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Nutrient Metabolism

Uptake, Hydrolysis, and Metabolism of Pyridoxine-5'-ß-D-Glucoside in Caco-2 Cells^1,2

Food Science and Human Nutrition Department, University of Florida, Gainesville, FL

³To whom correspondence should be addressed. E-mail: jfgy{at}ufl.edu.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
An important dietary source of vitamin B-6, pyridoxine-5'-ß-D-glucoside (PNG), exhibits only partial bioavailability, which is limited by the extent of enzymatic cleavage of the ß-glucosidic bond to release metabolically available pyridoxine (PN). This laboratory showed that the intestinal hydrolysis of PNG is catalyzed by cytosolic PNG hydrolase (PNGH) and brush border lactase-phlorizin hydrolase (LPH). LPH-catalyzed PNG hydrolysis in vitro is competitively inhibited by lactose. In the present study, the uptake and hydrolysis of PNG were examined in Caco-2 human colon carcinoma cells, which express a functional LPH but exhibit no PNGH activity. PNG uptake at 37°C was linear over 5–500 µmol/L PNG. Uptake was not significantly reduced when Na⁺ was substituted with K⁺, Li⁺, or Tris in the medium. Increasing PNG concentration in the medium did not change intracellular concentrations of PN, pyridoxamine (PM), pyridoxamine 5'-phosphate (PMP), or pyridoxal 5'-phosphate (PLP); however, intracellular pyridoxal (PL) concentration increased. Intracellular PNG concentration was not significantly reduced in the presence of lactose, but the concentration of PL declined in proportion to extracellular lactose (P = 0.01). These results indicate that PNG can be absorbed intact in a Na⁺-independent process and is taken up by passive diffusion. The presence of lactose in this in vitro model of intestinal uptake reduced the enzymatic hydrolysis of PNG by lactase.

KEY WORDS: • pyridoxine-5'-ß-D-glucoside (PNG) • Caco-2 cells • bioavailability • lactase-phlorizin hydrolase

An important source of vitamin B-6 in the human diet is pyridoxine-5'-ß-D-glucoside (PNG),⁴ which is found in many foods of plant origin. This glycosylated form provides 15% of total vitamin B-6 intake in a typical mixed diet; however, this percentage could increase markedly depending on food selection (1). The metabolic utilization of this form of vitamin B-6 is governed primarily by its partial hydrolysis by ß-glucosidases in the small intestine. Hydrolysis of PNG yields the products glucose and pyridoxine (PN). Relative to PN, PNG exhibits a 50% bioavailability in humans (2,3) and 25–30% in rats (4,5). This laboratory previously reported the intestinal hydrolysis of PNG to be catalyzed by a novel intracellular ß-glucosidase that was shown to be distinct from the intracellular broad specificity ß-glucosidase (6). This intracellular enzyme was shown to hydrolyze PNG and certain disaccharides and was later designated cytosolic PN-5'-ß-D-glucoside hydrolase (PNGH). More recently, we discovered that the brush border membrane ß-glucosidase, lactase phlorizin hydrolase (LPH), also catalyzed the hydrolysis of PNG (7).

LPH is the intestinal enzyme that is responsible for the hydrolysis of dietary lactose, a disaccharide that is important for energy derivation for developing mammals. Although kinetic analyses of purified rat LPH revealed that lactose was a better substrate than PNG, PNG was a secondary substrate for LPH (7). Further investigation of LPH-catalyzed PNG hydrolysis in vitro showed that lactose was a competitive inhibitor of PNG hydrolysis and therefore, the two substrates were hydrolyzed at the same active site. We speculated that the inhibition by lactose observed in vitro would have implications on the in vivo hydrolytic and absorptive processes of PNG. Intestinal hydrolysis of PNG in vivo might be reduced in the presence of lactose, which would reduce the bioavailability of PNG as a source of vitamin B-6. Simultaneous consumption of plant-derived foods with certain dairy products could decrease the hydrolytic release of free PN and, consequently, affect the vitamin B-6 nutritional status of the individual. Brush border LPH activity exceeds that of intracellular PNGH in the small intestine; thus, lactase insufficiency also might reduce PNG bioavailability. Subcellular fractionation of intestinal mucosa revealed that 50–60% of the hydrolytic activity toward PNG was localized to the brush border membrane in the rat small intestine, which was likely catalyzed by LPH (8).

In vivo absorption of vitamin B-6 was examined extensively in the intestine of rats. Intestinal absorption of PN occurs by a nonsaturable, passive diffusion process. PN is rapidly absorbed, mainly in the jejunum, where it is phosphorylated to form pyridoxine 5'-phosphate (PNP), which can be converted to pyridoxal 5'-phosphate (PLP) (9,10). Intracellular PLP in the intestine undergoes dephosphorylation to allow the release of PL into portal circulation (10). PNG hydrolysis occurs mostly in the small intestine to release free PN, which is passively absorbed; however, PNG, as the glucoside, can be absorbed and largely excreted intact in the urine (2,11). PNG also was shown to be taken up by isolated rat hepatocytes and to inhibit competitively the uptake of co-incubated pyridoxine (12).

Caco-2 human colon carcinoma cells were chosen as a model for the present investigation because these cells, when allowed to differentiate, exhibit the morphology and express many of the hydrolytic enzymes present in the small intestinal brush border (including LPH) (13), thus providing an environment that closely resembles the small intestine. Although Caco-2 cells express LPH, they were previously found not to exhibit cytosolic PNGH activity (McMahon, L. G. & Gregory, J. F., unpublished data). Hydrolytic activity toward PNG measured in these cells could, therefore, be attributed solely to the activity of brush border LPH. The purpose of the present study was to examine the uptake, hydrolysis, and metabolism of PNG in the absence and presence of lactose in a cell culture model using the established Caco-2 human colon carcinoma cell line.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Materials. Vitamin B-6 compounds (except PNG), lactose, HEPES, D-glucose, and L-glutamine were purchased from Sigma Chemical. Cell culture grade NaCl, CaCl₂, KCl, LiCl, MgSO₄, tris-hydroxymethylaminomethane hydrochloride (Tris-HCl), 2-(-4-morphlolino)-ethanesulfonic acid (MES), and plastic cell culture supplies were purchased from Fisher Scientific. Cell culture media, media supplements, and trypsin-EDTA were purchased from Invitrogen. PNG was synthesized biologically and purified chromatographically (14).

    Cell culture. Caco-2 human colon carcinoma cells, passage 18, were obtained from American Type Culture Collection. Cells were propagated and maintained at 37°C (95% air, 5% CO₂ atmosphere) in DMEM containing 4.5 g/L glucose, 25 mmol/L HEPES, 44 mmol/L sodium bicarbonate, and 4 mmol/L glutamine. The growth medium was supplemented with 1 mmol/L sodium pyruvate, 100 µmol/L nonessential amino acids, and 20% (v:v) fetal bovine serum plus 100 U/L penicillin, 100 U/L streptomycin, and 50 µg/L gentamycin (15). For routine subculturing, cells were washed with Ca^+2- and Mg^+2- free PBS and detached with 2.5 g/L trypsin with 1 mmol/L EDTA. For uptake experiments, cells were plated on 3-section 100-mm plates at 1.5 x 10⁵ cells/section. Growth medium was changed every 2–3 d with a change 24 h before an uptake experiment. Uptake studies were performed on monolayers at least 9 d postconfluency, a time when lactase activity is present in Caco-2 cells (16). Cells from passages 23–35 were used in the experiments.

    Uptake experiments. Uptake experiments were done by adding treatment medium containing different concentrations of PNG to the top of the cell monolayer with and without lactose. Treatment media were prepared with Krebs-Ringer buffer containing 123 mmol/L NaCl, 4.93 mmol/L KCl, 1.23 mmol/L MgSO₄, 0.85 mmol/L CaCl₂, 5 mmol/L glucose, 5 mmol/L glutamine, 10 mmol/L HEPES, and 10 mmol/L MES at pH 7.4. Monolayers were washed 3 times with 2–3 mL Krebs-Ringer buffer (37°C) before the addition of treatment media containing different concentrations of PNG and lactose. Treatment media were added to monolayers and incubated at 37°C in 5% CO₂ atmosphere for times as indicated in the text. Before the termination of incubation, a sample of treatment medium was collected for analysis. Incubation was then terminated at the desired time by the addition of 3 mL of Krebs-Ringer buffer (4°C), followed by two additional washes. Cell monolayers were mechanically lifted from the plates with a sterile plastic spatula into 0.5 mL PBS and homogenized using a Polytron homogenizer at medium speed for 15 s. An aliquot of crude homogenate was collected for protein measurement and the remaining crude cellular homogenate was centrifuged at 200,000 x g for 30 min to obtain a cytosolic subcellular fraction. Protein concentration was measured spectrophotometrically (17) using bovine serum albumin as the standard.

    Measurement of hydrolysis and uptake. Ratios of PN:PNG were calculated for the treatment media before and after each incubation period. Detection of PNG in the cytosolic compartment of the Caco-2 cells above that measured in cells receiving no additional PNG in the treatment medium was interpreted to be the amount of PNG taken up by the cells.

    Enzyme activity assays. PNG hydrolytic activity in the cytosolic fraction of Caco-2 cells was determined according to Nakano and Gregory (14) with modification of the assay buffer (7). Lactase activity present in the total membrane fraction obtained from a 200,000 x g, 30-min centrifugation was measured by a colorimetric assay as described by Dahlqvist (18) with modification of the maleate buffer to a phosphate buffer.

    Vitamin B-6 analyses. Treatment media were analyzed for PN and PNG both before and after the incubation period using reverse-phase fluorometric HPLC (14). Intracellular concentrations of PN, PNG, pyridoxamine (PM), and pyridoxamine 5'-phosphate (PMP) were measured using ion-pair reverse-phase HPLC with fluorometric detection (19). Intracellular concentrations of PL and PLP were measured by reverse-phase HPLC with fluorometric detection of PL- and PLP-semicarbazones (20).

    Statistical analyses. Data are presented as means ± SEM of multiple experiments done at separate times and are expressed as pmol B-6 vitamer/mg protein. Data for the PN:PNG ratios and intracellular concentrations of B-6 vitamers were evaluated using 1-way ANOVA (21). Data for PNG uptake and metabolism in the presence or absence of lactose were analyzed by a 2-way ANOVA using concentrations of PNG and lactose as factors. In all analyses, pairwise comparisons were performed using the Student-Newman-Keuls test. Statistical analyses were conducted using SigmaStat statistical software (Jandel Corporation). An -level of 0.05 was chosen as the level of statistical significance.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Uptake of PNG as a function of concentration. PNG uptake in Caco-2 cells was examined at concentrations ranging from 0.5 to 500 µmol/L (Fig. 1). After 30 min at 37°C, PNG was detected in the intracellular compartment of the cells at 5 µmol/L, whereas no PNG was detected below this concentration. The intracellular concentration of PNG increased (P < 0.0001) with increasing concentrations of PNG in the incubation media during these 30-min incubations. PNG concentrations between 5 and 500 µmo/L were taken up in an apparently nonsaturable (i.e., linear) process (Fig. 1).

View larger version (10K): [in this window] [in a new window]   FIGURE 1 Pyridoxine-5'-ß-D-glucoside (PNG) absorption by Caco-2 cells as a function of concentration during a 30-min incubation. Absorption of PNG was nonsaturable at 5–500 µmol/L. Each data point represents a mean of at least two replicates from 3 independent experiments. Data are presented as means ± SEM.

View larger version (11K): [in this window] [in a new window]   FIGURE 2 Absorption of pyridoxine-5'-ß-D-glucoside (PNG) at 50 (A) and 200 (B) µmol/L by Caco-2 cells as a function of time. Each data point represents at least two replicates from three independent experiments. Data are expressed as means ± SEM.

View larger version (8K): [in this window] [in a new window]   FIGURE 3 Distribution of intracellular concentrations of B-6 vitamers in Caco-2 cells without added PNG. Abbreviations: pyridoxine (PN), pyridoxal-5'-phosphate (PLP), pyridoxal (PL), pyridoxamine (PM), and pyridoxamine-5'-phosphate (PMP). Bars represent means ± SEM from at least 3 replicates from 4 independent experiments.

View larger version (15K): [in this window] [in a new window]   FIGURE 4 Effect of lactose (0, 75, or 150 mmol/L) on the intracellular concentration of PL during a 30-min incubation of Caco-2 cells with 0, 50, 100, or 200 mmol/L PNG. Intracellular concentrations of PL were significantly lower in cells incubated with 75 and 100 mmol/L lactose (P = 0.01) than in cells incubated with no lactose. Bars represent means ± SEM of at least 2 replicates from 2 independent experiments. Bars with different letters are significantly different, P < 0.05.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
The results from this study provide new insight into the intestinal absorption and metabolism of PNG. PNG uptake and metabolism were not examined in any intestinal epithelial cell culture before this investigation. After differentiation, Caco-2 cells function similarly to enterocytes in the small intestine, which provided a model by which the intestinal processing of PNG could be evaluated.

PNG was taken up by Caco-2 cell monolayers in a pattern that is consistent with passive diffusion across a wide range of PNG extracellular concentration. The exchange of sodium for other monovalent cations did not change PNG uptake by Caco-2 cells; however, the concentration of PNG used in these experiments exceeded the physiologic concentrations of PNG that would be encountered by the small intestine. The relatively high concentration of PNG was used to ensure adequate detection of intracellular concentrations of PNG. At PNG concentrations <100 µmol/L, there is the potential for a Na⁺-dependent uptake process to occur.

Collectively, the results from the present study suggest that PNG is taken up by a Na⁺-independent process that is similar to the absorption of PN as previously determined in rat small intestine (22,23). This observation is also consistent with PN uptake by isolated rat hepatocytes (24), isolated rat renal proximal tubular cells (25), and cultured opossum kidney cells (26). However, we cannot reconcile the difficulty that PNG, as a very hydrophilic molecule, would encounter in crossing the highly charged and hydrophobic plasma membrane. There is also recent evidence that other glycosylated compounds derived from the diet such as quercetin ß-glucosides either interact with or are transported by a sodium-dependent glucose transporter (27–30). Although the absence of sodium did not cause a significant decrease in PNG uptake at 100 µmol/L, we cannot rule out the possibility that PNG is taken up by a carrier-mediated process in intestinal mucosal cells. Zhang et al. (12) reported that PNG competitively inhibited the passage of PN through the plasma membrane of isolated rat hepatocytes, and that PNG uptake was only 20% that of PN under equivalent conditions. These results strongly support the existence of a vitamin B-6 carrier in hepatocytes, but such a mechanism has not been established in intestinal mucosa. The intestinal transport mechanism is further confounded by the dual role ascribed to LPH. Arts and colleagues (31) recently discussed the observation that LPH not only hydrolyzes ß-glucosidic bonds, but also is thought to transport aglycones in a Na⁺-independent process. Although this may explain the transport of PN derived from PNG into the cell, the process by which the intact glucoside, PNG, enters into the cell remains unclear. The detection of intact PNG inside the Caco-2 cells supports previous observations made by this laboratory that PNG can be absorbed intact (2,4).

As indicated in Figure 3, the most abundant form of vitamin B-6 inside Caco-2 cells was pyridoxine. This was not wholly unexpected because these cells were maintained in DMEM that had a high concentration (4 mg/L) of pyridoxine-HCl as the source of vitamin B-6. Consequently, it was difficult to measure the small changes in intracellular PN concentrations that likely were occurring in response to increasing concentrations of PNG against the very high background of intracellular PN and other B-6 vitamers. However, even the smallest change in intracellular PNG concentrations was easily detected because PNG is not present in DMEM, and there is no endogenous synthesis of PNG by mammalian cells. We expected that the ratio of PN:PNG and its change over time would provide a rough estimate of the PNG that is hydrolyzed at the apical surface of the cells. We did not observe any significant changes in these ratios, but there are several complicating factors that might affect this measurement. Once hydrolyzed, PN could be taken up by the cell and metabolized or released into the treatment medium. Alternatively, over the incubation period, intracellular PN might efflux out of the cell into the treatment medium. The PN measured in the treatment medium could contain PN originating from the hydrolysis of PNG and that released from intracellular pools. We did obtain indirect evidence of PNG hydrolysis. Intracellular concentrations of PL significantly increased as PNG concentration increased in the treatment medium (Table 1). PN taken up from the hydrolysis of PNG would be metabolized inside the cell to form PLP, which then undergoes partial hydrolysis to form PL. PL is reported to be the major metabolic product of the small intestine released into portal blood (10) and is thought to be an important transport form of vitamin B-6. Because intracellular PL was present in relatively low concentrations in the Caco-2 cells without added PNG, small increases in PL were easily detected. We did not measure the concentration of PL in the treatment media after the 30-min incubation, but we suspect that intracellular PL also would efflux into the media.

The addition of lactose to the treatment media did not directly inhibit the uptake of PNG. However, we previously showed that in the presence of lactose, purified LPH is competitively inhibited with regard to its PNG hydrolytic activity with a K_i of 56 mmol/L (12). The decline in intracellular PL concentration with increasing lactose (75 and 150 mmol/L) is evidence of the reduced release of available PN from PNG due to the inhibition of LPH in the Caco-2 cell culture studies. The concentration of 150 mmol/L was chosen because it is the approximate concentration of lactose in milk. We believe that PL was the sole form of vitamin B-6 affected because PL is the most abundant intestinal vitamin B-6 metabolite released into portal circulation (10), and its intracellular concentration might be most dramatically affected if the metabolic flux of PN to PL were reduced. Although PL concentrations declined, PLP concentrations did not change significantly in the present study. Our results are consistent with those of Middleton (32) who showed that, under conditions of high PN intake, PLP concentrations remained quite constant in the rat intestine. This effect is likely due to product inhibition by PLP on pyridoxine:pyridoxamine 5'-phosphate oxidase, which is an important means of regulating intracellular PLP concentration (33).

In summary, this study provides in vitro evidence that PNG is taken up intact by intestinal epithelial cells in a Na-independent process that is not saturable, which is similar to the absorption of PN. Lactose diminished the metabolic utilization of PNG in Caco-2 cells, which might translate into a reduction of the in vivo bioavailability of PNG.

	FOOTNOTES

 
¹ Presented in part at Experimental Biology Meeting 03, April 2003, San Diego, CA [Mackey, A. D., McMahon, R. J., Townsend, J. H. & Gregory, J. F. (2003) Pyridoxine-5'-ß-D-glucoside (PNG) uptake, hydrolysis, and metabolism in Caco-2 human colon carcinoma cells. FASEB J. 17: A717 (abs.)].

² Supported by National Institutes of Health grants # DK 37481 and T32 DK07667. This paper is Florida Agricultural Experiment Station Journal Series No. R-09589.

⁴ Abbreviations used: LPH, lactase phlorizin hydrolase; MES, 2-(-4-morphlolino)-ethanesulfonic acid; PL, pyridoxal; PLP, pyridoxal-5'-phosphate; PN, pyridoxine; PNG, pyridoxine-5'-ß-D-glucoside; PNGH, PNG hydrolase.

Manuscript received 27 October 2003. Initial review completed 16 November 2003. Revision accepted 6 January 2004.

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TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

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