuterium-labeled (d2) PN-glucoside in human subjects. In these studies, the mean bioavailability of labeled PN-glucoside fed orally mixed with oatmeal (i.e., by extrinsic enrichment) was similar to that of labeled P N administered under identical condition^.^^,^' Although the ability of humans metabolically to utilize PN-glucoside appears to vary substantially, the mean bioavailability of PN-glucoside in humans appears to be substantially greater than observed in the rat. Further application of
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these highly specific techniques will provide greater clarification of this and other questions of vitamin B, bioavailability. Utilization of PN-glucoside in vitamin B, metabolism requires in vivo hydrolysis of the glycosidic bond. The extent of utilization of PN-glucoside has been shown to be greater when fed orally than when injected in ratsi4 and humans.” These results suggest a role of the intestine in the partial release of biologically active P N from dietary PN-glucoside. The site of this hydrolysis, whether microbial or mucosal, is unclear a t present. P-Glucosidase activity capable of hydrolyzing PN-glucoside in vitro has been detected in several mammalian tissues.46 Later comparative studies showed that &glucosidase activity existed in the intestine of the rat, guinea pig, and human.52 The significance of this enzyme with respect to the bioavailability of PN-glucoside is unclear at present. Chemically ModiJied Forms of Vitamin B, Found in Foods The processing and storage of foods inevitably causes some losses of nutrients including vitamin B,. Whether this influences the bioavailability of the remaining vitamin B, requires a knowledge of the chemical identity and nutritional properties of the remaining B, vitamers, the influence of the food matrix on their absorption, as well as the identity and nutritional properties of the “degradation products” formed. The potential conversion of various B, vitamers to compounds exhibiting little or no mammalian vitamin B, activity has been proposed to explain certain reported effects of food processing or storage on the concentration of biologically available vitamin B, in certain foods. In situations involving microbiological assay of total vitamin B, in foods, the existence of vitamin B, reaction products that elicit a response of the assay microorganism but not in animals or humans, the apparent bioavailability of the vitamin B, in the food would be incomplete. The recent development of specific HPLC methods for food analysis should minimize problems of this type. Considerable effort has been directed toward determining the effect of thermal processing of foods on the bioavailability of vitamin B,, in part, as a result of incidents involving vitamin B, deficiency in infants fed unfortified formulas in the 1 9 5 0 ~ ’ ~ Tomarelli et al. reported that the thermal sterilization of milk or unfortijed infant formula caused a reduction in the apparent bioavailability as determined by rat bioassay, while added PN underwent no reduction in its bi~availability.’~I t was proposed that the thermal effect was due to the conversion of naturally occurring vitamin B, in milk to bis-4-pyridoxyl-dis~Ifide,~~ a compound that exhibits greater activity in Saccharomyces uvarum assays than in rat bioa~says.’~ Later studies using chick bioassays did not confirm this reduction in thermally sterilized milk products,” and the issue has not been resolved. We have examined the formation of bis-4-pyridoxyl-disulfide and other thermal reaction products of vitamin B,. When milk containing [’4C]pyridoxal (PL) or [‘4C]pyridoxalphosphate (PLP) was subjected to thermal sterilization, HPLC analysis revealed no formation of the disulfide c o m p o ~ n d . ’Alternatively, ~ losses of free PL or PLP were largely due to reductive binding of the vitamin B, aldehydes to protein as epsilon-pyridoxyllsyl resid~es.~’ Similar results were found in intrinsically enriched chicken liver and muscle tissues. The nutritional significance of this observation concerns our previous findings that epsilon-pyridoxyllysyl complexes can be partially
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metabolized in the rat to yield P L P via PN (PL) kinase and pyridoxine (pyridoxamine) phosphate oxidase reactions.59360 Thus, epsilon-pyridoxyllysine is a paradoxical “degradation product” because it partially contributes to the total vitamin B, although it is not usually measured in most methods of food analysis. I t should also be noted that this compound can also exhibit anti-vitamin B, effects when administered to B,-deficient rats.,’ This effect may have been responsible, in part, for the possibly reduced bioavailability of the vitamin in the unfortified infant formulas. The identity and nutritional properties of other vitamin B, derivatives found in foods have not been fully determined. Although the stability of PN is generally high, the formation of a previously unknown degradation product, 6-hydroxy-pyridoxine, has been reported in thermal treatment of various fruits and vegetables.6’ The hydroxylation of P N was associated with the oxidative degradation of ascorbic acid6’ and was apparently mediated by generation of hydroxyl radicals.62 Rat bioassays conducted to evaluate the nutritional characteristics of 6-hydroxy-PN indicated no vitamin B, activity (Leatham and Gregory, 1988, unpublished). This reaction does not appear to be a significant mechanism for the loss of biologically active and available vitamin B, in foods.
SUMMARY AND CONCLUSION Further clarification of the bioavailability of dietary vitamin B, requires better analytical data concerning the forms of vitamin B, in foods as well as the overall composition of foods. The application of radioisotopic and stable-isotopic studies such as those described will provide useful information concerning the inherent bioavailability of the various vitamin B, compounds. Additional studies should then address the influence of other dietary components on the utilization of the vitamin B, compounds, using intrinsic and extrinsic enrichment techniques. Care must be taken in interpretation of the results of animal bioassays in the determination of the bioavailability of vitamin B, in complex diets.
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ANNALS NEW YORK ACADEMY OF SCIENCES bound form of vitamin B, and its identification as 5’-u-(8-D-glUCOpyranOSyl)pyridoxine. Agri. Biol. Chem. 41: 1061-1067. TADERA,K., M. NAKAMURA & A. KOBAYASHI. 1978. Biosynthesis of S’-U-(p-oglucopyranosyl) pyridoxine in germinating rice seeds. Vitamins 5 2 17-22. 1979. A particulate glucosyltransTADERA, K., M. NAKAMURA, F. YAGI& A. KOBAYASHI. ferase catalyzing the formation of 5‘-U-(P-D-glucopyranosyl)pyridoxine from pyridoxine: The occurrence in the seedlings - of Pisum sativum L. J . Nutr. Sci. Vitaminol. 2 5 347350. GREGORY, J. F. & S. L. INK.1987. Identification and quantification of pyridoxine-@ glucoside as a major form of vitamin B, in plant-derived foods. J. Agri. Food Chem. 35: 76-82. SUZUKI, Y., K. UCHIDA& A. TSUBOI.1386. Enzymatic formation of pyridoxine pglucosides by wheat bran P-glucosidase. Nippon Nogeikagaku Kaishi 5 3 189-196. TADERA,K., F. YAGI & A. KOBAYASHI. 1982. Specificity of a particulate glucosyltransferase in seedlings of Pisurn sativum L. which catalyzes the formation of ~’-U-(@-Dglucopyranosy1)pyridoxine. J. Nutr. Sci. Vitaminol. 28: 359-366. IWAMI,K. & K. YASUMOTO.1986. Synthesis of pyridoxine-0-glucoside by rice bran P-glucosidase and its in situ adsorption in rat small intestine. Nutr. Res. 6 407414. SUZUKI, Y., H. ISHII,K. SUGA & K. UCHIDA.1986. Formation of 0-glucosylpyridoxines in soybean and rice callus. Phytochemistry 2 5 1331-1 332. TADERA, K., K. NAGANO,F. YAGI,A. KOBAYASHI, K. IMADA& S. TANAKA. Isolation and structure of a new metabolite of pyridoxine in seedlings of Pisum sativum L. Agri. Biol. Chem. 47: 1357-1359. TADERA, K., E. MORI,F. YAGI,A. KOBAYASHI, K. IMADA& M. IMABEPPU. 1985. Isolation and structure of a minor metabolite of pyridoxine in seedlings of Pisum sativum L. J. Nutr. Sci. Vitaminol. 31: 403-408. TADERA, K., T. KANEKO & F. YAGI.1988. Isolation and structural elucidation of three new pyridoxine-glycosides in rice bran. J. Nutr. Sci. Vitaminol. 3 4 167-177. TADERA, K., T. KANEKO & F. YAGI.1986. Evidence for the occurrence and distribution of a new type of vitamin B, conjugate in plant foods. Agri. Biol. Chem. 5 0 2933-2934. J. F. 1988. Methods for determination of vitamin B, in foods and other biological GREGORY, materials: A critical review. J. Food Comp. Anal. 1: 105-123. KABIR,H., J. LEKLEM& L. T. MILLER.1983. Measurement of glycosylated vitamin B, in foods. J. Food Sci.: 1422-1425. TSUJI,H., J. OKADA,K. IWAMI,K. YASUMOTO & H. MITSUDA.1977. Availability of vitamin B, and small intestinal absorption of pyridoxine-P-D-glucosidein rats. Vitamins 51: 153-159. 1988. Incomplete utilization of TRUMBO,P. R., J. F. GREGORY& D. B. SARTAIN. pyridoxine-8-glucoside as vitamin B, in the rat. J. Nutr. 118: 17CL175. KABIR,H., J. E. LEKLEM& L. T. MILLER.1983. Relationship of the glycosylated vitamin B, content of foods to vitamin B, bioavailability in humans. Nutr. Rept. Int. 28: 709-715. BILLS,N. D., J. E. LEKLEM& L. T. MILLER.1987. Vitamin B, bioavailability in plant foods is inversely correlated with percent glycosylated vitamin B,. Fed. Proc. 4 6 1487. TRUMBO,P. R., J. F. GREGORY, J. P. TOTH, L. B. BAILEY& J. J. CERDA.1988. The bioavailability of pyridoxine-beta-glucosidein the rat and human. FASEB J. 2 A1086. GREGORY,J. F., P. R. TRUMBO,L. B. BAILEY,J. P. TOTH & J. J. CERDA.1989. Bioavailability of oral and intravenous deuterium-labeled pyridoxine-beta-glucosidein human subjects. FASEB J. 3 A454. TRUMBO, P. R. & J. F. GREGORY. 1989.8-Glucosidaseactivity and the metabolic utilization of dietary pyridoxine-beta-glucoside.FASEB J. 3: A454. COURSIN,D. B. 1954. Convulsive seizures in infants with pyridoxine deficient diet. J. Am. Med. Assoc. 154 406-408. TOMARELLI, R. M., E. R. SPENCE & F. W. BERNHART. 1955. Biological availability of vitamin B, in heated milk. J. Agri. Food Chem. 3 338-341. BERNHART, F. W., E. D’AMATO& R. M. TOMARELLI. 1960. The vitamin B, activity of heat sterilized milk. Arch. Biochem. Biophys. 88: 267-269.
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56. WENDT,G. & F. W. BERNHART. 1960. The structure of a sulfur-containingcompound with vitamin B, activity. Arch. Biochem. Biophys. 8 8 270-272. & K. G. HENRY.1959. The effect of heat on the vitamin B, 57. DAVIES,M. K., M. E. GREGORY of milk. J. Dairy Res. 2 6 215-220. J. F., S. L. INK& D. B. SARTAIN. 1986. Degradation and binding to food proteins 58. GREGORY, of vitamin B, compounds during thermal processing. J. Food Sci. 51: 1345-1351. J. F. 1980. Effects of e-pyridoxyllysineand related compounds on liver and brain 59. GREGORY, pyridoxal kinase and liver pyridoxamine (pyridoxine) S’-phosphate oxidase. J. Biol. Chem. 2 2 5 2355-2359. J. F. 1980. Effects of c-pyridoxyllysinebound to dietary protein on the vitamin B, 60. GREGORY, status of rats. J. Nutr. 110 995-1005. 1986. Conversion of 61. TADERA,K., M. ARIMA,S. YOSHINO,F. YAGI & A. KOBAYASHI. pyridoxine into 6-hydroxypyridoxine by food components, especially ascorbic acid. J. Nutr. Sci. Vitaminol. 32 267-277. K., M. ARIMA& F. YAGI.1988. Participation of hydroxyl radical in hydroxylation 62. TADERA, of pyridoxine by ascorbic acid. Agri. Biol. Chem. 5 2 2359-2360.
INTRODUCTION The nutritional status of a person, with respect to vitamin B,, is a function of the quantity of the vitamin ingested and its bioavailability. Although various definitions of bioavailability have been proposed, the concept of bioavailability is best used in the broadest sense in reference to the extent of intestinal absorption and metabolic utilization of all dietary forms of the vitamin. This paper is a review of recent findings in the study of vitamin B, bioavailability. Emphasis will be directed toward research techniques for the study of vitamin B, bioavailability and various factors known to influence the bioavailability of the vitamin. Much of the discussion will be focused on the development and application of in vivo isotopic methods and recent studies concerning the nutritional properties of glycosylated forms of vitamin B, found in plant-derived foods. Additional information concerning the bioavailability of vitamin B, may be found in several reviews.'-5 In spite of recent improvements in analytical methods, there is a great need for more precise data concerning the quantity and forms of vitamin B, present in the diet. Even more fragmentary is present knowledge of the bioavailability of the vitamin, which is not sufficient to permit an accurate a priori assessment of dietary adequacy. Variability in digestive function among individuals may also influence the extent or rate of absorption of vitamin B, from foods, which would further complicate attempts to tabulate the bioavailability of nutrients. As a result, the development of data bases concerning the bioavailability of vitamin B, in various foods does not appear feasible a t this time.
EXPERIMENTAL PROCEDURES Bioassay Methods The concentration of biologically available vitamin B, in foods is generally determined by bioassays using the rat or chick. Traditional bioassay procedures have been limited by the potential for inaccuracy caused by the influence of dietary components on the production of vitamin B, by intestinal microorganisms. Animal bioassays are 'The author gratefully acknowledges support of this research by Grant No. 83-CRCR-1-1240 from the Competitive Research Grants Office, United States Department of Agriculture, and Grant No. DK37481 from the National Institutes of Health. 86
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subject to variable accuracy when applied to the measurement of available vitamin B, in foods. Both the rat and the chick are sensitive to influences of diet composition, especially with respect to fermentable carbohydrate, on the production of vitamin B, by the intestinal microflora!.' Large differences in the concentration of fecal vitamin B, have been detected in rats fed diets containing various dried foods as the source of vitamin B,.8 Recycling of this fecal vitamin B, via coprophagy or direct intestinal absorption could bias quantitative bioassays, even in procedures in which coprophagy is prevented.' Bioassays of available vitamin B, in individual foods or mixed diets also have been performed using human subjects. These procedures are lengthy, limited in precision, and often require relatively large quantities of the vitamin to elicit a measurable Recent applications of radioisotopic and stable-isotopic methods have provided an important alternative for the study of vitamin B, bioavailability. Attempts are frequently made to calculate the vitamin B, balance (ingested versus urinary and fecal excretion) of B, vitamers and 4'-pyridoxic acid (4PA) in bioassays employing human subjects. In such experiments, the use of data concerning fecal vitamin B, has little validity. Fecal material contains microbiologically synthesized B, vitamers in addition to unabsorbed vitamin B, from dietary sources. The microbial contribution to fecal vitamin B, would depend on the influence of dietary components on the number and types of intestinal microorganisms. Isotopic Methods
Significant advances have been made recently in the development of procedures, particularly isotopic, for the assessment of vitamin B, bi~availability.~ Advantages in the use of isotopic methods include: (a) the ability to determine specifically the fate of the administered labeled compound, (b) the ability to examine the kinetics of turnover and metabolism of the labeled compound, and (c) lack of interference by vitamin B, produced by intestinal microflora. A major question to be addressed in the design of isotopic methods in bioavailability studies is how to administer the labeled c o m p o ~ n d ( s ) Extrinsic .~ enrichment involves direct addition of the labeled compound to the diet or tested food. This approach requires the assumption that the labeled compound behaves in an analogous manner to that naturally present in the food. Alternatively, intrinsic enrichment involves introduction of the labeled compound into a plant or animal tissue by in vivo administration of the compound. In this manner the labeled compound undergoes metabolism and cellular distribution analogous to the endogenous forms of the vitamin. Radioisotopes Radioisotopic methods offer a powerful alternative to bioassays for the study of vitamin B, bioavailability using animal models. Typically a single test meal containing the labeled compound(s) is administered. By analysis of excreta and tissues for total radioactivity and the distribution of labeled B, vitamers, the extent of absorption, and metabolism of the test compounds can be determined. In studies involving the metabolism of intraperitoneally injected with ['4C]pyridoxine
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(PN), only 3% of the isotope was found in feces and intestinal contents over 24 hours po~t-dose.'~ These findings indicate that, when using an oral dose of labeled vitamin B,, most of the radioactivity in feces and intestinal contents would represent unabsorbed dietary vitamin B,, and would be a direct measurement of the extent of absorption of the test compound. Fecal isotopic excretion following oral administration of labeled P N represents approximately 10-17% of the dose over 24 hours when administered to rats in typical diets.'"'' The simultaneous administration of two isotopically labeled forms of the vitamin, for example, labeled with 'H and I4C,permits direct comparisons of the bioavailability of various forms of vitamin B, as well as determination of the effect of the route of
Stable Isotopes
The application of stable-isotopic methods to the study of vitamin B, bioavailability permits the use of isotopically labeled compounds in human subjects without the potential hazards of radioisotopes. Additional advantages of stable isotopic studies have been discussed in recent Deuterium-labeled forms of vitamin B, may be readily synthesized by methods developed by Coburn et aL2' These labeling procedures permit the synthesis of all forms of vitamin B, in bideutero (d2) form, with two deuterium atoms a t the 5'-methylene position, or in pentadeutero (d5) form, with two deuterium atoms at the 5'-position and three a t the 2'-methyl position. Quantitative analysis in stable isotopic studies involves measurement of the molar ratio of various labeled and unlabeled species of vitamin B, or 4PA in urine or other biological materials usually by gas chromatography-mass spectrometry (GCMS). Hachey et a1.*' developed procedures for G C M S (positive-ion electron-impact mode) which have been applied to assess the in vivo kinetics and pool sizes of vitamin B, by analysis of deuterium labeling of urinary 4PA in humans.23 Alternatively, G C M S analysis in the negative-ion chemicalionization mode provides greater ~ e n s i t i v i t yBecause .~~ vitamin B, compounds can be synthesized in d2 and d5 form, dual-label studies can be performed with experimental design analogous to those performed in animals with 'H and I4C for the evaluation of bioavailability and in vivo kinetics.
FACTORS AFFECTING THE BIOAVAILABILITY OF VITAMIN B, Most evidence indicates that the intestinal absorption of vitamin B, occurs by passive diffusion of vitamin B, compounds in nonphosphorylated form.25Diet composition could influence the extent of absorption of the vitamin by affecting the access of dietary B, compounds to the absorptive surface, either by binding or entrapment phenomena or by retarding diffusion through effects on the environment of the brush-border membrane. Whether food components (e.g. dietary fiber) retard the absorption of vitamin B, through such mechanisms is unclear. Tarr et al. determined the net bioavailability of vitamin B, in a mixed American diet using a long-term bioassay method." They observed a mean bioavailability of approximately 75% relative to P N in a formula diet. The factors responsible for this incomplete bioavailabil-
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ity were not determined. The study of Tarr et al. was important because it indicated that, although incomplete, the overall bioavailability of vitamin B, in a typical diet is reasonably high. The factors responsible for the incomplete bioavailability have not been determined. Dietary Fiber The influence of various forms of dietary fiber ozf vitamin B, bioavailability has been examined in animals and humans. N o physical interaction occurs in vitro between B, vitamers and purified neutral and anionic polysaccharides.26 Purified forms of dietary fiber have little or no effect on the bioavailability of P N in bioassays using rats and chicks and little effect in intestinal perfusion s t ~ d i e s .The ~ , ~results ~ of studies with human subjects also indicate little or no adverse effect of dietary fiber.‘z328
OH
FIGURE 1. Structural formula of 5’-O-(&D-glucopyranosyl)-pyridoxine, the primary form of glycosylated vitamin B, in foods.
Glycosylated Forms of Vitamin B, Although evidence of “bound” forms of vitamin B, had been reported for many
year^,'^-^' the first isolation and structural identification of a glycosylated B, compound was reported in 1977. In these studies, Yasumoto et al. isolated a compound from rice bran, which was identified as 5’-0-(P-D-glucopyranosyl)-pyridoxine(PN-glucoside; FIG. This compound was also found in germinating rice,33 peas,34 and alfalfa sprouts.35Incubation of germinating alfalfa seeds with P N was found to be a convenient method of biological synthesis of PN-glucoside in unlabeled, radiolabeled, or deuterium-labeled form. PN-glucoside appears to be formed in plants by enzymatic tran~glycosylation.~~,~~~~~ The glucose moiety of PN-glucosides has been detected a t the 5’ and 4‘ positions of the P N ring in various plants and in vitro enzymatic reaction^.^^.".^^ The presence of the 5’-isomer as the primary naturally occurring form of PN-glucoside suggests that the transglycosylation reaction exhibits specificity for this position in most plant tissue^.^' The existence of several derivatives of PN-glucoside has been reported. Tadera et al. isolated and identified forms of 5‘-O-(P-~-glucopyranosyl)-pyridoxine in which the C-6 position of the glucosyl moiety was esterified to either malonic acid or 3-hydroxy-
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3-methyl-glutaric acid (HMG).40s41 These compounds were found to be minor components of the total vitamin B, in pea seedlings grown in the presence of 1 m M PN, with observed molar ratios of PN: PN-glucoside : HMG-PN-glucoside : malonyl-PNglucoside of 7 : 30 : 7 : 1. Three additional PN-glycosides have been reported in rice bran, including 5’-0-(~-cellobiosyl)-pyridoxine,4‘-0-(@-D-glucosyl)-5’-~ -(@-cellobiosy1)-pyridoxine, and 5’-0-(P-~ellotriosyl)-pyridoxine.~~ The significance of these compounds as components of the total vitamin B, in foods is unclear; however, PN-5’glucoside has been the only sibnificant glycosylated form of vitamin B, detected in chromatographic studies of a variety of foods to date.35Tadera et nl. have reported the existence of an unidentified minor class of vitamin B, conjugate termed “B6X” that yields a response in microbiological assays only after sequential alkaline treatment and hydrolysis with @ - g l u c ~ s i d a s ePresumably, .~~ B6X comprises the esterified forms of PN-glucoside, in which the esters must be removed by alkaline hydrolysis to render the glycoside susceptible to enzymatic hydrolysis. The quantitative analysis of glycosylated forms of vitamin B, in foods can be performed by microbiological or HPLC methods.44A microbiological method devised by Kabir et al. involves measurement of microbiologically active vitamin B, before and after P-glucosidase treatment of an aqueous extract of the food sample.45This method may underestimate the concentration of glycosylated vitamin B, during analysis of raw samples that contain endogenous P-glucosidase activity. Alternatively, HPLC with fluorometric detection permits either the direct quantification of PN-glucoside in intact form or its measurement following enzymatic hydr~lysis?~ P N has been found to be the sole glycosylated form of vitamin B, in foods analyzed to date. Although PN-glucoside comprises 25-75% of the total vitamin B, in many plant-derived food^:^,^^ its bioavailability for humans has not been determined conclusively. PN-glucoside has been found to undergo intestinal absorption in intact form in the rat in ~ i t r o ”and . ~ ~in vivo.I6 Tsuji et al. reported that chemically synthesized PN-glucoside was biologically active as vitamin B, in a rat bioassay?6 In contrast, a rat bioassay by Trumbo et nl. indicated that PN-glucoside isolated from alfalfa sprouts exhibited only 10-30% bioavailability relative to PN.47 Studies conducted using radiolabeled PN-glucoside in the rat have shown that, while PN-glucoside is effectively absorbed, it undergoes little metabolic utilization as vitamin B, and is rapidly excreted in the ~ r i n e . ’ ~The . ’ ~extent of absorption of [3H]PN-glucoside from diets containing intrinsically enriched alfalfa sprouts was slightly less than that of purified [3H]PNglucoside,’6 which suggests an inhibitory effect of plant tissues. Initial studies using bioassays with human subjects indicated that the percentage of glycosylated vitamin B, in several foods was inversely related to the bioavailability of the vitamin.48However, further studies indicated that this correlation was inconsistent when additional foods were Stable-isotopic techniques have been employed to evaluate the bioavailability of purified deuterium-labeled (d2) PN-glucoside in human subjects. In these studies, the mean bioavailability of labeled PN-glucoside fed orally mixed with oatmeal (i.e., by extrinsic enrichment) was similar to that of labeled P N administered under identical condition^.^^,^' Although the ability of humans metabolically to utilize PN-glucoside appears to vary substantially, the mean bioavailability of PN-glucoside in humans appears to be substantially greater than observed in the rat. Further application of
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these highly specific techniques will provide greater clarification of this and other questions of vitamin B, bioavailability. Utilization of PN-glucoside in vitamin B, metabolism requires in vivo hydrolysis of the glycosidic bond. The extent of utilization of PN-glucoside has been shown to be greater when fed orally than when injected in ratsi4 and humans.” These results suggest a role of the intestine in the partial release of biologically active P N from dietary PN-glucoside. The site of this hydrolysis, whether microbial or mucosal, is unclear a t present. P-Glucosidase activity capable of hydrolyzing PN-glucoside in vitro has been detected in several mammalian tissues.46 Later comparative studies showed that &glucosidase activity existed in the intestine of the rat, guinea pig, and human.52 The significance of this enzyme with respect to the bioavailability of PN-glucoside is unclear at present. Chemically ModiJied Forms of Vitamin B, Found in Foods The processing and storage of foods inevitably causes some losses of nutrients including vitamin B,. Whether this influences the bioavailability of the remaining vitamin B, requires a knowledge of the chemical identity and nutritional properties of the remaining B, vitamers, the influence of the food matrix on their absorption, as well as the identity and nutritional properties of the “degradation products” formed. The potential conversion of various B, vitamers to compounds exhibiting little or no mammalian vitamin B, activity has been proposed to explain certain reported effects of food processing or storage on the concentration of biologically available vitamin B, in certain foods. In situations involving microbiological assay of total vitamin B, in foods, the existence of vitamin B, reaction products that elicit a response of the assay microorganism but not in animals or humans, the apparent bioavailability of the vitamin B, in the food would be incomplete. The recent development of specific HPLC methods for food analysis should minimize problems of this type. Considerable effort has been directed toward determining the effect of thermal processing of foods on the bioavailability of vitamin B,, in part, as a result of incidents involving vitamin B, deficiency in infants fed unfortified formulas in the 1 9 5 0 ~ ’ ~ Tomarelli et al. reported that the thermal sterilization of milk or unfortijed infant formula caused a reduction in the apparent bioavailability as determined by rat bioassay, while added PN underwent no reduction in its bi~availability.’~I t was proposed that the thermal effect was due to the conversion of naturally occurring vitamin B, in milk to bis-4-pyridoxyl-dis~Ifide,~~ a compound that exhibits greater activity in Saccharomyces uvarum assays than in rat bioa~says.’~ Later studies using chick bioassays did not confirm this reduction in thermally sterilized milk products,” and the issue has not been resolved. We have examined the formation of bis-4-pyridoxyl-disulfide and other thermal reaction products of vitamin B,. When milk containing [’4C]pyridoxal (PL) or [‘4C]pyridoxalphosphate (PLP) was subjected to thermal sterilization, HPLC analysis revealed no formation of the disulfide c o m p o ~ n d . ’Alternatively, ~ losses of free PL or PLP were largely due to reductive binding of the vitamin B, aldehydes to protein as epsilon-pyridoxyllsyl resid~es.~’ Similar results were found in intrinsically enriched chicken liver and muscle tissues. The nutritional significance of this observation concerns our previous findings that epsilon-pyridoxyllysyl complexes can be partially
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metabolized in the rat to yield P L P via PN (PL) kinase and pyridoxine (pyridoxamine) phosphate oxidase reactions.59360 Thus, epsilon-pyridoxyllysine is a paradoxical “degradation product” because it partially contributes to the total vitamin B, although it is not usually measured in most methods of food analysis. I t should also be noted that this compound can also exhibit anti-vitamin B, effects when administered to B,-deficient rats.,’ This effect may have been responsible, in part, for the possibly reduced bioavailability of the vitamin in the unfortified infant formulas. The identity and nutritional properties of other vitamin B, derivatives found in foods have not been fully determined. Although the stability of PN is generally high, the formation of a previously unknown degradation product, 6-hydroxy-pyridoxine, has been reported in thermal treatment of various fruits and vegetables.6’ The hydroxylation of P N was associated with the oxidative degradation of ascorbic acid6’ and was apparently mediated by generation of hydroxyl radicals.62 Rat bioassays conducted to evaluate the nutritional characteristics of 6-hydroxy-PN indicated no vitamin B, activity (Leatham and Gregory, 1988, unpublished). This reaction does not appear to be a significant mechanism for the loss of biologically active and available vitamin B, in foods.
SUMMARY AND CONCLUSION Further clarification of the bioavailability of dietary vitamin B, requires better analytical data concerning the forms of vitamin B, in foods as well as the overall composition of foods. The application of radioisotopic and stable-isotopic studies such as those described will provide useful information concerning the inherent bioavailability of the various vitamin B, compounds. Additional studies should then address the influence of other dietary components on the utilization of the vitamin B, compounds, using intrinsic and extrinsic enrichment techniques. Care must be taken in interpretation of the results of animal bioassays in the determination of the bioavailability of vitamin B, in complex diets.
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GREGORY, J. F. & J. R. KIRK.1981. The bioavailability of vitamin B, in foods. Nutr. Rev. 39 1-8. GREGORY, J. F. & S . L. INK.1985. The bioavailability of vitamin B,. In Vitamin B,: Its Role in Health and Disease. R. D. Reynolds & J. E. Leklem, Eds. Current Topics in Nutrition and Disease. Vol. 1 3 3-23. Alan R. Liss. New York. SAUBERLICH, H. E. 1985. Bioavailability of vitamins. Prog. Food Nutr. Sci. 9 1-33. GREGORY, J. F. 1988. Recent developments in methods for the assessment of vitamin bioavailability. Food Technol. 4 2 230-238. LEKLEM, J. E. 1988. Vitamin B, bioavailability and its application to human nutrition. Food Technol. 4 2 194-196. SARMA, P. S., E. E. SNELL & C. A. ELVEHJEM. 1947. The bioassay of vitamin B, in natural materials. J. Nutr. 3 3 121-128. WAIBEL,P. E., W. W. CRAVENS & E. E. SNELL. 1952. The effect of diet on the comparative activities of pyridoxal, pyridoxamine and pyridoxine for chicks. J. Nutr. 48: 53 1-538. NGUYEN, L. B. & J. F. GREGORY. 1983. Effects of food composition on the bioavailability of vitamin B, in the rat. J. Nutr. 1 1 3 1550-1560.
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9. GREGORY, J. F. & S . A. LITHERLAND. Efficacy of the rat bioassay for the determination of biologically available vitamin B,. J. Nutr. 116 87-97. 10. TARR,J. B., T. TAMURA & E. L. R. STOKSTAD. 1981. Availability of vitamin B, and pantothenate in an average American diet in man. Am. J. Clin. Nutr. 3 4 1328-1337. 11. KABIR,H., J. E. LEKLEM& L. T. MILLER.Comparative vitamin B, bioavailability from tuna, whole wheat bread and peanut butter in humans. J. Nutr. 113 2412-2420. 12. LINDBERG, A. s.,J. E. LEKLEM& L. T. MILLER.1983. The effect of wheat bran on the bioavailability of vitamin B, in young men. J. Nutr. 113: 2578-2586. 13. WOZENSKI, J. R., J. E. LEKLEM& L. T. MILLER. 1980. The metabolism of small doses of vitamin B, in men. J. Nutr. 110 275-285. P. R. & J. F. GREGORY. 1988. Metabolic utilization of pyridoxine-8-glucoside in 14. TRUMBO, rats: Influence of vitamin B, status and route of administration. J. Nutr. 118: 1336-1342. 15. INK, S. L., J. F. GREGORY& D. B. SARTAIN.1986. Determination of vitamin B, bioavailability in animal tissues using intrinsic and extrinsic labeling in the rat. J. Agri. Food Chem. 3 4 998-1004. & D. B. SARTAIN. 1986. Determination of pyridoxine P-glucoside 16. INK, S. L., J. F. GREGORY bioavailability using intrinsic and extrinsic labeling in the rat. J. Agri. Food Chem. 3 4 857-862. S . P., J. D. MAHUREN, B. S. WOSTMANN, D. L. SNYDER & D. W. TOWNSEND. 17. COBURN, 1989. Role of intestinal microflora in the metabolism of vitamin B, and 4’-deoxypyridoxine examined using germfree guinea pigs and rats. J. Nutr. 119 181-188. P. R. & J. F. GREGORY. The fate of dietary pyridoxine-0-glucoside in the lactating 18. TRUMBO, rat. J. Nutr. 119: 36-39. 19. BIER, D. M. 1987. The use of stable isotopes in metabolic investigation. Bailliere’s Clin. Endocrinol. Metab. 1: 817-836. 20. HACHEY,D. L., W. W. WONG,T. W. BOUTTON& P. D. KLEIN. 1987. Isotope ratio measurements in nutrition and biomedical research. Mass Spectrom. Rev. 6: 289-328. 21. COBURN, S. P., C. C. LIN, W. E. SCHALTENBRAND & J. D. MAHUREN. 1982. Synthesis of deuterated vitamin B, compounds. J. Labelled Compd. Radiopharm. 1 9 703-716. 22. HACHEY,D. L., S. P. COBURN,L. T. BROWN,W. R. ERBELDING, B. DEMARK& P. D. KLEIN.1985. Quantitation of vitamin B, in biological samples by isotope dilution mass spectrometry. Anal. Biochem. 151: 159-168. 23. COBURN, S. P., J. D. MAHUREN, W. F. ERBELDING, D. W. TOWNSEND, D. L. HACHEY& P. D. KLEIN.1984. Measurement of vitamin B, kinetics in vivo using chronic administration of labelled pyridoxine. In Chemical and Biological Aspects of Vitamin B, Catalysis, Part A: 43-54. Alan R. Liss. New York. J. F. & J. P. TOTH.1986. Rapid injection port derivatization and negative ion 24. GREGORY, GCMS detection of deuterium-labelled 4-pyridoxic acid. Proc. 34th Annual Conference on Mass Spectrometry and Allied Topics. p. 3 16. 25. HENDERSON, L. M. 1985. Intestinal absorption of B, vitamers. In Vitamin B,: Its Role in Health and Disease. R. D. Reynolds & J. E. Leklem, Eds. Current Topics in Nutrition and Disease. Vol. 1 3 25-33. Alan R. Liss. New York. L. B., J. F. GREGORY, C. W. BURGIN& J. J. CERDA. 1981. In vitro binding of 26. NGUYEN, vitamin B, by selected polysaccharides, lignin and wheat bran. J . Food Sci. 4 6 18601862. L. B., J. F. GREGORY & J. J. CERDA.1983. Effect of dietary fiber on absorption of 21. NGUYEN, B, vitamers in a rat jejunal perfusion study. Proc. SOC.Exp. Biol. Med. 173: 568-573. 1980. Bioavailability of 28. LEKLEM,J. E., L. T. MILLER,A. D. PERERA& D. E. PEFFERS. vitamin B, from wheat bread in humans. J. Nutr. 110: 1819-1828. 1947. The vitamin B, group. Extraction procedures for 29. RABiNowITz, J. C. & E. E. SNELL. the microbiological determination of vitamin B,. Ind. Eng. Chem. Anal. Ed. 1 9 277-280. K., K. IWAMI,H. TSUJI,J. OKADA& H. MITSUDA.1976. Bound forms of 30. YASUMOTO, vitamin B, in cereals and seeds. Vitamins 5 0 327-333. 31. NELSON,E. W., C. W. BURGIN & J. J. CERDA.1977. Characterization of food binding of vitamin B, in orange juice. J. Nutr. 107: 2128-2134. K., H. TSUJI,K. IWAMI & H. MITSUDA.1977. Isolation from rice bran of a 32. YASUMOTO,
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ANNALS NEW YORK ACADEMY OF SCIENCES bound form of vitamin B, and its identification as 5’-u-(8-D-glUCOpyranOSyl)pyridoxine. Agri. Biol. Chem. 41: 1061-1067. TADERA,K., M. NAKAMURA & A. KOBAYASHI. 1978. Biosynthesis of S’-U-(p-oglucopyranosyl) pyridoxine in germinating rice seeds. Vitamins 5 2 17-22. 1979. A particulate glucosyltransTADERA, K., M. NAKAMURA, F. YAGI& A. KOBAYASHI. ferase catalyzing the formation of 5‘-U-(P-D-glucopyranosyl)pyridoxine from pyridoxine: The occurrence in the seedlings - of Pisum sativum L. J . Nutr. Sci. Vitaminol. 2 5 347350. GREGORY, J. F. & S. L. INK.1987. Identification and quantification of pyridoxine-@ glucoside as a major form of vitamin B, in plant-derived foods. J. Agri. Food Chem. 35: 76-82. SUZUKI, Y., K. UCHIDA& A. TSUBOI.1386. Enzymatic formation of pyridoxine pglucosides by wheat bran P-glucosidase. Nippon Nogeikagaku Kaishi 5 3 189-196. TADERA,K., F. YAGI & A. KOBAYASHI. 1982. Specificity of a particulate glucosyltransferase in seedlings of Pisurn sativum L. which catalyzes the formation of ~’-U-(@-Dglucopyranosy1)pyridoxine. J. Nutr. Sci. Vitaminol. 28: 359-366. IWAMI,K. & K. YASUMOTO.1986. Synthesis of pyridoxine-0-glucoside by rice bran P-glucosidase and its in situ adsorption in rat small intestine. Nutr. Res. 6 407414. SUZUKI, Y., H. ISHII,K. SUGA & K. UCHIDA.1986. Formation of 0-glucosylpyridoxines in soybean and rice callus. Phytochemistry 2 5 1331-1 332. TADERA, K., K. NAGANO,F. YAGI,A. KOBAYASHI, K. IMADA& S. TANAKA. Isolation and structure of a new metabolite of pyridoxine in seedlings of Pisum sativum L. Agri. Biol. Chem. 47: 1357-1359. TADERA, K., E. MORI,F. YAGI,A. KOBAYASHI, K. IMADA& M. IMABEPPU. 1985. Isolation and structure of a minor metabolite of pyridoxine in seedlings of Pisum sativum L. J. Nutr. Sci. Vitaminol. 31: 403-408. TADERA, K., T. KANEKO & F. YAGI.1988. Isolation and structural elucidation of three new pyridoxine-glycosides in rice bran. J. Nutr. Sci. Vitaminol. 3 4 167-177. TADERA, K., T. KANEKO & F. YAGI.1986. Evidence for the occurrence and distribution of a new type of vitamin B, conjugate in plant foods. Agri. Biol. Chem. 5 0 2933-2934. J. F. 1988. Methods for determination of vitamin B, in foods and other biological GREGORY, materials: A critical review. J. Food Comp. Anal. 1: 105-123. KABIR,H., J. LEKLEM& L. T. MILLER.1983. Measurement of glycosylated vitamin B, in foods. J. Food Sci.: 1422-1425. TSUJI,H., J. OKADA,K. IWAMI,K. YASUMOTO & H. MITSUDA.1977. Availability of vitamin B, and small intestinal absorption of pyridoxine-P-D-glucosidein rats. Vitamins 51: 153-159. 1988. Incomplete utilization of TRUMBO,P. R., J. F. GREGORY& D. B. SARTAIN. pyridoxine-8-glucoside as vitamin B, in the rat. J. Nutr. 118: 17CL175. KABIR,H., J. E. LEKLEM& L. T. MILLER.1983. Relationship of the glycosylated vitamin B, content of foods to vitamin B, bioavailability in humans. Nutr. Rept. Int. 28: 709-715. BILLS,N. D., J. E. LEKLEM& L. T. MILLER.1987. Vitamin B, bioavailability in plant foods is inversely correlated with percent glycosylated vitamin B,. Fed. Proc. 4 6 1487. TRUMBO,P. R., J. F. GREGORY, J. P. TOTH, L. B. BAILEY& J. J. CERDA.1988. The bioavailability of pyridoxine-beta-glucosidein the rat and human. FASEB J. 2 A1086. GREGORY,J. F., P. R. TRUMBO,L. B. BAILEY,J. P. TOTH & J. J. CERDA.1989. Bioavailability of oral and intravenous deuterium-labeled pyridoxine-beta-glucosidein human subjects. FASEB J. 3 A454. TRUMBO, P. R. & J. F. GREGORY. 1989.8-Glucosidaseactivity and the metabolic utilization of dietary pyridoxine-beta-glucoside.FASEB J. 3: A454. COURSIN,D. B. 1954. Convulsive seizures in infants with pyridoxine deficient diet. J. Am. Med. Assoc. 154 406-408. TOMARELLI, R. M., E. R. SPENCE & F. W. BERNHART. 1955. Biological availability of vitamin B, in heated milk. J. Agri. Food Chem. 3 338-341. BERNHART, F. W., E. D’AMATO& R. M. TOMARELLI. 1960. The vitamin B, activity of heat sterilized milk. Arch. Biochem. Biophys. 88: 267-269.
GREGORY BIOAVAILABILITY
95
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