This research was conducted to investigate 1) the bioavailability of pyridoxine-5'-beta-D-glucoside (PN-glucoside) relative to that of pyridoxine (PN) in human subjects, and 2) the competitive effect of PN-glucoside on the metabolism of co-ingested PN. To evaluate PN-glucoside bioavailability, the subjects were administered a single oral dose of either deuterium-labeled ([2H2]) PN (Trial 1) or [2H2] PN-glucoside (Trial 2), and the urinary excretion rates of labeled 4-pyridoxic acid (4PA) were measured. The [2H2]4PA derived from [2H2] PN or [2H2]PN-glucoside was excreted mainly in the first 8 h after the dose. Excretion of [2H2]4PA during the 48-h postdose period indicated that the bioavailability of PN-glucoside was approximately 50% relative to PN, which is consistent with our previous report of 58% bioavailability determined using a different protocol and fewer subjects. To assess the effects of PN-glucoside on PN utilization, the subjects were administered different ratios of nonlabeled PN-glucoside with [2H2]PN in Trials 3 and 4. Comparing Trial 1 with Trials 3 and 4, the quantity of nonlabeled PN-glucoside, as a fraction of total vitamin B-6 administered, ranged from 0 to 40% (on the basis of pyridoxine equivalents), with a constant dose of [2H2]PN in each. In these trials, the rate but not the total extent of the excretion of [2H2]4PA derived from [2H2]PN was inversely related to the proportion of co-ingested nonlabeled PN-glucoside. Thus, antagonistic effects of PN-glucoside on PN metabolism do occur in humans, although the effect is less pronounced than that seen previously in rats. Such interactive effects must be considered in evaluating the net bioavailability of dietary forms of vitamin B-6.

During studies of the nutritional utilization of pyridoxine 5-␤-D-glucoside, a major form of vitamin B6 in plants, we detected two cytosolic ␤-glucosidases in jejunal mucosa. As expected, one was broad specificity ␤-glucosidase that hydrolyzed aryl ␤-D-glycosides but not pyridoxine ␤-D-glucoside. We also found a previously unknown enzyme, designated pyridoxine-␤-D-glucoside hydrolase, that efficiently hydrolyzed pyridoxine ␤-Dglucoside. These were separated and purified as follows: broad specificity ␤-glucosidase 1460-fold and pyridoxine-␤-D-glucoside hydrolase 36,500-fold. Purified pyridoxine-␤-D-glucoside hydrolase did not hydrolyze any of the aryl glycosides tested but did hydrolyze cellobiose and lactose. Pyridoxine-␤-D-glucoside hydrolase exhibited a pH optimum of 5.5 and apparent molecular mass of 130 kDa by SDS-polyacrylamide gel electrophoresis and 160 kDa by nondenaturing gel filtration, in contrast to 60 kDa for native and denatured broad specificity ␤-glucosidase. Glucono-␦-lactone was a strong inhibitor of both enzymes. Ionic and nonionic detergents were inhibitory for each enzyme. Conduritol B epoxide, a potent inhibitor of lysosomal acid ␤-glucosidase, inhibited pyridoxine-␤-D-glucoside hydrolase but not broad specificity ␤-glucosidase, but both were inhibited by the mechanism-based inhibitor 2-deoxy-2-fluoro-␤-D-glucosyl fluoride. Our findings indicate major differences between these two cytosolic ␤-glucosidases. Studies addressing the role of vitamin B6 nutrition in regulating the activity and its consequences regarding pyridoxine glucoside bioavailability are in progress.A naturally occurring glycosylated derivative of vitamin B6, pyridoxine 5Ј-␤-D-glucoside (PNG), 1 was first isolated from rice bran (1). PNG is now known to exist in most fruits, vegetables, and cereal grains, in which it comprises from 5-75% of total vitamin B6 (2, 3). Because of the prevalence of PNG in plantderived foods, its bioavailability as a source of available vitamin B6 is a matter of nutritional concern. The bioavailability of this glycosylated form of vitamin B6, relative to pyridoxine (PN), is ϳ25% in rats (4, 5) and ϳ50% for humans (6, 7), as estimated from urinary excretion of 4-pyridoxic acid after oral administration of isotopically labeled PNG. The rate-limiting phase of PNG utilization in vitamin B6 metabolism is the hydrolysis of the ␤-glycosidic bond in both rats and humans. These findings indicate that the hydrolysis of PNG is a major factor governing its bioavailability.Glucosidases exist widely in nature. The primary ␤-glucosidases in mammalian tissues consist of a lysosomal membranebound acid ␤-glucosidase (EC 3.2.1.45; N-acylsphingosyl-␤-Dglucopyranoside glucohydrolase) which is responsible for the hydrolytic cleavage of glucosphingolipids (for review see Ref. 8), and a cytosolic ␤-glucosidase capable of hydrolyzing a variety of aryl ␤-D-glycosides (for review see Ref. 9). This cytosolic enzyme has been detected in a variety of mammalian tissues and has been designated broad specificity ␤-glucosidase. A...

We have previously identified and purified a novel beta-glucosidase, designated PNGH (pyridoxine-5'-beta-D-glucoside hydrolase), from the cytosolic fraction of pig intestinal mucosal. PNGH catalyses the hydrolysis of PNG (pyridoxine-5'-beta-D-glucoside), a plant derivative of vitamin B6 that exhibits partial nutritional bioavailability in humans and animals. Preliminary amino acid sequence analysis indicated regions of close similarity of PNGH to the precursor form of LPH (lactase-phlorizin hydrolase), the beta-glucosidase localized to the brush-border membrane. We report in the present study amino acid sequence data for PNGH and results of Northern blot analyses, upon which we propose a common genomic origin of PNGH and LPH. Internal Edman sequencing of the PNGH band isolated by SDS/PAGE yielded data for 16 peptides, averaging 10.8 amino acids in length. These peptides from PNGH (approx. 140 kDa) were highly similar to sequences existing over most of the length of the >200 kDa precursor of rabbit LPH; however, we found no PNGH sequences that corresponded to approx. 350 amino acids between positions 463 and 812 of the LPH precursor, a region encoded by exon 7 of the LPH precursor gene (amino acids 568-784), and no sequences that corresponded to regions near the N-terminus. MS analysis of tryptic peptides yielded 25 peptides, averaging 15 amino acids, with masses that matched segments of the rabbit LPH precursor. Northern blot analysis of pig and human small intestinal polyadenylated mRNA using a non-specific LPH cDNA probe showed an expected approx. 6 kb transcript of the LPH precursor, but also an approx. 4 kb transcript that was consistent with the size predicted from the PNGH protein mass. Using a probe specific to the region encoded by exon 7, hybridization occurred only with the 6 kb transcript. Based on these observations, we propose that both PNGH and LPH enzymes have the same genomic origin, but differ in transcriptional and, possibly, post-translational processing.

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