INTRODUCTION

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THIAMINE (vitamin B₁), a water-soluble vitamin, plays a vital role in many metabolic reactions and is thus essential for normal cellular functions, growth, and development (21). Thiamine deficiency in humans occurs under different conditions (alcoholism, diabetes mellitus, celiac disease, aging) and leads to a variety of clinical abnormalities, including cardiovascular and neurological disorders (21). Additionally, in alcoholic and celiac disease patients, this deficiency has been suggested to be due to impairment of intestinal absorption of thiamine (21).

Humans and other mammals cannot synthesize thiamine and thus depend on the exogenous supply of the vitamin via intestinal absorption (21). Dietary thiamine exists mainly in the phosphorylated forms [predominantly as thiamine pyrophosphate (TPP)], which are hydrolyzed to free thiamine before absorption in the small intestine (12, 16, 20). The mechanism of absorption of dietary thiamine in the small intestine has been studied in animal models using a variety of intestinal preparations. The absorption was found to involve a specialized carrier-mediated system (5, 9). Furthermore, the thiamine transported across the intestinal epithelia has been shown to undergo some degree of phosphorylation inside the enterocytes (mainly to TPP) via the action of the cytoplasmic thiamine pyrophosphokinase (2, 12, 16). The thiamine that exits from the enterocyte was shown, however, to be in the form of free thiamine (4, 16).

In contrast to the available information regarding thiamine transport in the small intestine of animal models, little is known about the mechanism of thiamine transport in the human small intestine. In vivo studies in healthy humans performed by analyzing serum and urinary radioactivity levels after oral administration of [³H]thiamine (22) have suggested the involvement of a carrier-mediated system for the intestinal absorption process. This suggestion was confirmed by subsequent studies using human intestinal surgical and biopsy specimens (10, 15, 17) and more recently in studies using the human-derived cultured intestinal epithelial cells Caco-2 (19). Although these studies have provided important information regarding the human intestinal thiamine uptake process at the tissue/cellular level and its regulation, no study is available describing the mechanism of thiamine transport across the individual membrane domains of the polarized human intestinal absorptive cells, i.e., the brush-border membrane (BBM) and the basolateral membrane (BLM) domains. For a substrate that undergoes some degree of intracellular metabolism, as is the case with thiamine (2, 12, 16), such studies are best performed using purified membrane vesicle preparations that are devoid of intracellular components. Using purified BBM vesicle (BBMV) preparations isolated from the small intestine of organ donors, the current study was, therefore, undertaken to elucidate the mechanism of thiamine transport across the luminal BBM. The results demonstrated the existence of a Na⁺-independent, pH-dependent, amiloride-sensitive, carrier-mediated exchange process for thiamine uptake across the human intestinal BBM.

	MATERIALS AND METHODS

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[³H]thiamine (sp act 10 Ci/mmol; radiochemical purity > 97%) was obtained from American Radiolabeled Chemicals (St. Louis, MO). Unlabeled thiamine, amprolium, oxythiamine, pyrithiamine, tetraethylammonium (TEA), N-methylnicotinamide (NMN), valinomycin, and amiloride were obtained from Sigma Chemical (St. Louis, MO). All other chemicals and reagents were obtained from either Fisher Scientific (Fairlawn, NJ) or Sigma Chemical (unless otherwise stated) and were of the highest purity available. To determine the degree of thiamine metabolism after uptake by intestinal BBMV, a thin-layer chromatography procedure employing cellulose gel-precoated plates and a solvent system of isopropanol/0.5 M acetate buffer (pH 4.5)/water (65/15/20, vol/vol/vol) was used (11).

Isolation of human small intestinal BBMV and [³H]thiamine uptake studies. These investigations were approved by the Institutional Review Board of the University of Illinois at Chicago. Small intestines from 10-12 healthy adult organ donors were obtained after the harvest of transplantable organs. The upper one-third jejunum and lower one-third ileum sections of the small bowel were cleaned with an ice-cold 0.9% NaCl solution, and scraped mucosa was frozen away at 80°C and utilized for membrane preparations. Jejunal and ileal BBM were purified from the frozen mucosal scrapings utilizing divalent cation (Mg²⁺) chelation and differential centrifugation technique (18). The purity of intestinal BBMV preparations was assessed by measuring the activities of the appropriate marker enzymes. The membrane vesicles demonstrated approximately 15- to 20-fold enrichment in alkaline phosphatase and sucrase activities (BBM markers) compared with crude homogenate. The degree of contamination by other cell organelles was minimal as estimated by the activity of their appropriate marker enzymes (18). These vesicles are predominantly right side out (~80%) as assessed by the activity of the marker enzyme alkaline phosphatase in the presence and absence of 0.05% Triton X-100 to open up the vesicles. For studies where Mg²⁺-ATP was loaded into the purified membrane vesicles, the Mg²⁺-ATP was included in the buffer of final spin as well as in the resuspension/vesiculation buffer.

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Effect of pH gradient on time course of [3H]thiamine uptake. Jejunal brush-border membrane vesicles (BBMV) preloaded with 280 mM mannitol and 20 mM Tris/2-(N-morpholino)ethanesulfonic acid (MES) (pH 5.5) were incubated in a reaction medium containing 280 mM mannitol, and either 20 mM Tris/MES (pH 5.5) or 20 mM Tris/HEPES (pH 7.5) and 0.1 µM [3H]thiamine (final concn). Uptake was determined at 25°C for the indicated time periods. Values are means ± SE for 3 separate membrane preparations.

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Effect of medium osmolarity on [3H]thiamine uptake. Jejunal BBMV preloaded with 280 mM mannitol and 20 mM Tris/MES (pH 5.5) were incubated with buffer containing 20 mM Tris/HEPES (pH 7.5), 0.1 µM [3H]thiamine, and increasing concentrations of mannitol to give the indicated osmolarity. The uptake was determined at 25°C for a 90-min time period. Results are presented as means ± SE for 3 separate experiments.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Effect of Mg2+-ATP on [3H]thiamine uptake. Jejunal BBMV were preloaded with 100 mM K-gluconate, 20 mM Na-gluconate, 40 mM mannitol, and 20 mM Tris/HEPES (pH 7.5) ± 1 mM Mg2+-ATP. The uptake was measured at the indicated time points at 25°C by incubating the vesicles in reaction medium containing 100 mM Na-gluconate, 20 mM K-gluconate, 40 mM mannitol, 20 mM Tris/HEPES (pH 7.5), and 0.1 µM [3H]thiamine, ±1 mM Mg2+-ATP. Values are expressed as pmol/mg protein and represent means ± SE of 3 separate membrane preparations.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Effect of amiloride on H+ gradient-stimulated [3H]thiamine uptake. Jejunal BBMV were preloaded with 280 mM mannitol and 20 mM Tris/MES (pH 5.5). The uptake was determined for 10 s at 25°C by diluting the vesicles in medium containing 280 mM mannitol, 20 mM Tris/HEPES (pH 7.5), 0.1 µM [3H]thiamine, and increasing concentrations of the cation transport inhibitor amiloride (0.05-1.0 mM). Values are means ± SE for 3 separate experiments.

View larger version (13K): [in this window] [in a new window]   Fig. 5.   Effect of structural analogs on H+ gradient-stimulated [3H]thiamine uptake. Jejunal BBMV were preloaded with 280 mM mannitol and 20 mM Tris/MES (pH 5.5). The uptake was determined at 25°C for 10 s by incubating the vesicles in medium containing 280 mM mannitol, 20 mM Tris/HEPES (pH 7.5), 0.1 µM [3H]thiamine, and 50 µM of the various structural analogs, amprolium, cold-thiamine, and pyrithiamine. Values are expressed as percentage of control and represent means ± SE of 3 separate membrane preparations. * P  0.05 compared with control.

View larger version (12K): [in this window] [in a new window]   Fig. 6.   Effect of organic cations on H+ gradient-stimulated [3H]thiamine uptake. Jejunal BBMV were preloaded with 280 mM mannitol and 20 mM Tris/MES (pH 5.5). The uptake was determined at 25°C for 10 s by incubating the vesicles in medium containing 280 mM mannitol, 20 mM Tris/HEPES (pH 7.5), 0.1 µM [3H]thiamine, and 50 µM of the specific organic cations, tetraethylammonium (TEA), N-methylnicotinamide (NMN), and choline. Values are presented as means ± SE for 3 separate experiments.

View larger version (13K): [in this window] [in a new window]   Fig. 7.   Kinetic analysis of [3H]thiamine uptake. The initial rates of thiamine uptake in the presence of increasing extravesicular concentrations of total thiamine over the range of 0.1-2.0 µM were determined. BBMV were preloaded with 280 mM mannitol and 20 mM Tris/MES (pH 5.5). Uptake was measured at 10 s by diluting the vesicles in medium containing 280 mM mannitol, 20 mM Tris/HEPES (pH 7.5), [3H]thiamine, and varying concentrations of unlabeled thiamine. Results shown are representative of 5 separate membrane preparations.

	RESULTS

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Effect of Na+ and the presence and absence of a pH gradient on thiamine uptake as a function of time. To examine if the thiamine uptake in the human jejunal BBMV involved a Na⁺-dependent process similar to a number of other nutrient transporters, our initial studies were aimed at elucidating the effect of an inwardly directed Na⁺ gradient (100 mM Na/0 mM Na) on [³H]thiamine uptake compared with an inwardly directed K⁺ gradient (100 mM K/0 mM K). As shown in Table 1, there was no significant effect of an inwardly directed Na⁺ gradient compared with a K⁺ gradient on early linear time point (10 s) or equilibrium (90 min) uptake into these vesicles (P > 0.05).

Effect of temperature and incubation medium osmolarity on [³H]thiamine uptake. In this study, we examined the effect of temperature on the H⁺ gradient-driven uptake of thiamine (pH 5.5_in/pH 7.5_out) by jejunal BBMV. The results showed that thiamine uptake was markedly higher at 37°C compared with uptake at 0°C (0.12 ± 0.08 at 0°C and 1.11 ± 0.04 at 37°C, expressed as pmol · mg protein¹ · 10 s¹, n = 3, P < 0.001).

Effect of ATP on [³H]thiamine uptake. Previous studies by Laforenza et al. (13) have shown the thiamine transport across rat small intestinal basolateral membranes to be ATP dependent. To examine whether thiamine uptake in our human jejunal BBM preparations depends on ATP, the effect of 1 mM Mg²⁺-ATP was examined in this study. As shown in Fig. 3, ATP in the extravesicular medium and intravesicular medium failed to influence thiamine uptake at different time intervals, indicating that thiamine uptake across the human jejunal BBMV does not utilize ATP and thus does not display primary active transport.

Effect of transmembrane potential on [³H]thiamine uptake. To determine whether a H⁺ gradient-stimulated transport is an electroneutral process rather than a membrane potential-dependent mechanism, the effect of K⁺/valinomycin-induced membrane potential on thiamine uptake was determined. The [³H]thiamine uptake was measured after imposition of an intravesicular negative or positive membrane potential and compared with uptake under voltage-clamped conditions. As shown in Table 2, the changes in transmembrane potential did not affect thiamine uptake in these membranes.

Effect of amiloride on H⁺ gradient-stimulated [³H]thiamine uptake. Previous studies have shown that amiloride inhibits thiamine uptake by the cultured intestinal epithelial cell line Caco-2 (19), as well as in neuroblastoma cells (1). Therefore, to further characterize the mechanism of inhibition of amiloride on the uptake of the monovalent cation thiamine, the effect of increasing concentrations of amiloride (0.05-1.0 mM) on H⁺ gradient-driven [³H]thiamine uptake by human jejunum BBMV was determined. The results, presented in Fig. 4, demonstrated a dose-dependent inhibition of [³H]thiamine uptake into these vesicles by amiloride. An inhibitor constant of ~0.12 mM for amiloride was calculated.

Effect of thiamine structural analogs and unrelated organic cations on H+ gradient-stimulated [³H]thiamine uptake. To assess the specificity of the H⁺ gradient-dependent thiamine uptake process, the effects of the various structural analogs, i.e., amprolium, cold thiamine, and pyrithiamine, on [³H]thiamine uptake were examined. As shown in Fig. 5, there was a significant inhibition (70-75%) of the H⁺ gradient-stimulated thiamine uptake into the vesicles in the presence of the various structural analogs (50 µM) in the incubation medium.

Kinetics of [³H]thiamine uptake. In this study, we examined the uptake of thiamine by jejunal BBMV as a function of increasing the thiamine concentration in the incubation medium. As shown in Fig. 7, the H⁺ gradient-dependent thiamine uptake into the vesicles demonstrated saturation in the presence of increasing concentrations of unlabeled thiamine (0.1-2.0 µM) in the incubation media. Kinetic analysis of the thiamine uptake data (after correcting for the diffusion/binding component) using Lineweaver-Burk plots (n = 6 independent membrane preparations) yielded an apparent Michaelis-Menten constant (K_m) of 0.61 ± 0.08 µM for thiamine and a maximal velocity (V_max) of 1.00 ± 0.47 pmol · mg protein¹ · 10 s¹. We also examined thiamine uptake by BBMV isolated from human ileum as a function of thiamine concentration. Similar to the findings with jejunal BBMV, saturation was also observed in thiamine uptake with ileal BBMV, with an apparent K_m of 1.2 ± 0.5 µM and a V_max of 2.2 ± 1.2 pmol · mg protein¹ · 10 s¹ (n = 7, separate membrane preparations).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Previous studies have characterized certain aspects of the mechanism of thiamine uptake by human small intestine using surgical and biopsy specimens and cultured intestinal epithelial Caco-2 cells (10, 15, 17, 19). Very little, however, is known about the mechanism of thiamine transport across the individual membrane domains of the functionally polarized human intestinal epithelial cells. In the present study, we examined the mechanism of thiamine transport across the apical BBM of native human enterocytes using purified BBMV isolated by a well-validated technique from organ donor jejunal mucosa. This membrane preparation was utilized to avoid possible changes in the metabolic form of the transported thiamine into the intestinal BBMV. Our data provided evidence for the existence of a distinct pH-dependent, amiloride-sensitive, carrier-mediated system for an electroneutral thiamine transport process across the human intestinal BBM.

The uptake of thiamine by human jejunal BBMV was found to be similar in the presence and absence of a Na⁺ gradient, indicating that the process is Na⁺ independent in nature. An outwardly directed H⁺ gradient (pH 5.5_in/pH 7.5_out), however, was found to result in significant stimulation of the thiamine uptake with a distinct overshoot phenomena being observed during the initial phase of uptake. The overshoot phenomena in thiamine uptake indicated movement of the vitamin against a concentration gradient in the intravesicular space. These data could also be explained by a possible involvement of a thiamine/H⁺ exchange mechanism.

Uptake of thiamine by the human jejunal BBMV was found to involve a carrier-mediated system. This conclusion is based on a number of observations, including temperature dependence of the uptake process, inhibition by unlabeled thiamine and related compounds, and saturation of the uptake process as a function of increasing the substrate concentration in the incubation medium. Kinetic parameters of the thiamine uptake system in jejunal BBMV were an apparent K_m of 0.61 ± 0.08 µM and a V_max of 1.00 ± 0.47 pmol · mg protein¹ · 10 s¹. Similarly, saturation in thiamine uptake as a function of concentration was also observed in studies with human ileal BBMV with an apparent K_m of 1.2 ± 0.5 µM and V_max of 2.2 ± 0.5 pmol · mg protein¹ · 10 s¹. These findings suggest that both the proximal and the distal areas of the human small intestine are capable of transporting thiamine.

The identified pH-dependent uptake system for the monovalent cation thiamine across the human intestinal BBM was found to be potential insensitive in nature. This finding suggested that the process of thiamine uptake by the human intestinal BBMV was electroneutral in nature and further supported the earlier stated suggestion that a thiamine/H⁺ exchange mechanism may be involved in the uptake process. The carrier-mediated system for thiamine uptake was found to be specific for the vitamin and is different from the transport systems described for organic cations in human renal BBM and BLM and in the human liver BLM (7, 23). The latter conclusion is based on the finding that the presence of high concentration of the organic cations, e.g., NMN, TEA, and choline, in the incubation media failed to influence the uptake of a physiological concentration of [³H]thiamine. In addition, uptake of thiamine by the human intestinal BBMV was found to be ATP independent. This finding suggests that thiamine transport across this membrane domain of the polarized enterocyte displays secondary active transport. This is in contrast to the previous finding of Laforenza et al. (13) with rat small intestinal BLM vesicles, in which thiamine uptake was reported to be an ATP-dependent process.

An interesting observation was the ability of the diuretic amiloride to cause a significant and dose-dependent inhibition in thiamine uptake by the native human intestinal BBMV. This finding confirms our recent observation with cultured intestinal epithelial Caco-2 cells, where amiloride was found to cause a competitive inhibition in thiamine uptake by these cells (19). Similar interaction between thiamine uptake and amiloride at the level of membrane transport has also been observed in another cellular system, namely, neuroblastoma cells (1). Together, these findings highlight the need for studies to evaluate the nutritional implication of such vitamin-drug interactions on thiamine body homeostasis, especially in patients on long-term therapy with amiloride.

The above-described findings on Na⁺ independence of thiamine uptake are similar to those reported recently with human cultured intestinal epithelial Caco-2 cells (19) and with human biopsy specimens (15). These findings are, however, different from those reported earlier by Hoyumpa et al. (10) with human intestinal biopsies, in which uptake was reported to be Na⁺ dependent in nature (10). Our results with human intestinal BBMV are also similar to the recently reported findings of Laforenza et al. (14) with rat intestinal BBMV.

In summary, our current results provide strong evidence for the existence of a specific, Na⁺-independent, pH-dependent, electroneutral, carrier-mediated system for thiamine transport across the human intestinal BBM. Additionally, this system appears to be inhibited by the diuretic amiloride in a concentration-dependent manner. Further studies to characterize the molecular identity of this transporter and its molecular regulation will be of importance.