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*
Laboratoire de Biochimie, Biologie Moléculaire et Nutrition, EA2416 Faculté de Pharmacie et Centre de Recherche en Nutrition Humaine, 63001 Clermont-Ferrand, France and
Contseat Jeune Formation Institut National de la Santé et de la Recherche Médicale 95–09, Institut de Recherche su le Cancer de l'Appareil Digestif, 67091 Strasbourg Cedex, France and
**
INSERM U458, Hôpital Robert Debré, 75019 Paris, France.
1To whom correspondence and reprint requests should be addressed
 |
ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • rats • glycine • alanine • casein • endotoxemia
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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were the first to classify AA into two categories, nonessential
or essential, according to whether their carbon skeleton could or could
not be synthesized from other AA. This classification of AA has been
extensively refined over the years (Young and El-Khoury 1995
). Shortage of some AA may occur in special situations,
such as markedly increased demand or decreased synthesis, even though a
synthetic pathway has been demonstrated. This led to the concept of
conditionally essential AA, occurring during growth, trauma or
sepsis (Grimble 1993
). In addition, it has been shown
that some AA or peptides exhibit pharmacological properties when given
at doses far above the amounts supplied in an ordinary diet
(Grimble 1995
, Young and El-Khoury 1995
). However, to demonstrate their effects, these compounds
have to be compared to an isonitrogenous control, the choice of which
is delicate. Ideally, the nitrogenous control must be a "placebo,"
i.e., as inert as possible. However, the concept of inert nitrogen is
still largely controversial (Young and El-Khoury 1995
). A high dose of any nitrogenous compound is liable to
have pharmacological or deleterious effects that could invalidate the
study of the effectiveness of a pharmaconutrient. Mostly, nonessential
AA, such as glycine or alanine, or proteins, such as casein, are used
as isonitrogenous controls (Chen et al. 1994
,
Gianotti et al. 1993
and 1995
, Grimble et al. 1992
, Katayama et al. 1996
, Ziegler et al. 1992
). However, we are not aware of any work on the
rationale of such a choice.
To evaluate the potential metabolic and nutritional effects of the most
popular, currently used nitrogenous control compounds, a
well-defined experimental model that induces alterations in protein
metabolism is required. Animal and human studies have demonstrated that
a single dose of endotoxin evokes many signs of the response observed
in severe bacterial infection (Fink and Heard 1990
).
Also, sepsis leads to numerous metabolic disturbances, of which
alterations in AA and protein metabolisms are the most severe
(Jeevanandam 1995
).
Therefore, to define a rationale for the choice of an inert nitrogenous control, metabolic and nutritional effects of a diet enriched with glycine, alanine or casein were studied in young, endotoxemic rats. A control group receiving no supplement was also studied to determine whether the supplements had positive or deleterious effects.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Young male Sprague-Dawley rats (Iffa-Credo, L'Arbresle, France), weighing 65 ± 4 g, were housed in individual cages in an animal room maintained at 23°C with a 12 h light/12 h dark cycle (dark from 0800 h to 2000h). Before the experiments, the rats were acclimated for 1 wk with free access to powdered, nonpurified diet (AO3, UAR, Villemoisson-sur-Orge, France) (energy: 766 kJ/kg; proteins: 235 g/kg; lipids: 50 g/kg; carbohydrates: 498 g/kg; minerals, vitamins, fibers and water: 217 g/kg). The rats had free access to water throughout the experiments.
The laboratory was authorized by the French Ministry of Agriculture and Forestry to perform animal experiments, and the National Research Council Recommendations for the care and use of laboratory animals were followed.
Experimental design.
The experimental design follows an established model (Lasnier et al. 1996
). At the end of the acclimation period, the rats were
randomly assigned to four groups. Rats were weighed daily before
starting force feeding. All the rats were given at d 0 an
intraperitoneal injection of endotoxin [3 mg lipopolysaccharide
(LPS)/kg body weight] from E. coli (serotype 0127:B8,
Sigma, Saint-Quentin-Fallavier, France) in 9 g saline
buffer/L. After being food deprived for 24 h (d 0 to d 1),
the rats were enterally refed for 48 h (d 1 to d 3) 3 times per
day, with a liquid diet (Osmolite®, Abbott-Ross,
Rungis, France) (energy: 4.18 MJ/L, proteins: 35.2 g/L, lipids: 35 g/L,
carbohydrates: 136.5 g/L) as previously described (Lasnier et al. 1996
). Feeding was performed at the same time of day every
day. This hypocalorico-hyponitrogenous diet (878
kJ · kg-1 · d-1, 1.18 g
N · kg-1 · d-1) was administered without
supplement (LPS group, n = 6), or after
supplementation with 0.19 g
N · kg-1 · d-1 in the form of glycine (1
g · kg-1 · d-1, LPS-GLY group,
n = 6) or alanine (1.21
g · kg-1 · d-1, LPS-ALA group,
n = 7) or casein (1.33
g · kg-1 · d-1, LPS-CAS group,
n = 7). This dose of 0.19 g
N · kg-1 · d-1, equivalent to 1 g
GLY · kg-1 · d-1, corresponds to an average
dose commonly used in studies testing the effects of AA
supplementations (Ziegler et al. 1992
). This nitrogenous
supplementation represents 16% of total nitrogen intake. Glycine,
alanine and casein were from Sigma. At d 3 and 6 h after the last
gavage, the rats were killed by decapitation.
Blood samples, collected in heparinized tubes, were promptly spun, and the plasma deproteinized with sulfosalicylic acid (50 g/L). Supernatants were then stored at -80°C until AA analysis.
The abdominal cavity was opened, and the liver quickly removed. The small intestine, extending from the ligament of Treitz to the ileocecal junction, was promptly resected. The intestine was cut at its middle, thus affording two portions, the jejunum and ileum, which were flushed with ice-cold 9 g saline buffer/L. Two pieces (1 cm in length) were removed in the proximal part of the jejunum and ileum for histomorphometric examination. The following 20 cm of jejunum and ileum were divided into two equal segments, everted, and the mucosa scraped with a glass slide. The first 10 cm of jejunum and ileum were used for hydrolase activity determination and the second segment for protein and free AA analysis.
Two muscles from hindlimbs, soleus and extensor digitorum longus (EDL), were rapidly excised. All tissues were weighed and frozen in liquid nitrogen before storage at -80°C, until used.
Plasma amino acid assay.
Frozen plasma samples were analyzed for AA concentrations by ion-exchange chromatography with ninhydrin detection (Model 6300, Beckman Instruments, Palo Alto, CA) after dilution of the sample with a lithium citrate buffer (pH 2.2) containing D-glucosaminic acid and S2-amino-ethylcystein as external standards (Sigma).
Tissue amino acid and protein content determinations.
Frozen tissues (liver, muscles, intestinal mucosa) were pulverized and
homogenized in ice-cold 10 g trichloroacetic acid/L (1 mL/100
mg) by using an Ultra-Turrax T25 tissue disrupter (Ika
Labortechnik, Staufen, Germany). The acid soluble fraction containing
free AA was separated from the protein pellet by spinning. Free AA
concentrations were then measured by ion-exchange chromatography as
described above. The precipitate containing total proteins was
delipided with ethanol/ether (50/50) and dissolved in 1 N NaOH (4
mL/100 mg of tissue) for 12 h at 40°C. Total protein was then
assayed by Gornall's method as previously described (Fleury and Aberham 1951
).
Intestinal morphometry.
After fixation in Bouin's solution, the intestine pieces were dehydrated and embedded in paraffin. Sections (5 µm thick) were stained with hematoxylin and eosin. Villus height was taken as the distance from crypt-villus junction to villus tip, and crypt depth was the distance from crypt-villus junction to the bottom of the crypt. Villus height and crypt depth were measured with a semiautomatic image analyzer (Biocom®, Lyon, France). Total height was the sum of villus height and crypt depth.
Intestinal enzyme assay.
Enzyme assays were performed on frozen mucosa after dilution in a 20
mmol NaH2PO4- K2HPO4
buffer/L, pH 6.1. Sucrase, lactase and glucoamylase activities were
determined by Dahlquist's modified technique (Cézard et al. 1979
). Leucine aminopeptidase was measured as previously
described (Ahnen et al. 1982
). Enzyme activities were
expressed as total activities per segment (mU/cm).
Statistical analysis.
Results are expressed as mean ± SEM. Statistical analysis was performed with a one-way ANOVA followed by a Newman-Keüls test. The PCSM software package (Deltasoft, Meylan, France) was used. A P-value of <0.05 was considered as a significant difference.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Body weight.
At d 0, and before endotoxin injection, rat body weight was 113 ± 1 g (n = 26). Intraperitoneal injection of LPS and starvation significantly decreased body weight, with an average body weight loss of 9% at d 1 (body weight at d 1: 103 ± 1 g). At the end of the refeeding period (d 3), the body weight was also significantly decreased in all groups compared to d 1, but with no significant difference caused by supplementation (body weight at d3: LPS: 97 ± 2 g; LPS-GLY: 100 ± 4 g; LPS-ALA: 101 ± 2 g; LPS-CAS: 96 ± 1 g).
Tissue weight and protein content.
The supplemented diets did not affect tissue weight compared to LPS-treated rats refed with Osmolite® only (data not shown). No significant variation in total protein content occurred among groups in any of the tissues (data not shown).
Intestinal morphometry and hydrolase activities.
An intestinal histomorphometric study showed no significant difference
among groups in the jejunum or ileum (data not shown). In jejunum,
sucrase activity was greater in LPS-ALA and LPS-CAS groups
compared to LPS and LPS-GLY groups (Table 1
). Lactase activity did not significantly differ among any of the
groups. Lower glucoamylase activity was observed in the LPS-GLY
group than in the LPS group. The LPS-CAS group had greater
glucoamylase activity than did the LPS-GLY and LPS-ALA groups.
The total activity of aminopeptidase was significantly lower in the
LPS-GLY and LPS-ALA groups compared to that in the LPS and
LPS-CAS groups. In ileum, no significant variation of any of the
enzyme activities was observed (data not shown).
|
Significant effects of glycine, alanine and casein on plasma
and tissue-free AA concentrations are presented in Table 2
, except for the plasma and tissue glycine concentrations depicted in
Figure 1
. Other AA concentrations were not significantly different among groups
and so are not presented. In plasma and tissues, the LPS-GLY group
had a greater free glycine concentration than did the other three
groups. Plasma serine concentration was significantly greater in the
LPS-GLY group compared to that in the LPS-CAS group. In
soleus, the LPS-GLY group had a greater serine
concentration than did the LPS group. In EDL, the alanine
concentration was lower in the LPS-GLY group compared to that in
the other groups, and glutamine concentration was lower in the
LPS-GLY group than that in the LPS-ALA and LPS-CAS groups.
Hepatic glutamine concentration was significantly lower in the
LPS-GLY group compared to that in the other three; in ileum, the
alanine concentration was significantly lower in the LPS-CAS group
than it was in the other groups. Plasma and tissue-free AA levels
were not significantly different between the LPS-ALA group and the
LPS group. The LPS-CAS group differed from the other groups in
ileal alanine.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). It
closely mimics the changes encountered in septic animals and induces
anorexia and numerous metabolic alterations associated with
profound changes in whole-body AA metabolism (Fink and Heard 1990
, Garcia-Martinez et al. 1993
). The dose of
LPS to be injected has to be high enough to cause a measurable response
but low enough not to cause death. In the present study, we used a dose
of 3 mg/kg, as commonly described in the literature (Farges et al. 1996
, Jepson and Millward 1989
and 1991
,
Lasnier et al. 1996
). Because rats are very resistant to
endotoxemia, they were food deprived for 24 h prior to enteral
refeeding to exacerbate the catabolic response (Jepson et al. 1986
, Lasnier et al. 1996
). We observed that LPS
injection and starvation induced a rapid, marked body weight loss (9%
at d 1). Thereafter, rats were fed by enteral gavage with a liquid
diet, assuring that each rat received exactly the same quantity of diet
per unit of body weight. Rats were thus refed for 48 h with a
hypocaloric hyponitrogenous diet (Osmolite®) so
that the deleterious effects of endotoxemia were not fully corrected
and the potential effects of nitrogenous supplementation could be
easily detected. Under these conditions, the rats continued to lose
weight (total body weight loss: -13%). None of the nitrogenous
supplementations limited this loss.
In our endotoxemic rat model, none of the three molecules under study
(glycine, alanine and casein) improved tissue weights or tissue protein
contents compared to Osmolite® alone, thereby
suggesting that they could be considered inert with regard to these
variables. At the same level of supplementation, pharmaconutrients,
such as glutamine or ornithine 
-ketoglutarate, improve nitrogen
metabolism (Ziegler et al. 1992
).
Disturbed plasma and tissue AA patterns after LPS administration
were also extensively described (Jeevanandam 1995
).
However, only slight modifications in plasma and tissue free AA levels
were observed between our four LPS-treated groups. The main effect
of glycine supplementation concerned plasma and tissue glycine and
serine concentrations. Glycine concentrations in muscles and plasma
were twice as high in the LPS-GLY group as in the other groups and
were also markedly increased in splanchnic tissues. A massive
accumulation (sixfold increase) of glycine in blood was previously
described in healthy and LPS-treated rats after they were fed a
glycine-enriched (5%) diet (Ikejima et al. 1996
).
Our results reflect a net accumulation of this AA, probably resulting
from its limited metabolism. A higher serine concentration in the
soleus and a tendency for a greater concentration in plasma of the
LPS-GLY group compared to the LPS group may be related to
the interconversion of glycine to serine by serine
hydroxymethyltransferase (Jackson 1991
, Yoshida and Kikuchi 1970
). Alanine is the main substrate for
gluconeogenesis in the liver and that gluconeogenesis from alanine is
increased in septic patients compared to fed or fasted controls
(Young and El-Khoury 1995
). Despite an increased
supply of alanine, concentrations of this AA in blood and tissues were
not modified in the LPS-ALA group compared to the LPS group,
suggesting that alanine is readily metabolized, probably by entry in
gluconeogenesis as shown in septic patients (Jeevanandam 1995
). Supplementation of the diet with casein induced only
very limited modifications in free AA levels (a decrease of alanine in
the ileum).
Sepsis induces major impairments in the structure and functions of the
gastrointestinal tract, and in particular alters mucosal enzyme
activities (Gardiner and Barbul 1993
). Of the three
compounds we tested, none affected either jejunal or ileal
trophicity. It has been shown that after gastric infusion,
glycine does not affect, in terms of mucosa weight and protein and DNA
contents, the intestinal mucosa of rats fed parenterally
(Spector et al. 1981
). Several studies have compared the
effects of casein or casein hydrolysate on nutritional recovery and
intestine function after a stress, such as severe starvation or
intestinal resection (Boza et al. 1995
, Ribeiro et al. 1998
, Sales et al. 1995
). These studies
demonstrated that both casein and degraded casein improve intestine
trophicity in stressed rats (Sales et al. 1995
). In our
study, we found no trophic effect of casein, suggesting that under
these conditions, casein is inert like glycine and alanine. Refeeding
after starvation was shown to produce quick repair of enterocyte
atrophy and rapid restoration of brush border enzyme activities
(Poullain et al. 1989
). These responses depend on the
amount of food intake (proteins and energy) and also, for the brush
border enzyme activities, on the quantities of their specific
stimulatory substrates (Poullain et al. 1989
). After
2 d of refeeding endotoxemic rats, we observed only small
variations in jejunum trophicity in terms of sucrase, glucoamylase and
aminopeptidase activities. On the other hand, disaccharidase and
aminopeptidase activities were not significantly altered in the ileum.
This discrepancy between the jejunum and ileum may be related to the
prominence of substrate-dependent stimulation of hydrolase
activity in the jejunum (Holt and Kotler 1987
,
Williamson and Chir 1978
). Although all the groups
received the same quantity of Osmolite®, supplementation
of this diet with AA, such as glycine and alanine, partly inhibited
jejunal aminopeptidase activity. These effects of nitrogen supplements
on jejunal disaccharidase activities are difficult to explain because
the carbohydrate moiety of the diet was the same in all the groups.
None of the nitrogenous compounds we tested, representing a dietary supplementation of 16% total nitrogen intake, modified body and tissue weights, tissue protein contents or intestinal trophicity in endotoxemic rats. Alanine and casein induced only minor variations in free AA patterns and intestine enzyme activities, although larger effects were observed after glycine supplementation. Hence, glycine as a nitrogenous control should be used with care. On the other hand, alanine and casein, having no marked metabolic or nutritional effects in endotoxemic rats, can be considered inert. We conclude that either alanine or casein may be used as "placebo," with the choice depending on the study to be performed.
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FOOTNOTES |
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3 Abbreviations used: AA, amino acids; EDL,
extensor digitorum longus; LPS, lipopolysaccharide; GLY, glycine;
ALA, alanine; CAS, casein
Manuscript received September 30, 1998. Initial review completed February 16, 1999. Revision accepted June 11, 1999.
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