Transport of thiamine in human intestine: mechanism and
regulation in intestinal epithelial cell model Caco-2
Hamid M.
Said,
Alvaro
Ortiz,
Chandira K.
Kumar,
Nabendu
Chatterjee,
Pradeep K.
Dudeja, and
Stanley
Rubin
Veterans Affairs Medical Center, Long Beach 90822; University of
California Irvine, Irvine 92717; Wadsworth Veterans Affairs Medical
Center and University of California Los Angeles, Los Angeles,
California 90073; and Westside Veterans Affairs Medical Center and
University of Illinois-Chicago, Chicago, Illinois 60612
 |
ABSTRACT |
The present study
examined the intestinal uptake of thiamine (vitamin
B1) using the human-derived
intestinal epithelial cells Caco-2 as an in vitro model system.
Thiamine uptake was found to be 1)
temperature and energy dependent and occurred with minimal metabolic
alteration; 2) pH sensitive;
3)
Na+ independent;
4) saturable as a function of
concentration with an apparent Michaelis-Menten constant of 3.18 ± 0.56 µM and maximal velocity of 13.37 ± 0.94 pmol · mg
protein
1 · 3 min
1;
5) inhibited by the thiamine
structural analogs amprolium and oxythiamine, but not by unrelated
organic cations tetraethylammonium, N-methylnicotinamide, and choline; and
6) inhibited in a competitive manner
by amiloride with an inhibition constant of 0.2 mM. The role of
specific protein kinase-mediated pathways in the regulation of thiamine
uptake by Caco-2 cells was also examined using specific modulators of
these pathways. The results showed possible involvement of a
Ca2+/calmodulin (CaM)-mediated
pathway in the regulation of thiamine uptake. No role for protein
kinase C- and protein tyrosine kinase-mediated pathways in the
regulation of thiamine uptake was evident. These results demonstrate
the involvement of a carrier-mediated system for thiamine uptake by
Caco-2 intestinal epithelial cells. This system is
Na+ independent and is different
from the transport systems of organic cations. Furthermore, a
CaM-mediated pathway appears to play a role in regulating thiamine
uptake in these cells.
thiamine transport; human intestinal cells; transport mechanism; transport regulation
 |
INTRODUCTION |
THIAMINE (vitamin
B1) plays a critical role in
normal carbohydrate metabolism and thus is essential for normal
cellular functions and growth. Thiamine deficiency in humans leads to a
variety of clinical abnormalities, including cardiovascular disorders
(e.g., peripheral vasodilatation, biventricular myocardial failure,
edema, and potentially acute fulminant cardiovascular collapse) and
neurological disorders (e.g., confusion, disordered ocular motility,
neuropathy, ataxia of gait) (2, 34, 38). Thiamine deficiency occurs in
a high percentage of alcoholics (8, 19, 21, 33, 36, 37), where
impairment in the intestinal absorption of the vitamin is believed to
be a contributing factor (37). Deficiency of thiamine also occurs in
patients with diabetes, coeliac disease (22), renal diseases (26), and
in patients fed intravenously for long periods (22), as well as in the
elderly (23). Furthermore, thiamine deficiency has also been reported
in thiamine-responsive megaloblastic anemia (1, 28), where impairment
in thiamine intestinal absorption and cellular uptake is believed to be
a contributing factor (28).
Humans and other mammals cannot synthesize thiamine but instead must
obtain the vitamin from exogenous sources via intestinal absorption.
Thus the intestine plays a central role in maintaining normal thiamine
body homeostasis. Dietary thiamine exists mainly in the phosphorylated
forms (predominantly as thiamine pyrophosphate) that are
hydrolyzed to free thiamine in the intestinal lumen before absorption
(26). The mechanism of intestinal thiamine transport has been studied
in a number of animal species (5, 9, 11, 13, 16), but less is known
about the mechanism of thiamine transport in the human intestine.
Furthermore, nothing is known about the cellular regulation of the
intestinal thiamine uptake process. This is despite the fact that
recent studies have shown that intestinal transport of a variety of
substrates (including that of the water-soluble vitamins folate,
biotin, and riboflavin) is regulated by specific intracellular
regulatory mechanisms (4, 6, 7, 17, 30). Our aim in this study was,
therefore, to study the mechanism and cellular regulation of thiamine
uptake by the human intestine using the human-derived cultured
intestinal epithelial cell line Caco-2 as a model. We chose these cells
because previous studies have shown that postconfluent and
differentiated Caco-2 cells possess many of the structural and
functional characteristics of the native enterocyte, including similar
transport mechanisms and regulatory pathways (20, 25). Our results
showed that thiamine uptake by these cells occurs via a specialized,
Na+-independent, carrier-mediated
process that appears to be under the regulation of a
Ca2+/calmodulin (CaM)-mediated
intracellular pathway.
 |
MATERIALS AND METHODS |
Custom-made
[3H]thiamine (sp act
555 GBq/mmol; radiochemical purity >98%) was purchased from American
Radiolabeled Chemicals, St Louis, MO. The radiochemical purity of
[3H]thiamine was
checked routinely before use by means of TLC with the use of
cellulose-precoated thin-layer plates and a solvent system of
isopropanol-acetate buffer (0.5 M, pH 4.5)-water (65:15:20, vol/vol/vol) (15). The same chromatography procedure was used to
determine the degree of thiamine metabolism during uptake by Caco-2
cells. All chemicals and reagents used in these studies were of
analytical grade and were obtained from commercial sources. Caco-2
cells were obtained from ATCC (Manassas, VA). DMEM, trypsin, fetal
bovine serum, and other cell culture reagents were obtained from Life
Technologies (Grand Island, NY).
Caco-2 cells were grown as we described previously (30). Uptake studies
were performed on confluent monolayers 3-5 days following
confluence. Uptake of thiamine was examined in cells incubated in
Krebs-Ringer buffer (in mM: 133 NaCl, 4.93 KCl, 1.23 MgSO4, 0.85 CaCl2, 5 glucose, 5 glutamine, 10 HEPES, and 10 MES, pH 7.4, unless otherwise specified) at 37°C.
Labeled and unlabeled thiamine were added to the incubation medium at
the onset of the uptake experiment. In certain experiments, cells were
pretreated with the compound under study for a specific period of time
before the addition of thiamine and the start of uptake experiments. Uptake was examined over a period of 3 min (unless otherwise
specified), and the reaction was terminated by the addition of 2 ml of
ice-cold buffer followed by immediate aspiration. Cells were then
rinsed twice with ice-cold buffer, digested with 1 ml of 1 N NaOH,
neutralized with HCl, and then counted for radioactivity. The protein
content of cell digests was measured in parallel wells using a Bio-Rad kit (Bio-Rad, Richmond, VA).
Data are means ± SE of multiple separate uptake determinations and
were expressed in term of picomoles or femtomoles per milligram protein
per unit time. Statistical differences were analyzed by ANOVA or
Student's t-test, with statistical
significance being set at 0.05 (P < 0.05). Quantitative variations in the absolute amounts of thiamine
uptake were observed in certain experiments, especially those in which
cells were preincubated for 1 h before initiation of thiamine uptake
measurement. For this reason, appropriate controls were simultaneously
performed with each set of experiments. Kinetic parameters of thiamine
uptake [i.e., the apparent Michaelis-Menten constant
(Km) and
maximal velocity
(Vmax)]
were calculated using a computerized model of the Michaelis-Menten
equation as described previously by Wilkinson (39).
 |
RESULTS |
Uptake of thiamine as a function of time, temperature, incubation
buffer pH, and
Na+.
Figure 1 depicts the uptake of thiamine as
a function of time at two concentrations: low (0.1 µM) and high (10 µM). Uptake was found to be linear with time for 10 min of incubation
at both concentrations, and rates were 0.19 and 3.28 pmol · mg
protein
1 · min
1,
respectively. A 3-min incubation time was used as the standard time in
all subsequent studies.

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Fig. 1.
Uptake of thiamine by confluent monolayers of Caco-2 cells as a
function of time. Cells were incubated (37°C) in Krebs-Ringer
buffer (pH 7.4) for different periods of time in the presence of 0.1 µM (A) and 10 µM
(B) thiamine. Results are means ± SE of 4-7 separate uptake determinations performed on 2 separate occasions. When not apparent, the error bars are smaller than
the symbol. A:
y = 190.7x + 187, r = 0.99. B: y = 3.3x + 12.7, r = 0.97.
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We also examined the metabolic form of the transported radioactivity
following 3- and 10-min incubations of cells with 0.04 µM
[3H]thiamine.
Cellulose-precoated TLC plates and a solvent system of
isopropanol-acetate buffer (0.5 M, pH 4.5)-water (65:15:20, vol/vol/vol) were used (see MATERIALS AND
METHODS). The results showed that the majority (96%
in both 3- and 10-min incubations) of the radioactivity taken up by the
cells was in the form of intact thiamine.
In a separate experiment, the effect of incubation temperature (37, 22, and 4°C) on thiamine (0.02 µM) uptake was examined. Uptake was
found to decrease progressively with decreasing incubation temperature
(223.7 ± 3.6, 134.82 ± 8.2, and 38.4 ± 4.3 fmol · mg
protein
1 · 3 min
1 at 37, 22, and
4°C, respectively).
The effect of varying the incubation buffer pH over the range of
5-8 on thiamine (0.02 µM) uptake was also tested. The results showed a progressive decrease in thiamine uptake as a function of
decreasing the incubation buffer pH from 8 to 5 (Fig.
2). We also investigated the role of
Na+ in the incubation medium on
thiamine uptake by Caco-2 cells. This was performed by examining the
effect of replacing Na+ in the
incubation medium with an equimolar concentration of
NH+4 or mannitol. The results showed a slight
but statistically insignificant decrease in thiamine uptake on
Na+ removal [198.6 ± 5.3, 175.1 ± 19.2, and 174.7 ± 12.4 fmol · mg protein
1 · 3 min
1 in the presence of
Na+ (control) as well as in the
absence of Na+ and the presence of
NH+4 and mannitol, respectively]. The
role of Na+ in thiamine uptake was
also investigated by examining the effect of pretreating the cells (for
1 h) with the
Na+-K+-ATPase
inhibitor ouabain (1 mM) on the uptake of the substrate. The result
showed no inhibition of thiamine (0.02 µM) uptake by this compound
(114.6 ± 3.2 and 112 ± 1.2 fmol · mg
protein
1 · 3 min
1 for control and
ouabain-pretreated cells, respectively).

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Fig. 2.
Effect of incubation buffer pH on thiamine uptake by confluent
monolayers of Caco-2 cells. Cells were incubated (37°C) for 3 min
in Krebs-Ringer buffer of varying pH.
[3H]thiamine (0.02 µM) was added at the onset of incubation. Results are means ± SE
of 6-7 separate uptake determinations performed on 2 separate
occasions. When not apparent, error bars are smaller than symbols.
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|
Uptake of thiamine as a function of
concentration. Figure 3
depicts the results on the initial rate of thiamine uptake as a
function of increasing the vitamin concentration in the incubation medium (0.02-10 µM). Uptake of thiamine was found to include a saturable component. Uptake by this component was calculated by subtracting uptake by simple diffusion from total uptake at each thiamine concentration. Uptake by diffusion was calculated from the
slope of the uptake line between uptake at high thiamine concentration (1 mM) and the point of origin. Apparent
Km and
Vmax of the
saturable component were then calculated as described in
materials and methods and found to be
3.18 ± 0.56 µM and 13.37 ± 0.94 pmol · mg
protein
1 · 3 min
1, respectively.

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Fig. 3.
Uptake of thiamine by confluent monolayers of Caco-2 cells as a
function of concentration. Cells were incubated at 37°C in
Krebs-Ringer buffer (pH 7.4) for 3 min (initial rate) in the presence
of different concentrations of thiamine. Results are means ± SE of
6-10 separate uptake determinations performed on 3 separate
occasions.
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Effect of thiamine structural analogs and unrelated organic cations
on the uptake of [3H]thiamine.
In these studies, we examined the effect of different concentrations of
the thiamine structural analogs amprolium and oxythiamine on the uptake
of [3H]thiamine. The
results showed that both compounds caused a concentration-dependent inhibition of
[3H]thiamine uptake;
the inhibition was found (by the Dixon method) to be competitive in
nature with calculated inhibition constants (Ki) of 7.8 and
28.7 µM for amprolium and oxythiamine, respectively (Fig.
4).

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Fig. 4.
Dixon plot of the effect of amprolium
(A) and oxythiamine
(B) on thiamine uptake by confluent
monolayers of Caco-2 cells. Cells were incubated at 37°C in
Krebs-Ringer buffer (pH 7.4) in the presence of 0.1 µM ( ) and 4.0 µM ( )
[3H]thiamine and
different concentrations of the structural analog under study. Thiamine
uptake was measured during initial rate of uptake (3 min), and results
were applied to Dixon plot as shown. Results are means ± SE of
3-7 separate uptake determinations performed on 2 separate
occasions.
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In other studies, we examined the effect of different concentrations of
the organic cations tetraethylammonium (TEA),
N-methylnicotinamide (NMN), and
choline on
[3H]thiamine (0.02 µM) uptake. The effect of unlabeled thiamine (25 µM) in these
studies served as a positive control. With the exception of unlabeled
thiamine, none of the other compounds examined significantly affected
[3H]thiamine uptake
(Table 1).
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Table 1.
Effect of unrelated organic cations and that of unlabeled thiamine
on the uptake of [3H]thiamine by confluent monolayers
of Caco-2 cells
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Effect of metabolic and membrane transport
inhibitors. The effect of pretreating the cells (for 30 min) with the metabolic inhibitors dinitrophenol (DNP; 10 mM),
p-chloromercuriphenylsulfonate (pCMPS;
1 mM), and sodium azide (10 mM) on the uptake of 0.02 µM thiamine was
examined. The results showed significant inhibition (P < 0.01) in thiamine uptake by
each inhibitor tested (114.6 ± 3.2, 52.7 ± 1.6, 66.6 ± 3.3, and 78.9 ± 2.2 fmol · mg
protein
1 · 3 min
1 for control and in
cells pretreated with DNP, pCMPS, and azide, respectively).
In another study, we examined the effect of various transport
inhibitors DIDS, probenecid, furosemide, and amiloride (all at 1 mM
concentration) on the uptake of 0.02 µM thiamine by Caco-2 cells. The
results showed that, although DIDS, probenecid, and furosemide do not
have an effect on thiamine uptake, amiloride was found to cause a
marked inhibition of the vitamin uptake [238 ± 6.3, 223 ± 12.5, 220.3 ± 6.7, and 72.4 ± 5.8 fmol · mg
protein
1 · 3 min
1
(P < 0.01), for control and in the
presence of DIDS, probenecid, furosemide, and amiloride,
respectively]. Amiloride inhibition was found (by the Dixon
method) to be competitive in nature with a
Ki value of 0.27 mM (Fig. 5).

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Fig. 5.
Dixon plot of the effect of amiloride on thiamine uptake by confluent
monolayers of Caco-2 cells; same as Fig. 4, except that different
concentrations of amiloride were used.
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Regulation of thiamine uptake: role of intracellular
protein kinase-mediated pathways. In these studies we
examined the possible regulation of the thiamine uptake process by
specific intracellular protein kinase-mediated pathways. Specifically,
we investigated the possible role of CaM-, protein kinase C (PKC)-, and
protein tyrosine kinase (PTK)-mediated pathways in the regulation of
thiamine uptake by examining the effect of pretreating Caco-2 cells
(for 1 h) with specific modulators of these pathways on the vitamin uptake process.
Pretreatment of cells with the inhibitors of the CaM-mediated pathway
trifluoroperazine (TFP), calmidazolium, and
1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine (KN-62) led to a significant inhibition of thiamine (0.02 µM) uptake
(Table 2). To further characterize the
effect of these inhibitors on thiamine uptake, we examined the effect
of a representative compound, TFP, on kinetic parameters of the
carrier-mediated thiamine uptake process. The results (Fig.
6) showed that TFP (75 µM) caused a
significant (P < 0.01) inhibition of
Vmax but not of
the apparent Km
of the thiamine uptake process
(Vmax = 12.4 ± 0.78 and 8.32 ± 0.35 pmol · mg
protein
1 · 3 min
1 and apparent
Km = 2.6 ± 0.44 and 3.15 ± 0.34 µM for control and TFP-pretreated cells,
respectively).

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Fig. 6.
Effect of pretreatment of confluent monolayers of Caco-2 cells with
trifluoroperazine (TFP) on thiamine uptake as a function of
concentration; same as Fig. 3, except that cells were pretreated (1 h)
with TFP (75 µM) at 37°C in Krebs-Ringer buffer, pH 7.4. ,
TFP; , control.
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|
The possible role of a PKC-mediated pathway in the regulation of
thiamine uptake by Caco-2 cells was investigated by examining the
effect of pretreatment of the cells with modulators of PKC activity
[phorbol 12-myristate 13-acetate (PMA), staurosporine, and
bisindolylmaleimide (Bis I)] on thiamine (0.02 µM)
uptake. The results showed that none of the PKC modulators had an
effect on thiamine uptake (119 ± 5.4, 114.9 ± 3.1, 129.9 ± 7.1, and 112.6 ± 5.2 fmol · mg
protein
1 · 3 min
1 for control and in
cells pretreated with 10 µM PMA, 10 µM staurosporine, and 1 µM
Bis I, respectively). We also examined the effect of pretreating Caco-2
cells with inhibitors of PTK activity on thiamine (0.02 µM) uptake.
The results showed that, although the PTK inhibitors genistein (100 µM) and tyrphostin A25 (10 µM) inhibit thiamine uptake, equimolar
concentrations of their negative controls genistin and tyrphostin A1,
respectively, also cause the same degree of inhibition (112 ± 6.6, 89.4 ± 6.1, 87.1 ± 2.9, 78.2 ± 13.7, and 74.3 ± 9.7 fmol · mg
protein
1 · 3 min
1 for control cells and
in cells pretreated with genistein, tyrphostin A25, genistin, and
tyrphostin A1, respectively). These findings indicate that the
inhibition caused by genistein and tyrphostin A25 is nonspecific in nature.
 |
DISCUSSION |
In this study, we examined the mechanism and cellular regulation of the
thiamine uptake process by the human intestine using the human-derived
intestinal epithelial cell line Caco-2 as an in vitro model system. The
results showed that thiamine uptake by Caco-2 cells is linear for up to
10 min incubation and occurred with minimal metabolic alteration in the
transported substrate. Uptake was also found to be temperature and
energy dependent, as indicated by the significant inhibition of
thiamine uptake caused by decreasing incubation temperature and by
metabolic inhibitors, respectively. Decreasing incubation buffer pH
from 8 to 5 was found to lead to a progressive inhibition of thiamine
uptake. Although the exact cause of this inhibition is not clear, it
cannot be attributed to changes in the ionic state of the thiamine
molecule, since thiamine exists as a monovalent cation over the pH
range examined (27). Further studies are required to clarify this issue. The uptake process of thiamine was found to be
Na+ independent in nature, as
indicated by the lack of significant inhibition of substrate uptake
when Na+ was removed from the
incubation medium, as well as by the lack of effect on thiamine uptake
when the cells were pretreated with the
Na+-K+-ATPase
inhibitor ouabain. This conclusion is in agreement with recent findings in our laboratory using purified human jejunal brush-border membrane vesicles (unpublished observations) and with the
findings of Laforenza et al. (18) using human intestinal biopsy
specimens. It is, however, in contrast with the findings of Hoyumpa et
al. (14) who reported that thiamine uptake by human intestinal biopsies
is Na+ dependent.
Uptake of thiamine by Caco-2 cells was found to involve a
carrier-mediated process. This is indicated both by the saturation of
thiamine uptake as a function of concentration and by the inhibition of
[3H]thiamine uptake
caused by the thiamine structural analogs amprolium and oxythiamine.
The apparent Km
of the carrier system of 3.18 ± 0.56 µM suggests that this system
is capable of efficiently absorbing dietary thiamine, which has been
estimated to exist in the lumen of the human intestine at a
concentration range of 0.1-2.0 µM (13). The observation that
oxythiamine is a weaker inhibitor of the thiamine uptake process
compared with amprolium suggests that the amino group at carbon-4 of
the pyrimidine moiety of the thiamine molecule is important for the
interaction of thiamine with its carrier protein, whereas the sulfur of
the thiazol moiety (the sulfur exists in the molecule of oxythiamine
but not in the molecule of amprolium) is not important. The identified
carrier system for transport of the cationic thiamine appears to be
different from the transport systems described for organic cations in
human renal and liver cells (10, 40). This conclusion is based on the
observation that high concentrations of the organic cations TEA, NMN,
and choline (substrates for the organic cation transport systems)
failed to inhibit the uptake of physiological concentrations of thiamine.
The uptake process of thiamine was found to be insensitive to the
effect of the membrane transport inhibitors DIDS, probenecid, and
furosemide. The finding of a lack of effect by furosemide on thiamine
uptake suggests that the observed thiamine deficiency and suboptimal
conditions observed in patients on long-term therapy with this diuretic
agent (32) are not due to inhibition of thiamine transport at the
intestinal level. Other mechanisms such as altered renal handling of
thiamine could be involved, as has been previously suggested (32). In
contrast to the lack of effect of these inhibitors on thiamine uptake,
the diuretic amiloride, a classical inhibitor of
Na+/H+
exchangers, was found to cause a concentration-dependent, competitive inhibition of thiamine uptake with a
Ki value of 0.27 mM. Such an interaction between thiamine and amiloride at the level of cell membrane transport has also been observed in neuroblastoma cells
(3). Together, these findings highlight the need for further studies to
assess the nutritional significance of such an interaction on the
normal thiamine body homeostasis, especially in patients treated
chronically with amiloride.
After the characterization of the mechanism of thiamine uptake by
Caco-2 cells, we used these cells to study possible regulation of the
vitamin uptake process by specific intracellular regulatory pathways.
We focused on the possible role of protein kinase-mediated pathways,
especially those that have been shown to play a significant regulatory
role in the regulation of transport of other substrates in intestinal
and other epithelia (4, 6, 7, 17, 30). The results showed that
inhibition of the CaM-mediated pathway resulted in a significant
inhibition of thiamine uptake by these cells. The effect of one such
inhibitor (TFP) was further characterized by testing its effect on the
kinetic parameters of the carrier-mediated thiamine uptake process. The
results showed that treatment with TFP decreases
Vmax but not the
apparent Km of
the thiamine uptake process. These findings suggest that the TFP effect
is mediated via inhibition of the activity (and/or number) of the
thiamine carriers at the luminal membrane, with no effect on carrier
affinity. In contrast to the possible role of the CaM-mediated pathway, no role for PKC- and PTK-mediated pathways in the regulation of thiamine uptake was observed. This conclusion was based on the observation that pretreatment of cells with specific modulators of
these pathways did not significantly affect thiamine uptake.
In summary, our findings demonstrate the involvement of a specialized,
Na+-independent, carrier-mediated
system for thiamine uptake by Caco-2 cells. Furthermore, this system
appears to be under the regulation of a CaM-mediated pathway.
 |
ACKNOWLEDGEMENTS |
This study was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grant DK-56061 and by a grant from the
Department of Veterans Affairs.
 |
FOOTNOTES |
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: H. M. Said,
Veterans Affairs Medical Center-151, Long Beach, CA 90822 (E-mail:
hmsaid{at}uci.edu).
Received 15 March 1999; accepted in final form 1 June 1999.
 |
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