Vol. 273, Issue 6, E1052-E1058, December 1997
Leucine metabolism in TNF-
- and endotoxin-treated rats:
contribution of hepatic tissue
M.
Hole
ek1,
L.
prongl2,
F.
Skopec3,
C.
Andrýs4, and
M.
Pecka5
Departments of 1 Physiology,
4 Immunology,
5 Medicine, and
3 Radioisotope Laboratory,
Charles University School of Medicine, 500 01 Hradec
Králové; and
2 University Hospital Motol, 150 00 Prague, Czech Republic
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ABSTRACT |
The effects of tumor necrosis factor-
(TNF-; cachectin) and lipopolysaccharide of
Salmonella
enteritidis (LPS; endotoxin) on
leucine metabolism in rats were evaluated in the whole body using
intravenous infusion of
L-[1-14C]leucine
and in isolated perfused liver (IPL) using the single-pass perfusion
technique with
-keto[1-14C]isocaproate
as a tracer for measurement of ketoisocaproic acid (KIC) oxidation, and
the recirculation technique for measurement of hepatic amino acid
exchanges. The data obtained in TNF- and LPS groups were compared
with those obtained in controls. Both TNF- and LPS treatment induced
an increase of whole body leucine turnover, oxidation, and clearance.
As the result of a higher increase of leucine oxidation than of
incorporation into the pool of body proteins, the fractional oxidation
of leucine was increased. The fractional rate of protein synthesis
increased significantly in the spleen (both in TNF- and LPS rats),
in blood plasma, liver, colon, kidneys, gastrocnemius muscle (in LPS
rats), and in lungs (TNF--treated rats), whereas it decreased in the
jejunum (LPS rats). In IPL of TNF-- and LPS-treated rats a decrease
of KIC oxidation and higher uptake of branched-chain amino acids (BCAA; valine, leucine, and isoleucine) were observed when compared with control animals. We hypothesize that the negative consequences of
increased whole body proteolysis and of increased oxidation of BCAA
induced by TNF- and/or LPS are reduced by decreased activity of hepatic branched-chain ketoacid dehydrogenase that can help resupply
BCAA to the body.
cytokines; liver; ketoisocaproate; protein metabolism; branched-chain ketoacid dehydrogenase; systemic inflammatory response
syndrome
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INTRODUCTION |
CRITICAL ILLNESS is often associated with
skeletal muscle proteolysis, increased muscle amino acid oxidation, and
release of amino acids into the bloodstream. This results from the
systemic inflammatory response syndrome (SIRS), which in turn is a
result of the host response to severe infection, trauma, thermal
injury, and cancer (10, 27). The mobilized amino acids are extracted by
many tissues, particularly by the liver, where they are used for
gluconeogenesis and for the production of acute-phase proteins. The
principal mediators of these reactions are cytokines, particularly tumor necrosis factor- (TNF-; cachectin) (2). The synthesis of
cytokines may be stimulated experimentally by administration of
bacterial lipopolysaccharides or their toxins. However, the precise
metabolic role and physiological significance of cytokines are not
completely understood. This shortcoming in our knowledge prevents the
use of cytokines and cytokine agonists or antagonists as therapeutic
tools in clinical practice.
The effects of cytokines on the metabolism of branched-chain amino
acids (BCAA) leucine, isoleucine, and valine are of special interest
because these amino acids have a crucial role in skeletal muscle
protein metabolism and because the BCAA have a beneficial effect in the
treatment of sepsis or trauma (6, 18). The oxidation of BCAA usually
involves more than one tissue. The first step is reversible
transamination catalyzed by BCAA aminotransferase. The formed
corresponding branched-chain keto acids (BCKA) are then the substrates
for the irreversible oxidative decarboxylation by BCKA dehydrogenase.
Because of the high level of transaminase and the low level of BCKA
dehydrogenase, skeletal muscle releases a significant amount of BCKA
into the circulation (9, 20). These BCKA are oxidized in tissues with
high BCKA dehydrogenase activity, primarily in liver and adipose tissue
(25), and/or used as substrates for the resynthesis of BCAA.
There are a number of controversial and unsolved issues related to the
effect of endotoxin and cytokines, especially TNF- on protein and
BCAA metabolism. The administration of TNF- has been associated with
increased muscle proteolysis and activated BCKA dehydrogenase activity
(4, 19). On the contrary, in other studies, TNF- did not increase
muscle protein breakdown (8) and it has been suggested that protein
wasting in SIRS could be accounted for by anorexia (16). Another
unsolved problem is alteration of BCAA and protein metabolism in
visceral tissues, especially in liver. The infusion of TNF- has been
associated with reduced proteolysis in hepatic tissue (4) that is
consistent with the gain in liver mass observed in SIRS. However, there
are no data about changes in hepatic oxidation of BCAA. The main
purpose of this study was to estimate the hepatic contribution to
changes of whole body leucine metabolism after endotoxin or TNF-
treatment in rats.
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METHODS |
Animals.
Male Wistar rats weighing 210-240 g were obtained from Velaz,
Prague, CR. Rats were housed in standardized cages in quarters with a
controlled temperature and a 12:12-h light-dark cycle and received
Velaz-Altromin 1320 (Velaz) laboratory chow and drinking water ad
libitum. All procedures involving animals were performed according to
the guidelines set by the Institutional Animal Use and Care Committee
of Charles University.
Materials.
L-[1-14C]leucine
and
-keto[1-14C]isocaproate
were obtained from Amersham International (Buckinghamshire, UK),
[14C]bicarbonate was
purchased from Du Pont-NEN (Bad Homburg, Germany). Leucine, sodium salt
of -ketoisocaproic acid (KIC), lipopolysaccharide from
Salmonella enteritidis (LPS;
endotoxin), methylbenzethonium hydroxide, Folin and Ciocalteu's phenol
reagent, and bovine serum albumin were purchased from Sigma Chemical
(St. Louis, MO). Amino acid solution AMINO-MEL, 10% pure, was obtained
from Leopold Pharma GesmbH (Graz, Austria). Human recombinant TNF-
was purchased from Promega (Madison, WI). Mouse and human TNF-
enzyme-linked immunosorbent assay (ELISA) kits were obtained from
Genzyme (Cambridge, MA). We used a mouse TNF- ELISA kit, which is
specific also for natural rat TNF- for determination of endogenous
TNF- production, and a human TNF- ELISA kit for determination of
changes in levels of administered human TNF-. The remaining
chemicals were obtained from Lachema (Brno, CR). The radioactivity of
the samples was measured with the liquid scintillation radioactivity
counter LS 6000 (Beckman Instruments, Fullerton, CA). Amino acid
concentrations in deproteinized samples of blood plasma or tissues were
determined with high-performance liquid chromatography (Waters) after
precolumn derivatization with
o-phthaldialdehyde.
Experimental design.
Experiments were started between 7 and 8 AM to minimize the influence
of diurnal variations of food intake and plasma hormone levels. To
exclude nutritional effects, all rats were fasted overnight before the
experiment. The parameters of leucine metabolism in whole body and in
the isolated perfused liver (IPL) were evaluated in two separate
studies.
Study 1: Effect of TNF- and LPS on whole body
leucine metabolism and protein synthesis in specific tissues.
The parameters of whole body leucine metabolism were evaluated
essentially by the procedure as described in detail previously (11). A
polyethylene cannula was inserted into the jugular vein under light
diethyl ether narcosis. This procedure is very easy, gentle, and rapid,
and the physical condition of these animals is very good immediately
after awakening from anesthesia. We suppose that all animals
experienced similar stress stimuli before the examination of changes in
whole body leucine metabolism. Each animal was then placed in a glass
metabolic cage to enable the collection of expired air and infused with
L-[1-14C]leucine
solution (control) or with
L-[1-14C]leucine
solution containing TNF- (10 µg/kg body wt) or LPS (2 mg/kg body
wt). A priming dose of 0.7 ml (i.e., 1.4 µCi of L-[1-14C]leucine and 17% of the
dose of TNF- or LPS) infused over a 30-s period was followed by a
constant infusion at a rate 0.36 ml/h (0.72 µCi/h) for 200 min. The
expired CO2 was trapped at 10-min
intervals between 125th and 185th min of infusion by monoethanolamine. The average value of six measurements of
14CO2
radioactivity in expired air at steady-state condition (see Fig.
1) was used for calculations of leucine
oxidation rate. In the NaH14CO3 infusion
experiments, the
14CO2
recovery factor (FR) of LPS and control animals was the same (we did
not perform recovery study using TNF-). The same
CO2 FR was used for control and
TNF--treated rats by others (4). Thus the same FR (0.9) has been
used for all three groups in our study. The blood plasma leucine
specific activity was measured in the blood collected at termination of
the infusion. The rats were killed by exsanguination via the abdominal
aorta exactly at the 201st min from the beginning of the
L-[1-14C]leucine
infusion. Leucine specific activity
(SALeu), turnover (QLeu), clearance
(CLeu), and decarboxylation
(DLeu) rates were calculated by
the following formulas
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where
dpm is disintegrations per minute. Whole body leucine metabolism was
considered to take place within a common metabolic pool, represented by
free plasma leucine. Leucine leaves the pool and is either incorporated
into protein (In) or oxidized (D). Leucine enters the pool from protein
breakdown (B) or exogenous dietary sources (E). Due to the fact that
exogenous leucine intake was zero in our protocol, leucine turnover (Q)
estimates the leucine released from protein, i.e., the protein
breakdown. These parameters of leucine metabolism are described by the
following equation
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With the use
of this formula, rates of leucine incorporation into protein, the
oxidized fraction of leucine, and the fraction of leucine incorporated
into protein were calculated.
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Fig. 1.
Recovery of labeled CO2 at expired
air of rats during continuous infusion of
L-[1-14C]leucine
solution without (Control) or with tumor necrosis factor- (TNF-;
10 µg/kg body wt) or lipopolysaccharide (LPS; 2 mg/kg body wt); dpm,
disintegrations/minute. Values are means ± SE. Significance from
Control: * P < 0.1,  P < 0.05,  P < 0.01.
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It has been suggested that blood plasma specific activity of KIC
instead of leucine is more relevant for calculating the rate of leucine
oxidation (15). However, it has been shown that the ratio of KIC to
leucine specific activity is constant after a number of experimental
maneuvers, and no qualitative differences were observed whether the KIC
or leucine specific activities were used to calculate parameters of
leucine metabolism (23). The similar changes in leucine and KIC
enrichments were demonstrated also after LPS treatment (5). In
addition, the measurements of leucine enrichments were more
reproducible than those of KIC, probably because KIC is very unstable,
in our conditions. Thus we used the specific activity of leucine for
the calculation of parameters of leucine metabolism as was also done by
others (13).
For the measurement of the fractional protein synthesis rate, samples
of blood plasma, liver, spleen, kidney, small intestine, large
intestine, lungs, gastrocnemius muscle, and heart were quickly removed
and immediately frozen in liquid nitrogen after the animals were
killed. Small pieces of tissue (about 0.5 g) were rinsed and
homogenized in perchloric acid (2%). The precipitated proteins were
collected by centrifugation. The supernatant was used for the
measurement of
L-[1-14C]leucine
radioactivity and leucine concentration. Free
L-[1-14C]leucine
specific activity was determined after treatment with 30% hydrogen
peroxide, which causes the carboxyl carbon of KIC to be released as
CO2. The pellet was washed three
times and then hydrolyzed in 2 N NaOH. Aliquots were taken for protein
analysis (14) and radioactivity measurement. The fractional protein
synthesis rates were calculated by using the equation derived by
Garlick et al. (7)
where
Sb and
Si are the specific activities of
the protein-bound and free acid-soluble tissue leucine pools,
respectively, in disintegrations per minute per micromole;
i is the rate constant for the
rate of rise of specific activity of leucine in the acid-soluble amino
acid pool per day; t is the duration
of
L-[1-14C]leucine
infusion in days; and Ks is the
the fraction of protein mass renewed each day, in percent per day. The
value of 38/day was taken to represent
i in different tissues of rats
under different treatments on the basis of literature. Accurate
determination of the value of i
is unnecessary, and the approximate values are adequate, since quite
large variations in the value of
i result in the small
variations in the value of Ks.
Study 2: Effect of TNF- and LPS on KIC oxidation
and amino acid utilization by the IPL.
The overnight-fasted rats were anesthetized with pentobarbital sodium
(35 mg/kg body wt ip), and the livers were prepared for perfusion as
described recently (11). Briefly, after laparotomy, the bile duct was
cannulated and 1,000 IU/kg of heparin were injected into the saphenous
vein. Then the portal vein was cannulated with a polyethylene catheter
(ID 1.5 mm), and the hepatic artery was ligated. During portal
perfusion with Krebs-Henseleit solution (20°C), the liver was
quickly removed. The perfusate was Krebs-Henseleit bicarbonate buffer
containing amino acids (996 ml of Krebs-Henseleit solution mixed with 4 ml of AMINO-MEL, 10% pure; for concentration of individual amino acids
in perfusion medium see the 1st column of Table 6), 1 mM KIC, and
tracer amounts of [1-14C]KIC, pH 7.4, saturated
with an
O2-CO2
mixture (95%-5%). The perfusion was carried out at 37°C in a
thermostatically controlled cabinet. A peristaltic pump took the
perfusate from the reservoir through an oxygenator and a bubble trap to
the liver. The membrane oxygenator was made from thin-walled silicone
tubing (Silastic; ID 0.058 in., OD 0.077 in.) 25 ft in length, enclosed
in a glass cylinder continually gassed with a mixture of
O2 and
CO2 (95%-5%) at a flow rate of
500 ml/min. The glass bubble trap also served as a peristaltic wave
compensator. Flow rates were maintained at 3.5 ml · g
liver
1 · min1.
Viability of the perfused livers was monitored by their appearance and
by the stability of the bile flow. Before the start of the experimental
protocol, livers were perfused with a tracer-free perfusion medium for
a period of 15 min to ensure stabilization of the liver and washout of
endogenous hormones. At the 16th min, the perfusion medium containing a
tracer was infused for 15 min (single pass no. 1), and samples of the
effluent perfusate were collected in 20-ml flasks equipped with
stoppers and center wells containing 0.4 ml of methylbenzethonium
hydroxide at 1-min intervals to monitor
14CO2
production (21). Labeled carbon dioxide in the perfusate, which was
produced from the infused [1-14C]KIC, was
released by injection of 0.5 ml of 5 N sulfuric acid through the
stopper into the flasks. Metabolic flux through the BCKA dehydrogenase
was calculated using the specific activity of the KIC in perfusion
solution,
14CO2
production rate, the flow rate, and the liver weight and expressed as
micromoles of substrate oxidized per gram of dry liver weight per hour.
The procedure of measurement of KIC oxidation by IPL was followed at
the 31st min by recirculation with solution containing TNF- (1.5 µg/200 ml) or LPS (0.2 mg/200 ml) or with solution without these
substances (control), for 90 min. Samples of perfusion solution were
collected at 30-min intervals for measurements of TNF- and enzyme
concentrations. Changes of perfusate amino acid concentrations were
measured at the end of the recirculation phase of the experiment. Amino
acid exchanges were calculated as follows
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where
Ct90 and
Ct0 are
amino acid concentrations at the end and at the beginning of perfusion,
V is the total volume of perfusate in liters (corrected for the sample removal), t is the duration of
recirculation in hours and Wtdry is the liver dry weight in grams. Results are expressed as micromoles per grams dry liver per hour. Negative values mean net amino acid uptake; positive values indicate net release. At the 121st min, the
influence of TNF- and endotoxin on KIC oxidation in IPL were evaluated using a single-pass technique (single pass no. 2), as described above. In the separate IPL study. the labeled
CO2 recovery was determined using
[14C]bicarbonate on
three animals. The
14CO2
recovery was 97.8 ± 0.8% before (single pass no. 1) and 97.0 ± 3.3% after recirculation with perfusion solution containing LPS
(single pass no. 2). The same correction factor (0.97) for each group
of rats was used on the basis of the assumption that the effect of LPS
on IPL is provided in part by TNF- and the observations of
negligible effect of reincorporation of
14CO2
in the perfused liver and insignificant effect of LPS on
14CO2
recovery.
The main reason for using KIC as a substrate instead of leucine for
evaluation of the position of liver in leucine oxidation by the IPL
system was based on the fact that BCAA transamination is reversible and
that the substrate of the first irreversible reaction in leucine
oxidation is KIC. Because the direction and the rate of transamination
are regulated significantly by concentrations of substrates (28), the
results obtained by IPL model using leucine as a substrate cannot give
a clear idea about the situation in the whole body, and the
interpretation of the results may be misleading. The ability of the
liver to reaminate BCKA was demonstrated by several investigators (1,
3). In addition, it should be mentioned that a significant amount of
KIC is delivered into the liver from peripheral tissues, primarily from
muscle (9). The evaluation of CO2
release using labeled KIC indicates changes in BCKA dehydrogenase
activity, i.e., the first irreversible step in BCAA degradation.
Statistical analysis.
Results are expressed as the means ± SE. Data analysis was
performed by one-way analysis of variance. For pairwise comparison of
means with control Bonferroni method (procedure P7M) was used with
restriction for two comparisons (control vs. TNF and control vs. LPS).
The effects of TNF- and LPS on KIC oxidation by IPL were evaluated
by the F-test and then by Student's
t-test for paired data.
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RESULTS |
Leucine metabolism in vivo (Study 1).
With the use of a mouse TNF- ELISA kit an increase of rat TNF-
was observed in blood plasma of LPS-infused rats and insignificant changes of rat TNF- concentrations were detected in rats treated by
human TNF-. Insignificant differences in red blood cell number, hematocrit, and leukocyte number were observed between experimental and
control animals. However, LPS treatment resulted in a decrease in the
platelet number in the blood caused probably by disseminated intravascular coagulation. The decrease in the platelet number in the
TNF--treated group was insignificant (Table
1).
We did not observe a conspicuous effect of TNF- and/or LPS
infusion on blood plasma amino acid levels in this study (Table 2). Nevertheless, it should be noted that
some amino acids decreased or had a tendency to decrease (glutamine and
threonine), whereas other amino acids had a tendency to increase
(tryptophan and ornithine).
Both TNF- and endotoxin treatment induced an increase of leucine
turnover, oxidation, and clearance (Table
3). However, the increase of leucine
incorporation into the pool of body proteins was insignificant. These
changes caused a marked increase of leucine oxidized fraction and a
decrease of leucine fraction incorporated into proteins.
The fractional rates of protein synthesis (Fig.
2) of TNF-- and LPS-treated animals
increased significantly in the spleen (both in TNF- and LPS rats),
blood plasma, liver, colon, kidneys, gastrocnemius muscle (LPS rats),
and in the lungs (TNF--treated rats), whereas it decreased in the
jejunum (LPS rats).
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Fig. 2.
Fractional rate of protein synthesis in various tissues of rats infused
with
L-[1-14C]leucine
solution without (Control) or with TNF- (10 µg/kg body wt) or LPS
(2 mg/kg body wt). Results are given in %/day, i.e., proportion of
protein mass that is replaced each day. Values are means ± SE.
Significance from Control: * P < 0.1, P < 0.05, and P < 0.01.
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KIC and leucine metabolism in isolated perfused liver
(Study 2).
Neither TNF- nor LPS affected the liver viability assessed by bile
production, liver dry-to-wet weight ratios, or by the enzyme release
(Table 4). In both TNF- and LPS groups a
decrease of KIC decarboxylation was observed (Table
5). In the TNF--treated animals a
significantly higher uptake of BCAA was observed during the
recirculation phase of this experiment. In the LPS group, there was a
trend, although not statistically significant, for higher uptake of
BCAA, also (Table 6).
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DISCUSSION |
In the first study we observed an increase of whole body leucine
turnover, indicating increased protein breakdown, an increase of
leucine oxidation, and an increase of oxidized leucine fraction in LPS-
and TNF--treated rats in a postabsorptive state. Because the
physiological role and the catabolic pathways of the BCAA have several
features in common (9), the observed changes in leucine metabolism
indicate similar perturbations in the metabolism of all three BCAA. The
tissue substantially responsible for the increase of whole body leucine
oxidation is undoubtedly skeletal muscle. A significant increase of the
activity of skeletal muscle BCKA dehydrogenase, the rate-limiting
enzyme for BCAA oxidation in skeletal muscle, was observed after both
TNF- and LPS treatments (4, 19, 26, 30). The increased BCAA
oxidation in skeletal muscle is associated with the release of alanine
and glutamine into the blood stream. This response to trauma or
infection is undoubtedly beneficial for the body because alanine is an
important substrate for gluconeogenesis and glutamine is used at a very high rate by the intestine and cells of the immune system. An increased
demand of the body for glutamine is demonstrated by the decreased
glutamine plasma level in LPS-treated rats in this study. Because the
administration of LPS elicits increased plasma concentrations of
various cytokines, some metabolic changes may differ from those
observed after administration of TNF- alone (e.g., differences in
plasma taurine and alanine levels).
The increase of BCAA oxidation in skeletal muscle is probably one of
the mechanisms decreasing BCAA levels in SIRS (29). However, it should
be noted that unchanged and even elevated plasma BCAA levels have also
been demonstrated in septic patients (24) and that the decrease of
blood plasma BCAA levels was not significant in this study. These
different patterns in plasma BCAA levels are caused mainly by changes
in food intake, proteolysis, protein synthesis, and BCAA oxidation.
Increased proteolysis, BCAA oxidation, and protein synthesis were
demonstrated in our study. Because the activated proteolysis increases,
whereas activated protein synthesis and oxidation decrease BCAA levels,
the decreased, unchanged, and increased levels of BCAA may be observed
as a result of small changes in proportions between proteolysis and
oxidation and/or protein synthesis.
In consideration of the pivotal position of BCAA in the metabolism of
proteins, the decreased food intake that is common in SIRS plus
increased BCAA oxidized fraction may result in the lack of these
essential amino acids. These circumstances may cause the significant
disturbances in protein homeostasis that are involved in the wasting of
skeletal muscle tissue. Thus the excessive oxidation of BCAA by
skeletal muscle may be disastrous if it lasts a long time.
The increased protein synthesis rates in many tissues of LPS- and
TNF--treated animals associated with accelerated whole body protein
breakdown demonstrate a rapid response of the body to altered
circumstances. The obvious advantage of this recycling of nitrogen is
increased synthesis of acute-phase proteins in hepatic tissue, as
indirectly demonstrated by the increased synthesis of plasma proteins
in this study. The jejunum was the only tissue in which a decrease in
protein synthesis was observed. This finding is in good agreement with
observations of gut atrophy in SIRS and lower weight of the
gastrointestinal tract of rats continuously infused with TNF- for 10 days (12). The decrease of protein synthesis in small intestine is
probably the main cause of insignificant changes in incorporation of
leucine into the pool of whole body proteins in TNF-- and
LPS-infused rats.
The principal new finding of this study is the observation of the
decreased rate of KIC oxidation, indicating decreased hepatic BCKA
dehydrogenase activity, in the IPL of TNF-- and LPS-treated rats.
Because the transamination of BCAA is reversible and the ability to
reaminate BCKA was demonstrated both in liver and other tissues (1, 3),
the decrease of hepatic BCKA decarboxylation should result both in
decreased hepatic leucine oxidation and in increased resynthesis of
BCAA from BCKA. Unfortunately, it is not known which tissues have a
crucial role in resynthesis of BCAA in SIRS. The lower activity of BCKA
dehydrogenase may explain also an increase of hepatic venous level of
KIC in humans after endotoxin administration observed by Fong et al.
(5).
With consideration that SIRS is usually associated with anorexia, the
resynthesis of BCAA can be one of the mechanisms preventing the rapid
development of negative nitrogen balance. Increased uptake of BCAA by
IPL and increased incorporation of
[14C]leucine into
plasma proteins observed in vivo caused by TNF- indicate the higher
hepatic utilization of BCAA in protein synthesis. It was demonstrated
that the synthesis of some proteins (e.g., albumin) is reduced in
sepsis by >50% (22), whereas the synthesis of some acute-phase
proteins, such as C-reactive protein and serum amyloid, is activated
(27). Unfortunately, we did not measure the synthesis of particular
types of proteins in this study. The increased rates of protein
synthesis indicating the higher utilization of BCAA have been observed
also in the colon, kidneys, spleen, and even in skeletal muscle.
In conclusion, the increase of leucine turnover, clearance, oxidation,
and oxidized fraction in TNF-- and LPS-treated rats exhibits the
significant perturbation of protein metabolism that is involved in
muscle wasting in SIRS. We hypothesize that the observed decrease of
KIC oxidation by IPL demonstrates an important adaptive response of the
body that can resupply essential BCAA to the body.
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ACKNOWLEDGEMENTS |
We are grateful for the technical support of I. Altmannová,
J. Hofmanová, L. Kriesfalusyová, H. Mancová, R. Ry
avá, I. pri
arová,and H. Weisbauerová. Many thanks for help in revision of the English to
Karl Wagner.
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FOOTNOTES |
This study was supported by a grant from the Grant Agency of the Czech
Republic (no. 306-94-1873), by a grant from the Internal Grant Agency of Ministry of Health of the Czech Republic (no. 3772-3), by a grant from Charles University (no. 258), and by a
grant from the European Society of Parenteral and Enteral Nutrition.
Address for reprint requests: M. Holeek, Dept. of Physiology,
Charles Univ. School of Medicine, imkova 870, 500 01 Hradec
Králové, Czech Republic.
Received 18 February 1997; accepted in final form 8 August 1997.
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Abstract
Introduction
