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Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322
3To whom correspondence should be addressed at Rollins Research Center, Room 4131, 1510 Clifton Road NE.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-glutamyltransferase. GSSG (250 µmol/L) was
reduced to GSH, with the luminal redox potential (Eh) for
GSSG/2GSH changing from >0 to -111, -132 and -143 mV at 10, 20 and
30 min, respectively. The Eh for luminal cystine/2cysteine
was 
20 mV more reducing than that for GSSG/2GSH at each time point,
suggesting that cysteine could function in the reduction of GSSG in the
lumen. Measurements in specific regions showed that GSSG reduction was
more rapid in the duodenum and proximal jejunum than in the distal
jejunum. Preparations without acivicin treatment showed that
Eh values were unaffected by inhibition of
-glutamyltransferase despite differences in GSH and cysteine pool
sizes. Rat intestine has a mechanism to adjust the luminal
thiol-disulfide redox. In principle, dysfunction of this mechanism
could contribute to malabsorption or other nutritional disorders.
KEY WORDS: • glutathione • cysteine • redox • small intestine • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Aw and Williams 1992
, Kowalski et al. 1990
) and to detoxify reactive electrophils in mucus and
mucosal epithelium (Samiec et al. 2000
). Cysteine (Cys)
functions as a reductant in the absorption of iron (Van Campen 1973
) and selenium (Scharrer et al. 1992
), and
both GSH and Cys can, in principle, function as reductants in the
maintenance of mucus fluidity (Snary et al. 1970
) and
the protection of enzymes and transport systems that contain critical
thiol groups (Gilbert 1990
). Although some of these
functions (e.g., GSH peroxidase and GSH S-transferase
reactions) are likely to depend on the thiol concentrations per se,
others (e.g., reduction in ferric iron or selenite, maintenance of
mucus fluidity, maintenance of thiols in proteins) are likely to depend
on the redox potential (Eh) of the respective
thiol-disulfide couple, i.e., glutathione disulfide (GSSG)/2GSH or
cystine (CySS)/2Cys.
Eh, which is also termed "electromotive
force," is a quantitative expression of the tendency of a
redox-active couple to donate or accept electrons relative to a
standard hydrogen electrode (Clark 1960
). Consumption of
foods with divergent GSH/GSSG ratios (Jones et al. 1992
,
Wierzbicka et al. 1989
) is associated with a relatively
large variation in Eh, and therefore mechanisms
are needed to adjust the luminal thiol-disulfide redox state to an
appropriate range for intestinal functions. Two mechanisms that supply
thiols to the intestinal lumen could function in the control of luminal
Eh: the supply of GSH in bile (Ballatori and Rebbeor 1998
, Kaplowitz et al. 1996
) and the
release of thiols from the intestinal epithelium (Wien and Van Campen 1991a
and 1991b
). GSH is present at millimolar
concentrations in bile, and calculations of Eh of
the GSSG/2GSH pool from reported concentrations of GSH and GSSG in
human bile (Eberle et al. 1981
), assuming a 10-fold
dilution of bile into the lumen, indicate that Eh
would be -150 mV (using an Eo value of -240
mV for pH 7.0; Rost and Rapoport 1964
). This value is
very similar to the estimated Eh in the lumen of
rat small intestine (-137 to -164 mV, calculated from data of
Hagen et al. 1990
). The release of thiols from the
epithelium could also function to control luminal redox because the
Eh of the GSSG/2GSH pool in the jejunum is
-203 mV (Jonas et al. 1999
), and an analysis of
thiols with HPLC showed that both GSH and Cys are released into the
lumen of isolated, perfused rat small intestine (Dahm and Jones 1994
).
The purposes of the current study were to determine whether the small intestinal epithelium can reduce a disulfide in the lumen and to determine the Eh value that is established for the resulting thiol-disulfide pool. An isolated, vascularly perfused rat small intestinal preparation was used with the bile duct ligated to minimize the contribution from biliary thiols. Added GSSG was reduced to GSH to bring the luminal GSSG/2GSH pool from >0 to -132 mV within 20 min. Thus, the rat intestinal epithelium contains a mechanism to reduce a disulfide to maintain luminal thiol-disulfide redox.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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GSH, GSSG, CySS, glutamate (sodium salt), aspartate (sodium salt), glutamine, glucose, acivicin, HEPES, dithiothreitol, iodoacetic acid, 1-fluoro-2,4-dinitrobenzene (Sanger’s reagent) and heparin (sodium salt) were obtained from Sigma Chemical Co. (St. Louis, MO). Cys-GSH disulfide (CySSG) was from Toronto Research Chemicals (North York, Ontario, Canada). Cys hydrochloride and perchloric acid were purchased from Kodak (Rochester, NY) and Mallinckrodt (Paris, KY), respectively. SOLVABLE tissue solubilizer was obtained from New England Nuclear Research Products (Boston, MA). Sodium pentobarbital (Wyeth-Ayerst, Princeton, NJ) was obtained from the Emory University Hospital. All other reagents were reagent grade or better.
Intestinal preparation.
Animal use was reviewed and approved by the Emory University Institutional Animal Care and Use Committee. Male Sprague-Dawley rats (VAF/1; Sasco, Omaha, NE) weighing 200–320 g were housed in the animal care facility at Emory University under conditions of controlled temperature and humidity. A 12-h light/dark cycle was maintained. Rats consumed tap water and rat food (Purina 5001; Purina, St. Louis, MO) ad libitum.
Isolated, vascularly perfused rat jejunal preparations were prepared as
previously used for studies of the transepithelial transport of GSH and
GSSG (Hagen and Jones 1987
). This preparation was
developed with a Krebs-Henseleit balanced salt solution for
vascular perfusion to simplify the analytic measurement of transported
species. Integrity was shown by the lack of release of lactic
dehydrogenase, exclusion of luminal inulin and polyethylene glycol from
the perfusate, maintenance of sodium-dependent transport and
ability of transport inhibitors to block transepithelial transport
(Hagen and Jones 1987
). This preparation was also found
to retain responsiveness to hormonal stimulation of transport
(Hagen et al. 1991
). Thus, the preparation provides an
intact system for evaluation of the ability of the epithelium, without
contribution of bile or blood components, to reduce luminal GSSG.
However, because this preparation is nonphysiological and no systematic
evaluation of components (e.g., amino acids) normally supplied by bile
or blood was made, the rates and regulation of GSSG reduction by the
small intestinal epithelium in vivo may differ from the values obtained
in this isolated system.
Briefly, rats were anesthetized with sodium pentobarbital (50 mg
IP/kg), and heparin (70 U) was injected into the left
femoral vein. The proximal 40 cm of jejunum was isolated and cleared of
its contents by flushing with 40 mL of 37°C saline (9 g/L); only a
small volume (i.e., 1 mL) of saline remained in the lumen. For all
studies except those that measured total luminal thiols with Ellman’s
reagent, 10 mL of 37°C saline containing 250 µmol acivicin/L, an
inhibitor of -glutamyltransferase, was added to the lumen and, after
20 min, flushed with a gentle stream of air. The bile duct, celiac
artery, renal arteries and aorta proximal to the emergence of the
celiac artery were ligated, and the rat was killed while anesthetized
by cutting the vena cava. The aorta was cannulated distal to the renal
arteries and perfused retrograde so that the intestinal vasculature was
perfused via the superior mesenteric artery. The perfusion rate was 1
mL/min with oxygenated Krebs-Henseleit buffer (pH 7.4, 37°C)
containing 12.5 mmol HEPES/L.
The perfusate was collected via an 18-gauge cannula in the portal vein.
Temperature was maintained at 37–38°C with a heat lamp.
Krebs-Henseleit buffer containing 7 mmol glutamine/L and 1 mmol
glucose/L without or with 250 µmol GSSG/L was used as the luminal
buffer and was deoxygenated by bubbling with N2 for 20 min
before use to minimize the oxidation of any thiols formed during
incubation. The pH was adjusted to 7.4 with HCl after bubbling with
N2. Glutamine was included because it is a preferred energy
substrate of small intestine, and the concentration that we used
approximates that in the lumen under postprandial conditions
(Windmueller 1982
).
At time points after the addition of GSSG, luminal fluid was drained with a gentle stream of air from a syringe. An aliquot was spun immediately in a microcentrifuge for 30 s and acidified with perchloric acid (1 mol/L) for analysis of GSSG, GSH and other thiols and disulfides. A second aliquot was reduced with dithiothreitol (5 mmol/L final concentration) before acidification for the measurement of total Cys equivalents. To assess GSSG reduction in regions of the small intestine, 10-cm lengths were isolated; the first were in large part duodenum, and an additional five lengths were taken from the successive 50 cm of jejunum. Experiments were analogous to the larger segment, but 2 mL of medium was used for each length.
To verify inhibition of -glutamyltransferase, the mucosa from
intestines without and with acivicin treatment was scraped from the
proximal 40 cm of jejunum with a glass microscope slide, homogenized in
Krebs-Henseleit buffer (pH 7.4) to make a 200 g/L homogenate and
analyzed spectrophotometrically with
-glutamyl-p-nitroanilide (Orlowski and Meister 1963
). There was substantial (68–96%) but incomplete
inhibition of -glutamyltransferase with this treatment.
Analysis of thiols and disulfides.
GSSG, GSH, CySSG, cystinyl-bis-glycine, cysteinyl-glycine, Cys and
CySS in luminal fluid were analyzed with HPLC as described by
Fariss and Reed (1987)
. Compounds were identified by
retention times of authentic standards, and concentrations were
determined by integration relative to standards. For some
chromatograms, CySS did not resolve from glutamate, and CySS was
calculated from the difference in integrals between samples without and
with dithiothreitol treatment (Ellman and Lysko 1979
).
The error due to this indirect approach was estimated to be <10% and
resulted in a <2-mV error in the calculation of Eh.
Eh values were calculated with the Nernst equation with
Eo values at pH 7.4 of -264 mV for GSSG/2GSH, -257 mV for
CySSG/(Cys · GSH) and -250 mV for CySS/2Cys (Jones et al. 2000
).
Total thiols were measured with 1 mmol/L 5,5'-dithio-bis(2-nitrobenzoic acid) prepared in the luminal incubation buffer. After a 5-min incubation at room temperature, absorbance was read at 412 nm, and the total thiol concentration was calculated from a standard curve prepared with Cys.
Statistical analysis.
Results are expressed as means ± SEM. Homogeneity of
variance was tested using the F-max test. Log
transformations were performed on nonhomogeneous data. If the variances
were homogeneous, data were analyzed with Student’s t
test, one-way ANOVA or completely randomized factorial ANOVA as
appropriate. Individual comparisons between treatment means after ANOVA
were made with Tukey’s 
test (Steel and Torrie 1980
). When variances were nonhomogeneous after log
transformations of data, the data were analyzed with the nonparametric,
distribution-free, multiple comparison test (Gad and Weil 1986
). The criterion for significance was P
< 0.05 for all comparisons.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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GSH accumulated in the lumen approximately linearly for the first 20
min (Fig. 1A
) with a rate of 28 nmol ·
segment-1 · min-1. In
control intestines without added GSSG, GSH increased at an average rate
of <6 nmol · segment-1 ·
min-1. Thus, the inclusion of GSSG in the lumen
resulted in a substantially higher rate of appearance of GSH in the
lumen. During this time, there was no significant increase in GSH in
scrapings from the intestinal mucosa (Table 1
), but there was an increase in GSH in the portal vein from 5.5 ± 1.5 to 12 ± 2 µmol/L (Fig. 2
).
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) after 30 min without or with luminal GSSG. The total
thiol concentration was 174 ± 23 µmol/L without GSSG and was
significantly higher (246 ± 5 µmol/L) with GSSG. The average
total difference between these total thiol values due to the addition
of GSSG was 23.3 nmol · segment-1 ·
min-1. Because the average increase in GSH
during the first 30 min was 20 nmol ·
segment-1 · min-1 (Fig. 1A
), the greater total thiol in the presence of GSSG can in
large part be explained by the greater amount of GSH.
Measurements of GSSG loss showed that initially there was a rapid
decrease in GSSG, followed by a linear loss between 10 and 30 min (Fig. 1B
). The basis for the initial loss was likely due to
redistribution within accessible aqueous compartment and/or binding to
surfaces and was not pursued. However, the rate of loss of GSSG during
the linear phase [30 nmol · segment-1
· min-1] was similar to the rate of GSH
appearance (Fig. 1A
). Because direct reduction of GSSG to
2GSH should result in 2GSH for each GSSG lost, only half of the GSH
equivalents in GSSG were recovered as GSH. Analysis of mucosal
scrapings showed that GSSG was not detectable (<0.02 nmol/mg protein).
Similarly, there was no detectable GSSG in the vascular perfusate
during the 30-min time course. Thus, a process other than direct
two-electron reduction of GSSG to 2GSH appeared to be involved in
the loss of GSSG.
Formation of the disulfide of Cys and GSH, CySSG.
We previously found that Cys is released at a substantial rate into the
lumen of the vascularly perfused small intestine (Dahm and Jones 1994
), and Cys reacts with GSSG to form CySSG and GSH. Thus,
the reduction of GSSG to GSH could involve the reduction of GSSG by
Cys. To determine the rate of appearance of CySSG in the lumen, we
measured CySSG with HPLC as a function of time after the addition of
GSSG. CySSG was at a low concentration (<1 µmol/L) in the lumen
without added GSSG but increased during the first 20 min with a rate of
25 nmol · segment-1 ·
min-1 in the presence of GSSG (Fig. 3A
). Thus, the rate of CySSG formation was approximately equal
to the rate of GSH formation and accounted for the loss of GSSG via the
reaction Cys + GSSG 
CySSG + GSH.
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To determine whether the reaction of Cys with GSSG caused a decrease in
luminal Cys, Cys was measured as a function of time after the addition
of GSSG. Cys accumulated within the lumen at a rate of 52 nmol ·
segment-1 · min-1
either without or with added GSSG (Fig. 3B
). However, when
samples were reduced with dithiothreitol before analysis, total Cys
equivalents (average values for three or four intestinal preparations)
increased 86 nmol · segment-1 ·
min-1 in the presence of GSSG but only 60 nmol
· segment-1 · min-1
without GSSG (Fig. 3C
). Thus, there was a higher amount of
total Cys equivalents [26 nmol · segment-1
· min-1], all of which were in oxidized
forms. This rate of GSSG-dependent increase in total Cys
equivalents approximated the rate of GSSG loss (Fig. 1B
) and
the rates of GSH (Fig. 1B
) and CySSG formation (Fig. 1A
). CySS was also present (Fig. 3D
) but
accumulated only to a relatively low concentration and at a relatively
slow rate above that without GSSG [6 nmol ·
segment-1 · min-1]. Thus, the addition of GSSG to the lumen
resulted in more luminal Cys equivalents that were principally due to
CySSG.
Redox potential of the luminal thiol-disulfide pools.
To determine the change in redox associated with the reduction of GSSG,
the Eh value for the luminal GSSG/2GSH pool was
calculated with the Nernst equation. In the GSSG solution added to the
lumen, the Eh was 
6 mV. By 10 min, the value
was shifted to -112 mV and then to -132 and -143 mV at 20 and 30
min, respectively (Fig. 4
). The value achieved by 20 min was similar to that found in the lumen
in vivo and similar to the Eh for GSSG/2GSH in
bile (see Introduction). Thus, the changes in GSSG and GSH
concentrations in the lumen of the jejunum are associated with an
adjustment in Eh of luminal thiol-disulfide
pools toward in vivo luminal and biliary Eh
values.
|
. Eh for CySSG/(GSH · Cys) was
intermediate between the values for GSSG/2GSH and CySS/2Cys (Fig. 4)
.
Thus, Eh values show that the energetics in the
lumen of the small intestine are appropriate for Cys reduction of GSSG.
Eh changes in jejunal preparations without inhibition
of -glutamyltransferase.
To determine whether the capacity to reduce luminal disulfides was
affected by inhibition of -glutamyltransferase, we calculated
Eh values for thiol-disulfide pools from
perfused jejunal preparations without or with acivicin pretreatment.
Eh was not significantly different for luminal
GSSG/2GSH, CySS/2Cys or CySSG/(Cys · GSH) pools after 30 min
(Table 2
). Total GSH equivalents remaining after 30 min were only 1.2 ± 0.2 µmol/segment in untreated jejunum compared with 2.7 ± 0.2
µmol/segment in pretreated jejunum (Table 2)
. Total Cys equivalents
were 2.6 ± 0.3 µmol/segment in untreated and 1.8 ± 0.1
µmol/segment in pretreated jejunum. Thus, even though the pool sizes
were very different as a consequence of inhibition of
-glutamyltransferase, there was no effect on the capacity to reduce
the disulfides to establish thiol-disulfide redox.
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To assess whether modulation of luminal redox was specific to a region
of the jejunum, studies were conducted in 10-cm segments of intestine
incubated without or with 250 µmol GSSG/L for 10 min. The first
segment was in large part duodenum, and the successive five segments
were of jejunum. In jejunal segments that received buffer alone,
luminal GSH was <10 µmol/L (Fig. 5A
). In contrast, the duodenum had 30 µmol GSH/L, which
may have been due to remaining GSH supplied from the bile duct despite
ligation or due to more active GSH secretion than was found in the
jejunum. In the presence of added GSSG, GSH was substantially higher in
all regions, indicating that all regions had the capacity to reduce
GSSG. However, the amount of GSH was successively lower from proximal
to distal jejunum, indicating that the greatest reductive capacity was
in the proximal region.
|
). As with the 40-cm
jejunal preparation at 10 min, there was no difference in luminal Cys
concentration between preparations without or with GSSG. CySSG
accumulation was significantly higher in regions with than without
GSSG, but there was no significant difference between different regions
(Fig. 5C
). Calculations of Eh values
for GSSG/2GSH showed that the lumina of duodenum and proximal jejunum
were more reduced than the distal regions (Fig. 5D
).
Eh for CySSG/(GSH · Cys) similarly showed that
proximal jejunum was more reduced than distal jejunum. These values
were 15 mV more reduced than that for GSSG/2GSH (Fig. 5D
). Therefore, the capacity to reduce GSSG is greatest in
the proximal jejunum, i.e., the region of the jejunum with the greatest
rate of Cys release into the lumen. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and 1998
).
In contrast, relatively little is known about the control of
extracellular thiol-disulfide redox. In human plasma, GSSG/2GSH
redox appears to be tightly regulated because interindividual variation
is small. Measurements in 24 healthy, young individuals showed a mean
of -137 mV with 1 SD of only ±9 mV (Jones et al. 2000
). The CySS/2Cys pool was considerably more oxidized (-80
mV) but also had a relatively small standard deviation (±9 mV). GSH is
released at a substantial rate from the liver, and this process appears
to be a major mechanism for controlling plasma thiol-disulfide
redox.
The current data show that the rat jejunum has the capacity to reduce a
luminal disulfide to bring the Eh to a range
similar to that found in vivo. However, unlike in plasma, the
Eh of the CySS/2Cys pool was more reduced than
that for the GSSG/2GSH pool, indicating that the reductant to maintain
the luminal redox is Cys rather than GSH. Because Cys is released into
the lumen at a substantial rate (Dahm and Jones 1994
)
and CySS is rapidly taken up across the brush border (Neil 1959
), the combination of these processes with intracellular
reduction of CySS could provide a Cys–CySS shuttle for the control of
luminal thiol-disulfide redox (Fig. 6
). The intestinal mucosa has a relatively high Cys content (25% of
the GSH concentration; Dahm and Jones 1994
), and this
Cys could be released to the lumen via a system that is sensitive to
luminal redox. However, if such a system exists, it would function in
opposition to the known intestinal absorption of Cys. Thus, regulation
of the direction of transport would be necessary to avoid an
energy-wasting futile cycle. For instance, uptake of Cys could
occur within a certain redox range, whereas release of Cys could occur
if the luminal redox is too oxidized.
|
).
Although this GSH may be used for detoxification of electrophils by the
GSH S-transferase associated with intestinal mucus
(Samiec et al. 2000
), most is degraded to Cys by
-glutamyltransferase and dipeptidases associated with the brush
border (Dahm and Jones 1994
). Stimulated GSH release in
response to oxidized luminal conditions could thereby supply Cys and
support a shuttle mechanism analogous to that shown in Figure 6
.
The control of luminal redox by such a shuttle mechanism could be very
important for the maintenance of intestinal homeostasis and absorptive
functions. In rat, processes such as iron and selenium absorption are
thiol dependent and may, in fact, be redox dependent (Scharrer et al. 1992
, Wien and Van Campen 1991b
and 1994
). The inability to regulate luminal redox may therefore
limit absorption and result in a requirement for added Cys or other
thiols to support absorption. Disulfide reduction may facilitate
protein digestion by disrupting disulfide-linked peptides.
Disulfide cross-links increase the viscosity of mucus (Snary et al. 1970
), and thiols function as mucolytic agents
(Rhee et al. 1999
). Thus, redox could affect mucus
thickness or inappropriate sloughing and this could be particularly
important under oxidative conditions, such as immune activation.
In summary, the results of the current study show that the jejunum can
reduce luminal GSSG to GSH with concomitant formation of the disulfide
of GSH and Cys. Within 20 min, the redox potential of the GSSG/2GSH
pool was brought to a value similar to that found in the lumen in vivo.
The Eh of the CySS/2Cys pool was consistently
20 mV more reduced than that for the GSSG/2GSH pool, indicating that
disulfide reduction in the lumen could involve a Cys–CySS shuttle
mechanism. GSSG reduction decreased from proximal to distal jejunum.
This reductive mechanism may be important for the absorption of
redox-sensitive nutrients and the maintenance of conditions for
digestion, and failure of this mechanism could contribute to
malabsorption and digestive disorders.
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FOOTNOTES |
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2 Current address: Pfizer, Inc., Eastern Point Road, Groton, CT 06340.
4 Abbreviations used: Cys, cysteine; CySS, cystine; CySSG, cysteine–glutathione disulfide; Eh, redox potential; GSH, glutathione; GSSG, glutathione disulfide.
Manuscript received April 10, 2000. Initial review completed May 26, 2000. Revision accepted July 12, 2000.
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) or with (•) 250 µmol GSSG/L for 10–30
min. At appropriate times, luminal buffer was collected and analyzed by
HPLC for GSH and GSSG determinations. GSSG was not detected in the
lumen of intestines without added GSSG. Values are means
± SEM, n = 5.
aSignificantly different from respective control time
point. bSignificantly different from respective 10-min time
point. cSignificantly different from initial incubation
medium.
) and with (
, 
) being determined by HPLC
after reduction by dithiothreitol. Values are means ± SEM, n = 3–4 for each point.
aSignificantly different from respective control.
bSignificantly different from respective 10-min time point.
cSignificantly different from respective 20-min time
point.
), CySSG/(Cys · GSH) (
) or with (•, 