Nephrology Section, Veterans Affairs Medical Center, New York University School of Medicine, New York, New York 10010
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ABSTRACT |
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Short-chain fatty acid (SCFA) transport across
the colon may occur by nonionic diffusion and/or via apical
membrane
SCFA/
exchange. To examine the relative importance of these processes,
stripped segments of rat (Ratus
ratus) proximal and distal colon were studied in
Ussing chambers, and the unidirectional fluxes of radiolabeled SCFA
butyrate, propionate, or weakly metabolized isobutyrate were measured.
In
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) or 1 or 5 mM
Ringer,
decreases in mucosal pH stimulated mucosal-to-serosal flux
(Jm
s)
of all SCFA, decreases in serosal pH stimulated serosal-to-mucosal flux
(Js
m),
and bilateral pH decreases stimulated both fluxes equally. These
effects were observed whether the SCFA was present on one or both sides
of the tissue, in both proximal and distal colon, in the absence of
luminal Na+, and in the presence
of either luminal or serosal ouabain. Changes in intracellular pH or
intracellular [
] did
not account for the effects of extracellular pH. Luminal
Cl
removal, to evaluate the
role of apical membrane
Cl
/SCFA
exchange, had no effect on
Jm
s but
decreased
Js
m 32%
at pH 6.5 and 22% at 7.2. Increasing SCFA concentration from 1 to 100 mM, at pH 6.4 or 7.4, caused a linear increase in
Jm
s. We
conclude that SCFA are mainly transported across the rat colon by
nonionic diffusion.
butyrate; propionate; in vitro; flux; pH; intracellular pH
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INTRODUCTION |
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SHORT-CHAIN FATTY ACIDS (SCFA) are produced by
bacterial metabolism of unabsorbed carbohydrates in the
mammalian colon. They provide the predominant anions in the colonic
lumen and include acetic (60-75%), propionic (15-25%), and
butyric acids (10-15%). Once absorbed, SCFA stimulate
Na+ and
Cl absorption, contribute
to the maintenance of cell pH and volume, and contribute potential base
to the systemic acid-base pool (5). The transport and metabolic
pathways by which each of these functions is achieved are well
described.
Until recently, the colonic absorption of SCFA was believed to occur
through nonionic diffusion. This mechanism of SCFA passage across the
luminal and serosal membranes is consistent with the functions noted
above and with partial recycling of SCFA across the luminal membrane
via
Cl/SCFA
exchange (4, 25). This passive transport process is stimulated by
decreases in bulk fluid or microclimate luminal pH consistent with the
fact that the acid dissociation constant
(pKa) of the most abundant SCFA in the
colonic lumen is approximately two pH units lower (6).
Recently, an
SCFA/
exchange process was identified in apical brush-border membrane
vesicles prepared from the rat colon (24). This process also is
stimulated by reductions in luminal pH and exhibits a Michaelis
constant (Km) for butyrate of 27 mM, near or below typical SCFA concentrations found
in the colonic lumen. It was suggested that at least in this segment of this species, the major mechanism by which SCFA are
absorbed is luminal membrane anion exchange (24). This mechanism of
SCFA absorption had been suggested for the human ileum (21), and in the
absence of an identified exchanger, for the rat jejunum (3) and rabbit
and guinea pig proximal colon (18, 30). To examine these possibilities
in the rat colon, we studied SCFA transport under in vitro conditions
designed to test the functional importance rather than the presence of
anion exchange. We systematically examined the effects of altering
extracellular and intracellular pH
(pHe and
pHi, respectively) and examined
the tenets of carrier-mediated transport. Our intent was to determine
to what extent passive movement of SCFA across the luminal and serosal
cell membranes could account for transepithelial transport in rat
colon.
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METHODS |
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Male Sprague-Dawley rats weighing 250-350 g were maintained on a standard diet with free access to water. Under pentobarbital sodium anesthesia (5 mg/100 g body wt), the proximal or distal 10 cm of colon were removed and rinsed with 0.9% saline. The serosa was stripped while the tissue was mounted on a glass rod.
Ion flux measurements. Details of the method were previously described (9, 10). Briefly, tissue pairs were mounted in modified Ussing half-chambers exposing 1.12 cm2 surface area. The transepithelial potential difference (PD) was referenced to the mucosal side. Tissues were studied under short-circuit conditions except for 1-s intervals every 100 s, during which bipolar pulses of 0.5 mV yielded electrical current values that were used to calculate tissue conductance (G). Tissues were paired for ion flux studies on the basis of differences in G no greater than 25%. The short-circuit current (Isc) divided by G yielded the active transport PD.
The fluxes of SCFA were measured by adding 1 µCi 14C-SCFA (10-20 mCi/mM specific activity; NEN, Boston, MA) to the mucosal side of one member of each tissue pair and to the serosal side of the other. Mucosal-to-serosal (JmSolutions and acid-base conditions.
The composition of the solutions is shown in Table
1. The
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid (HEPES)-Ringer solutions (A and
F) were gassed with 100%
O2, and pH was titrated using 2 M
H2SO4
or 1 M NaOH. The solutions (B-E)
were gassed with 1% CO2
(PCO2 = 7 mmHg), 3%
CO2 (PCO2 = 21 mmHg), 5%
CO2
(PCO2 = 35 mmHg), 11% CO2
(PCO2 = 75 mmHg), or 14%
CO2
(PCO2 = 95 mmHg) (balance
O2) to obtain various pH values.
All solutions were maintained at 37°C. The solutions were so
designed that after the addition of the salt of an SCFA or gluconic
acid, similar final osmolality and, where appropriate,
Na+ concentration (always <150
mM) were achieved.
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pHi measurements. The method is described in detail elsewhere (9-11, 13). Briefly, a segment of stripped distal colon (described previously) was mounted as a flat sheet over a 1-cm2 hollow ring assembly. It was first incubated in Ringer containing 2 mM DL-dithiothreitol (Sigma), a mucolytic agent, for 10 min. It was then bathed in Ringer containing 9.68 µM 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-acetoxymethyl ester (BCECF-AM; Molecular Probes, Eugene, OR) for 40 min. Intracellular cleavage of BCECF-AM by endogenous esterases produces the poorly permeable pH-sensitive dye BCECF. The mounted tissue was then washed three times in fresh Ringer solution (as designated by the experimental protocol) to remove extracellular dye. Fluorescence microscopy localized the dye primarily to surface epithelial cells (9).
A Perkin-Elmer LS-5B spectrofluorometer (South Plainfield, NJ) equipped with a thermoregulated cuvette holder was used. The mounted tissue was placed in a fixed position at the bottom of a 4-ml cuvette (Markson Science, Phoenix, AZ) with the mucosal surface facing the excitation beam at a 45° angle. In the experiments with a pH gradient a modified tissue holder was used (10). It consisted of a plastic divider that spanned two opposite inside corners of the cuvette. Stripped colonic tissue was placed over a plastic ring designed to snugly fit a 9-mm hole in the divider. The divider was snapped over the ring and tissue. Silicone grease was used to provide a water-tight seal between the divider and the cuvette. When acid-base conditions were altered, all readings were performed after pHi reached a plateau, but no less than 12 min after the acid-base condition was changed. During this time the solution in the cuvette was exchanged rapidly with fresh aliquots every 2 min. When ![]() |
RESULTS |
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Effect of pH on butyrate flux in distal colon.
Initially the effect of unilateral and bilateral changes in bathing
solution pH on butyrate flux in HEPES Ringer were examined. In these
experiments butyrate was present at 25 mM on both sides of the tissue.
As shown in Fig. 1, at pH 7.38 the net flux
of butyrate was 0.1 ± 0.3 µeq · cm
2 · h
1.
As mucosal solution pH was decreased in steps from 7.38 to 5.47, Jm
s
increased from 3.4 to 7.2 µeq · cm
2 · h
1
and net absorption was observed (3.5 ± 0.3 µeq · cm
2 · h
1).
As shown when the luminal pH was then increased to 7.38, the increase
in Jm
s
was completely reversible. Luminal pH changes had no effect on the
Js
m of
butyrate.
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Effect of pH on propionate flux in distal colon.
Similar effects of pH changes on propionate fluxes in HEPES Ringer were
observed. Propionate was present at 25 mM on both sides of the tissue.
Decreases in luminal pH in steps from 7.36 to 5.46 selectively
increased
Jms of
propionate from 2.7 ± 0.2 to 6.3 ± 0.4 µeq · cm
2 · h
1,
n = 4, P < 0.001. As serosal solution pH
was decreased in steps from 7.36 to 5.46, Js
m
selectively increased from 4.1 ± 0.2 to 5.4 ± 0.5 µeq · cm
2 · h
1,
n = 4, P < 0.05. Minimal net propionate
secretion was observed at bilateral pH 7.36 (
1.4 ± 0.3 µeq · cm
2 · h
1,
P < 0.02), and zero net transport
was found at pH 5.46 (0.9 ± 0.4 µeq · cm
2 · h
1,
P < 0.01 compared with flux at pH
7.36).
Effect of butyrate gradient on butyrate flux.
We then examined the effects of unilateral pH changes in HEPES Ringer
on butyrate fluxes in the presence of a 25 mM luminal to 0 mM serosal
butyrate concentration gradient. As mucosal solution pH was decreased
in steps from 7.39 to 5.58, Jms
increased from 1.0 ± 0.1 to 2.4 ± 0.2 µeq · cm
2 · h
1,
n = 4, P < 0.005. When pH was then
increased to 7.39, the increase in
Jm
s was
reversed to 1.1 ± 0.1 µeq · cm
2 · h
1,
P < 0.005. When serosal pH changes
were studied in the presence of a 25 mM serosal to 0 mM luminal
butyrate concentration gradient, similar results were obtained. As
serosal solution pH was decreased in steps from 7.39 to 5.56, Js
m of
butyrate increased from 2.0 ± 0.1 to 4.1 ± 0.1 µeq · cm
2 · h
1,
n = 4, P < 0.007. When pH was then
increased to 7.39, the increase in
Js
m was
reversed to 2.3 ± 0.1 µeq · cm
2 · h
1,
P < 0.007.
Effect of pH on butyrate flux in
Ringer.
The effect of bilateral changes in bathing solution pH on butyrate flux
across distal colon also was examined in
Ringer where pH changes were
induced by changing PCO2. In these
experiments butyrate was present at 25 mM on both sides of the tissue.
In 1 mM
Ringer at
PCO2 = 7 mmHg, pH 6.79, the net flux
of butyrate was 0.1 ± 0.2 µeq · cm
2 · h
1,
n = 6. As solution pH was decreased in
steps to 6.08 by increasing PCO2 to
95 mmHg, net flux was little changed:
0.5 ± 0.2 µeq · cm
2 · h
1.
Jm
s increased from 4.2 ± 0.2 to 4.8 ± 0.3 µeq · cm
2 · h
1
and Js
m
increased from 4.1 ± 0.3 to 5.3 ± 0.5 µeq · cm
2 · h
1,
n = 6, P < 0.05.
Effect of butyrate metabolism on butyrate flux.
We then examined whether the metabolism of SCFA influenced the pattern
of their transepithelial transport. We studied the effect of pH in
HEPES Ringer on the flux across distal colon of isobutyrate, a weakly
metabolized SCFA (22). Isobutyrate was present at 25 mM on both sides
of the tissue. Decreases in luminal pH in steps from 7.35 to 5.45 increased
Jms of
isobutyrate from 2.1 ± 0.1 to 4.9 ± 0.1 µeq · cm
2 · h
1,
n = 4, P < 0.0001. Js
m flux
increased slightly from 2.2 ± 0.1 to 2.8 ± 0.1 µeq · cm
2 · h
1,
n = 4, P < 0.04. In a separate experiment,
when pH was reduced in both bathing solutions in steps, net isobutyrate
flux remained unchanged:
1.0
µeq · cm
2 · h
1
at pH 7.34, 0.0 µeq · cm
2 · h
1
at pH 6.68,
0.8
µeq · cm
2 · h
1
at pH 6.04, and
0.5
µeq · cm
2 · h
1
at pH 5.55. These changes in unidirectional fluxes were completely reversible.
Effect of luminal
Na+ removal and
ouabain.
To determine whether apical
Na+/H+
exchange activity was necessary for SCFA transport, the exchanger was
inhibited by substituting choline for
Na+ in the mucosal bathing
solution. In HEPES Ringer, with butyrate present at 25 mM on both sides
of the tissue, bilateral reductions in pH in steps stimulated
Jms and
Js
m of
butyrate equivalently. At pH 7.41 and 5.68, net flux was unchanged and near zero, and
Jm
s was
1.3 ± 0.1 and 5.1 ± 0.1 µeq · cm
2 · h
1
and Js
m
was 2.1 ± 0.2 and 4.1 ± 0.1 µeq · cm
2 · h
1,
respectively, n = 2, P < 0.05. In 5 mM
Ringer, similar results were
obtained. At pH 7.32 and 6.51, net fluxes were unchanged and near zero,
and Jm
s
was 1.5 ± 0.1 and 2.1 ± 0.1 µeq · cm
2 · h
1
and Js
m
was 2.7 ± 0.1 and 3.4 ± 0.3 µeq · cm
2 · h
1,
respectively, n = 3, P < 0.05. The effects of pH in both
HEPES and
Ringer were completely
reversible.
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Effect of luminal Cl removal on
butyrate flux.
To evaluate the role of apical membrane
Cl
/SCFA
exchange, we examined the effect of substituting isethionate for
Cl
in the mucosal bathing
solution. In 5 mM
Ringer, the
absence of luminal Cl
at pH
7.21 did not significantly increase
Jm
s of
butyrate [2.6 ± 0.1 vs. 3.2 ± 0.3 µeq · cm
2 · h
1,
n = 5, not significant (NS)] but
did decrease
Js
m 22% (3.6 ± 0.2 vs. 2.4 ± 0.2 µeq · cm
2 · h
1,
n = 5, P < 0.05). At pH 6.50, the removal
of luminal Cl
did not
increase
Jm
s (4.0 ± 0.3 vs. 4.7 ± 0.5 µeq · cm
2 · h
1,
n = 5, NS) and again decreased
Js
m
~32% (4.4 ± 0.2 vs. 3.0 ± 0.3 µeq · cm
2 · h
1,
n = 5, P < 0.005). The absence of luminal
Cl
also altered the effect
of pH on butyrate flux. A reduction in pH from 7.21 to 6.50 stimulated
net flux from 0.8 ± 0.2 to 1.7 ± 0.4 µeq · cm
2 · h
1,
n = 5, P < 0.01, as a consequence of a
greater increase in
Jm
s (3.2 ± 0.3 vs. 4.7 ± 0.5 µeq · cm
2 · h
1,
P < 0.01) than in
Js
m (2.4 ± 0.2 vs. 3.0 ± 0.3 µeq · cm
2 · h
1,
P < 0.02).
Cl
removal had similar
effects on fluxes in HEPES Ringer, and the effects of pH in both
and HEPES Ringer were completely
reversible.
Effect of SCFA concentration.
Carrier-mediated transport processes, unlike nonionic diffusion,
exhibit saturation as evidenced by flattening of the curve describing
the relationship between substrate concentration and flux. We examined
for saturation by measuring
Jms of
butyrate in HEPES or 5 mM
Ringer
at pH 6.40 or 7.40 in the presence of a mucosal-to-serosal butyrate
gradient. As shown in Fig. 5, when the
mucosal butyrate concentration was progressively increased from 1 to
100 mM, regardless of the Ringer or pH,
Jm
s increased in a linear fashion. Furthermore, the weakly metabolized SCFA
isobutyrate exhibited similar transport behavior as its concentration was increased in
Ringer at pH
6.40.
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Effect of pHi and
[]i.
To determine whether the relation of SCFA flux to solution pH changes
could be accounted for by changes in intracellular acid-base conditions, we measured pHi and
calculated
[
]i in distal colon during the various experimental conditions. As shown in
Table 2, in HEPES Ringer containing 25 mM
butyrate and gassed with 100% O2,
CO2 tension and therefore
[
]i were zero. pHi mirrored
pHe whether the pH change was
unilateral or bilateral. However, when the
pHe decrease was unilateral, the decrease in pHi was less than when
the pHe change was bilateral. Furthermore, mucosal changes in
pHe seemed to have a somewhat greater effect on pHi than serosal
changes.
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Effect of pH on butyrate flux in proximal colon.
We then examined whether the pattern of pH effects on SCFA transport
was similar in the proximal colon. Butyrate flux was measured when
present at 25 mM on both sides of the tissue in HEPES Ringer. Bilateral
decreases in pH from 7.39 to 5.69 in steps increased
Jms from
2.9 ± 0.2 to 4.5 ± 0.3 µeq · cm
2 · h
1,
n = 4, P < 0.01, and
Js
m from
2.2 ± 0.4 to 3.3 ± 0.4 µeq · cm
2 · h
1,
n = 4, P < 0.002. Net butyrate fluxes were
minimal at bilateral pH 7.39 (0.8 ± 0.5 µeq · cm
2 · h
1)
and pH 5.69 (1.2 ± 0.5 µeq · cm
2 · h
1,
n = 4, NS). Reductions in pH
also had no effect on G but decreased Isc. All of these
changes were qualitatively and quantitatively similar to those observed
in distal colon.
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DISCUSSION |
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The importance of SCFA in colonic energy metabolism (5, 26), ion
transport (1, 2, 4, 12, 17, 26, 28, 32),
pHi regulation (7, 14, 15), and
systemic acid-base balance (5) has been recognized for some time. SCFA
absorption precedes and is required for these functions. For many years
the mechanism of SCFA absorption by the colon was believed to be by nonionic diffusion. The basis for this were the findings that chain
length, luminal pH, and the concentration gradient from lumen to blood
(or tissue or serosa) affected the absorption rate (5, 29, and see Ref.
30 for a review of these considerations). However, such characteristics
are compatible with SCFA absorption through apical and basolateral
membrane
SCFA/
exchange processes. Indeed, these process have recently been identified
in colonic membrane vesicles (20, 24, 27).
Our experiments were designed to determine the relative importance of
nonionic diffusion and anion exchange as mechanisms by which SCFA cross
the colonic mucosa. We considered the
Km of the apical
anion exchanger (27 mM for butyrate in rat colon) (24), the
pKa of the SCFA under consideration
(4.8-4.9), metabolism of SCFA by the colonic mucosa, the
cell-to-lumen flux of SCFA by apical
Cl/SCFA
exchange (25), and the possibility that various SCFA or segments of the
colon had different transport characteristics (30). Thus experiments
were performed at unilateral or bilateral butyrate concentrations of 25 mM; with butyrate, propionate, and weakly metabolized isobutyrate (22);
over a pH range of 7.4 to 5.3; and in the presence and absence of
luminal Na+ or
Cl
, or mucosal or serosal
ouabain. Our findings strongly suggest that a passive transport
pathway, presumably nonionic diffusion, accounts for SCFA transport
across rat colon.
The most compelling evidence for nonionic diffusion includes the
findings that unilateral reductions in
pHe had selective and
quantitatively equivalent effects on
Jms and
Js
m, that
the effects of pHe were similar in
HEPES and
Ringer, and that
transepithelial transport was unsaturable at SCFA concentrations up to
100 mM. None of these findings would be expected or accounted for by a
carrier-mediated epithelial transport process and by apical membrane
SCFA
/
exchange in particular. It is otherwise difficult to explain how SCFA
could move at equivalent rates in both directions across colonic tissue
if the transport process were not passive, how SCFA could traverse the
tissue via a
SCFA
/
exchange process in the apparent absence of intracellular bicarbonate
(in HEPES Ringer), and why transport saturation would not be observed
at substrate concentrations almost four times greater than the
Km (observed in
brush-border membrane vesicles).
The measurements of pHi also shed light on the mechanism of SCFA transport. In HEPES Ringer, the effects of pHe on unidirectional fluxes were equivalent whether the changes in pHe were unilateral or bilateral. Moreover, the absolute values for the unidirectional fluxes were equivalent at similar values for unilateral pHe and bilateral pHe. However, in HEPES Ringer (Table 2), the effects of unilateral and bilateral changes in pHe on pHi were not equivalent. This suggests that the effect of pHe on SCFA flux was primarily localized to the mucosal or serosal compartment in which it occurred rather than through the effects of pHe on pHi. As discussed previously, such an effect would be more compatible with the transport process of nonionic diffusion than anion exchange.
We also found that luminal removal of Na+ to inhibit apical Na+/H+ exchange did not alter SCFA transport in rat colon or the effects of pHe. Furthermore, neither luminal ouabain, which may inhibit colonic apical membrane H+-K+-ATPase (8, 16, 19, 23), nor serosal ouabain, which inhibits all active transport processes, affected SCFA transport or the effects of pHe. Epinephrine stimulation of apical Na+/H+ exchange has been shown to stimulate propionate absorption in rabbit proximal but not distal colon (29, 30). A pH gradient (presumably luminal microclimate pHe < pHi) was suggested as the mechanism of these effects (29). Because we could not confirm a role for apical Na+/H+ exchange in SCFA absorption, we believe that the reported requirement for this exchanger may be species specific. The presence of a microclimate pH, of course, is consistent with both SCFA absorption by nonionic diffusion and anion exchange.
Metabolism of SCFA by the colonic mucosa certainly occurs, and in preliminary experiments we found that ~7% of the butyrate that entered cells was metabolized to CO2. In the rabbit proximal colon in vitro, from 4 to 7% of absorbed propionate was metabolized to CO2 under similar experimental conditions (29). We do not believe that SCFA metabolism affected our flux measurements or their interpretation. First, in our studies the absolute fluxes and the effects of pH and substrate concentration were similar for butyrate, propionate, and weakly metabolized isobutyrate. Second, the presence of glucose in our studies reduced the fraction of colonic energy derived from SCFA (5). Third, our unidirectional flux calculations were based on the appearance of radiolabeled butyrate in the unlabeled "cold" bathing solution rather than on the disappearance of butyrate from the labeled "hot" side. Thus it is irrelevant to the calculation of unidirectional flux that ~7% more butyrate entered cells than exited into the opposite bathing solution.
In addition to transport across the cell, SCFA may be recycled across
the apical membrane. Recycling presumably occurs via the
Cl/SCFA
exchange process described by Rajendran and Binder (25). In our studies
of rat colon, the fraction recycled was estimated by comparing the
Jm
s of
butyrate in the presence and absence of luminal
Cl
. We found that
Jm
s was
not significantly affected by the absence of this anion.
Js
m,
however, was decreased 22-32%, depending on
pHe. Apparently, under the
experimental conditions described (5 mM
Ringer at
pHe between 6.50 and 7.21) apical
membrane
Cl
/SCFA
exchange has a greater role in the transcellular secretory flux of SCFA
than in apical membrane recycling of absorbed SCFA.
Our findings do not rule out a contribution of anion exchange at the
apical and basolateral membranes to SCFA absorption in situ. The
effects on SCFA transport of intact tissue layers, blood flow, cell
membrane potential, competing substrates, and varying energy stores and
demands are unknown. Moreover, the complexity of the in situ
environment suggests that the relative importance of active and passive
transport of SCFA may not be fixed. Nevertheless, the experimental
conditions examined here do mirror in situ conditions where the
[]i
is very low and a lumen-to-blood pH gradient and SCFA concentration
gradient exist. Indeed, such conditions favor net absorption of SCFA by
nonionic diffusion. Our studies suggest that at least in the rat colon
nonionic diffusion is the most important if not the only mechanism of
SCFA absorption.
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ACKNOWLEDGEMENTS |
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The authors appreciate the technical assistance of Matthew Jenkins.
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FOOTNOTES |
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This material is based on work supported by the Office of Research and Development, Medical Research Service, Department of Veterans Affairs.
Address for reprint requests: A. N. Charney, Nephrology Section, VA Medical Center, 423 East 23rd St., New York, NY 10010.
Received 19 August 1997; accepted in final form 3 December 1997.
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