Mechanism of thiamine uptake by human
jejunal brush-border membrane vesicles
Pradeep K.
Dudeja1,
Sangeeta
Tyagi1,
Reena J.
Kavilaveettil1,
Ravinder
Gill1, and
Hamid M.
Said2
1 Department of Medicine, West Side Veterans Affairs Medical
Center and University of Illinois at Chicago, Chicago, Illinois
60612; and 2 Departments of Medicine and Physiology/Biophysics,
University of California Irvine, and Veterans Affairs Medical
Center, Long Beach, California 90822
 |
ABSTRACT |
Thiamine, a water-soluble vitamin, is essential for
normal cellular functions, growth and development. Thiamine deficiency leads to significant clinical problems and occurs under a variety of
conditions. To date, however, little is known about the mechanism of
thiamine absorption in the native human small intestine. The objective
of this study was, therefore, to characterize the mechanism of thiamine
transport across the brush-border membrane (BBM) of human small
intestine. With the use of purified BBM vesicles (BBMV) isolated from
the jejunum of organ donors, thiamine uptake was found to be
1) independent of Na+ but markedly stimulated by
an outwardly directed H+ gradient (pH 5.5in/pH
7.5out); 2) competitively inhibited by the
cation transport inhibitor amiloride (inhibitor constant of 0.12 mM);
3) sensitive to temperature and osmolarity of the incubation medium; 4) significantly inhibited by thiamine structural
analogs (amprolium, oxythiamine, and pyrithiamine), but not by
unrelated organic cations (tetraethylammonium,
N-methylnicotinamide, or choline); 5) not
affected by the addition of ATP to the inside and outside of the BBMV;
6) potential insensitive; and 7) saturable as a
function of thiamine concentration with an apparent Michaelis-Menten constant of 0.61 ± 0.08 µM and a maximal velocity of 1.00 ± 0.47 pmol · mg protein
1 · 10 s
1. Carrier-mediated thiamine uptake was also found in
BBMV of human ileum. These data demonstrate the existence of a
Na+-independent, pH-dependent, amiloride-sensitive,
electroneutral carrier-mediated mechanism for thiamine absorption in
native human small intestinal BBMV.
thiamine transporter; human small intestine; brush-border membranes
 |
INTRODUCTION |
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 |
[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.
Uptake of [3H]thiamine into intestinal BBMV was measured
using a rapid filtration technique (8), as previously
described in our laboratory (3, 18). The BBMV (20 µl
containing 60-80 µg protein) were incubated in incubation media
(80 µl) with known buffer composition (described in detail in the
legends to Figs. 1-7) and containing 0.10 µM radioactive
[3H]thiamine (unless stated otherwise). The transport was
studied at 25°C in a water bath with uniform temperature. The uptake
was stopped at various time points using 5 ml of ice-cold stop solution containing 280 mM mannitol and 20 mM Tris-HEPES, pH 7.5. The diluted sample was rapidly filtered using a rapid filtration technique, employing 0.45-µm nitrocellulose filters. Filters were further washed
twice with 5 ml ice-cold stop solution. The filters were then dissolved
in Filtercount, and the radioactivity was measured in a Packard TR1600
liquid scintillation counter (Packard, Downers Grove, IL). All values
were corrected for nonspecific [3H]thiamine binding to
filters and/or vesicles by subtracting radioactivity present in
time 0 vesicle blank.

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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.
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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.
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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.
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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.
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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.
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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.
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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.
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Data presented in this study are means ± SE of 3-7
independent preparations. Kinetic parameters of the saturable component of thiamine uptake was determined by Lineweaver Burk plot using Graphpad Software (PRISM). Statistical comparisons between control and
experimental groups were performed using Student's t-test.
 |
RESULTS |
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).
Previous studies have suggested the possible involvement of a
H+ gradient-driven cation-exchange process for thiamine
uptake in the rat intestinal and renal BBMV (6, 13, 14).
To determine whether thiamine uptake by the human jejunal BBMV also
involved a H+ gradient-driven carrier-mediated mechanism,
the effect of an outwardly directed H+ gradient on
[3H]thiamine uptake was examined. As shown in
Fig. 1, imposition of an outwardly
directed H+ gradient (pH 5.5in/pH
7.5out) significantly stimulated the
[3H]thiamine uptake (at early linear time points, e.g.,
10 and 15 s) into the human jejunal BBMV, compared with uptake in
the absence of a pH gradient (pH 5.5in/pH
5.5out). Additionally, the [3H]thiamine
uptake into BBMV demonstrated a time-dependent increase and was linear
for up to 15 s of incubation time. [3H]thiamine
uptake exhibited an overshoot phenomenon, as the peak uptake at the
15-s time point was markedly higher compared with the 90-min
equilibrium uptake. The uptake of thiamine at conditions of pH
7.5in/pH 7.5out was not significantly different
from uptake at pH 5.5in/pH 5.5out but was lower
than uptake at pH 5.5in/pH 7.5out (data not
shown). These data, therefore, clearly indicate the existence of a pH
gradient-dependent uptake process for thiamine across the human jejunal BBM.
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
5.5in/pH 7.5out) 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
protein
1 · 10 s
1, n = 3, P < 0.001).
To differentiate the nonspecific binding of [3H]thiamine
to the vesicular membrane from its transport into the intravesicular space, the effect of varying medium osmolarities on
[3H]thiamine uptake was examined. The osmolarity of the
incubation medium was altered by increasing the concentration of the
mannitol. As shown in Fig. 2,
[3H]thiamine uptake into the vesicles at the equilibrium
time (90 min) was sequentially reduced parallel to increasing the
osmolarity of the incubation media. These data indicated that the
vesicles were intact and responsive to variations in medium osmolarity, and [3H]thiamine associated with the membrane vesicles
was predominantly due to its uptake into the closed intravesicular
space rather than due to nonspecific binding to the membrane surface. A
linear relationship between the uptake and the reciprocal of osmolarity was seen (Fig. 2). Extrapolation of the straight line to zero (infinite
osmolarity) indicated that the binding to the membrane surface of
vesicles was minimal (~10%).
In another preliminary study, we examined the metabolic form of the
radioactivity taken up by human jejunal BBMV after a 90-min incubation
with 0.42 µM [3H]thiamine using a thin-layer
chromatography procedure as described in MATERIALS AND
METHODS (11). The results showed 96% of the radioactivity taken up by the BBMV to be in the form of intact thiamine.
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.
Thiamine exists as a monovalent cation at the pH range of 5 to 7.4. Thus to further distinguish between the thiamine uptake system of
jejunal BBM and that of the previously characterized organic cations,
the effect of various organic cations, e.g., TEA, NMN, and choline, on
the uptake of [3H]thiamine was investigated. As shown in
Fig. 6, the presence of various organic
cations in the extravesicular medium (50 µM) failed to inhibit
thiamine uptake into the human jejunal BBMV.
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 (Km) of
0.61 ± 0.08 µM for thiamine and a maximal velocity
(Vmax) of 1.00 ± 0.47 pmol · mg
protein
1 · 10 s
1. 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 Km of 1.2 ± 0.5 µM and a
Vmax of 2.2 ± 1.2 pmol · mg
protein
1 · 10 s
1 (n = 7, separate membrane preparations).
 |
DISCUSSION |
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 5.5in/pH
7.5out), 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
Km of 0.61 ± 0.08 µM and a
Vmax of 1.00 ± 0.47 pmol · mg
protein
1 · 10 s
1. Similarly,
saturation in thiamine uptake as a function of concentration was also
observed in studies with human ileal BBMV with an apparent Km of 1.2 ± 0.5 µM and
Vmax of 2.2 ± 0.5 pmol · mg
protein
1 · 10 s
1. 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.
 |
ACKNOWLEDGEMENTS |
This study was supported by grants from the Department of Veterans
Affairs and by National Institute of Diabetes and Digestive and Kidney
Diseases Grants DK-33349, DK-54016, DK-56061, and DK-58057.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: P. K. Dudeja, Dept. of Medicine, Univ. of Illinois at Chicago, Medical Research Service (600/151), Veterans Affairs Medical Center, 820 South
Damen Ave., Chicago, IL 60612 (E-mail: pkdudeja{at}uic.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 30 October 2000; accepted in final form 18 April 2001.
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