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Food Science and Human Nutrition Department, University of Florida, Gainesville, FL 32611
4To whom correspondence should be addressed. E-mail: jfgy{at}ufl.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • vitamin B-6 • pyridoxine-5'-ß-D-glucoside • lactase-phlorizin hydrolase • rats • bioavailability
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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–3
). PN-5'-ß-D-glucoside (PNG) was first identified as a predominant glycosylated form of vitamin B-6 in rice bran (4
) and in a variety of fruits, vegetables and cereal grains (2
,3
). Glycosylated species account for a mean of 
15% of total vitamin B-6 in mixed diets (5
), but actual intakes of glycosylated vitamin B-6 ingested vary widely depending on food selection.
The bioavailability of PNG for human adults is 50–60% relative to that of free PN (6
,7
), and the proportion of PNG in foods is a determinant of overall vitamin B-6 bioavailability (8
). The nutritional effect of the incomplete bioavailability of PNG was demonstrated in a study in which chronic intake of a diet high in PNG (27%) led to a reduction in indicators of vitamin B-6 status (9
). The incomplete bioavailability of glycosylated vitamin B-6 undoubtedly contributes to the incomplete bioavailability (75%) of total vitamin B-6 in typical diets (10
). However, the factors governing the bioavailability of PNG and its relation to the overall bioavailability of vitamin B-6 in humans have not been fully determined (11
).
The rate-limiting step in the nutritional use of PNG as a partially available source of vitamin B-6 for humans and rodents is the enzymatic hydrolysis of the ß-glycosidic bond between PN and the sugar moiety (6
,12
). Previous studies have detected ß-glucosidase(s) capable of catalyzing PNG hydrolysis in rat small intestinal contents (presumably of microbial origin) and in soluble fractions of jejunal mucosa of humans, pigs and rodents (13
,14
). Our initial assumption (13
) that PNG hydrolysis was catalyzed by cytosolic broad specificity ß-glucosidase was later shown to be incorrect (15
). Further investigation led to the identification and purification of a distinct mucosal cytosolic ß-glucosidase designated PN-5'-ß-glucoside hydrolase (PNGH) (15
), and the cytosol appeared to be the main locus of PNG hydrolyzing activity (13
). Unlike many other dietary glycosides, PNG can undergo absorption without prior deglycosylation (6
,12
); thus, cytosolic PNGH seems to function in the release of available PN from dietary PNG. Initial characterization of cytosolic PNGH (15
) indicated that molecular mass and pH optimum were similar to those of brush border lactase-phlorizin hydrolase (LPH) (16
,17
). Extensive amino acid sequence homology also has been found to exist between cytosolic PNGH and brush border LPH (C.-W. Tseung, L. G. McMahon and J. F. Gregory, unpublished data); however, several aspects of PNGH sequence, along with a lack of sucrase activity (15
), rule out the possibility that cytosolic PNGH activity is an artifact caused by contamination of isolated cytosol with the brush border proteins. The data reported herein and a recently published paper (18
) constitute a quantitative reassessment of the role of the brush border membrane as a site of PNG hydrolysis.
We hypothesized that the hydrolytic activity of brush border membrane toward PNG would parallel the age-dependent activity of brush border enzymes, particularly LPH. It was further hypothesized that brush border hydrolytic activity toward PNG would decline in concert with the developmental decline in brush border LPH activity (19
,20
). LPH and cytosolic PNGH have similarities and differences in substrate specificity (15
,21
). Both catalyze the hydrolysis of PNG and lactose. Brush border LPH (21
) but not cytosolic PNGH (15
) can hydrolyze the synthetic substrate para-nitrophenyl-ß-D-glucoside (p-NPGlc). Furthermore, broad specificity ß-glucosidase hydrolyzes p-NPGlc but not PNG (15
) or disaccharides (22
). Soluble intracellular ß-galactosidase activity has been reported previously, although its origin and molecular characteristics are unclear (23
).
The objectives of the study were to document the hydrolytic activities of intestinal brush border and cytosolic subcellular fractions toward PNG and lactose in rats of different ages to determine the degree of relatedness of their enzymatic activities and the potential role of enzymes in the brush border membrane in PNG hydrolysis over the course of rat development.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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PNG was prepared by biological synthesis and purified as described previously (24
). Common chemicals were obtained from Fisher Scientific (Suwanee, GA); p-NPGlc, EDTA and protease inhibitor cocktail (catalogue no. P2714) were obtained from Sigma-Aldrich (St. Louis, MO); and halothane was obtained from Alocarbon Laboratories (River Edge, NJ).
Animals and diet
This protocol was approved by the University of Florida Institutional Animal Care and Use Committee. Experimental groups consisted of Sprague Dawley rats (Harlan Sprague Dawley, Indianapolis, IN) of different ages: newborn (1–2 d) and 2, 4, 8, 12 and 24 wk (n = 9 rats per group). Rats that were 4–24 wk were male only, whereas 1- to 2-d-old and 2-wk-old rats were both male and female. For those 4–24 wk of age each rat was considered to be one observation, whereas the litter mean was considered as one observation for newborn and 2-wk-old rats.
Pregnant dams were received at least 4 d prior to the birth of their pups. Nonsuckling rats were housed in individual wire mesh cages, while lactating dams were kept with their pups in individual plastic shoebox cages at 22°C with a 12-h light/dark cycle. All nonsuckling rats, including the dams, consumed the AIN-93M purified diet (25
) (Dyets, Bethlehem, PA) ad libitum at least 4 d prior to killing and tissue removal.
Tissue preparation
Nonsuckling rats were food-deprived overnight and pups were separated from dams 1 h before killing and tissue removal. The rats were killed under halothane anesthesia either by decapitation (for newborn rats) or by exsanguination and/or removal of the heart. The entire small intestine was washed with 9 g/L ice-cold saline until fluid ran clear. Each intestine was longitudinally cut and placed on a cold glass plate, and mucosa was removed by scraping with a glass microscope slide.
Intestinal mucosa was added to 15 mL homogenizing buffer consisting of 25 mmol/L sodium phosphate (pH 8), 50 mmol/L mannitol and 1 mmol/L EDTA, with protease inhibitor cocktail added as directed by the manufacturer. Due to the difficulty of scraping the intestinal mucosa of the newborn and 2-wk-old rats, the small intestine was homogenized in intact form. Homogenization was performed with a Potter-Elvehjem homogenizer device (10 strokes) in 15 mL of the buffer.
A cytosolic fraction of intestinal mucosa was prepared by centrifuging 5 mL of this homogenate at 200,000 x g for 30 min (Optima LE-80K Ultracentrifuge; Beckman, Fullerton, CA). Brush border membrane vesicles were prepared by the method of Kessler et al. (26
) with modifications (27
). The final brush border membrane isolate was resuspended in 3 mL of buffer and dispersed with a glass Dounce homogenizer. This procedure yielded 10- to 14-fold enrichments of specific activities of lactase and sucrase (28
), which are brush border membrane markers. Portions of brush border and cytosolic fractions were stored at -80°C until analysis.
Analytical methods
Hydrolytic activity toward PNG was assayed by a minor modification of the method described previously (24
). This assay was performed using 0.25 mmol/L PNG (under nonsaturating conditions to conserve substrate) in a 4 mmol/L sodium phosphate buffer (pH 6) with PN measured by fluorometric high performance liquid chromatography (HPLC). Activity toward lactose was determined using a modification of the Dahlqvist method (28
) with 25 mmol/L lactose in 50 mmol/L maleate buffer (pH 5.5) containing 0.1 mmol/L p-hydroxymercuribenzoate (17
), with a glucose oxidase assay kit (kit no. 510; Sigma-Aldrich). All assays were conducted at 37°C under conditions that provided linearity in product formation vs time and with rates proportional to enzyme concentration. Protein concentration was determined spectrophotometrically (29
). To verify the adequacy of vitamin B-6 status, the plasma pyridoxal phosphate (PLP) concentration was determined by HPLC (30
).
Statistical analysis
The effect of rat age on the specific activity of cytosolic and brush border membrane mucosal fractions was determined for each substrate (PNG and lactose) using analysis of variance (31
) with a value of P < 0.05 regarded as significant. The Student-Newman-Keuls method was used for pairwise comparisons among groups, with differences considered significant at P < 0.01 to protect against unequal variances (32
). The relationships among the enzymatic activities were observed by pooling the data for all age groups and conducting Pearson product moment correlation analysis (31
). All calculations were performed using SigmaStat for Windows software (Jandel Scientific, San Rafael, CA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Newborn rats exhibited the highest cytosolic activity (P < 0.01) toward PNG, but subsequent activity remained relatively stable during development up to 24 wk. The specific activity toward PNG in the brush border fraction greatly exceeded that of cytosolic fractions, with a range of 10.5–36.8 nmol · h-1 · mg protein-1 (Table 1)
. Brush border specific activity using PNG as substrate in newborn rats exceeded that of all other groups (P < 0.0001). Throughout subsequent development, the specific activity for brush border hydrolysis of PNG remained at approximately half of that in the newborn rats.
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). This activity decreased after weaning, with 4-wk-old rats exhibiting the least lactase activity (P < 0.0001). Lactase specific activity in brush border fractions ranged from 4480 to 21,400 nmol · h-1 · mg protein-1 (Table 2)
; it was greater in newborn rats than in the other age groups (P < 0.0001). Enzymatic activity in 2-wk-old rats unexpectedly decreased by 63% and decreased further in 4-wk-old rats, but from 8 wk of age the hydrolytic activity remained at approximately half the activity observed in the newborns. Lactase specific activity in the brush border greatly exceeded that of cytosolic fractions.
|
). Similarly, brush border specific activities toward lactose and PNG were positively correlated (r = 0.85; P < 0.0001) (Fig. 1)
.
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), we measured plasma PLP in this study to verify vitamin B-6 adequacy. Plasma PLP concentration (data not shown) increased steadily with age (121–615 nmol/L for rats at age 2 and 24 wk, respectively) and confirmed a lack of vitamin B-6 deficiency at all ages. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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,12
) before partial intracellular hydrolysis by cytosolic PNGH (15
).
In interpreting the specific activities for PNG hydrolysis, it is important to note that the assays described here were conducted under nonsaturating conditions. Irrespective of that, specific activities for lactose greatly exceeded those for PNG in both brush border and cytosolic fractions. Because lactose hydrolysis occurs before absorption, there is probably little physiological importance of intracellular lactase activity. At the present time, the relationship between brush border LPH and cytosolic PNGH is unclear, and we can not exclude the possibility that cytosolic PNGH is a secondary product of the LPH gene. The moderate correlation between specific activities for cytosolic PNG hydrolysis and lactose hydrolysis suggests that at least part of the intracellular lactase activity is associated with cytosolic PNGH, as would be predicted from our previous observation of hydrolytic activity toward ß-linked disaccharides by this enzyme (15
).
The strong correlation between brush border specific activities toward lactose and PNG and the parallel physiological changes shown here confirm our concurrent findings that purified rat LPH can catalyze PNG hydrolysis (18
) and is consistent with the report that LPH also can hydrolyze ß-glucosides of several plant flavonoids and isoflavonoids (33
). The data reported herein and the results of Mackey et al. (18
) indicate that lactose is a preferred substrate over PNG for hydrolysis by LPH and, as predicted, a competitive inhibitor of PNG hydrolysis. This finding raises the question of whether the bioavailability of dietary PNG would be reduced by concurrently ingested lactose.
The ability of intestinal enzymes to hydrolyze ingested PNG during infancy (i.e., prior to weaning) would have physiological importance only if PNG were present in milk or infant formula in large quantities. However, several studies have shown that little or no PNG appears in milk (2
,5
,34
). Consequently, the findings of this study are not particularly relevant to nursing infants, but they are much more pertinent to all others (i.e., weaned infants, children and adults). The fact that residual lactase activity is apparently involved in PNG hydrolysis makes further physiological characterization of importance in our understanding of vitamin B-6 bioavailability. A key question to be addressed is the relative contribution of LPH and cytosolic PNGH to the hydrolysis of dietary PNG, especially as affected by the extent of LPH expression and the intake of inhibitors of PNG hydrolysis by LPH. Whether lactose consumption in infants consuming fruits, vegetables and/or cereal grains affects PNG bioavailability is a question of particular interest.
Both brush border and cytosolic hydrolytic activities toward PNG were lower after weaning than in the neonatal period in the present study. We hypothesize that the bioavailability of PNG at all stages past the neonatal period would be related to the net retention of both brush border LPH activity and cytosolic PNGH activity. To our knowledge this is the first study to document the effect of age on enzymatic activities capable of catalyzing PNG hydrolysis. This study has shown that brush border membrane lactase and PNG hydrolytic activities followed the expected decline associated with reduced expression of LPH with development (20
,35
–38
). Our findings are also consistent with those of others who showed a reduction in LPH in the first 2 wk of life (37
,39
). The reasons for variation among older age groups are unclear but may be attributed in part to possible age-related differences in the rate of adaptation to effects of dietary carbohydrate on LPH expression (40
) and length of dietary acclimation before tissue collection. Extensive existing literature addresses the effect of age on the specific activity of LPH in the brush border (19
,20
,37
,39
). LPH activity decreases at the typical weaning age (21 d for rats) regardless of whether nursing continues (37
).
Although the effects of vitamin B-6 status were not examined in this study, we determined plasma PLP concentration to ensure that the rats were of adequate nutritional status. Plasma PLP concentration was adequate in all groups and unexpectedly increased throughout the study, with a fourfold increase between ages 2 and 24 wk, in contrast to the age-dependent decline reported in rats between 7 and 31 mo of age (41
). This increase in plasma PLP may be a function of the length of time during which the rats had moderately high vitamin B-6 intake prior to their acquisition for this study. The physiological implications of these observations, if any, are unclear.
In summary, this study has shown a role of the intestinal brush border membrane in catalyzing the partial hydrolysis of PNG and, thus, its involvement in the bioavailability of a common dietary form of vitamin B-6. The relative roles of brush border and cytosolic sites of PNG hydrolysis, the relationships of these enzymes and the regulation of their activities in the context of PNG bioavailability are currently being considered.
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FOOTNOTES |
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2 Supported in part by National Institutes of Health grants DK37481 and T32 DK07667.
3 Journal Series No. R-08774 of the Florida Agricultural Experiment Station.
5 Abbreviations used: HPLC, high performance liquid chromatography; LPH, lactase-phlorizin hydrolase; p-NPGlc, para-nitrophenyl-ß-D-glucoside; PLP, pyridoxal phosphate; PN, pyridoxine; PNG, PN-5'-ß-D-glucoside; PNGH, PNG hydrolase.
Manuscript received 14 May 2002. Initial review completed 29 May 2002. Revision accepted 27 June 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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