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*
Laboratoire d’Etude du Métabolisme Azoté, INRA, Clermont-Ferrand Theix, 63122 Saint Genès Champanelle, France, and
Centre de Recherches Nestlé, Vers chez les blanc, Lausanne 26, Switzerland
3To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-glutamyl-cysteine synthetase and
glutathione reductase were significantly greater in liver of infected
rats than of pair-fed rats. From the present study, we estimate
that glutathione synthesis accounts for at least 40% of the enhanced
cysteine utilization during infection. This increased utilization may
be the primary cause of an enhanced cysteine requirement in
infection.
KEY WORDS: • rats • glutathione synthesis rate • cysteine infusion • glutathione-related enzymes
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-glutamyl-L-cysteinyl-glycine,
GSH)4
is a tripeptide that is present in nearly all animal cells and is the
predominant intracellular low-molecular-weight thiol compound. GSH has
many important roles in the cell (Beutler 1989
,
Deneke et al. 1985
). It is involved in DNA and protein
synthesis (Kan et al. 1988
, Suthanthiran et al. 1990
) and the synthesis of leukotrienes, which are important
mediators of inflammation (Rouzer et al. 1982
). GSH also
maintains the reduced state of the sulfhydryl groups of many proteins,
which is required for their normal function
(Gérard-Monnier and Chaudière 1996
). It acts
as a cysteine reserve during food deprivation (Cho et al. 1981
) and is a major source of cysteine for lymphocytes.
Indeed, the normal activity of lymphocytes is dependent on cellular
availability of this sulfur amino acid (Dröge et al. 1986
). GSH is also very important in the protection of cells
against toxic insult (Beutler 1989
, Deneke et al. 1985
). It participates in the detoxification of
electrophilic metabolites of xenobiotics and is a very efficient free
radical scavenger, protecting cells from the toxic effects of reactive
oxygen compounds (Beutler 1989
). During injury,
oxidative stress is increased (Fagan et al. 1996
,
Peralta et al. 1993
). This stress is of prime importance
for the organism’s defense but may have deleterious effects on tissues
if it is not tightly controlled by antioxidants. This can be seen very
clearly in GSH-depleted rats where hyperoxia induces an increased
mortality rate (Deneke et al. 1985
).
GSH is synthesized by nearly all organs and tissue beds in a
two-step metabolic pathway. The first step, conversion of glutamic
acid and cysteine in -glutamyl-cysteine, is catalyzed by the enzyme
-glutamyl-cysteine synthetase. This step is rate-limiting and is
feed-back inhibited by GSH. The second step is the conversion of
-glutamyl-cysteine to GSH. Numerous other enzymes participate in GSH
metabolism. Glutathione peroxidase catalyzes the formation of oxidized
glutathione (GSSG) during the reduction of hydroperoxides. GSH can be
regenerated from GSSG by GSSG reductase. During the detoxification
processes, toxic compounds are attached to GSH by glutathione
transferase. This is followed by further reactions which result in a
net loss of glutathione. The glutathione status of cells is therefore
dependent on the relative activity of these reactions. Indeed,
increased activity of enzymes involved in GSH synthesis and of GSH
reduction can lead to an increased GSH concentration. Conversely,
increased activity of GSH peroxidase and GSH transferase decreases GSH
concentration.
The glutathione level is altered in many inflammatory conditions. A
fall in GSH concentration has been reported in human diseases such as
HIV infection and trauma (Luo et al. 1998
, Staal et al. 1992
). In animal models, during the initial phase of
septic shock (during the first hours after infection), tissue GSH
concentrations are decreased whereas GSSG concentrations are increased
(Ikegami et al. 1994
, Keller et al. 1985
). In contrast, during the first days following infection,
tissue glutathione levels are elevated in treated animals compared with
controls (Hunter and Grimble 1997
, Malmezat et al. 1998
). However, in the later stages of infection, depletion
of tissue GSH levels occurs (Breuillé et al. 1994a
, Colomb et al. 1995
).
Variations in glutathione levels during oxidative conditions may result
from modification in synthesis and/or loss. To determine the relative
importance of these two potential mechanisms, it is necessary to
measure glutathione kinetics in vivo. However, mechanisms responsible
for maintenance of GSH homeostasis in different tissues are poorly
documented, especially in pathological conditions. The aim of this
study was to measure glutathione concentrations, glutathione synthesis
rates and the activities of several enzymes involved in glutathione
metabolism in various tissues of infected rats. Infection induces a
strong anorexia. Because glutathione concentrations are decreased by
food deprivation (Hunter and Grimble 1997
, Jahoor et al. 1995
), pair-fed rats were used as controls.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Male Sprague-Dawley rats (Iffa-Credo, Lyon, France), 270–280 g
body weight, were individually housed in wire-bottom cages and
consumed ad libitum a semisynthetic diet containing 12 g protein/100 g
diet (Malmezat et al. 1998
). After an acclimation period of 5 d,
rats in the post-absorptive state were anesthetized with ketamine
(50 mg/kg; Imalgene®, Rhône Mérieux, Lyon, France) and
acepromazine (2.5 mg/kg; Vetranquil®, Sanofi, Libourne, France). Using
sterile techniques, silicone catheter (Silastic tubing; Sigma Medical,
Nanterre, France) was inserted into the right jugular vein and flushed
with heparinized normal saline. The free-end of the catheter was
tunnelled subcutaneously and exteriorized dorsally on the head, through
a flexible spring secured to the top of the head with dental cement.
The infusion line passed through the spring and was connected to a
swivel suspended from the top of the cage, which allowed free movement
of the rat. During a 10-d recovery period, rats were continuously
infused with saline at 0.1 mL/h. Animals grew ~6 g/d during the
recovery period.
The rats were then injected via a tail vein, with either live
Escherichia coli (6.6 x 108 bacteria
per rat, infected group, n = 13) or saline (control
group, n = 8) as described previously
(Malmezat et al. 1998
). Since infection induces a strong
anorexia, control rats were pair-fed the mean intake of the
infected rats.
Forty-eight hours after injection of bacteria or saline, food was withdrawn. A primed-continuous infusion of 15N cysteine (Cambridge Isotope Laboratories, Andover, MA) into the jugular vein was started. The priming dose was 1.5 µmol/100 g, and the isotope was continuously infused at 2.4 µmol/(100 g · h), for 4 h in five infected rats (Group 1) and for 6 h in eight infected rats (Group 2). Blood samples were taken from a tail vein at 30 min, 1, 2 and 4 h after the beginning of the infusion in Group 1, and 5 h and 5.5 h after the beginning of the infusion in Group 2. Pair-fed rats were fed a priming dose of 1.1 µmol/100 g and 6 h infusion of 15N cysteine at a rate of 1.8 µmol/(100 g · h). Different priming dose and infusion rates were used in pair-fed rats and in infected rats to obtain the same cysteine enrichment in free amino acid pool in both groups. Blood samples were taken from a tail vein 5 h and 5.5 h after the start of the infusion. At the end of the infusion, rats were anesthesized (pentobarbital, 60 mg/kg). Blood was withdrawn from the vena cava, and liver, spleen, lungs, heart, small and large intestine and muscles from the hind leg were rapidly excised. Liver, spleen, heart and digestive tract were flushed and all tissues frozen in liquid nitrogen and kept at -80°C until analysis.
The protocol was approved by the Ethics Committee of the Institute and conducted in conformity with the guiding principles in the care and use of laboratory animals.
Analytical procedures.
Tissues were finely pulverized in liquid nitrogen in a ball mill
(Dangoumeau; Prolabo, Paris, France). An aliquot of frozen powder was
homogenized by sonication in 0.2 mol/L perchloric acid containing 5
mmol/L EDTA. The homogenate was then centrifuged (20 min, 8000 x g), and the supernatant assayed for glutathione content,
using a standard enzymatic recycling procedure as described previously
(Malmezat et al. 1998
). Blood glutathione has been
measured by the same method on 10% (v/v) hemolysates prepared in
water.
For enzymatic activity measurements in liver, frozen powders were
homogenized in phosphate buffer (phosphate 75 mmol/L, dithiotreitol 2
mmol/L, sucrose 0.3 mol/L). Homogenates were centrifuged (1 h, 100,000
x g), and enzymatic activities were measured in
the supernatants. Glutathione reductase (EC 1.6.4.2) was measured in
the presence of GSSG by following the oxidation of NADPH at 340 nm
(Carlberg and Mannervik 1975
). Glutathione peroxidase
(EC 1.11.1.9) was measured in the presence of GSH and
H2O2 by following the disappearance of NADPH at
340 nm (Paglia and Valentine 1967
). The activity of
-glutamyl-cysteine synthetase (EC 6.3.2.2) was measured by the
coupled enzyme assay of Seelig and Meister (1985)
, using
pyruvate kinase and lactate dehydrogenase. Glutathione
S-transferase (EC 2.5.1.18) was measured in the presence
of GSH and 1-chloro-2,4-dinitrobenzene (CDNB) by following the
formation of GSH-CDNB at 340 nm (Habig et al. 1974
).
Liver protein concentration was determined using the bicinchoninic acid
procedure (Pierce Chemical Co., Rockford, IL) (Smith et al. 1985
).
Determination of free and glutathione-bound 15N cysteine enrichment.
Free and glutathione-bound 15N cysteine enrichments
were measured on their N,S ethoxyl carbonyl methyl
esters, using a method adapted from Kataoka et al. (1995)
. An aliquot of plasma or blood was diluted (1/2) in a
0.2 mol/L phosphate buffer (EDTA 5 mmol/L, dithiotreitol 80 mmol/L, pH
8.5). Aliquots of powdered frozen tissues were homogenized by
sonication in the same phosphate buffer. The mixture was left at room
temperature for 15 min and then the pH was adjusted to between 7 and 8.
The N,S-ethoxycarbonyl methyl esters of cysteine and
glutathione were prepared as follows. Ethyl chloroformate (0.1 mL)
(Acros Organics, Geel, Belgium) was added to the sample and mixed by
rotation at 100 rpm for 15 min at room temperature (tube rotator; Poly
Labo, Strasbourg, France). The reaction mixture was then acidified to
pH 1–2 with 2 mol/L HCl, saturated with NaCl and extracted twice with
3 mL of peroxide-free diethyl ether. The ether extracts were
evaporated to dryness at 80°C, and then 0.2 mL of 1 mol/L HCl in
methanol was added to the residue and the mixture was incubated at
80°C for 10 min. The residual solvent was evaporated to dryness at
80°C under a stream of air and the residue was finally dissolved in
0.1 mL of ethyl acetate. Free- and glutathione-bound cysteine
enrichments were then measured by gas chromatography-mass
spectrometry, with an HP 5890 gas chromatograph coupled with a HP 5972
organic mass spectrometer quadripole (Hewlett-Packard, Les Ulis,
France). Gas chromatograph analysis was conducted on a crosslinked 5%
diphenyl 95% dimethyl siloxane capillary column (0.25 mm i.d. x 0.25
mm film thickness) (HP S M 5; Hewlett-Packard). The length of the
column was 30 m for cysteine and 10 m for glutathione. Free
15N cysteine and 14N cysteine were monitored at
m/z = 221 [M + 1 - 59]- and at
m/z = 220 [M - 59]-,
respectively. Glutathione-bound 15N cysteine and
glutathione-bound 14N cysteine were monitored at
m/z = 364 [M + 1 - 116]-
and at m/z = 363 [M - 116]-,
respectively.
Calculations.
The fractional synthesis rate (ks) of tissue glutathione was calculated
according to the precursor-product relationship. When a labeled
amino acid such as 15N cysteine is given by constant
infusion, the isotopic enrichment (atom % excess, APE) of the
precursor pool reaches a constant value with time. By measuring the
enrichment of the labeled amino acid in the product (glutathione) when
there is a steady state in the precursor pool, ks can be obtained using
the following equation:
 | (1) |
where dEbt is the increase in isotopic enrichment of reduced glutathione-bound cysteine in tissues over the period dt, and Ef is the isotopic enrichment of free cysteine in tissues.
Ef is not constant during the entire infusion period and the plateau
value (Ef max) is not reached immediately. However, the time course of
Ef can be described by a simple exponential of the form
(Waterlow et al. 1978
):
 | (2) |
where Ef max is tracer/tracee ratio during the steady state and
is an empirically-fitted rate constant.
The time course of the enrichment of free cysteine in plasma can also
be described by a simple exponential analogous to the equation
presented above for tissues. If we assume that the value of in
tissues can be taken as equal to the value determined in plasma
(Waterlow et al. 1978
), equation 1
becomes:
 |
and after integration,
 |
then
 |
In a previous study we found that the value of was similar
in infected and pair-fed rats.
The absolute synthesis rate (ASR) of tissue reduced glutathione was calculated as the product of tissue reduced glutathione concentration and ks.
The standard steady-state equation was used to calculate plasma
cysteine flux in the circulation as follows:
 |
where I is the infusion rate of 15N cysteine, Etr the enrichment of cysteine tracer and Epl the enrichment of plasma cysteine at plateau.
Statistics.
Values are given as means ± SD. The statistical significance of differences between means was assessed by one-way ANOVA. Unpaired Student’s t tests were used to compare data between the two groups of infected rats and between infected and pair-fed rats. Differences were accepted as significant when P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Before infection, rats consumed about 26 g of dry matter per day. On the day of infection, food was mainly consumed in the morning, before infection (4.4 ± 3.9 g). One day after infection, the rats ate 10.6 ± 5.7 g, i.e., about 60% less than before injection of bacteria.
Food-intake restriction produced a body weight loss in pair-fed
rats. However, infected rats lost significantly more weight than
pair-fed controls (Fig. 1
).
|
).
|
Total glutathione concentration was significantly greater in septic
rats than in pair-fed controls in all tissues except small
intestine and blood (Table 2
). Because liver and spleen weights were also greater in infected rats
than in pair-fed rats, the total amount of glutathione in these two
tissues in the septic rats was more than double that of the
pair-fed rats. There was no difference in small intestine
glutathione concentration between the two groups, whereas blood
glutathione concentrations were significantly lower in infected rats
than in pair-fed rats (Table 2)
.
|
) liver and spleen glutathione concentrations (5.18
± 0.42 µmol/g liver and 2.82 ± 0.19 µmol/g spleen) were
significantly greater than in pair-fed rats and significantly lower
than in infected rats. By contrast, both infection and food restriction
induced a fall in gastrocnemius glutathione concentration when compared
to glutathione concentration values in control rats (0.91 ± 0.12
µmol/g). In the total digestive tract, glutathione concentration in
control rats (2.46 ± 0.26 µmol/g) was significantly greater
than in pair-fed rats (2.22 ± 0.16 µmol/g) and not
different from infected rats (2.44 ± 0.19 µmol/g). Activities of enzymes involved in glutathione metabolism.
The specific activity of -glutamylcysteine synthetase was not
significantly different between the infected group and the pair-fed
group (Table 3
). In contrast, since liver weight and protein content were
significantly greater in infected rats, the total activity of
-glutamyl-cysteine synthetase was 50% greater in infected compared
to pair-fed rats (Table 3)
. The hepatic activity of glutathione
reductase was significantly higher in infected compared to pair-fed
rats, when expressed as specific (16% increase), or total activity
(60% increase) (Table 3)
.
|
. The specific activity of GSH
transferase was also not different in two groups of rats, but its total
activity was 23% greater (P < 0.05) in the infected
compared to the pair-fed rats (Table 3)
. Cysteine and glutathione kinetics.
The empirically fitted rate constant for cysteine was determined
from serial samples of blood taken in infected rats from Group 2
(Fig. 2
) and was estimated to be 30 d-1.
|
. This steady state was
maintained until the end of the infusion in infected and pair-fed
rats (Fig. 3
). We assume that steady state was reached also in the liver, since the
ratio of liver to plasma enrichment was not significantly different in
infected rats at 4 h (Group 1) and 6 h (Group 2) of infusion
(0.25 ± 0.04 and 0.28 ± 0.04, respectively, Table 4
).
|
|
. It is likely
that the glutathione synthesis rate calculated in these rats was
probably underestimated, as illustrated by the values obtained in the
infected rats infused for 4 h (Group 1). Nevertheless, the value
obtained in infected rats infused for 6 h (Group 2) was
significantly greater than in pair-fed rats.
Glutathione ks was significantly enhanced in liver (+39%),
spleen (+37%), muscle (+68%) lungs (+49%) and large intestine
(+95%) of septic rats compared to pair-fed rats (Table 5
). In heart, there was a trend (P < 0.09) for a greater
glutathione ks in septic rats than in pair-fed rats. Infection had
no effect on glutathione ks in small intestine and blood (Table 5)
.
|
. The ASR for glutathione in the small intestine
did not significantly differ in the two groups (Table 3)
. Blood
glutathione ASR was significantly lower in septic rats than in
pair-fed rats (-47%) (Table 5)
. Cysteine flux was significantly greater in infected rats [18.3 ± 2.2 µmol/(h · 100 g body)] than in pair-fed rats [12.3 ± 1.7 µmol/(h · 100 g body)].
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Hunter and Grimble 1997
, Jahoor et al. 1995
). However, the
mechanisms that lead to these metabolic modifications are still poorly
understood. The major aim of the present study was therefore to
determine if changes in tissue glutathione concentrations observed
during the early phase of sepsis in rats (2 d) could be partly
explained by changes in glutathione ks.
Numerous studies have shown that tissue and plasma glutathione
concentrations are depleted during either protein deficiency or food
restriction (Cho et al. 1981
and 1984
, Hum et al. 1992
, Hunter and Grimble 1997
, Jahoor et al. 1995
). Such a decrease was also observed in this experiment
in liver, since the level determined previously in well-fed rats
was 5.18 µmol/g (Breuillé et al. 1994a
). The
decrease in glutathione concentration probably results from the
decreased availability of substrates needed for glutathione synthesis,
and particularly cysteine which is considered to be the most limiting
amino acid (Deneke et al. 1983
, Grimble et al. 1992
). It has been demonstrated that protein-deficient
diets produce a decrease in plasma, erythrocytes and liver glutathione
ks (Hum et al. 1992
, Hunter and Grimble 1997
, Jahoor et al. 1995
).
In spite of strong anorexia, glutathione concentration of septic rats
was greater than pair-fed rats in nearly all tissues. Only the
blood exhibited a decrease in glutathione content. Liver glutathione
concentrations were even greater than the values we had measured in
healthy fed rats (Breuillé et al. 1994a
). Thus the
inflammatory challenge overcame the influence of food restriction.
Others have described unchanged or increased glutathione status in
different tissues 24 and 48 h after induction of stress in animal
models (Hunter and Grimble 1997
, Jahoor et al. 1995
). These results suggest that the organism is able to
improve glutathione status through an increase in glutathione
concentration in response to infection, that may represent a mechanism
to protect against oxidative damage. Indeed, a higher rate of mortality
has been observed in rats exposed to hyperoxia and glutathione
depletion (Deneke et al. 1985
).
Changes in the activities of glutathione reductase, peroxidase and
transferase may have a bearing on the concentration of glutathione
within tissues. A higher activity of glutathione reductase was observed
in liver of septic compared to pair-fed rats. This agrees with the
results of Hunter and Grimble (1997)
who also showed
that rats treated with tumor necrosis factor (TNF) exhibited an
increase in the glutathione reductase activity. This response of liver
to an inflammatory challenge may allow for the maintenance of a high
GSH/GSSG ratio providing a high antioxidative potential. Peroxidase
activities were little affected by infection. Thus, changes in the
activity of reductase can have a greater potential impact on recycling
of glutathione than alterations in peroxidase activity. The increased
total liver GSH transferase activity in infected rats suggests an
accelerated loss of glutathione from the body by the excretion of
reduced glutathione conjugates, to protect cells against toxic
compounds. However, as glutathione levels were greater in infected
animals compared to pair-fed rats, rate of glutathione synthesis
was probably more than adequate to support a potential higher rate of
utilization. In the present study, the activity of
-glutamyl-cysteine synthetase (limiting enzyme in glutathione
synthesis) in whole liver was 50% higher in infected rats than in
pair-fed controls. The difference in all these enzyme levels were
small even when significant. However, there was no decrease of these
enzymes, as is observed for negative acute phase proteins
(Schreiber et al. 1982
). Furthermore, measurements of
enzyme activities indicate the capacity of the tissue for synthesis and
does not consider the flow of available substrates through the
enzymatic reaction and the presence of other modulating substances in
vivo.
Few studies have been performed in vivo on glutathione synthesis in
animal tissues. The methods used are often indirect (Hunter and Grimble 1997
, Potter and Tran 1993
). Only
Jahoor et al. (1995
and 1999
) have measured glutathione
fractional ks using 2H and
13C glycine infusion. We have developed a
selective and rapid procedure to determine enrichment of glutathione as
a whole, and used labeled cysteine infusion to measure the glutathione
fractional ks in tissues. However, the values are most likely lower
than the actual rates, because of the uncertainties of the infusion
method. The purpose of a constant infusion is to produce a constant
enrichment of the precursor. As the calculation of ks requires the
determination of the area under the curve describing the time course of
the precursor enrichment, it can be very simply calculated if this
constancy is achieved immediately. For cysteine, this was not the case.
However, the area under the curve can be calculated more precisely,
taking into account the time course of the rise of enrichment to
plateau. The rate at which the plateau is reached depends upon the pool
size of the labeled amino acid and is characterized by an empirically
fitted rate constant. For lysine and tyrosine, this constant is 15 and
80 d-1, respectively (Waterlow et al. 1978
). As plasma cysteine concentration is between plasma
lysine and tyrosine concentration, the value for cysteine that we have
estimated to be 30 d-1 seems to be reasonable.
In liver of infected rats infused for 6 h, glutathione enrichment
was very similar to free cysteine enrichment. There are several
potential reasons for this. The enrichment of the immediate precursor
pool for glutathione synthesis is different from that of the general
free cysteine pool. Another possibility can be that glutathione ks is
very fast in these rats, allowing glutathione enrichment to reach a
plateau value at the same level as free cysteine within 6 h of
infusion. In these conditions, the values obtained for glutathione ks
underestimated the real values because we used the intracellular free
cysteine as precursor for glutathione synthesis in the calculations.
Therefore, we calculated liver glutathione ks in rats infused for
4 h. At this time, glutathione-bound cysteine enrichment has
not reached a plateau, and this allowed us to obtain a better estimate.
However, with both 4 and 6 h infusion, infection increased
cysteine utilization for liver glutathione synthesis. Finally, the
calculations do not take into account glutathione excretion and
cysteine recycling from glutathione catabolism which are probably both
important. Regardless, our data in liver are consistent with values
previously reported. Using 13C glycine infusion,
Jahoor et al. (1995)
reported a fractional ks of 175%/d
in piglet liver 2 d after injection of turpentine. The apparent
glutathione turnover in liver, determined from the decrease of
glutathione-specific radioactivity after injection of
35S cysteine, is about 350%/day in young rats
(Potter and Tran 1993
). By inhibiting glutathione
synthesis using buthionine sulfoximine and assuming that the rate of
glutathione disappearance reflects glutathione synthesis, Hunter and Grimble (1997)
indirectly estimated glutathione synthesis
in rat liver to be 440%/day.
Glutathione fractional ks were similar in the small intestine and blood
of infected and pair-fed rats. Thus, there is a positive
correlation in these tissues between fractional ks and glutathione
concentrations, both not altered by infection. Similar results were
observed in the intestine in pigs fed a protein-deficient diet
(Jahoor et al. 1995
). In other tissues, especially
liver, glutathione ks was dramatically increased after infection. This
is in keeping with the increased glutathione concentration observed in
this tissue, and is in accordance with the results of Hunter and Grimble (1997)
in lipopolysaccharide injected rats. These
authors have also shown that lipopolysaccharide treatment prevented the
fall in liver glutathione level and synthesis due to the reduced food
intake associated with the treatment. In our study, we have not
included a well-fed group of rats and we cannot conclude if
glutathione synthesis was restored to normal levels or enhanced above
them. However, because glutathione levels in infected rats were close
to those found in rats eating ad libitum (Breuillé et al. 1994a
), we can assume that, despite lower food intake, the main
effect of infection was to maintain glutathione concentration and
synthesis.
The total amount of glutathione synthesized in the whole body can be
estimated by summing the total glutathione synthesis of the tissues
examined and extrapolating the value to the body weight of the rat.
Calculated in this way, infection stimulated total glutathione
synthesis 3.4-fold. From data previously obtained in our laboratory, we
can estimate that 2.20 mmol of cysteine was incorporated daily into
whole protein of septic rats vs. 1.39 mmol in pair-fed rats
(Breuillé et al. 1994b
and 1996
). Thus, using
these values, glutathione synthesis represents 38 and 18% of the
amount of cysteine used for protein synthesis in infected and
pair-fed rats, respectively. Therefore, glutathione synthesis
accounts for 42% of the increased cysteine utilization 2 d after
infection. Furthermore, it appears that about 64% of the plasma
cysteine flux in septic rats is sustained by the turnover of
glutathione compared to only 28% in pair-fed rats. In healthy men,
glutathione synthesis may account for >50% of cysteine flux
(Fukagawa et al. 1996
).
Grimble et al. (1992)
has suggested that sulfur amino
acids play a key role in the economy of the body under inflammatory
conditions. Following fractures and burns, urinary nitrogen excretion
is enhanced to a greater extent than is sulfur excretion
(Cuthbertson 1931
, Larsson et al. 1982
).
Cysteine catabolism is decreased in numerous tissues following
infection (Breuillé et al. 1996
, Malmezat et al. 1998
), suggesting a preferential retention of this amino
acid in stress. Thus, taken together, these results suggest that
cysteine requirements are increased during inflammatory states.
Moreover, the present study shows that increased glutathione synthesis
may be the primary cause of the increased cysteine requirement.
Glutathione homeostasis has been studied in numerous human diseases. In
HIV infection, concentration of erythrocyte glutathione is decreased.
This is due in part to a reduced ks secondary to a shortage in cysteine
availability (Jahoor et al. 1999
). As shown in our study
and by Hunter and Grimble (1997)
, in the first days
following an inflammatory challenge, tissue glutathione amounts were
elevated in treated animals compared to control animals. However later
on (data not shown) we observed depletion of tissue glutathione levels.
Further studies on glutathione metabolism are therefore required to
determine mechanisms responsible for glutathione deficiency in the late
phase of infection and the effect of cysteine supplementation.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
2 This study was supported by the Institut
National de la Recherche Agronomique, France, and Nestlé,
Switzerland.
4 Abbreviations used: APE, atom % excess; ASR,
absolute synthesis rate; CDNB, 1-chloro-2,4-dinitrobenzene; GSH,
glutathione; GSSG, oxidized glutathione; ks, synthesis rate; TNF, tumor
necrosis factor.
Manuscript received October 7, 1999. Initial review completed November 29, 1999. Revision accepted January 13, 2000.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1. Beutler E. Nutritional and metabolic aspects of glutathione. Ann. Rev. Nutr. 1989;9:287-302[Medline]
2. Breuillé D., Arnal M., Rambourdin F., Rosé F., Obled C. Increased cysteine requirements induced by sepsis. Clin. Nutr. 1996;15:21
3. Breuillé D., Malmezat T., Rosé F., Pouyet C., Obled C. Assessment of tissue glutathione status during experimental sepsis. Clin. Nutr. 1994a;13:5
4. Breuillé D., Rosé F., Arnal M., Melin C., Obled C. Sepsis modifies contribution of different organs to whole-body protein synthesis in rats. Clin. Sci. 1994b;86:663-669[Medline]
5.
Carlberg I., Mannervik B. Purification and characterization of the flavoenzyme glutathione reductase from rat liver. J. Biol. Chem. 1975;250:5475-5480
6. Cho E. S., Johnson N., Snider B. C. Tissue glutathione as a cyst(e)ine reservoir during cystine depletion in growing rats. J. Nutr. 1984;114:1853-1862
7. Cho E. S., Sahyoun N., Stegink L.D. Tissue glutathione as a cyst(e)ine reservoir during fasting and refeeding of rats. J. Nutr. 1981;111:914-922
8. Colomb V., Petit J., Matheix-Fortunet H., Heecketsweiler B., Kaeffer N., Lerebours E., Colin R. Influence of antibiotics and food intake on liver glutathione and cytochrome P-450 in septic rats. Br. J. Nutr. 1995;73:99-110[Medline]
9. Cuthbertson D. P. The distribution of nitrogen and sulfur in the urine during conditions of increased catabolism. Biochem. J. 1931;25:236-244
10.
Deneke S. M., Fanburg B. L. Regulation of cellular glutathione. Am. J. Physiol. 1989;257:L163-L173
11.
Deneke S. M., Gershoff S. N., Fanburg B. L. Potentiation of oxygen toxicity in rats by dietary protein or amino acid deficiency. J. Appl. Physiol. 1983;54:147-151
12. Deneke S. M., Lynch B. A., Fanburg B. L. Effects of low protein diets or feed restriction on rat lung glutathione and oxygen toxicity. J. Nutr. 1985;115:726-732
13. Dröge W., Pottmeyer-Gerber C., Schmidt H., Nick S. Glutathione augments the activation of cytotoxic T lymphocytes in vivo. Immunobiology 1986;172:151-156[Medline]
14. Fagan J. M., Ganguly M., Tiao G., Fischer J. E., Hasselgren P. O. Sepsis increases oxidatively damaged proteins in skeletal muscle. Arch. Surg. 1996;131:1326-1331[Abstract]
15.
Fukagawa N. K., Ajami A. M., Young V. R. Plasma methionine and cysteine kinetics in response to an intravenous glutathione infusion in adult humans. Am. J. Physiol. 1996;270:E209-E214
16. Gérard-Monnier D., Chaudière J. Metabolism and antioxidant function of glutathione. Pathol. Biol. 1996;44:77-85[Medline]
17. Grimble R. F., Jackson A. A., Persaud C., Wride M. J., Delers F., Engler R. Cysteine and glycine supplementation modulate the metabolic response to tumor necrosis factor alpha in rats fed a low protein diet. J. Nutr. 1992;122:2066-2073
18. Habig W. H., Pabst M. J., Jakoby W. B. Glutathione S-transferases. The first enzymatic step in mercapturic acid formation. J. Biol. Chem. 1974;249:7130-7139
19. Hum S., Koski K. G., Hoffer L. J. Varied protein intake alters glutathione metabolism in rats. J. Nutr. 1992;122:2010-2018
20. Hunter E.A.L., Grimble R. F. Dietary sulphur amino acid adequacy influences glutathione synthesis and glutathione dependent enzymes during the inflammatory response to endotoxin and tumour necrosis factor-alpha in rats. Clin. Sci. 1997;92:297-305[Medline]
21. Ikegami K., Lalonde C., Youn Y. K., Picard L., Demling R. Comparison of plasma reduced glutathione and oxidized glutathione with lung and liver tissue oxidant and antioxidant activity during acute inflammation. Shock 1994;1:307-314[Medline]
22.
Jahoor F., Jackson A., Gazzard B., Philips G., Sharpstone D., Frazer M. E., Heird W. Erythrocyte glutathione deficiency in symptom-free HIV infection is associated with decreased synthesis rate. Am. J. Physiol. 1999;276:E205-E211
23. Jahoor F., Wykes L. J., Reeds P. J., Henry J. F., Del Rosario M. P., Frazer M.E. Protein-deficient pigs cannot maintain reduced glutathione homeostasis when subjected to the stress of inflammation. J. Nutr. 1995;125:1462-1472
24.
Kan B., London I. M., Levin D. H. Role of reversing factor in the inhibition of protein synthesis initiation by oxidized glutathione. J. Biol. Chem. 1988;263:15652-15656
25. Kataoka H., Takagi K., Makita M. Determination of glutathione and related aminothiols by gas chromatography with flame photometric detection. Biomed. Chromatogr. 1995;9:85-89[Medline]
26. Keller G. A., Barke R., Harty J. T., Humphrey E., Simmons R. L. Decreased hepatic glutathione levels in septic shock. Arch. Surg. 1985;120:941-945[Abstract]
27. Larsson J., Liljedahl S., Martenson J., Nordstrom H., Schildt B., Sarbo B. Urinary excretion of sulfur amino acids and sulfur metabolites in burned patients receiving parenteral nutrition. J. Trauma 1982;2:656-663
28.
Luo J. L., Hammarqvist F., Andersson K., Wernerman J. Surgical trauma decreases glutathione synthetic capacity in human skeletal muscle tissue. Am. J. Physiol. 1998;275:E359-E365
29.
Malmezat T., Breuillé D., Pouyet C., Patureau Mirand P., Obled C. Metabolism of cysteine is modified during the acute phase of sepsis. J. Nutr. 1998;128:97-105
30. Miller S. A. Protein metabolism during growth and development. Munro H. N. eds. Mammalian Protein Metabolism 1969;vol. 3:183-233 Academic Press New York, NY.
31. Paglia D. E., Valentine W. N. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J. Lab. Clin. Med. 1967;70:158-169[Medline]
32. Peralta J. G., Llesuy S., Evelson P., Carreras M. C, Flecha B., G & Poderoso J. J. Oxidative stress in skeletal muscle during sepsis in rats. Circ. Shock 1993;39:153-159[Medline]
33. Potter D. W., Tran T. B. Apparent rates of glutathione turnover in rat tissues. Toxicol. Appl. Pharmacol. 1993;120:186-192[Medline]
34.
Rouzer C. A., Scott W. A., Hamill A. L., Liu F. T., Katz D. H., Cohn Z. A. Secretion of leukotriene C and other arachidonic acid metabolites by macrophages challenged with immunoglobulin E immune complexes. J. Exp. Med. 1982;156:1077-1086
35.
Schreiber G., Howlett G., Nagashima M., Millership A., Martin H., Urban J., Kotler L. The acute phase response of plasma protein synthesis during experimental inflammation. J. Biol. Chem. 1982;257:10271-10277
36.
Seelig G. F., Meister A. Glutathione biosynthesis; -glutamyl-cysteine synthetase from rat kidney. Meth. Enzymol. 1985;113:379-390[Medline]
37. Smith P. K., Krohn R. I., Hermanson G. T., Gartner A. K., Provenzano M. D., Fuijimoto E. K., Goeke N. M., Olson B. J., Klenk D. C. Measurement of protein using bicinchoninic acid. Anal. Biochem. 1985;150:76-85[Medline]
38. Staal F.J.T, Ela S. W., Roederer M., Anderson M. T., Herzenberg L. A., Herzenberg L. A. Glutathione deficiency and human immunodeficiencyvirus infection. Lancet 1992;339:909-912[Medline]
39.
Suthanthiran M., Anderson M. E., Sharma V. K., Meister A. Glutathione regulates activation-dependent DNA synthesis in highly purified normal human T lymphocytes stimulated via the CD2 and CD3 antigen. Proc. Natl. Acad. Sci. USA 1990;87:3343-3347
40. Waterlow J. C., Garlick P. J., Millward D. J. Protein turnover in mammalian tissues and in the whole body 1978:804 North-Holland Publishing Company Amsterdam.
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