Effect of alanyl-glutamine on leucine and protein metabolism in irradiated rats.

The mechanism by which glutamine produces a favorable effect in the treatment of sepsis, injury, burns and abdominal irradiation is not completely understood. The main aim of this study was to evaluate the effect of alanyl-glutamine (AlaGln) administration on the metabolism of proteins in irradiated rats. The rats were exposed to whole-body irradiation (8Gy) and then fed intragastrically with a mixture of glucose and amino acids either with AlaGln or without AlaGln. At 48 hours after irradiation, parameters of whole-body protein metabolism and DNA synthesis in intestinal mucosa were investigated using a primed, continuous infusion of [1-14C]leucine and [3H]thymidine. In addition, we evaluated the effect of irradiation and AlaGln on gut morphology, blood count and amino acid concentrations in blood plasma and skeletal muscle. Control rats were not irradiated but were given identical treatment. An increase in whole-body leucine oxidation, and insignificant changes in whole-body proteolysis and in protein synthesis were observed after irradiation. In irradiated rats we observed a decrease in muscle glutamine concentration, a decrease in protein synthesis in jejunum, colon and heart, and an increase in synthesis of proteins of blood plasma and spleen. Morphological examination and measurement of DNA synthesis failed to demonstrate any favorable effect of AlaGln supplementation on irradiated gut. However, administration of AlaGln resulted in a decrease in whole-body proteolysis and leucine oxidation which caused an increase in the fraction of leucine incorporated into the pool of body proteins. We conclude that the data obtained demonstrate that irradiation induces metabolic derangement associated with increased oxidation of essential branched-chain amino acids (valine, leucine and isoleucine) and that these disturbances can be ameliorated by administration of AlaGln.

The long-term changes in liver DNA and total protein contents following low level sarin exposure in rats.

1. The changes in contents of DNA and total protein in the liver of the rats exposed to low level sarin by inhalation at 3, 6 and 12 months following the exposure were studied. The influence of sarin on the DNA and protein metabolism in liver was determined by the measurements of incorporation of tritiated thymidine into DNA, the concentration of DNA and total protein. 2. Our results show that not only symptomatic level 3 but also asymptomatic levels 1 and 2 of sarin are able to significantly decrease the incorporation of radiolabelled thymidine without changing total concentrations of DNA as well as protein at three months following sarin exposure. On the other hand, the significant decrease in total contents of DNA and protein in liver without the changes in the incorporation of tritiated thymidine was determined in liver six months following sarin exposure. Practically no significant changes in the metabolism of DNA and protein were observed at 12 months following sarin exposure. 3. Thus, not only clinically manifested intoxication but also low-level, asymptomatic exposure to nerve agents such as sarin is able to influence the metabolism of nucleic acids as well as proteins even several months following the exposure.

Effect of alanyl-glutamine on leucine and protein metabolism in endotoxemic rats.

BACKGROUND: Branched-chain amino acids (BCAA; valine, leucine, and isoleucine) have a regulatory effect on protein metabolism and are the main donor for synthesis of alanine and glutamine in the skeletal muscle. This study was performed to investigate whether exogenous alanine or glutamine would affect leucine and protein metabolism in intact and endotoxemic rats. METHODS: Rats were injected with endotoxin of Salmonella enteritidis or saline. Thirty minutes later, the effects of endotoxemia and L-alanyl-L-glutamine (AG) on leucine and protein metabolism were evaluated using a primed constant infusion of [1-14C]leucine, endotoxin, and AG (200 mg/mL) solution or an infusion of [1-14C]leucine without endotoxin or AG. The specificity of the effect of exogenous alanine and glutamine was evaluated by a single infusion of alanine, glutamine, and glycine in a separate study. RESULTS: Endotoxin treatment induced more negative net protein balance caused mainly by an increase in whole-body proteolysis. Protein synthesis increased in kidneys, colon, and spleen, while a decrease was observed in skeletal muscle. The impressive effects of AG were the decrease in plasma branched-chain amino acid (BCAA) levels, decrease in leucine oxidized fraction, and improvement of protein balance associated with a decrease in whole-body proteolysis. Similar changes in leucine and protein metabolism were induced by infusion of alanine or glutamine but not by infusion of glycine. CONCLUSIONS: IV administration of alanine or glutamine improves protein balance and decreases leucine oxidized fraction in postabsorptive state and in endotoxemia. Decreased proteolysis is the main cause of decreased plasma BCAA levels after AG treatment.

Influence of buthionine sulfoximine, S-adenosylmethionine and glutathione on liver regeneration following partial hepatectomy.

This study was designed to evaluate the effect of derangement in methionine metabolism, and the effects of S-adenosylmethionine (SAM) and glutathione (GSH) on liver regeneration. Rats were treated immediately and 6 h after 70% hepatectomy (PH) as follows: (1) SAM, (2) GSH, (3) buthionine sulfoximine (BSO; inhibitor of SAM and GSH synthesis), (4) BSO + SAM, (5) BSO + GSH, and (6) buffer solution (B). The control group consisted of laparotomized animals treated with B. The increase in hepatic DNA synthesis, hepatocyte mitotic activity, and protein synthesis were observed in PH rats while in BSO rats, a delayed onset of liver regeneration was observed. SAM induced an earlier onset of hepatocyte mitotic activity, and reduced the inhibitory effect of BSO. GSH treatment exerted an inhibitory effect on liver regeneration and worsened the negative effect of BSO. It is concluded that derangement in methionine metabolism can impair the development of liver regeneration. The data also demonstrate the favourable effect of SAM and negative effect of GSH.

Effect of 7-methoxytacrine and L-carnitine on the activity of choline acetyltransferase.

Changes of choline acetyltransferase (ChAT) activity in the hippocampus and the basal ganglia were studied in rats treated i.p. with L-carnitine (CRT) and 7-methoxytacrine (7-MEOTA) (i.m.) separately or 3-days treated with L-carnitine and then with one administration of 7-MEOTA. Both compounds increased ChAT activity when administered separately. 3-day treatment of CRT followed by administration of 7-MEOTA normalized ChAT activity.

Leucine metabolism in partially hepatectomized rats.

BACKGROUND/AIMS: We hypothesized that the decrease in plasma branched-chain amino acids (i.e. valine, leucine and isoleucine) and the increase in the oxidized leucine fraction demonstrated in cirrhotic rats in our previous study were caused by the reduced liver cell mass. In the present study we have evaluated the influence of the loss of a substantial amount of the hepatic tissue on changes in leucine metabolism. METHODS: A two-thirds partial hepatectomy (PH) was performed in male Wistar rats, weighing 210-250 g. Sham-operated rats served as controls. Whole-body leucine kinetics and ketoisocaproate oxidation rates in the isolated perfused liver were investigated using continuous infusion of [1-14C]leucine and alpha-keto[1-14C]ketoisocaproate at 0 h, 24 h and 72 h after surgery. All groups were compared by analysis of variance, and differences were considered significant at the p < 0.05 level. RESULTS: A significant decrease in the sum of branched-chain amino acids in blood plasma was observed at 24 h after PH. The decrease in whole-body leucine utilization in protein synthesis observed at 24 h after PH was associated with a decrease in protein synthesis in the gastrocnemius muscle, in the small intestine and in the liver remnant (although protein synthesis per mg of liver protein was higher than in sham-treated animals). In contrast, the rate of whole-body leucine oxidation increased immediately after PH (PH: 4.5 +/- 0.7 vs. sham: 2.4 +/- 0.4; mumol .100 g b.w.-1.h-1). As a result of the opposite changes in protein synthesis and leucine oxidation, marked increases in oxidized leucine fraction were observed immediately (14.6 +/- 1.5%) and 24 h (15.1 +/- 1.6%) after PH in comparison to the sham-treated rats (7.1 +/- 0.8%). In isolated perfused livers of PH rats, an increase in ketoisocaproate oxidation per liver weight unit was observed at 24 h and 72 h in comparison to the sham group. The loss of liver capacity for ketoisocaproate oxidation was restored at 72 h after PH, although the liver weight did not reach the preoperative value. CONCLUSIONS: We conclude that the loss of hepatic tissue results in an increase in leucine oxidized fraction that is caused by both a decrease in protein synthesis and an increase in leucine oxidation. Both the liver remnant and the extrahepatic tissues are involved in this response.

Leucine metabolism in TNF-alpha- and endotoxin-treated rats: contribution of hepatic tissue.

The effects of tumor necrosis factor-alpha (TNF-alpha; 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 alpha-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-alpha and LPS groups were compared with those obtained in controls. Both TNF-alpha 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-alpha and LPS rats), in blood plasma, liver, colon, kidneys, gastrocnemius muscle (in LPS rats), and in lungs (TNF-alpha-treated rats), whereas it decreased in the jejunum (LPS rats). In IPL of TNF-alpha- 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-alpha and/or LPS are reduced by decreased activity of hepatic branched-chain ketoacid dehydrogenase that can help resupply BCAA to the body.

Mitogen-induced lymphocyte proliferation and systemic immune activation in cancer patients.

AIMS AND BACKGROUND: Proliferative response of lymphocytes to mitogens is known to be decreased in cancer patients. Depressed proliferation of lymphocytes in other disorders has been linked to systemic inflammatory response. METHODS: Lymphocyte proliferation in response to phytohemagglutinin (PHA) and concanavalin A, as well as urinary neopterin, plasma interferon-gamma and interleukin-4, were investigated in 41 patients with solid tumors. RESULTS: The proliferative response to PHA was significantly depressed in cancer patients at most of the concentrations examined. Urinary neopterin and plasma interferon-gamma were significantly elevated in cancer patients, whereas interleukin-4 was undetectable in all the subjects. A negative correlation was observed between urine neopterin and lymphocyte proliferative response to mitogens. CONCLUSIONS: Lymphocyte proliferative response to PHA is significantly diminished in cancer patients, and this depression appears to be partly linked to systemic inflammatory response. Plasma interferon-gamma was significantly increased in cancer patients, whereas interleukin-4 was undetectable.

Leucine metabolism in rats with cirrhosis.

BACKGROUND/AIMS: This study aimed to investigate the pathogenesis of reduced plasma levels of branched-chain amino acids leucine, isoleucine and valine in cirrhosis. METHODS: Cirrhosis was induced by intragastric administration of 36 doses of carbon tetrachloride in olive oil over a period of 12 weeks. Rats treated with oil alone served as controls. The rates of leucine turnover, clearance, oxidation and incorporation into proteins were evaluated using [1-14C]leucine, [4,5-3H]leucine and alpha-keto[1-14C]isocaproate 3 days after the last intragastric treatment in vivo and in the isolated perfused liver. RESULTS: In animals with cirrhosis we observed a profound fall in plasma branched-chain amino acid levels and significant decreases in leucine turnover, oxidation and incorporation into tissue proteins. A more pronounced fall in leucine incorporation in proteins resulted in a significant increase in the oxidized leucine fraction in rats with cirrhosis as compared to controls. Leucine clearance was higher in the cirrhosis group. Concomitant to the fall of whole body leucine turnover, decreases of leucine incorporation into protein and of ketoisocaproic acid decarboxylation were observed in the isolated perfused liver of rats with cirrhosis. However, leucine oxidation was increased compared with control rats. CONCLUSIONS: Our results indicate that the predominant mechanism of the decrease in plasma leucine levels in rats with cirrhosis is an increase in the oxidized leucine fraction associated with a decrease in leucine turnover. An increase in leucine oxidation in the cirrhotic liver is one of the mechanisms involved.

Toxicities of O-alkyl S-(2-dialkylaminoethyl) methyl phosphonothiolates (V-compounds).

Toxicities expressed as LD50 values of O-alkyl S-(2-dialkylaminoethyl) methyl phosphonothiolates in different species and different routes of administration were determined. Rats were more sensitive to these compounds than mice, rabbits and guinea-pigs were more sensitive than mice and rats. The most sensitive to these compounds were dogs. LD50 values varied in a wide range, however, a decrease of toxicity was observed in the following rank: i.v., i.m., s.c., i.p., p.o. and p.c., respectively. Depending on the route of administration, LD50 values varied from 5 g/kg (i.m., dogs) through tens of microgram/kg for the most part of compounds and routes of administration to hundreds, rarely thousands of g/kg (some chemicals, p.c. administration).

Protein metabolism in cirrhotic rats: effect of dietary restriction.

This paper evaluates the role of decreased food intake in protein metabolism in cirrhotic animals by comparing the changes with those observed in pair-fed controls. Rats were injected with [14C]leucine and then divided into 3 groups. Liver cirrhosis was induced in 1 group of rats by repeated intragastric administration of CCl4 in oil over a period of 8 weeks. Control animals were gavaged with oil and either pair-fed or given access to food ad libitum. Three days after the last intragastric dose, rats were injected with [3H]leucine and sacrificed 20 min later. The daily food intake of CCl4 rats declined to 60% of that of the ad libitum controls. Both the pair-fed control group and the cirrhotic group showed decreased body weight gain, and a decline in muscle and intestinal protein degradation. The pair-fed and the cirrhotic groups differed from one another in many metabolic abnormalities. In the cirrhotic group we observed higher levels of serine, asparagine, proline, methionine, tyrosine, phenylalanine, ornithine and histidine, and lower levels of valine, isoleucine and arginine. In these animals higher relative (per kilogram body weight) weights and protein content of the spleen, kidneys and heart were observed. Additionally higher liver weight despite lower protein concentration, as well as lower liver protein degradation and lower skeletal muscle protein synthesis were found.

Protein metabolism in specific tissues of endotoxin-treated rats: effect of nutritional status.

Rats received an injection of [14C]leucine and were then divided into four groups. Groups I and II consisted of ad libitum fed rats were administered saline or endotoxin of Salmonella enteritidis eight and twenty-two h after the [14C]leucine treatment. Animals of Group III (saline) and Group IV (endotoxin) fasted after [14C]leucine injection. Twenty three hours after [14C]leucine treatment rats were injected with [3H] leucine and sacrificed 20 min afterwards. Endotoxin administration decreased body weight in fed rats only. After endotoxin treatment, higher [3H]leucine specific activity in the blood plasma, decreased leucine incorporation into proteins and lowered plasma amino acid levels were observed. [14C]leucine radioactivity was significantly higher in the spleen and lower in skeletal muscles of endotoxin-treated rats. All changes were less expressed in fasted than in ad libitum fed animals. Our results indicate that endotoxin treatment results in (a) changes in host metabolism that are not mediated solely by anorexia; (b) a decrease of protein synthesis in the viscera and skeletal muscles; (c) an increase of protein degradation in skeletal muscles; (d) reutilization of leucine released from skeletal muscles in viscera, and (e) a slower disappearance rate of leucine from the blood.

Effects of essential phospholipids on the amino acid metabolism in whole body irradiated rats.

Ionizing radiation induces injury to cell membranes resulting in an increase of plasma amino acids concentration and catabolic response of the body. Essential phospholipids (EPL) administered before radiation protect the cell structures and can be used for their renewal. For this reason EPL are expected to have some effect on amino acid metabolism in radiation sickness. The adult male Wistar rats were injected with EPL in a dose of 125 mg EPL/kg b.w. (Essentiale) or saline solution. Three h later whole-body gamma irradiation with a single dose of 10 Gy was performed. Untreated pair-fed rats served as control. The rats were sacrificed 2 days after irradiation. In irradiated rats a decrease in body weight, increased concentrations of plasma amino acids, higher oxidation of branched chain keto acids in liver mitochondria, higher incorporation of leucine into liver proteins and decreased leucine incorporation into proteins of small intestine were observed. In EPL pretreated rats a longer survival time, a higher body weight, lower levels of plasma amino acids and higher protein synthesis in small intestine and in liver when compared with irradiated, saline treated rats were found. No significant changes were observed in branched chain amino acid oxidation measured by means of labeled ketoisocaproate in liver mitochondria.

Changes of cholinesterases in the blood and some tissues following administration of tacrin and its two derivatives to rats.

Tacrin, its 7-methoxy-(MEOTA) and 7-hydroxy-(HYOTA) derivatives were i.m. administered to rats in a dose of 1.2 x LD50 and acetylcholinesterase (blood, hippocampus, frontal cortex, basal ganglia, septum and diaphragm) or butyrylcholinesterase (liver) activities were detected. The inhibitory effect of the examined substances in vivo decreased in the following order: tacrin HYOTA > MEOTA. The marked inhibition of the enzymes studied following administration of all three compounds in the frontal cortex could suggest importance of this structure for action of these drugs.

Changes of rat blood and tissue cholinesterases following administration of tacrine derivatives in vivo.

Acetylcholinesterase (blood, hippocampus, frontal cortex, basal ganglia, septum and diaphragm) or butyrylcholinesterase (liver) activities following i.m. administration of tacrine (9-amino-1,2,3,4-tetrahydroacridine), its 7-methoxy- and 7-hydroxy derivatives to rats in dose of 1.2 x LD50 were detected. The most marked inhibition of the enzymes studied following tacrine administration was demonstrated in the frontal cortex, diaphragm, liver and blood. Inhibition of acetylcholinesterase in the frontal cortex and blood only was observed following administration of tacrine derivatives. The results indicate that inhibition of cholinesterases could be important but not unique part explaining the action of these drugs in general.

Anticholinesterase action of organophosphates: importance of the liver.

Acetylcholinesterase activity in the blood, pontomedullar area, basal ganglia of the brain and diaphragm following sublethal soman (i.m., 31 micrograms/kg) and 2-dimethylaminoethyl-(dimethylamido)-fluorophosphate (GV) poisoning (i.m., 6 micrograms/kg) was studied in groups of rats (n = 6) pretreated as follows: intact, hepatectomized, sham-operated and narcotized animals. Except hepatectomized rats, all animals in the groups survived; in hepatectomized rats, 2 animals died following soman poisoning. Acetylcholinesterase activity was decreased in each group. The highest and significant (p < 0.05-0.0001) decrease of this activity in the pontomedullar area and diaphragm following hepatectomy was demonstrated. The results indicate that undiminished liver functional capacity is an important factor influencing soman and GV anticholinesterase action.

Validity of two different methods to detect liver injury.

Following single administration of carbon tetrachloride (p.o., 200 microliters/200 g) to female rats, activities of transaminases AST and ALT were determined from 1 hr to 7 days after the intoxication. At the same time intervals, aminopyrine breath test (ABT) was applied. The results indicate that marked decrease of ABT was observed within the first 3 hrs of exposition and lasted 24 hrs. On the other hand, statistically significant elevation of plasma enzymes was demonstrated from the 3rd hr of administration and lasted also 24 hrs. The results indicate that ABT reacts more rapidly to carbon tetrachloride administration than the changes of plasma transaminase.

Effect of polyunsaturated phosphatidylcholine on liver regeneration onset after hepatectomy in the rat.

The purpose of this study was to examine the effect of polyunsaturated phosphatidylcholine (PPC, Essentiale) on the onset of liver regeneration after partial hepatectomy (PH). Hepatectomy was carried out on rats, and immediately after operation doses of 25, 50 or 250 mg PPC/kg b.w. were injected into the vena femoralis. The control group consisted of saline-injected PH rats. The rats were killed 18, 21, 24 and 30 h after PH. The course of liver regeneration was assessed by measuring the incorporation of labelled thymidine into DNA and the hepatocyte mitotic activity. After PPC injection no values of liver DNA specific activity differing from baseline were observed. However, higher values of hepatocyte mitotic activity and lower liver triglyceride levels were found as compared to the saline-treated rats. These observations indicate that the administration of PPC has a favourable effect on liver regeneration after PH.

An evaluation of continual determination of blood cholinesterase activity for inhibition studies.

Simple method of continual monitoring of the rat blood cholinesterase activity in vivo was used to demonstrate its inhibition following i. m. administration of acridine and carbamate inhibitors. Another type of inhibition was demonstrated for different routes of administration of highly toxic organophosphate, VX. Reactivation of the blood cholinesterase was also monitored following intoxication with VX and soman and treated with obidoxime and atropine. This continual determination of cholinesterase activity represents an useful approach to study effects of cholinesterase inhibitors.

2-Dialkylaminoalkyl-(dialkylamido)-fluorophosphates: acute toxicities.

Toxicities expressed as LD50 values of 2-dialkylaminoalkyl-(dialkylamid) o-fluorophophates for rats and mice (i. m. administration) were determined. Rats were more sensitive to these compounds than mice: LD50 values varied from 17 to 261 micrograms/kg for rats and from 30.5 to 1222 micrograms/kg for mice, respectively. Different routes of administration in one derivative of this group substituted by methyl groups only were compared. The highest toxicity (lowest LD50 value) in intravenous administration (11 micrograms/kg) and the lowest one in percutaneous (1366 micrograms/kg) were found.