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Food Science and Human Nutrition Department, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL
2To whom correspondence should be addressed. E-mail: jfgy{at}ufl.edu.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • bioavailability • intestinal hydrolysis • pyridoxine-5'-ß-D-glucoside • vitamin B-6
Pyridoxine-5'-ß-D-glucoside (PNG), a glycosylated form of pyridoxine (PN) may provide a significant dietary source of vitamin B-6. This form of vitamin B-6 was first isolated from rice bran (1
) and can be found in foods of plant origin (2
–4
). PNG accounts for 5–75% of the vitamin B-6 present in plant tissues. In a mixed American diet, it is estimated that half of the dietary vitamin B-6 intake is from plant sources (5
). Although PNG provides 
15% of the total vitamin B-6 in typical diets in the United States (6
, 7
), some patterns of food selection could lead to a much larger proportion of dietary vitamin B-6 as PNG.
The bioavailability of PNG in humans is 50–60% relative to PN, (8 ,9 ), which is greater than that estimated for rats (25–30%) (10 –13 ). The rate-limiting factor in the utilization of PNG as a source of vitamin B-6 is the enzymatic hydrolysis of the ß-glucosidic bond, rather than intestinal absorption of intact PNG or free PN derived from PNG hydrolysis (11 ,14 ). The variations in PNG bioavailability within and among species may be explained by differences in intestinal enzymatic activities toward PNG. Cytosolic broad specificity ß-glucosidase (BSßG) (15 ) was initially thought to be responsible for the hydrolysis of dietary PNG (16 –18 ). However, purification and kinetic analysis of BSßG from pig intestine revealed that this enzyme does not hydrolyze PNG (19 ). This observation led to the detection and purification of a novel intestinal cytosolic ß-glucosidase, designated pyridoxine-5'-ß-D-glucoside hydrolase (PNG hydrolase), which catalyzed the hydrolysis of PNG. Recent data from this laboratory indicate that PNG can be hydrolyzed in both the cytosolic and membrane subcellular fractions of rat small intestinal mucosa. In an investigation of the age dependence of enzymatic hydrolysis of lactose and PNG, Armada et al. (20 ) measured PNG hydrolytic activity in the cytosolic and brush border membrane subcellular fractions of intestinal mucosa in nursing, weaned and adult rats. A concurrent publication by Mackey et al. (21 ) reported the kinetic analysis of PNG hydrolysis as catalyzed by rat brush border membrane and purified lactase-phlorizin hydrolase (LPH), a ß-glucosidase in the intestinal brush border membrane. Although these investigations reported PNG hydrolysis to be catalyzed in both subcellular compartments of the intestinal mucosa, the contribution of these cytosolic and brush border membrane enzymes toward PNG hydrolysis in vivo is not fully known.
Previous investigations of the effects of vitamin B-6 nutritional status on cytosolic PNG hydrolase activity in the small intestine of guinea pigs and rats showed that PNG hydrolase activity was inversely related to vitamin B-6 status (17 ,18 ). Subsequent studies (Mackey et al., unpublished data) examined more closely the effect of vitamin B-6 status on the activity of cytosolic PNG hydrolase in rat intestine and showed that vitamin B-6 status had little or no reproducible effect on this enzymatic activity. This raised the question whether nutrient differences in the basal diet may have had an effect on the vitamin B-6 status of the rodents and perhaps intestinal ß-glucosidase activity. Our preliminary rat studies, which did not find an effect of dietary vitamin B-6 on intestinal ß-glucosidase activity, used the AIN-93G purified diet instead of the AIN-76A diet that was used in previous studies (17 ,18 ). Although there are several differences between the two diet formulations (22 ), we hypothesize that the carbohydrate source in the basal diet affected nutritional status. The carbohydrate source in the AIN-93G diet, mainly cornstarch, is a more fermentable carbohydrate for the microflora of the cecum and large intestine than sucrose, which is the major carbohydrate component of the AIN-76A formulation. In the presence of fermentable carbohydrate, the gut microflora actively synthesize protein, growth factors and other nutrients, including vitamin B-6 (23 ), which may enhance the vitamin B-6 and overall nutritional status of the rat.
The goals of the present study were as follows: 1) to determine the subcellular distribution of enzymatic activities toward PNG in rat intestinal mucosa as a function of vitamin B-6 status and 2) to examine the differences between the AIN-76A and AIN-93G purified diets in their effect on PNG hydrolase activity in the cytosolic and membrane-associated subcellular compartments.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Weanling male rats (Hsd:Sprague-Dawley SD; Harlan Laboratories, Indianapolis, IN), weighing 50 g were used in both studies. Rats were housed in hanging wire-mesh stainless steel cages and maintained at constant temperature with a 12-h light:dark cycle. All procedures for animal care and treatment were approved by the Institutional Animal Care and Use Committee at the University of Florida.
Both studies employed standard rodent diet formulations (AIN-76A and/or AIN-93G) modified only with respect to the vitamin B-6 content. The recommended fortification of PN, as PN-HCl, in purified rat diets is 7 mg PN/kg diet. The diets did not contain any PNG. In study 1, rats (n = 29) were randomly assigned to one of four AIN-93G purified diets (22 ) from Dyets (Bethlehem, PA) that differed only in the concentrations of PN. Rats had free access to diets that provided 7 mg/kg (n = 5), 1 mg/kg (n = 6), 0.5 mg/kg (n = 6) and 0 mg/kg (n = 6) PN-HCl. Another group of rats (n = 6) was pair-fed a diet containing 7 mg/kg PN (7 mg/kg, pair-fed) matched to the intake of rats fed the 0 mg/kg diet. Animal weight and food intake were monitored daily over the study period. In study 2, rats (n = 49) were fed one of 10 AIN-formulated purified diets (22 ,24 ) from Dyets (Bethlehem, PA). Rats were randomly assigned to either basal diet AIN-76A or AIN-93G: AIN-76A with 2 mg/kg (n = 5), 1 mg/kg (n = 4), 0.5 mg/kg (n = 5), 0.1 mg/kg (n = 5), or 0 mg/kg (n = 5) PN-HCl, or AIN-93G with 2 mg/kg (n = 5), 1 mg/kg (n = 5), 0.5 mg/kg (n = 5), 0.1 mg/kg (n = 5) or 0 mg/kg (n = 5) PN-HCl. Study 2 was conducted at a time separate from study 1.
After 5 wk, the rats in both studies were anesthetized by Halothane (Alocarbon Laboratories, River Edge, NJ) inhalation and exsanguinated by cardiac puncture. Blood was collected with heparinized needles and syringes and transferred to evacuated tubes containing EDTA as an anticoagulant. Plasma was obtained by centrifugation at 2000 x g for 20 min and stored at -80°C until analysis. The small intestine was removed, flushed with 9 g/L NaCl at 4°C to remove intraluminal contents, cut longitudinally, and mucosa was obtained by scraping with a glass slide; these steps took place on ice or at 4°C. All procedures were done under gold fluorescent light to minimize photochemical degradation of vitamin B-6.
HPLC analysis of vitamin B-6 concentrations.
Pyridoxal 5'-phosphate (PLP) in plasma and liver was measured as the semicarbazone derivative by reversed-phase fluorometric HPLC using a modification of the method of Ubbink et al. (25 ). The PN concentration of both the AIN-76A and AIN-93G diets was measured using reversed-phase fluorometric HPLC (26 ) after extraction as described previously (14 ). This analysis was not performed on the diets used in study 1.
Tissue preparation for enzyme activity assays.
The intestinal mucosa was homogenized using a Potter-Elvehjem tissue homogenizer in 5 volumes of homogenization buffer containing 25 mmol/L sodium phosphate, pH 7.4, and 50 mmol/L mannitol, 1 mmol/L EDTA, and a general use protease inhibitor cocktail (P 3684) from Sigma Chemical (St. Louis, MO). The cytosolic and total membrane fractions were obtained by centrifugation of the mucosal crude homogenate at 200,000 x g for 30 min at 4°C. The cytosolic supernatant was removed, and the resulting membrane pellet was resuspended in homogenization buffer using a Potter-Elvehjem homogenizer. The brush border membrane was isolated according to the method of Kessler et al. (27
) with some modification (28
). Briefly, non-brush border membranes were precipitated by the addition of solid MgCl2 (to yield a final concentration of 10 mmol/L) to a portion of mucosal crude homogenate, followed by centrifugation at 3000 x g for 10 min. The supernatant was centrifuged at 40,000 x g for 20 min to obtain a brush border pellet. The pellet was resuspended using a Potter-Elvehjem homogenizer and centrifuged again at 40,000 x g for 20 min. The final brush border membrane pellet was resuspended in homogenization buffer with a Potter-Elvehjem homogenizer. Using the specific activity of the brush border membrane enzyme sucrase, brush border membrane isolation consistently yielded an enrichment factor of 10–14, which is consistent with other published values (27
,29
). Using PNG as the substrate, the crude homogenate and cytosolic, total membrane, and brush border membrane subcellular fractions of intestinal mucosa were assayed for hydrolytic activity in study 1, and cytosolic and total membrane subcellular fractions were assayed for activity in study 2.
Measurement of PNG hydrolytic activity.
In vitro assays of enzyme activity were done on the different subcellular fractions of intestinal mucosa using PNG as the substrate. PNG was prepared by biological synthesis using alfalfa seeds and purified as described by Gregory and Nakano (26 ). All activity assays were done under conditions that allowed the measurement of the initial rate.
The assay for the hydrolysis of PNG was done using the method of Nakano and Gregory (18 ) with a minor modification. Reaction mixtures contained 80 mmol/L sodium phosphate, pH 6.0, and 0.25 mmol/L PNG. Reaction mixtures were incubated for 60 min at 37°C and terminated by incubation in a 100°C water bath for 5 min. The amount of PN released was measured by reversed-phase HPLC with fluorometric detection (18 ). Protein concentration was determined using a colorimetric assay (30 ).
Statistical analyses.
In study 1, differences in plasma PLP and PNG hydrolytic enzyme activities among the rats fed the diets containing different concentrations of PN were analyzed using one-way ANOVA (31 ) with the Student-Newman-Keuls pairwise comparison test and SigmaStat software (Jandel, San Rafael, CA). In study 2, linear regression was used to analyze raw or log-transformed data. This was done to account for small differences in measured PN concentration in the AIN-76A and AIN-93G diets. Rat weight gain, plasma and liver PLP concentrations, and enzymatic activities were analyzed by a t test comparison of linear regression line slopes and y-intercepts between the AIN-76A and AIN-93G diet groups (32 ) using Prism software (GraphPad Software, San Diego, CA). Plasma PLP concentration was log transformed to normalize the distribution and variance. Dietary PN concentrations were log transformed to linearize their relationship with the liver PLP concentrations. A P-value of < 0.05 was considered to be significant. Data are presented as means ± SEM.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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In study 1, rat growth increased (P < 0.0001) as dietary PN concentration increased, with final body weights of 255 ± 4 g (7 mg/kg, ad libitum), 197 ± 5 g (7 mg/kg, pair-fed), 229 ± 3 g (1 mg/kg), 222 ± 6 g (0.5 mg/kg) and 164 ± 4 g (0 mg/kg). Increases in dietary PN improved vitamin B-6 nutritional status among the different groups of rats. Concentrations of PLP in liver and plasma in study 1 were normally distributed with equal variance. PLP concentrations in plasma and liver increased (P < 0.0001) with increasing dietary PN (Table 1).
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In study 1, PNG hydrolytic specific activities of the small intestinal mucosa were determined in the cytosolic and brush border membrane subcellular fractions using PNG as the substrate (Table 1). The activity measured in the brush border membrane fraction was significantly greater in rats that were pair-fed the 7 mg/kg PN than rats fed the 7 mg/kg and 0 mg/kg diets (ad libitum). There was no effect of dietary PN concentration or pair-feeding on PNG hydrolytic specific activity measured in the cytosolic fraction (P = 0.35).
Enzymatic activity data from study 1 were used to make further comparisons among the intestinal mucosa subcellular fractions (crude homogenate, cytosol, total and brush border membrane) within the different dietary PN concentrations (Fig. 1
). With PNG hydrolytic activity expressed on the basis of mucosal weight, the subcellular distribution of activity could be assessed. Although dietary PN concentration did not affect the subcellular distribution of PNG hydrolysis, the activity measured in the total membrane fraction was greater than that measured in cytosolic and brush border membrane fractions (P < 0.001). Recovery of brush border membrane from the crude homogenate was 45% as measured by lactase activity. After adjusting PNG hydrolytic activity for its respective recovery, brush border membrane accounted for 50–60% of the total PNG hydrolytic activity measured in intestinal mucosa, whereas cytosolic activity contributed 10% of the total PNG hydrolytic activity.
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The AIN-76A and AIN-93G diets from study 2 were analyzed for PN concentrations at the conclusion of the study. At each intended level of fortification, PN concentrations were consistently higher in the AIN-76A diets than in the AIN-93G diets (Table 2). In study 2, weight gain [final weight (g) - initial weight (g)] increased with increasing concentrations of dietary PN in rats fed either the AIN-76A or -93G diet (P < 0.0001). The slopes of the regression lines describing the weight gain of rats fed AIN-76A or AIN-93G as a function of dietary PN did not differ (Fig. 2 ), but did exhibit different y-intercepts. The y-intercept of the regression line describing weight gain was higher (P < 0.001) for rats fed AIN-93G than the y-intercept of the regression line of the rats fed the AIN-76A diet (153.5 vs. 135.7 g).
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In study 2, PNG hydrolytic activity in the cytosolic fraction increased with increasing dietary PN in rats fed the AIN-76A (P = 0.02), but not the AIN-93G diet (P = 0.6547) (Fig. 4 ). PNG hydrolytic activity measured in the total membrane fractions was not influenced by dietary PN in rats fed either basal diet (regression line slopes that were not different from zero). The slopes of the regression lines describing the PNG hydrolytic activity in the cytosolic and total membrane fractions did not differ between groups fed the AIN-76A or AIN-93G diets (Fig. 4 ). However, rats fed the AIN-93G diet had greater hydrolytic activity toward PNG in the cytosolic (P < 0.0001) and total membrane (P = 0.006) fractions than rats fed the AIN-76A diet, irrespective of dietary PN concentration. This was concluded from the parallel slopes, but significantly different y-intercepts of the regression lines describing these data when fit to a linear regression model. The data do indicate some nonlinearity, and inspection of the data suggests that the plateau for specific activity for the rats fed AIN-93G diet exceeds that of rats fed AIN-76A diet. When PNG was used as a substrate, specific activities were consistently higher in the total membrane fraction than in the cytosolic fraction, regardless of diet formulation (AIN-76A or AIN-93G) or PN concentration, which is consistent with the data in study 1.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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We observed pronounced changes in plasma and liver PLP concentrations in response to variations in dietary PN concentration in both studies; these changes were in general agreement with other studies of rats fed PN-defined diets (12 ,33 –35 ). Both the AIN-76A and AIN-93G PN-defined diets yielded increased plasma and liver PLP concentrations as dietary PN increased. Liver, but not plasma PLP concentrations, appeared to be influenced by another dietary component in addition to dietary PN because the y-intercept of the regression line for the AIN-93G diet was significantly higher than that for the AIN-76A diet.
The absence of a robust effect of dietary PN on cytosolic PNG hydrolytic activity was contrary to earlier work done in this laboratory which showed an inverse relationship between dietary PN and cytosolic PNG hydrolase specific activity toward PNG in adult rats (18 ). In that study (18 ), mucosal cytosolic PNG hydrolytic activity in rats fed a purified diet (AIN-76A) providing 0 mg/kg PN was twice that measured in rats fed 2 mg/kg PN. Similarly, Banks et al. (17 ) reported the same magnitude of change in mucosal cytosolic PNG hydrolase activity, using PNG as a substrate, in guinea pigs fed diets providing 0 and 3 mg/kg dietary PN. Rats in the present investigation were chronically fed purified diets (either AIN-93G or AIN-76A) providing a wide range of dietary PN concentrations but exhibited no significant increase in mucosal cytosolic PNG hydrolase specific activity with decreasing dietary PN concentration. However, rats that were pair-fed an adequate concentration of PN (7 mg/kg) exhibited a mean PNG hydrolytic activity in the brush border membrane that was twice that measured in rats consuming 7 mg/kg PN ad libitum in study 1. This was likely to be a PN-independent effect of food intake patterns that might have altered mucosal mass or overall rates of mucosal protein turnover. The combined data from the two studies indicate a trend for increasing dietary PN concentration to increase PNG hydrolytic activity not only in the cytosolic fraction, but also in the brush border membrane subcellular fraction of intestinal mucosa.
We compared the AIN-76A and AIN-93G diets to test our hypothesis that the difference in carbohydrate composition of the diets would lead to differences in vitamin B-6 nutritional status and possibly intestinal enzymatic activities. The predominantly starch-based carbohydrate component of AIN-93G may be a more favorable energy source for cecal and colonic microflora that synthesize vitamin B-6, possibly making more vitamin B-6 available for absorption and improving other aspects of nutritional status. Rats fed the AIN-93G diets had a higher final body weight gain and exhibited higher specific activities for the intestinal hydrolysis of PNG in the cytosolic and total membrane fractions than rats fed AIN-76A, regardless of PN concentration. This supports our hypothesis of an effect of diet composition on the nutritional status of growing rats. A recent comparison of the AIN-76A and AIN-93G diets by Lien et al. (36 ) found only minor gastric histopathological differences in rats fed the purified diets, with more pathologies occurring in rats fed the AIN-76A diet. No weight differences between rats fed the AIN-93G or AIN-76A diet were detected; however, rats examined by Lien et al. (36 ) were at least 3 times larger and more mature than the rats used in our study. It is possible that rapidly growing rats respond differently to the two basal diet formulations.
Results from study 2 also indicated that changes in mucosal PNG hydrolytic activity in the rat small intestine are influenced by basal diet composition, not only dietary PN. Rats fed the AIN-93G diet had significantly greater cytosolic and total membrane hydrolytic activity than rats fed AIN-76A, regardless of PN concentration. In addition, rats in study 2 that were fed AIN-76A, but not those fed AIN-93G, exhibited a positive relationship between cytosolic PNG hydrolase activity and dietary PN concentration. To our knowledge, this is the first report of such effects of the composition of purified diets on intestinal enzymes.
In the present study, we were interested in determining the subcellular distribution of PNG hydrolytic activity in intestinal mucosa and the potential effects of diet composition on this distribution. Our laboratory recently discovered that the intestinal brush border membrane hydrolysis of PNG is catalyzed by LPH (21 ), which complements the intracellular hydrolysis of cytosolic PNG hydrolase reported previously (19 ). To make direct comparisons of PNG hydrolytic activities among subcellular fractions obtained from intestinal mucosa, in vitro activity from study 1 was expressed relative to mucosal weight. As expected, the greatest PNG hydrolytic activity was detected in the crude homogenate. Activity measured in the total and brush border membrane fractions was greater than the activity measured in the cytosolic fractions regardless of dietary PN concentration. Because our isolation of brush border membrane was incomplete, we adjusted the PNG hydrolytic activity in the brush border membrane to reflect 100% recovery. We found that 50–60% of hydrolytic activity toward PNG in the intestinal mucosa was localized to the brush border membrane of rat intestinal mucosa. Similarly, we found that nearly all of the activity measured in the total membrane fraction could be attributed to the brush border membrane. Although the primary function of LPH is to catalyze the hydrolysis of dietary lactose, this laboratory has found it to hydrolyze PNG, and further evidence of secondary substrates for LPH has been reported (37 ).
From results obtained in the present study, along with our recent kinetic analysis of LPH-catalyzed PNG hydrolysis (21 ), and partial characterization of the cytosolic PNG hydrolase (19 ), we have constructed a model to explain the intestinal absorption and processing of dietary PNG. A fraction of the PNG that enters the intestinal lumen may not be absorbed at all; consequently, it may be excreted in the feces. PNG may also be absorbed as the intact glucoside and excreted in the urine unchanged or undergo intracellular hydrolysis by cytosolic PNG hydrolase, which would release free PN. Alternatively, PNG could be hydrolyzed at the brush border membrane by LPH and absorbed as free PN and glucose. In addition to the partial hydrolysis of PNG that occurs in the intestine, there is evidence of limited hydrolysis by other organs. An investigation of PNG bioavailability in humans using stable isotopically labeled PNG reported the recovery of the label in the vitamin B-6 urinary metabolite, 4-pyridoxic acid (4-PA), from an intravenous dose, which suggested that some PNG was hydrolyzed outside of the intestine (8 ). These data are further supported by the observations of Nakano and Gregory (18 ). In vitro assays of PNG hydrolytic activity found that rat kidney, along with the intestinal mucosa, was able to partially hydrolyze PNG. This model, which could be applicable to both rodents and humans, extends our understanding of the absorption and metabolism of PNG.
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FOOTNOTES |
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3 Abbreviations used: BSßG, broad specificity ß-glucosidase; LPH, lactase phlorizin hydrolase; PLP, pyridoxal 5'-phosphate; PN, pyridoxine; PNG, pyridoxine-5'-ß-D-glucoside.
Manuscript received 17 October 2002. Initial review completed 7 November 2002. Revision accepted 15 February 2003.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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C. P. Lima, S. R. Davis, A. D. Mackey, J. B. Scheer, J. Williamson, and J. F. Gregory III Vitamin B-6 Deficiency Suppresses the Hepatic Transsulfuration Pathway but Increases Glutathione Concentration in Rats Fed AIN-76A or AIN-93G Diets J. Nutr., August 1, 2006; 136(8): 2141 - 2147. [Abstract] [Full Text] [PDF]
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J. B. Scheer, A. D. Mackey, and J. F. Gregory III Activities of Hepatic Cytosolic and Mitochondrial Forms of Serine Hydroxymethyltransferase and Hepatic Glycine Concentration Are Affected by Vitamin B-6 Intake in Rats J. Nutr., February 1, 2005; 135(2): 233 - 238. [Abstract] [Full Text] [PDF]
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A. D. Mackey, R. J. McMahon, J. H. Townsend, and J. F. Gregory III Uptake, Hydrolysis, and Metabolism of Pyridoxine-5'-{beta}-D-Glucoside in Caco-2 Cells J. Nutr., April 1, 2004; 134(4): 842 - 846. [Abstract] [Full Text] [PDF]
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