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Article

Glucose Modulates Vitamin C Transport in Adult Human Small Intestinal Brush Border Membrane Vesicles^{1 ,2}

Membrane Transport Research Group, Department of Physiology, Faculty of Medicine, Université de Montréal, Montréal, QC, Canada and ^* Department of Physiology, University of Western Ontario, London, ON, Canada

³To whom correspondence should be addressed.

	ABSTRACT

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The uptake of L-ascorbate (vitamin C) and its oxidized form, dehydro-L-ascorbic acid (DHAA), was evaluated in brush border membrane vesicles isolated from adult human duodenum, jejunum and ileum. Ascorbate was taken up along the entire length of the small intestine with a threefold higher initial uptake rate in distal than proximal segments. Ascorbate uptake was Na⁺-dependent, potential-sensitive and saturable (K_m, 200 µmol/L), whereas DHAA transport involved facilitated diffusion (K_m, 800 µmol/L). Pharmacologic experiments were conducted to characterize further these transport mechanisms. DHAA uptake was not mediated by the fructose carrier GLUT5, the uridine transporter or the 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)-sensitive anion exchanger of the apical membrane. DIDS and sulfinpyrazone , an inhibitor of the urate/lactate exchanger, both significantly reduced the initial rate of ascorbate uptake. Acidic pH inhibited ascorbate uptake, and this effect was not due to a transmembrane proton gradient. Increasing concentrations of glucose in the transport media also significantly inhibited ascorbate uptake, but no effect of glucose was seen when glucose internalization was blocked by phlorizin . Preloading the vesicles with glucose inhibited ascorbate uptake similarly, indicating that glucose interferes with the ascorbate transporter from the internal side of the membrane. The results of this study suggest that DHAA crosses the apical membrane by facilitated diffusion, whereas ascorbate transport is a Na⁺-dependent, electrogenic process modulated by glucose.

KEY WORDS: • vitamin C • ascorbate • glucose • human small intestine • absorption • transport regulation

	INTRODUCTION

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Humans, like other primates and guinea pigs, need to ingest vitamin C because they lack L-gulonolactone oxidase, which is the enzyme required for the final step in the de novo synthesis of ascorbate from glucose. Stewart and Booth (1964) concluded that the fastest absorption of radiolabeled vitamin C in human small intestine occurs at a proximal site. However, their experimental approach was indirect because they monitored urinary excretion of vitamin C after an oral dose. Furthermore, both proximal (Hornig et al. 1973 ) and distal segments (Stevenson and Brush 1969 ) have been proposed as the major site of ascorbate absorption in guinea pig intestine. Thus, it is uncertain which intestinal segment comprises the principal site of vitamin C uptake.

The typical human diet contains both vitamin C (ascorbate) and its oxidized form (dehydroascorbic acid, DHAA)⁴. Additional DHAA is produced by oxidation within the lumen of the gastrointestinal tract (Kyrtopoulos et al. 1991 ). DHAA can prevent scurvy (Todhunter et al. 1950 ), perhaps because it can be reduced to ascorbate by glutathione- or NADPH-dependent DHAA reductases within human enterocytes (Buffinton and Doe 1995 ). However, little is known about the transport mechanisms mediating ascorbate and DHAA absorption by the intestinal mucosa and their relative contributions to meeting the body’s need for vitamin C.

Some properties of vitamin C transport have been studied with the use of in vitro preparations and brush border membrane vesicles prepared from guinea pig small intestine. The observed K_m values for vitamin C varied from 300 to 1000 µmol/L (Bianchi et al. 1986 , Mellors et al. 1977 , Siliprandi et al. 1979 , Suzuki et al. 1991 ), indicating a much lower affinity than reported for cultured cells capable of concentrative ascorbate uptake (K_m values of 4–30 µmol/L) (Rose and Wilson 1997 , Rumsey and Levine 1998 ). Na⁺-dependent, electroneutral ascorbate uptake has been observed in human intestinal brush border membrane vesicles isolated from jejunum and ileum indiscriminately (Toggenburger et al. 1979 ). This putative electroneutral mechanism (Toggenburger et al. 1979 ) contrasts with the electrogenic Na⁺-ascorbate cotransport observed in rat kidney (Toggenburger et al. 1981 ) and various non-intestinal cell types studied in vitro (Rose and Wilson 1997 ).

DHAA uptake by brush border membrane vesicles prepared from guinea pig ileum appears to involve a saturable, facilitated diffusion pathway that does not depend on Na⁺ (Bianchi et al. 1986 ). There are some notable similarities between the intestinal absorption of vitamin C and glucose. The latter is mediated by the Na⁺-dependent glucose cotransporter, SGLT1, and by the facilitative hexose transporters, GLUT2 and GLUT5 (Wright 1993 ). Interactions between DHAA and other facilitative hexose transporters have been observed in various tissues. For example, GLUT1 mediates DHAA uptake in astrocytes (Siushansian et al. 1997 ), whereas both GLUT 1 and GLUT3 transport DHAA in the Xenopus laevis oocyte expression system (Rumsey et al. 1997 , Vera et al. 1993 ).

In a recent review, Rose and Wilson (1997) proposed a model for vitamin C uptake by polarized epithelial cells such as enterocytes. A Na⁺-coupled transport process takes up ascorbate across the apical surface. Meanwhile, DHAA enters into the cell at both apical and basolateral surfaces by a Na⁺-independent, saturable process and then it is reduced intracellularly to ascorbate. The rise in intracellular ascorbate concentration due to these mechanisms creates a gradient favoring the exit of ascorbate across the basolateral plasma membrane by facilitated diffusion. This model must be validated in human intestinal membranes, and the possible interactions between vitamin C and glucose transporters of the enterocyte’s membranes have to be determined.

This study was designed to achieve the following: 1) determine whether ascorbate and DHAA transport occurs along the entire length of the human small intestine; 2) compare the pharmacologic properties of ascorbate, DHAA and glucose uptake; and 3) investigate the possible interactions of vitamin C and glucose with each other’s transporters in the apical membrane of human enterocytes. A novel view of ascorbate transport in the human small intestine has arisen from our new observations.

	MATERIALS AND METHODS

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Chemicals.

L-[1-¹⁴C]Ascorbic acid (296 MBq/mmol), D-[1-³H(N)]glucose (370 GBq/mmol) and L-[4,5-³H(N)]leucine (2.44 TBq/mmol) were purchased from New England Nuclear (Mississauga, Canada). Ascorbic acid was dissolved in 4 mmol/L homocysteine. [¹⁴C]DHAA was prepared by incubating [¹⁴C]ascorbate with ascorbate oxidase at 37°C for 1 min, immediately before the beginning of the uptake assay (Siushansian et al. 1997 ). An HPLC-based electrochemical assay was used to confirm that all ascorbate was oxidized in this DHAA preparation (Siushansian et al. 1997 ). Ascorbate oxidase, valinomycin, phlorizin (PZ), sulfinpyrazone (SPZ) and diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) were obtained from Sigma-Aldrich Chemicals (Oakville, Canada). All salts and chemicals used for buffer preparation were of the highest purity available.

Preparation of brush border membrane vesicles.

Adult human small intestine was obtained from three healthy organ donors with the generous collaboration of Québec-Transplant after signature of consent forms. The entire procedure was approved by the Ethics Committee of the Faculty of Medicine, Université de Montréal. The small intestine was divided into 8 segments, namely, duodenum (first 25 cm), four jejunal segments of 30–35 cm each, designated J1 to J4, and three ileal pieces of equal length, designated I1 to I3. The tissues were rinsed with ice-cold saline solution and frozen at -80°C. For preparation of brush border membranes, the intestinal pieces were thawed and the mucosa was scraped with a spatula onto a cold glass plate. Brush border membranes were prepared as previously described (Malo 1988 ). The P₂ fractions were suspended in 50 mmol/L Tris-HEPES buffer, pH 7.5, containing 0.1 mmol/L MgSO₄, 250 mmol/L KCl and 125 mmol/L mannitol. These fractions were divided into aliquots and stored in liquid nitrogen until use. The final steps in the preparation of brush border membrane vesicles were done the day before the experiment after suspension of thawed P₂ fractions in transport medium. The final vesicle pellet (P₄) was divided into 25-µL aliquots and frozen in liquid nitrogen until the time of assay to prevent instability and loss of transport activity during the transport experiments, as previously shown (Malo and Berteloot 1991 ). For experiments performed under zero or negative membrane potential, the following two different approaches were used: 1) The vesicles were resuspended in the presence of 3 µmol/L valinomycin with 150 mmol/L (out of the usual 250 mmol/L) KCl replaced by an equivalent amount of choline chloride. The transport media contained 3 µmol/L valinomycin and 100 mmol/L KCl for 0 mV voltage-clamped condition or 100 mmol/L choline chloride for valinomycin-induced inside-negative K⁺-diffusion potential. 2) The highly permeant anion thiocyanate (SCN^-) replaced the 250 mmol/L Cl^- on both sides of the membrane (zero membrane potential) or only in the transport medium (inside-negative membrane potential).

Transport measurements.

Brush border membrane vesicles were diluted to a concentration of 30–40 g/L in suspension buffer and then 20-µL aliquots were combined with 0.5 mL transport medium. The exact compositions of the suspension buffers and transport media are given in the table and figure legends. All experiments were done according to the rapid filtration technique using a Fast Sampling, Rapid Filtration Apparatus (FSRFA), as previously described (Malo and Berteloot 1991 ). Initial rates of ascorbate and DHAA uptake were determined at 37°C using nine time points within a 30-s period. At each time point, an aliquot (50 µL) of incubated vesicles was injected into 1 mL ice-cold stop solution (composition adjusted to match the final concentrations of the different species in the transport medium), then applied to a prewet 0.65-µm nitrocellulose filter (Micro-Filtration System, Dublin, CA); the filter was washed three times with 1 mL ice-cold stop solution. Subsequently, filters were dissolved in minivials by 15 min incubation with 5 mL Beta-Blend (ICN Radiochemicals, Irvine, CA) and continuous shaking. Radioactivity was determined using a LS6000SC Beckman (Mississauga, Canada) scintillating counter. The protein content of vesicles was determined using BCA Protein Assay Reagent (Pierce Chemical, Rockford, IL) with bovine serum albumin as standard.

Data analysis.

Transport data are expressed as picomoles of solute taken up per milligram protein. The initial rates of transport were determined by linear regression analysis over the linear part of the uptake-time curves. Alternatively, when uptake-time curves deviated from linearity, second-degree polynomial analysis was carried out in which the initial rate (±SD of regression) was the first-degree coefficient of the polynomial. Kinetic parameters were determined using the Michaelis-Menten equation for one or two binding sites or by using linear transformation according to Eadie-Hofstee. Inhibition curves were fitted according to competitive or noncompetitive equations. Data were analyzed using FigureP version 2.7 (FigureP Software, Durham, NC). Statistical differences between two experimental conditions were evaluated using the one-tailed t test for paired data, with P < 0.05 considered significant.

	RESULTS

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Regional distribution of vitamin C transport

Uptake of glucose, ascorbate and DHAA was detectable along the entire length of the human small intestine (Fig. 1 ). However, the proximal to distal profiles of uptake rates differed among substrates. Unlike glucose uptake, ascorbate transport exhibited a reverse proximal to distal gradient with highest activity in the distal ileum, whereas DHAA uptake was higher in jejunal segments (Fig. 1) . The ratios of ascorbate uptake to DHAA uptake were 3.3 ± 1.2 in duodenum and jejunum but increased to 4.8, 7.2 and 30 in ileum segments I1, I2 and I3, respectively. Initial rate measurements were performed with the use of a much lower concentration of radiolabeled glucose (4 µmol/L) than radiolabeled ascorbate (200 µmol/L) because of the relatively low specific activity of the latter. When normalized to the same substrate concentration, the ratio of glucose uptake to ascorbate uptake declined progressively from duodenum to distal ileum, falling from 59 to 8.5.

View larger version (28K): [in this window] [in a new window]   Figure 1. Regional distribution of L-ascorbate, L-dehydroascorbic acid (DHAA) and D-glucose transport along the entire length of adult human small intestine. Initial rates of uptake ± SD of regression were measured in duodenum (D), four jejunal pieces (J1–J4) and three ileal segments (I1–I3) from one subject. Values are means ± SD for 3 or 4 replications. Brush border membrane vesicles were resuspended in 50 mmol/L Tris-HEPES buffer, pH 7.5, 0.1 mmol/L MgSO4, 125 mmol/L mannitol and 250 mmol/L KCl. The final concentrations in the transport medium were: 50 mmol/L Tris-HEPES buffer, pH 7.5, 0.1 mmol/L MgSO4, 125 mmol/L mannitol, 50 mmol/L KCl, 200 mmol/L NaCl. Concentrations of radiolabeled substrates were 4 µmol/L [3H]glucose, 200 µmol/L [14C]ascorbate and 200 µmol/L [14C]DHAA.

Characteristics of transport

    Concentration-dependence. The initial uptake rates of both ascorbate and DHAA were determined as a function of increasing substrate concentrations (50–500 µmol/L). Ascorbate transport clearly was saturable with an apparent K_m of 267 ± 33 µmol/L and a V_max of 28 ± 2 pmol/(s·mg protein) (Fig. 2A ). The goodness of fit for the 1-site model was confirmed by Eadie-Hofstee representation of the uptake data (shown in the inset). Similarly, DHAA uptake was also a curvilinear function of external DHAA concentration and could be described by a single Michaelian component, as confirmed by the linearity of the Eadie-Hofstee plot shown in the inset (Fig. 2B ). For DHAA, the apparent K_m was 805 ± 108 µmol/L and the V_max was 35 ± 3 pmol/(s·mg protein). For both ascorbate and DHAA, the 2-site model was rejected on the basis of either divergence or negative parameters.

View larger version (18K): [in this window] [in a new window]   Figure 2. Determination of kinetic parameters of L-ascorbate (Panel A) and L-dehydroascorbic acid (DHAA, Panel B) transport in an adult human jejunum. Experimental conditions were as described in the legend to Figure 1 . Values are means ± SD for 3 or 4 replications from one subject. Substrate concentrations varied from 10 to 500 µmol/L for ascorbate and from 50 to 700 µmol/L for DHAA. Osmolarity of the media was maintained with D-mannitol. Inset: data are displayed in a Eadie-Hofstee linear transformation showing that influx data can be resolved into one system with the following kinetic parameters ± SD of regression: Km = 267 ± 33 µmol/L; Vmax = 28 ± 2 pmol/(s·mg protein) for ascorbate and Km = 805 ± 108 µmol/L; Vmax = 35 ± 3 pmol/(s · mg protein) for DHAA.

Ascorbate uptake was decreased by 83% (P < 0.04, n = 2) when Na⁺ was removed from the transport medium, which revealed both major Na⁺-dependent and minor Na⁺-independent pathways (Fig. 3A ). External SPZ (P < 0.02, n = 4) and intravesicular DIDS (P < 0.05, n = 2) inhibited the Na⁺-dependent component of ascorbate uptake. Total ascorbate uptake was reduced by 33% (P < 0.002, n = 5) in the presence of PZ. However, ascorbate uptake was not affected by a high concentration of fructose, which is the specific substrate for GLUT5. Similarly, ascorbate uptake was not changed by either uridine, a substrate for the Na⁺-nucleoside transporter, or bumetanide, a Cl^- transport inhibitor. Unlike ascorbate transport, DHAA uptake was not affected by any of the conditions tested (Fig. 3B ).

View larger version (21K): [in this window] [in a new window]   Figure 3. Pharmacology of L-ascorbate (Panel A) and L-dehydroascorbic acid (DHAA, Panel B) transport. Initial rates of uptake were determined under various experimental conditions. Brush border membrane vesicles were resuspended in 50 mmol/L Tris-HEPES buffer, pH 7.5, 0.1 mmol/L MgSO4, 125 mmol/L mannitol and 250 mmol/L KCl with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDSin) or without (all other conditions) 3 mmol/L DIDS. Final concentrations in the transport media were 50 mmol/L Tris-HEPES buffer, pH 7.5, 0.1 mmol/L MgSO4, 250 mmol/L KCl (Na 0) or 50 mmol/L KCl and 200 mmol/L NaCl, and enough mannitol to equilibrate the osmolarity of the various media. Concentrations of radiolabeled substrates were 200 µmol/L [14C]ascorbate and 200 µmol/L [14C]DHAA. Drug concentrations were 1 mmol/L phlorizin (PZ), 3 mmol/L sulfinpyrazone (SPZ), 50 mmol/L fructose, 5 mmol/L uridine and 1 mmol/L bumetanide. Initial rates of uptake (±SD of regression) from 1 to 3 subjects were expressed as a percentage of their own control value set as 100%. *P < 0.05 compared with control for vesicles from the same subject.

View larger version (16K): [in this window] [in a new window]   Figure 4. Interactions between ascorbate and glucose uptake in human jejunal brush border membrane vesicles (BBMV). Panel A: Inhibition of L-ascorbate or L-dehydroascorbic acid (DHAA) uptake (±SD of regression) by increasing concentration of glucose in transport media. The estimated Ki value for competitive inhibition of ascorbate uptake by glucose was 1.1 ± 0.1 mmol/L. The 56% inhibition observed with 5 mmol/L glucose was significant (P < 0.003, n = 4 from 3 different subjects). Panel B: Inhibition of glucose uptake (±SD of regression) by increasing concentrations of ascorbate or DHAA in transport media. Estimated Ki values were 18 ± 5 and 42 ± 8 mmol/L for ascorbate and DHAA, respectively. Conditions were as described in the legend to Figure 1 with the use of mannitol to maintain the osmolarity of the different media.

View larger version (19K): [in this window] [in a new window]   Figure 5. Initial rates of ascorbate uptake as a function of increasing ascorbate concentrations without and with phlorizin (PZ, 1 mmol/L) in the transport medium. Conditions were as described in the legend to Figure 2 , but this assay was performed with another subject. At the ascorbate concentration of 200 µmol/L, the effect of the combinations PZ + glucose (100 mmol/L) and PZ + sulfinpyrazone (SPZ, 3 mmol/L) were tested. Nonlinear regression analysis gave Km values (±SD of regression) of 112 ± 16 and 240 ± 19 µmol/L and Vmax values of 21 ± 1 and 19 ± 0.7 pmol/(s·mg protein), for the control and PZ conditions, respectively. Inset: Lineweaver-Burk plot of the inhibition of ascorbate uptake at 50, 200 and 500 µmol/L in the absence or presence of 1 mmol/L PZ.

    Effects of anions. Thiocyanate (SCN^-) decreased the initial rate of ascorbate uptake whether it was present on both sides of the membrane (zero membrane potential) or only in the transport medium (inside-negative membrane potential) (Table 1) . When various concentrations of sodium azide (NaN₃) partially replaced NaCl in the transport media, increases in ascorbate and DHAA uptake were recorded (Fig. 6 ). These increases were dose dependent, with a maximal effect at ~50 mmol/L azide_out (37 and 388% for ascorbate and DHAA, respectively). The effects of 50 mmol/L azide on transport were studied in vesicles loaded with KN₃ or KCl (Table 1) . External azide stimulated ascorbate uptake (P < 0.03, n = 3), but internal azide inhibited ascorbate uptake by 68%. When azide was present on both side of the membrane, a 32% inhibition was still observed even though external azide partially reversed the effect of internal azide. DHAA uptake was influenced by external azide only.

View larger version (16K): [in this window] [in a new window]   Figure 6. Effects of increasing concentrations of azide in transport media on the initial uptake rates (±SD of regression) of L-ascorbate or L-dehydroascorbic acid (DHAA) in jejunal brush border membrane vesicles (BBMV) from one adult subject. BBMV were resuspended in 50 mmol/L Tris-HEPES buffer, pH 7.5, 0.1 mmol/L MgSO4, 125 mmol/L mannitol and 250 mmol/L KCl. Final concentrations in the transport media were 50 mmol/L Tris-HEPES buffer, pH 7.5, 0.1 mmol/L MgSO4, 125 mmol/L mannitol, 50 mmol/L KCl, NaCl and NaN3 up to a total concentration of 100 mmol/L and 200 µmol/L radiolabeled substrate. The line shown represents the best visual fit.

	DISCUSSION

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In this study, the regional distribution of human intestinal vitamin C absorption has been established and compared with the proximal-distal gradient for glucose absorption (Malo 1988 ). We found that ascorbate uptake is greatest in distal segments of human intestine. This appears to contrast with a study of everted sleeves of guinea pig intestine, whose authors reported that uptake rates for radiolabeled vitamin C were faster in jejunum than in ileum (Karasov et al. 1991 ). However, interpretation of the latter study is limited by the fact that vitamin C may have oxidized during the transport measurements, and the uptake periods probably were too long to measure the initial rates of transport. We observed that DHAA uptake occurs along the entire length of the human small intestine. Nevertheless, the relatively low affinity of DHAA transport compared with ascorbate transport indicates that most vitamin C is absorbed as ascorbate.

Intestinal uptake of ascorbate and DHAA uptake is not mediated by the hexose transporters SGLT1 or GLUT5, the uridine transporter or the DIDS-sensitive anion exchanger (Cabantchik et al. 1978 ). Furthermore, because ascorbate uptake is sensitive to PZ and SPZ but DHAA uptake is not, it is obvious that the reduced and oxidized forms of vitamin C are not transported by identical mechanisms in human intestine and in cultured cells (Welsh et al. 1995 ).

We observed Na⁺-dependent ascorbate uptake in human intestinal brush border membrane vesicles. Similarly, a study employing perfusion of the small intestine of guinea pigs showed that at least part of vitamin C transport is Na⁺ dependent because it could be inhibited partially by the Na⁺,K⁺-ATPase blocker, ouabain (Suzuki et al. 1991 ). Intestinal absorption of ascorbate evidently is mediated by Na⁺-ascorbate cotransporters because ascorbate uptake into brush border vesicles is inhibited by DIDS and SPZ, drugs that have been shown to block Na⁺-ascorbate cotransporters in other cell types (Rose and Wilson 1997 , Siushansian et al. 1997 ). External DIDS (100 µmol/L) does not inhibit glucose uptake by rat jejunum brush border membrane vesicles (Beesley et al. 1997 ) but we observed a partial inhibition of glucose uptake and a total inhibition of Na⁺-ascorbate uptake by internal DIDS (3 mmol/L). Our experiments were performed under zero-trans conditions for both Na⁺ and ascorbate, whereas Cl^- concentrations and pH were identical on both sides of the membrane. Thus, inhibition of ascorbate uptake cannot be due to changes in Cl^- conductance or blockade of an anion exchanger. As reported previously for inhibition of H⁺,K⁺-ATPase and Na⁺,K⁺-ATPase, our results indicate that DIDS exerts its inhibitory effect by modifying the transporters from the cytosolic side of the membrane (Vega et al. 1988 ).

It has been reported that Na⁺-dependent ascorbate transport was electroneutral in brush border membrane vesicles from human (Toggenburger et al. 1979 ) and guinea pig small intestine (Siliprandi et al. 1979 ), but was potential-sensitive in vesicles from rat kidney cortex (Toggenburger et al. 1981 ). Differences may arise depending on how the membrane potential is imposed, as discussed by Toggenburger et al. (1981) , who reported an inhibitory effect of thiocyanate but a stimulatory effect of K⁺-induced depolarization in the presence of valinomycin, as observed here. Ascorbate stimulates the tissue short-circuit current in isolated rat intestine (Barnett et al. 1978 ). An inside-negative membrane potential stimulates transport of ascorbate by intestinal brush border membrane vesicles of eel, and Hill analysis suggests a 2 Na⁺:1 ascorbate stoichiometry (Maffia et al. 1993 ). Our data also suggest a minimal stoichiometry of 2 Na⁺:1 ascorbate for transport in human intestinal mucosa, leading to the transfer of at least one positive charge inside the vesicles.

A stimulatory effect of azide on ascorbate uptake was reported for rat kidney cortex (Toggenburger et al. 1981 ); this study extends this finding to both ascorbate and DHAA in human intestine. Because ascorbate transport is Na⁺ dependent and DHAA uptake is Na⁺ independent, inhibition cannot be caused by an effect of azide on the Na⁺ gradient. N₃^- is a highly permeant anion that stimulates electrogenic glucose transport by changing membrane potential (Liedtke and Hopfer 1976 ). However, when N₃^- was present in an equal amount on both sides of the membrane, ascorbate uptake was inhibited and DHAA was stimulated, thus excluding membrane potential effects. Similarly, chemical modification of ascorbate and DHAA is unlikely to account for the effects of azide because we observed that internal N₃^- immediately affected the uptake of external ascorbate and DHAA.

Toggenburger et al. (1981) found that between pH 5 and 8, the initial rate of ascorbate transport is not affected, and no effect of an outward-directed proton gradient occurs in rat kidney cortex. In our experiments, both the inward- and the outward-directed pH gradients inhibited ascorbate uptake but were ineffective on DHAA uptake. Surprisingly, when pH 5.5 was present on both sides of the membrane, ascorbate and DHAA uptake were decreased by 88 and 58%, respectively. These results indicate that a proton gradient is not involved, that chemical modification of the substrates at low pH is excluded and that H⁺ interacts with the ascorbate and DHAA carriers on both sides of the membrane simultaneously.

The inhibition of intestinal ascorbate uptake by PZ that we observed is not likely caused by a direct interaction of the glucose moiety of PZ with the external portion of the Na⁺-ascorbate cotransporter because external glucose does not affect ascorbate uptake when glucose internalization through SGLT1 is blocked. However, internal glucose affects Na⁺-ascorbate uptake, which is consistent with the observation that inhibition of ascorbate uptake by glucose occurs only in the presence of Na⁺ (Siliprandi et al. 1979 ). The inhibitory effect of glucose cannot be due to a shift in membrane potential. Based on a V_max for glucose uptake of 2 nmol/(s·mg protein) (Malo and Berteloot 1991 ) and a stoichiometry of 2 Na⁺:1 glucose (Malo 1988 ), we estimate that glucose-coupled Na⁺ entry during the brief (30 s) ascorbate transport assay cannot increase internal Na⁺ concentration by >1 mmol/L, which is much smaller than the external Na⁺ concentration of 200 mmol/L. Thus, the transmembrane Na⁺ gradient is not affected significantly during the assays.

The possibility that ascorbate uptake proceeds through the Na⁺-glucose cotransporter, SGLT1, in intestinal and renal brush border membranes has been evoked (Goldenberg and Schweinzer 1994 ). Uptake of glucose and ascorbate share Na⁺ dependency, electrogenicity, and sensibility to PZ, SPZ and internal DIDS. However, the present data showing that glucose interferes with ascorbate uptake only from the internal side of the membrane and that SCN^- inhibits ascorbate uptake while stimulating glucose transport (Liedtke and Hopfer 1976 ) clearly rule out mediation of ascorbate transport by SGLT1. On the basis of this study, a model of vitamin C uptake in enterocytes can be proposed (Fig. 7 ). Ascorbate crosses the apical membrane with a minimum of 2 Na⁺ ions, whereas DHAA enters through a facilitated diffusion pathway. The electrochemical gradient for Na⁺ across the plasma membrane will allow Na⁺-coupled transport to concentrate ascorbate in mucosal cells, which explains why ascorbate concentrations are higher in biopsy specimens from duodenum compared with levels in plasma or gastric juice (Buffinton and Doe 1995 , Waring et al. 1996 ). Guinea pig jejunum mucosa contains 0.3 mg ascorbate/g wet weight, corresponding to an average intracellular concentration of ~2 mmol/L (Rose et al. 1988 ). Rat intestinal mucosa contains higher ascorbate concentration than does the underlying serosal tissue (Oelrichs and Kratzing 1980 ), and the vitamin may protect mucosa against dietary oxidants and ulcer-causing drugs such as aspirin (McAlindon et al. 1996 ).

View larger version (15K): [in this window] [in a new window]   Figure 7. A model of vitamin C transport in enterocytes. The apical membrane contains a Na+-ascorbate cotransporter involving a minimum of two 2 Na+ ions (square) and a facilitated diffusion pathway for L-dehydroascorbic acid (DHAA) (oval). The Na+-glucose transporter, SGLT1, allows rapid entrance of glucose, which is then released into blood by crossing the basolateral membrane via GLUT2. An increase of glucose concentration in the enterocyte inhibits the ascorbate carrier in the apical membrane. Inside the enterocyte, DHAA is reduced to ascorbate, which may leave the cell at the basal membrane by an unknown mechanism.

	ACKNOWLEDGMENTS

 
The authors thank Gregory Dubourg and Claudie Leroy for excellent assistance.

	FOOTNOTES

 
¹ Presented in part in abstract form at Experimental Biology 99, April 17–21, Washington, DC [Malo, C., Dubourg, G. & Wilson, J. X. (1999) Transport of ascorbic acid and dehydroascorbic acid in the human small intestine. FASEB J. 13: 349 (abs.)].

² Supported by the Natural Sciences and Engineering Research Council of Canada.

⁴ Abbreviations used: DHAA, dehydro-L-ascorbic acid; DIDS, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid; PZ, phlorizin; SPZ, sulfinpyrazone.

Manuscript received July 8, 1999. Initial review completed July 29, 1999. Revision accepted October 1, 1999.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Barnett G., Lockwood T., Arancibia A., Benet L. Z. Ascorbate stimulation of salicylate absorption in isolated rat intestine. J. Pharm. Sci. 1978;67:224-226[Medline]

2. Beesley A. H., Hardcastle J., Hardcastle P. T., Taylor C. J. Chloride conductance and sodium-dependent glucose transport in rat and human enterocytes. Gastroenterology 1997;112:1213-1220[Medline]

3. Bianchi J., Wilson F. A., Rose R. C. Dehydroascorbic acid and ascorbic acid transport systems in the guinea pig ileum. Am. J. Physiol. 1986;250:G461-G468[Medline]

4. Buffinton G. D., Doe W. F. Altered ascorbic acid status in the mucosa from inflammatory bowel disease patients. Free Radic. Res. 1995;22:131-143[Medline]

5. Cabantchik Z. I., Knauf P. A., Rothstein A. The anion transport system of the red blood cell. The role of membrane protein evaluated by the use of probes. Biochim. Biophys. Acta 1978;515:239-302

6. Goldenberg H., Schweinzer E. Transport of vitamin C in animal and human cells. J. Bioenerg. Biomembr. 1994;26:359-367[Medline]

7. Hornig D., Weber F., Wiss D. Site of intestinal absorption of ascorbic acid in guinea pigs and rats. Biochem. Biophys. Res. Commun. 1973;52:168-172[Medline]

8. Karasov W. H., Darken B. W., Bottum M. C. Dietary regulation of intestinal ascorbate uptake in guinea pigs. Am. J. Physiol. 1991;260:G108-G118[Abstract/Free Full Text]

9. Kyrtopoulos S. A., Pignatelli B., Karkanias G., Golematis B., Esteve J. Studies in gastric carcinogenesis V. The effects of ascorbic acid on N-nitroso compound formation in human gastric juice in vivo and in vitro. Carcinogenesis 1991;12:1371-1376[Abstract/Free Full Text]

10. Liedtke C. M., Hopfer U. Anion transport in brush border membranes isolated from rat small intestine. Biochem. Biophys. Res. Commun. 1976;76:579-585[Medline]

11. Maffia M., Ahearn G. A., Vilella S., Zonno V., Storelli C. Ascorbic acid transport by intestinal brush-border membrane vesicles of the teleost Anguilla anguilla. Am. J. Physiol. 1993;264:R1248-R1253[Abstract/Free Full Text]

12. Malo C. Kinetic evidence for heterogeneity in Na⁺-D-glucose cotransport systems in the normal fetal small intestine. Biochim. Biophys. Acta 1988;938:181-188[Medline]

13. Malo C., Berteloot A. Analysis of kinetic data in transport studies: new insights from kinetic studies of Na⁺-D-glucose cotransport in human intestinal brush-border membrane vesicles using a fast sampling, rapid filtration apparatus. J. Membr. Biol. 1991;122:127-141[Medline]

14. McAlindon M. E., Muller A. F., Filipowicz B., Hawkey C. J. Effect of allopurinol, sulphasalazine, and vitamin C on aspirin induced gastroduodenal injury in human volunteers. Gut 1996;38:518-524[Abstract/Free Full Text]

15. Mellors A. J., Nahrwold D. L., Rose R. C. Ascorbic acid flux across mucosal border of guinea pig and human ileum. Am. J. Physiol. 1977;233:E374-E379[Medline]

16. Oelrichs B. A., Kratzing C. C. Effect of barbiturate on the distribution of ascorbic acid in the gastrointestinal tract. Biochem. Pharmacol. 1980;29:1839-1841[Medline]

17. Rose R. C., Choi J. L., Koch M. J. Intestinal transport and metabolism of oxidized ascorbic acid (dehydroascorbic acid). Am. J. Physiol. 1988;254:G824-G828[Abstract/Free Full Text]

18. Rose R. C., Wilson J. X. Ascorbate membrane transport properties. Packer L. Fuchs J. eds. Vitamin C in Health and Disease 1997:143-162 Marcel Dekker New York, NY

19. Rumsey S. C., Levine M. Absorption, transport, and disposition of ascorbic acid in humans. Nutr. Biochem. 1998;9:116-130

20. Rumsey S. C., Kwon O., Xu G. W., Burant C. F., Simpson I., Levine M. Glucose transporter isoforms GLUT1 and GLUT3 transport dehydroascorbic acid. J. Biol. Chem. 1997;272:18982-18989[Abstract/Free Full Text]

21. Siliprandi L., Vanni P., Kessler M., Semenza G. Na⁺-dependent, electroneutral L-ascorbate transport across brush border membrane vesicles from guinea pig small intestine. Biochim. Biophys. Acta 1979;552:129-142[Medline]

22. Siushansian R., Tao L., Dixon S. J., Wilson J. X. Cerebral astrocytes transport ascorbic acid and dehydroascorbic acid through distinct mechanisms regulated by cyclic AMP. J. Neurochem. 1997;68:2378-2385[Medline]

23. Stevenson N. R., Brush M. K. Existence and characteristics of Na⁺-dependent active transport of ascorbic acid in guinea-pig. Am. J. Clin. Nutr. 1969;22:318-326[Abstract]

24. Stewart J. S., Booth C. C. Ascorbic acid absorption in malabsorption. Clin. Sci. (Lond.) 1964;27:15-22[Medline]

25. Suzuki E., Kurata T., Arakawa N. Comparison of absorption of erythorbic acid and ascorbic acid in guinea pig small intestine. J. Nutr. Sci. Vitaminol. 1991;37:453-459

26. Todhunter E. N., McMillan T., Ehmke D. A. Utilization of dehydroascorbic acid by human subjects. J. Nutr. 1950;42:297-308[Medline]

27. Toggenburger G., Haüsermann M., Mütsch B., Genoni G., Kessler M., Weber F., Hornig D., O’Neill B., Semenza G. Na⁺-dependent, potential-sensitive L-ascorbate transport across brush border membrane vesicles from kidney cortex. Biochim. Biophys. Acta 1981;646:433-443[Medline]

28. Toggenburger G., Landoldt M., Semenza G. Na⁺-dependent, electroneural L-ascorbate transport across brush border membrane vesicles from human small intestine: inhibition by D-erythorbate. FEBS Lett 1979;108:473-476[Medline]

29. Vega F. V., Cabero J. L., Mardh S. Inhibition of H,K-ATPase and Na,K-ATPase by DIDS, a disulphonic stilbene derivative. Acta Physiol. Scand. 1988;134:543-547[Medline]

30. Vera J. C., Rivas C. I., Fischbarg J., Golde D. W. Mammalian facilitative hexose transporters mediate the transport of dehydroascorbic acid. Nature (Lond.) 1993;364:79-82[Medline]

31. Waring A. J., Drake I. M., Schorah C. J., White K. L., Lynch D. A., Axon A. T., Dixon M. F. Ascorbic acid and total vitamin C concentrations in plasma, gastric juice, and gastrointestinal mucosa: effects of gastritis and oral supplementation. Gut 1996;38:171-176[Abstract/Free Full Text]

32. Welsh R. W., Wang Y., Crossman A., Jr, Park J. B., Kirk K. L., Levine M. Accumulation of vitamin C (ascorbate) and its oxidized metabolite dehydroascorbic acid occurs by separate mechanisms. J. Biol. Chem. 1995;270:12584-12592[Abstract/Free Full Text]

33. Wright E. M. The intestinal Na⁺/glucose cotransporter. Annu. Rev. Physiol. 1993;55:575-589[Medline]

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