Methionine transsulfuration is increased during sepsis in
rats
Thierry
Malmezat1,
Denis
Breuillé2,
Corinne
Pouyet2,
Caroline
Buffière2,
Philippe
Denis2,
Philippe Patureau
Mirand1, and
Christiane
Obled1
1 Laboratoire d'Etude du Métabolisme Azoté,
Institut National de la Recherche Agronomique, Clermont-Ferrand-Theix,
63122 Saint Genès Champanelle, France; and 2 Centre de
Recherches Nestlé, Vers chez les blanc, Lausanne 26, Switzerland
 |
ABSTRACT |
Methionine transsulfuration in plasma and liver, and plasma
methionine and cysteine kinetics were investigated in vivo during the
acute phase of sepsis in rats. Rats were infected with an intravenous
injection of live Escherichia coli, and control pair-fed rats were injected with saline. Two days after injection, the rats were
infused for 6 h with [35S]methionine and
[15N]cysteine. Transsulfuration was measured from the
transfer rate of 35S from methionine to cysteine. Liver
cystathionase activity was also measured. Infection significantly
increased (P < 0.05) the contribution of
transsulfuration to cysteine flux in both plasma and liver (by 80%)
and the contribution of transsulfuration to plasma methionine flux (by
133%). Transsulfuration measured in plasma was significantly
(P < 0.05) higher in infected rats than in pair-fed
rats (0.68 and 0.25 µmol · h
1 · 100 g
1, respectively). However, liver cystathionase specific
activity was decreased by 17% by infection (P < 0.05). Infection increased methionine flux (16%, P < 0.05) less than cysteine flux (38%, P < 0.05).
Therefore, the plasma cysteine flux was higher than that predicted from
estimates of protein turnover based on methionine data, probably
because of enhanced glutathione turnover. Taken together, these results
suggest an increased cysteine requirement in infection.
methionine flux; cystathionase; components of cysteine flux
 |
INTRODUCTION |
TRAUMA AND SEPSIS
markedly alter metabolism, and particularly amino acid and protein
metabolism (33). The roles of protein synthesis and
breakdown in mediating the response of whole body protein economy after
injury have been extensively studied. A net catabolism of protein
occurs primarily in muscle, and the amino acids are used to increase
synthesis of proteins, including acute-phase proteins and proteins of
the immune system (5, 23). However, the metabolism of
individual amino acids, especially methionine and cysteine, has
received less attention despite their important roles. Methionine
participates in methyl group metabolism and synthesis of polyamines,
creatine, and other sulfur amino acids, notably cysteine
(8). Cysteine is required for the synthesis of glutathione
and taurine, which are important compounds for host defense against
oxidative stress (19, 34).
Cysteine is formed principally in the liver by transsulfuration from
methionine to serine. The transsulfuration is initiated by the
conversion of homocysteine, formed by transmethylation of methionine,
into cystathionine by cystathionine synthase. Cystathionine is in turn
converted into cysteine and 2-ketobutyrate by cystathionase (8). Under normal circumstances, this pathway constitutes
a significant source of cysteine, and cysteine is appropriately called
nonindispensable. However, in certain clinical conditions, cysteine
biosynthesis is altered, and a diminished supply of cysteine may
reduce its further metabolism and the synthesis of important metabolites. In cirrhotics, methionine utilization and cysteine biosynthesis are impaired, and a source of cysteine has been suggested as a necessary component of the cirrhotic diet (6). In
premature infants, hepatic cystathionase is absent or in low
concentration, leading to decreased cysteine plasma levels and
glutathione synthesis rates in these subjects
(31). In rats exposed to surgical stress, cystathionase
activity was reduced by ~40%, inducing a low rate of cysteine
synthesis in isolated hepatocytes (30). These data suggest
that cysteine could be referred to as conditionally indispensable. Moreover, an exogenous supply of cysteine would be required for critically ill patients.
Methionine metabolism has been widely studied in healthy humans
(11, 13, 21, 27, 28). By contrast, to our knowledge, only
one study has explored methionine kinetics in burn patients; that study
showed an increased methionine flux and transsulfuration (35). The present study examines the influence of
infection on methionine transsulfuration and methionine and cysteine
kinetics, by use of labeled methionine and cysteine infusion. Because
cystathionase is the limiting enzyme in methionine transsulfuration,
its activity has been measured in liver. Because anorexia is a common
feature of the response to infection, the study was performed in
infected rats and control pair-fed rats.
 |
MATERIALS AND METHODS |
Animals and experimental design.
Male Sprague-Dawley rats (Iffa Credo, Lyon, France), ~250 g body
weight, were individually housed in wire-bottom cages and received ad
libitum a semi-liquid diet containing 12% protein, which has been
described in detail previously (16). The amount of
cyst(e)ine in the diet was 1.2 g/kg, and the amount of methionine was
4.7 g/kg. After an acclimatization of 5 days, rats were operated on as
described previously (15). Briefly, a silicone catheter was inserted into the right jugular vein, and the free end of the
catheter was tunneled 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 7-day recovery period, the rats,
which were continuously infused with saline at 0.1 ml/h, had a growth
rate of 6.09 ± 0.96 g/day.
The rats were then injected via a tail vein with either live
Escherichia coli (4.3 × 108 bacteria per
rat, infected group, n = 8) or saline (control group, n = 5), as described previously (5, 16).
Because infection induced a strong anorexia, control rats were pair-fed
to the infected rats.
In the morning of the 2nd day after injection of bacteria or saline,
food was withdrawn and a primed-continuous infusion of [15N]cysteine [97 atom percent excess (APE); Cambridge
Isotope Laboratories, Andover, MA] and [35S]methionine
(>37 TBq/mmol, Amersham, Les Ullis, France) was started. The priming
dose was 0.6 mg of [15N]cysteine and 6.4 × 107 dpm of [35S]methionine. The isotopes were
continuously infused for 6 h at 0.6 ml/h, 0.9 mg/h for
[15 N]cysteine and 6.4 × 107 dpm/h for
[35S]methionine. Blood samples were taken from a tail
vein 5 and 5.5 h after the start of the infusion. At the end of
the infusion, animals were anesthetized, and the liver was rapidly
excised. The samples were frozen in liquid nitrogen and conserved at
80°C until analysis.
The protocol was approved by The Ethics Committee of the Institute and
was conducted in conformity with the principles described in the
National Research Council's Guide for the Care and Use of
Laboratory Animals.
Cystathionase activity.
An aliquot of liver (the same lobe for each rat) was taken off
immediately after exsanguination of the rat and homogenized in ice-cold
buffer of 100 mM potassium hydrogen-phosphate and 1 mM EDTA, pH 7.0. Homogenates were centrifuged at 3,000 g and 4°C for 60 min, and glycerol was added to 20% (vol/vol). The cystathionase activity was measured as described by Vina et al. (30).
Dithiothreitol (5 µmol/tube) was added to bring all cystine to
the reduced form, and the amount of cysteine was determined by the
spectrophotometric method of Gaitonde (12). The soluble
protein concentration of tissue extracts was determined according to
Smith et al. (25) by the colorimetric reaction with
bicinchoninic acid.
Cystathionine concentrations.
Liver and plasma samples were extracted in 8 volumes of ice-cold TCA
(0.6 M) containing
-mercaptoethanol 2.5% (vol/vol). The soluble
fraction containing amino acids was separated from the protein
precipitate by centrifugation (20 min, 8,000 g) and then
chromatographed on cationic resin columns (resin AG50×8, 100-200
mesh, hydrogen form, Bio-Rad). Amino acids eluted with 4 M
NH4OH were dried and resuspended in 0.2 M lithium citrate buffer, pH 2.2. Cystathionine concentration was determined with an
amino acid analyzer (Alpha Plus, LKB, London, UK).
Plasma and liver methionine and cysteine specific
radioactivities.
Aliquots of plasma and liver were suspended in 1 vol of the mobile
phase used thereafter in the HPLC procedure (3.2 ml/l
O-phosphoric acid, 0.5 g/l heptane sulfonic acid, 30 ml/l
methanol, pH 2.4) and ultrafiltered. Separation of methionine and
cysteine-cystine in the ultrafiltrate was then carried out by
reversed-phase liquid chromatography as detailed previously
(16). Methionine concentration was measured with an
electrochemical detector (Coulochem II, ESA, Eurosep, France) placed
after the column. Fractions containing methionine or cysteine-cystine
were collected, and radioactivity was determined using a liquid
scintillation counter (Betamatic IV, Kontron). Cysteine-cystine
concentration in the ultrafiltrate was determined by the method of
Gaitonde (12).
Plasma cysteine enrichments.
To 500 µl of plasma were added 500 µl 10 mM dithiothreitol,
and the pH was adjusted to 8-9. The mixture was left at room
temperature for 20 min to recover cystine and cysteine bound to protein
as free cysteine. Then, 2 ml of 0.6 M TCA were added. The acid-soluble fraction containing free amino acids was separated by centrifugation (20 min, 4°C, 8,000 g), and TCA was removed by cation
exchange chromatography (resin AG50X8, 100-200 mesh, hydrogen
form, Bio-Rad). Amino acids, eluted with 4 M NH4OH, were
dried and resuspended in 20 µl acetonitrile + 15 µl
ethanethiol + 20 µl
N-(tert-butyldimethylsilyl) N-methyltrifluoroacetamide. Cysteine enrichment was then
measured with an HP 5890 gas chromatograph coupled with an HP
5972 organic mass spectrometer (Hewlett-Packard, Les Ullis, France).
Calculations.
Plasma methionine flux was determined as follows
where FMet is the methionine flux
(µmol/h), IMet is the infusion rate of
[35S]methionine (dpm/h), and SAMet is the
methionine specific activity at steady state in plasma (dpm/µmol).
Plasma cysteine flux was determined as follows
where FCys is the cysteine flux
(µmol/h), ICys is the infusion rate of
[15N]cysteine (µmol/h), Etr is the
enrichment of the cysteine tracer (97 APE), and Epl is the
enrichment of cysteine at the steady state in plasma (APE).
Because [35S]cysteine is produced from
[35S]methionine by the transsulfuration pathway, the
percentage of total entry into the plasma or liver cysteine pool that
originates from methionine, i.e., the contribution of transsulfuration
to cysteine flux in plasma (kpl) and in liver
(kliver) was calculated according to Shipley and
Clark (24) as follows
where SACys and SAMet are the
specific activities of [35S]cysteine and
[35S]methionine in plasma, and
SACys.liver and
SAMet.liver are the specific activities of
[35S]cysteine and [35S]methionine,
respectively, in liver.
In plasma, the rate of synthesis of cysteine from methionine, or
transsulfuration, was calculated as follows
where FCys is the cysteine flux
calculated with [15N]cysteine.
In steady-state conditions, the flux is the sum of inputs or the sum of
outputs. Hence, in the postabsorptive state
where BMet is the rate of methionine
appearance from protein breakdown, SMet is the rate of
methionine disappearance via nonoxidative metabolism, an index of the
rate of protein synthesis, RM is the remethylation rate, and TM is the
transmethylation rate. However, TS = TM
RM, and
Therefore, SMet is calculated from the difference
between FMet and TS, and
where BCys and BGSH are the rates of
cysteine appearance from protein breakdown and GSH breakdown,
respectively; ICys is the rate of
[15N]cysteine infusion; SCys is the rate of
cysteine disappearance via nonoxidative metabolism, an index of the
rate of protein synthesis; SGSH is the rate of cysteine
utilization for GSH synthesis; and CCys is the rate
of cysteine catabolism. BCys and SCys can be estimated from BMet and SMet by multiplying
these values by the molar ratio of cysteine to methionine in average
proteins in rat whole body. This ratio is assessed to be 1.36 (20). Therefore BGSH is calculated from the
difference between FCys and (TS + BCys + ICys), and (SGSH + CCys) is calculated from the difference between
FCys and SCys.
Statistics.
Values are given as means ± SD. The nonparametric Mann-Whitney
U-test was used to compare the infected with the pair-fed
groups. A value of P < 0.05 was accepted as
statistically significant.
 |
RESULTS |
Food consumption, rat body weight, and liver weight.
Before infection, rats consumed ~25 g of dry matter per day (Table
1). On the day of infection, the diet was
consumed mainly during the morning before the injection. One day after
injection, animals ate only ~35% of the amount consumed before
injection of bacteria. On day 2 after injection, rats had no
access to food, so they were in postabsorptive state during the
infusion. Food intake restriction produced a body weight loss over the
2 days in control rats (Table 1). Nevertheless, infected animals lost more weight than pair-fed rats (~30 and ~24 g, respectively, on day 2). Two days postinfection, liver weight of infected
rats was significantly higher than that of pair-fed animals (11.8 ± 1.6 and 8.9 ± 1.2 g, respectively, P < 0.05).
Amino acid concentrations and cystathionase activity.
Plasma cystathionine and methionine concentrations were significantly
higher in septic rats than in pair-fed rats (34 and 12%, respectively;
Table 2). However, there was no
difference in plasma cysteine + cystine levels (Table 2). As
observed in the plasma, liver cystathionine concentration was
significantly higher (+80%) in septic rats than in pair-fed rats
(Table 3). Because sepsis induced an
increase of liver weight, total liver cystathionine content was much
higher (+136%) in whole livers of septic rats than in whole livers of
pair-fed rats (Table 3). In whole liver, cysteine + cystine and
methionine contents were similar in septic rats to those in pair-fed
rats (Table 3). The whole liver content of total glutathione was
significantly increased (45%) by infection. Liver cystathionase
activity was significantly lower (17%) in infected rats, but whole
liver cystathionase activity was not significantly different between
infected and pair-fed rats (Table 3).
Cysteine and methionine metabolism.
Steady-state conditions for plasma isotopic enrichments of the two
tracers, [15N]cysteine and [35S]methionine,
had been achieved within the duration of infusion (Fig.
1). Infection induced a significant
increase of plasma methionine (16%) and cysteine (38%) fluxes in
infected rats compared with pair-fed rats (Table
4).

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Fig. 1.
Cysteine enrichment and methionine specific activity in
plasma of infected and control pair-fed rats, 5 and 5.5 h after
the beginning of the infusion. One group of rats was infected by an iv
injection of live Escherichia coli (4.3 × 108) (n = 8). A control group was injected with
saline and pair-fed with the infected rats (n = 5). Two days
after infection, rats were given a primed constant iv infusion of
[35S]methionine and [15N]cysteine for
6 h. Cysteine enrichment is expressed in atom percent excess
(APE). Values are means ± SD.
|
|
Infection increased the ratio of [35S]cysteine to
[35S]methionine specific radioactivities measured either
in plasma or in liver, indicating an increased percentage of total
entry into plasma or liver cysteine pool coming from methionine by
transsulfuration or the contribution of transsulfuration to cysteine
flux (Table 4). Because the plasma cysteine flux was measured, it was
possible to calculate the amount of cysteine coming from methionine in plasma, i.e., transsulfuration, which was calculated to be 2.7 times
greater in infected rats than in pair-fed rats. The percentage of
methionine flux entering the transsulfuration pathway was more than
doubled in infected rats compared with pair-fed animals (Table 5). Infection caused an increase of the
amount of cysteine produced by protein breakdown and glutathione
catabolism and of the amount of cysteine used for catabolism and
glutathione synthesis (Table 5).
 |
DISCUSSION |
There have been few attempts to explore cysteine synthesis
directly and quantitatively in pathological states, and the present study was developed to address this problem. Some data suggested that
cysteine synthesis was impaired in rats after surgery. As the metabolic
demand for cysteine is high in injury because of increased glutathione
synthesis (15), this finding could lead to redefining the
dietary recommendations for critically ill patients.
Cysteine synthesis from methionine in vivo in rats is poorly
documented. Methionine cycle and transsulfuration were determined in
humans under several conditions by use of a constant intravenous infusion of doubly labeled methionine
([methyl-2H3]- and
[1-13C]methionine) (11, 13, 27, 28). In
these studies, methionine transsulfuration was determined indirectly by
methionine oxidation measured from the rate of
13CO2 appearance. In the present study, two
labeled amino acids ([35S]methionine and
[15N]cysteine) were used to quantify methionine
transsulfuration in septic rats and pair-fed rats, and transsulfuration
was measured directly from the rate of synthesis of
[35S]cysteine. The method used requires steady-state
conditions (24). Using [15N]cysteine
infusion, we showed that [15N]cysteine enrichment
plateaued in the plasma cysteine pool after 2 h of infusion
(15). In the present study, we verified that plateau was
maintained in plasma cysteine and methionine pools during the last hour
of a 6-h infusion (Fig. 1). The rate at which plateau is reached
depends on the size of the free pool of the tracee and the turnover
rate of the tissue, i.e., the fractional turnover rate of the tracee
(32). Because the fractional turnover rate of plasma
methionine is three times higher than that of cysteine, it could be
expected that plateau could be reached more rapidly for methionine than
for cysteine. Furthermore, it is likely that steady state in liver is
reached at about the same time as in plasma because of the high
turnover in this tissue (32).
In the present study, methionine transsulfuration accounted for only
3% of total plasma methionine flux and 1.6% of total plasma cysteine
flux in pair-fed rats (Tables 4 and 5). However, the percentage of
total entry into the cysteine pool coming from methionine was greater
in liver than in plasma, reflecting its main localization in liver.
However, the values in plasma seem low when compared with those
obtained in humans, which, in the postabsorptive state, were
15-22% of methionine flux (11, 13, 27, 28) and
5-7% of cysteine flux (11, 13). Several causes could
explain these discrepancies. The capacity for cysteine synthesis can be
lower in rats than in humans. The methods used are also different.
Measuring transsulfuration from the oxidation of methionine can
overestimate this pathway, because methionine is also metabolized via a
transamination-decarboxylation route. However, this route seems to be
of minor importance in humans (3). It is also possible that our experimental conditions produce lower values. The rats were
restricted in feed on the day before the metabolic study, which was
performed in the postabsorptive state. Furthermore, the rats received
no methionine during the infusion (radioactive methionine was given in
trace amounts). On the other hand, stable isotopes are never infused in
trace amounts, and the amount of labeled cysteine infused in this study
(6 mg) accounted for ~53% of the intake of cysteine on day
1. It is well known that cysteine has a sparing effect on
methionine by reduction of the transsulfuration pathway in rats
(9, 26). Therefore, our conditions would favor a low
transsulfuration rate.
Nevertheless, transsulfuration was measured under the same conditions
in the two groups of rats, and our results clearly show that the
synthesis of cysteine from methionine was higher in the infected
animals than in the pair-fed rats (Table 4). Similar data were obtained
in burn patients (35). However, the rate of cysteine
synthesis from methionine determined in isolated hepatocytes was
decreased after 3 days of stress induced by surgery in rats (30). This decrease was attributed to a decline of the
activity of liver cystathionase, which is the limiting enzyme in the
transsulfuration pathway. We report here that infection increased
cysteine synthesis, despite inhibiting cystathionase activity, by 17%
(Table 3). However, cystathionase activity was more reduced by surgical
stress (30), and Rao et al. (22) showed that
the production of cysteine was not significantly affected when the
cystathionase activity was inhibited up to 63%.
An accumulation of cystathionine was observed in plasma of premature
infants due to cystathionase deficiency (31) and in plasma
of rats treated with propargylglycine, a cystathionase inhibitor
(7). By contrast, plasma and liver cystathionine concentrations were higher in infected rats than in pair-fed rats (Table 3). These results are in agreement with those of Rao et al.
(22), who observed an accumulation of cystathionine at all levels of cystathionase inhibition, including those resulting in no
reduction of cysteine synthesis. Infection generally induced no
modification of total cysteine (free cysteine and cystine and protein-bound cysteine) and methionine concentrations in plasma and
liver (Tables 2, 3), as generally found for plasma free cysteine and
methionine in patients (2, 10, 14, 29). These results
suggest that there is no impairment of the cystathionine pathway.
Total methionine and cysteine fluxes were higher in infected rats than
in pair-fed rats (Table 4). Moreover, the methionine released from
protein, but not the rate of incorporation of methionine into proteins,
was significantly increased by infection (Table 5). These findings are
in general agreement with those observed in injured patients by use of
other amino acid tracers. Measurements of protein turnover in patients
usually reveal an increase in whole body protein breakdown, with little
or no increase in protein synthesis (1, 17, 18, 33),
although the increase in protein breakdown is always greater than that
in protein synthesis, leading to a negative nitrogen balance.
Cysteine flux was stimulated by infection more than methionine flux,
suggesting a preferential utilization of cysteine during sepsis (Table
4). If we assume that the amino acid composition of whole body proteins
is not modified by infection, the amount of cysteine used for protein
synthesis or produced from protein breakdown can be calculated from the
corresponding methionine data (Table 5). The part of cysteine flux not
explained by protein turnover, which corresponds mainly to glutathione
synthesis and cysteine catabolism, was greatly enhanced by infection in
both absolute and relative terms (Fig.
2). We have shown previously that
cysteine catabolism (sulfate production) was decreased 2 days after
infection in rats (16). Taken together, these results indicate that glutathione synthesis was increased 2 days after infection, which is in keeping with our previous results demonstrating that glutathione turnover was stimulated during the acute phase of
sepsis (15). Therefore, the increase of cysteine flux
observed in sepsis is probably determined by the stimulation of
glutathione turnover.

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Fig. 2.
Contribution of various pathways (%) to cysteine plasma
flux in infected and pair-fed rats. SCys, cysteine
incorporated into proteins; CCys, cysteine catabolism;
SGSH, cysteine incorporated into glutathione;
BCys, cysteine released from proteins; BGSH,
cysteine released from glutathione; TS, cysteine synthesis by
transsulfuration. See MATERIALS AND METHODS for description
of rat groups, treatments, and calculations.
|
|
In conclusion, this study has shown that cysteine kinetics are
profoundly altered by infection, with a decrease of the contribution of
protein turnover. Cysteine synthesis from methionine is increased during sepsis, both in absolute amounts and relative to methionine flux, in agreement with the results of Yu et al. (35) in
burned patients. However, Yu et al. observed an increased activity of the various components of the methionine cycle in these patients, i.e.,
transmethylation and remethylation, with a relative reduction of
homocysteine entering the transsulfuration pathway. This increased methionine cycle, probably due to increased methyl group transfer and
utilization, can indicate enhanced requirements of various compounds,
such as polyamines, choline, and carnitine. On the other hand, our
results show that infection greatly enhances cysteine demand for
glutathione synthesis (15). This increased utilization of
cysteine can promote increased cysteine synthesis from methionine. However, taken together, these data suggest that a competition can
exist at the homocysteine locus of methionine metabolism. Because
cysteine synthesis is probably not sufficient to respond to the
increased demand, a consequence would be additional depletion of body
proteins to provide limiting amino acids. Therefore, an exogenous
supply of cysteine could improve protein homeostasis and body defenses
in critically ill patients. In septic rats, we have shown beneficial
effects of cysteine supplementation on recovery, N balance, and muscle
protein stores (4). Further metabolic and nutritional
studies are needed to explore the importance of sulfur amino acid
requirements in human patients.
 |
ACKNOWLEDGEMENTS |
We thank G. Bayle for amino acid measurements, P. Capitan for gas
chromatography-mass spectrometry analysis, and D. Bonin and H. Lafarge
for literature management.
 |
FOOTNOTES |
This study was supported by the Institut National de la Recherche
Agronomique, France, and by Nestec, Switzerland.
Address for reprint requests and other correspondence: C. Obled, Laboratoire d'Etude du Métabolisme Azoté,
INRA, Clermont-Ferrand Theix, 63122 Saint Genès
Champanelle, France (E-mail: obled{at}clermont.inra.fr).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 20 April 2000; accepted in final form 24 August 2000.
 |
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