The effect of N-stearoylethanolamine on free amino acid levels in plasma and liver of rats with an experimental burn

The effect of an endocannabinoid congener, N-stearoylethanolamine (NSE), on the content of plasma and liver pools of free amino acids (AA) was studied in burned rats. After application of a thermal skin burn (stage III) animals perorally received an aqueous suspension of NSE (10 mg/kg of body weight) during 7 days or were treated with the aqueous NSE suspension (10 mg/ml) applied onto the burn wound, or received a combined treatment. It has been originally demonstrated for the first time, that the treatment of burned rats with NSE prevented the decrease in total AA concentration in blood plasma and the increase in hepatic AA concentration due to modulation in concentrations of glycogenic AA. In burned animals the ratio of plasma and liver homogenate Phe/Tyr and Gly/Val increased while the Fischer ratio (Ile+Leu+Val/Phe+Tyr) decreased, and after the treatment with NSE these parameters remained at the level of intact animals. These data demonstrate that NSE possesses adaptogenic properties, and it is involved in the organism response to the burn. This prevents changes in blood plasma and hepatic pools of free AA of NSE-treated rats with the burn wound compared with untreated animals.     

Metabolism of vitamin A in the heart increases after a myocardial infarction

The supply of vitamin A to the myocardium by storage organs during increased oxidative stress subsequent to myocardial infarction (MI) was examined in hemodynamically assessed rats using compartment analysis of a radio-labeled vitamin A. 3H-Vitamin A was injected into two groups of rats: an MI group and a control group. There were no differences in the plasma or myocardial content of total vitamin A (unlabeled + labeled) between the two groups. However, the proportion of 3H-vitamin A was greater in the myocardium as well as plasma of MI rats. Rats with MI also had significantly lower 3H-vitamin A levels in liver and kidney than sham controls. The greatest difference in vitamin A content was in the concentrations of 3H-labeled storage forms of vitamin A in the liver of MI animals. Activity of bile salt-dependent retinyl ester hydrolase, an enzyme responsible for hydrolyzing vitamin A storage forms, was significantly increased in the liver of MI animals. These data indicate that analysis of plasma concentrations of vitamin A to ascertain links to cardiac conditions may be inappropriate. Specifically, during MI, increased amounts of vitamin A are mobilized from the liver to the heart without changing plasma concentrations. This is facilitated by an increase in the activity of an enzyme that hydrolyzes vitamin A storage forms.     

Carnitine metabolism in the vitamin B-12-deficient rat.

In vitamin B-12 (cobalamin) deficiency the metabolism of propionyl-CoA and methylmalonyl-CoA are inhibited secondarily to decreased L-methylmalonyl-CoA mutase activity. Production of acylcarnitines provides a mechanism for removing acyl groups and liberating CoA under conditions of impaired acyl-CoA utilization. Carnitine metabolism was studied in the vitamin B-12-deficient rat to define the relationship between alterations in acylcarnitine generation and the development of methylmalonic aciduria. Urinary excretion of methylmalonic acid was increased 200-fold in vitamin B-12-deficient rats as compared with controls. Urinary acylcarnitine excretion was increased in the vitamin B-12-deficient animals by 70%. This increase in urinary acylcarnitine excretion correlated with the degree of metabolic impairment as measured by the urinary methylmalonic acid elimination. Urinary propionylcarnitine excretion averaged 11 nmol/day in control rats and 120 nmol/day in the vitamin B-12-deficient group. The fraction of total carnitine present as short-chain acylcarnitines in the plasma and liver of vitamin B-12-deficient rats was increased as compared with controls. When the rats were fasted for 48 h, relative or absolute increases were seen in the urine, plasma, liver and skeletal-muscle acylcarnitine content of the vitamin B-12-deficient rats as compared with controls. Thus vitamin B-12 deficiency was associated with a redistribution of carnitine towards acylcarnitines. Propionylcarnitine was a significant constituent of the acylcarnitine pool in the vitamin B-12-deficient animals. The changes in carnitine metabolism were consistent with the changes in CoA metabolism known to occur with vitamin B-12 deficiency. The vitamin B-12-deficient rat provides a model system for studying carnitine metabolism in the methylmalonic acidurias.     

Low methylcobalamin in liver tissues is an artifact as shown by a revised extraction procedure

BackgroundVitamin B12 (cobalamin, Cbl) is represented by several molecular variants distinguished by the exchangeable ligand X coordinated to cobalt ion (XCbl). The most typical XCbl-forms are cyanocobalamin (CNCbl), hydroxocobalamin (HOCbl), methylcobalamin (MeCbl) and 5′-deoxydeoxyadenosylcobalamin (AdoCbl). Cells convert the “inactive” vitamins CNCbl and HOCbl to the two critically important coenzymes AdoCbl or MeCbl. Surprisingly, little or no MeCbl is usually uncovered in the tissue samples, as compared to AdoCbl and HOCbl. We hypothesized that a low level of MeCbl is an artifact of “harsh” extractions, leading to degradation of MeCbl and/or its conversion to other XCbl-forms.MethodsWe designed a “mild” extraction protocol, including homogenization of rat liver in ammonium acetate (pH 4.6), dilution with EtOH (final 60%) and heating for 10 min at 70 °C. The XCbls were separated by HPLC and quantified by isotope dilution assays.ResultsA “mild” extraction revealed the following composition of Cbls: 37% AdoCbl, 35% MeCbl, 15% HOCbl and 13% CNCbl. The usual “harsh” protocol (pH 7, 20 min at 80 °C) changed this balance to 33%, 5%, 43% and 17%, respectively. A model assay revealed that MeCbl underwent demethylation and conversion to HOCbl at pH 3 and pH > 7, when heated with thiols. Other changes included decyanation of CNCbl and destruction of HOCbl.ConclusionsOur procedure reveals a high content of MeCbl in rat liver.General significanceThis result challenges previous data and pinpoints the need for new studies to characterize the endogenous Cbl-forms in health and disease.     

Glycosaminoglycan content in tissues and body fluids of rats treated with atherogenic diet.

An increase of total glycosaminoglycan content in aortic wall and liver as well as changes in the concentration of glycosaminoglycan fractions in aorta, skin, liver, and blood serum were found in white rats fed with atherogenic diet. Urinary excretion of glycosaminoglycans was increased in experimental animals.     

Age-related changes of protein- and RNA-synthetic processes in experimental hyper- and hypothyroidism

The rate of liver and plasma protein synthesis and the activity of liver RNA polymerases 1 and 2 were investigated in rats of various age under experimental hyper- and hypothyroidism. The rate of plasma protein synthesis decreased with age more dramatically than that of liver proteins. Hyper- and hypothyroidism exerted opposite effects on protein synthesis in rats: stimulation and inhibition, respectively. The manifestation of these effects was age related. The thyroid status of animals also influenced the balance of protein synthesis. Thyroxin administration caused preferential incorporation of a label into blood plasma proteins. Changes of thyroid status of old animals insignificantly affected the absolute values of the label incorporation into proteins and the ratio of the label incorporation into local and secreted liver proteins. Age-related decrease of total hepatic nuclear RNA-polymerase activity was due to reduction of the template-bound functionally active forms of RNA-polymerases 1 and 2. Administration of thyroxin caused initial redistribution of the enzyme activity between template-bound and free fractions accompanied by the increase of template bound RNA-polymerases. Prolonged hormonal stimulus also caused an increase of free RNA-polymerases, which reflects the increased synthesis of these enzymes. Mecrazolyl administration reduced the activity of RNA-polymerase 1 and 2. All age groups were characterized by preferential reduction of the bound form. RNA-polymerase 2 activity decreased to a greater extent than that of RNA-polymerase 1. The data suggest age-determined reactions of the body to altered thyroid status.     

Antioxidant system activation in rats with experimental cirrhosis after injection of cryopreserved fetal liver cells

The possibility to recover the antioxidant system in rats with experimental liver cirrhosis (LC) after allo- and xenotransplantation of cryopreserved fetal liver cells (FLC) was investigated. It was shown that the content of lipid peroxidation products in the blood serum of animals with LC four weeks after FLC transplantation decreased significantly as compared to control group. Such changes were accompanied by a significant increase of catalase (CAT), glutathione reductase (GR), Se-dependent glutathione peroxidase (GP) activity in the liver and total anti-oxidative activity (AOA) of blood. Obtained results demonstrate that the main direction of FLC effects in animals with LC agree with that we observed previously in other experimental models (partial hepatectomy, chronic alcohol poisoning and hypercholesterolemia). In conclusion, cell therapy may be considered as the universal method for correction of disorders in regulation of free-radical processes in various experimental pathologies.     

Decreased Brain Levels of Vitamin B12 in Aging,Autism and Schizophrenia

Many studies indicate a crucial role for the vitamin B₁₂ and folate-dependent enzyme methionine synthase (MS) in brain development and function, but vitamin B₁₂ status in the brain across the lifespan has not been previously investigated. Vitamin B₁₂ (cobalamin, Cbl) exists in multiple forms, including methylcobalamin (MeCbl) and adenosylcobalamin (AdoCbl), serving as cofactors for MS and methylmalonylCoA mutase, respectively. We measured levels of five Cbl species in postmortem human frontal cortex of 43 control subjects, from 19 weeks of fetal development through 80 years of age, and 12 autistic and 9 schizophrenic subjects. Total Cbl was significantly lower in older control subjects (> 60 yrs of age), primarily reflecting a >10-fold age-dependent decline in the level of MeCbl. Levels of inactive cyanocobalamin (CNCbl) were remarkably higher in fetal brain samples. In both autistic and schizophrenic subjects MeCbl and AdoCbl levels were more than 3-fold lower than age-matched controls. In autistic subjects lower MeCbl was associated with decreased MS activity and elevated levels of its substrate homocysteine (HCY). Low levels of the antioxidant glutathione (GSH) have been linked to both autism and schizophrenia, and both total Cbl and MeCbl levels were decreased in glutamate-cysteine ligase modulatory subunit knockout (GCLM-KO) mice, which exhibit low GSH levels. Thus our findings reveal a previously unrecognized decrease in brain vitamin B₁₂ status across the lifespan that may reflect an adaptation to increasing antioxidant demand, while accelerated deficits due to GSH deficiency may contribute to neurodevelopmental and neuropsychiatric disorders.     

Lipid peroxidation and regulation of it in rat blood and liver under the experimental alimentary radionuclide effect

Effect of alimentary radionuclide load (137Cs, 700 Bq for animal per day during 7, 14 and 22 days) on the lipid peroxidation intensity and blood and liver enzymatic and non-enzymatic antioxidant system in the Wistar male-rats was investigated. It was found that considerable change of antioxidant system activity in plasma and erythrocytes of experimental animals was already noticeable on the 7th day of radionuclide load. After 22 days of experiment the reliability of glutathione-dependent antioxidant system in blood was essentially decreased and lipid hydroperoxide content was increased. The increase of lipid peroxidation intensity was also found in the experimental animals liver but at the same time the activities of all studied enzymes of antioxidant system were significantly higher than they were in the control rats.     

Stereoselective pharmacokinetics of flurbiprofen and formation of covalent adducts with plasma protein in adjuvant-induced arthritic rats

To determine the effect of arthritis on the disposition of flurbiprofen (FP) and its acyl glucuronide (FPG) as well as formation of covalent adducts with plasma protein, a pharmacokinetic study was carried out in adjuvant-induced arthritic (AA) rats. In control animals the pharmacokinetics of FP were stereoselective following intravenous bolus injection of rac-FP: (-)-(R)-FP showed higher plasma clearance (CL(tot)) and shorter mean residence time (MRT) compared to (+)-(S)-FP. The CL(tot) and clearance for the glucuronide formation (CL(glu)) of both enantiomers in AA rats were extremely increased compared to those in control rats. Increased total clearance in AA rats was due, at least in part, to a remarkable increase in the plasma unbound fraction of FP, consistent with a decrease in the plasma albumin level. The yield of covalent binding of FP to plasma protein in AA rats was less than that in controls, being consistent with the decrease in the plasma acyl glucuronide level.     

Effects of Sterylglycosides from Soybean on Lipid Indices in the Plasma,Liver, and Feces of Rats

The effects of soybean oil (SOO, control), soybean lecithin (SOL), and of sterylglycocides (STG) and phospholipids (PL) fractionated from SOL on lipid indices in the plasma, liver, and feces were examined for male Wistar rats fed with diets containing these lipids for 3 weeks. The body weight gain and liver weight decreased or tended to be reduced in the animals given the diet containing a 5% STG mixture (STGM) compared with the values in the other dietary groups. The plasma lipid concentration in general declined in the rats fed with the diets supplemented with 5% SOL, STGM, or the PL mixture (PLM), and with 1% of STGM, acylated STG (ASTG), or non-acylated STG (NSTG). The triacylglycerol level was significantly depressed in the rats fed with the diets including 1 or 5% of STGM, ASTG, or NSTG when compared to the level of the SOO—fed animals. The total cholesterol and triacylglycerol contents in the liver were lower in the rats provided with the diets containing 5% of SOL, PC, or PLM than in the SOO- or STGM-diet-fed animals. The rats given the diets supplemented with 1 or 5% of STGM, ASTG, or NSTG had a decreased content of liver triacylglycerol compared with the content of the SOO—fed animals. The amounts of total lipids and total cholesterol excreted into the feces were higher in the rats fed with the diets supplemented with 5% SOL, or with 1% of STGM, ASTG, or NSTG, or especially with 5% STGM than in the SOO—fed animals. The present results suggest that STG suppressed the absorption of cholesterol and fatty acids in the intestines.     

Effect of salvipholin on the carbohydrate and lipid metabolism in the rat liver in alloxan diabetes]

Peroral administration of salvipholin in a dose of 50 mg/kg to intact male rats (the body weight 180-220 g) had a positive effect on the carbohydrate-lipid metabolism in the liver and blood serum of animals. Administration of the same dose to rats with alloxan diabetes induced a significant decrease in the content of glucose, free fat acids, triglycerides and lysophospholipids of the liver and blood serum. The level of these components has sharply increased after subcutaneous alloxan administration in a dose of 150 mg/kg, the content of glycogen and pyruvic acid being normalized and insulin deficiency removed. These changes are closely related to salvipholin-promoted restoration of phospholipid spectra of the blood serum and liver of experimental animals disturbed under conditions of insulin deficiency. The possible mechanism of the salvipholin action is analyzed.     

Influence of magnesium deficiency on the bioavailability and tissue distribution of iron in the rat

We investigated the effect of dietary magnesium (Mg) deficiency on the nutritive utilization and tissue distribution of iron (Fe). Wistar rats were fed an Mg-deficient diet (56 mg/kg) for 70 days. Absorbed Fe, Fe balance, number of the erythrocytes [red blood cells (RBC)] and leukocytes white blood cells (WBC)], hemoglobin (Hb), and Fe content were determined in samples of plasma, whole blood, skeletal muscle, heart, kidney, liver, spleen, femoral bone, and sternum obtained on experimental days 21, 35, and 70. The Mg-deficient diet significantly increased Fe absorption and Fe balance from week 5 until the end of the experimental period. This effect was accompanied by a significant decrease in the concentration of RBC and Hb from day 35, which caused the decrease in whole blood Fe seen on day 70. However, WBC were significantly increased from day 21 until the end of the experimental period. Mg deficiency significantly increased plasma and liver Fe at all three time points investigated. Spleen, heart, and kidney Fe were significantly increased only at the end of the study. However, on day 70, Fe concentration in the sternum had decreased significantly. No changes were found in skeletal muscle or femur Fe content. Mg deficiency led to increased intestinal absorption of Fe and decreased RBC counts, possibly as a result of increased fragility of the erythrocytes. Intestinal interactions between Fe and Mg, together with activation of erythropoiesis as a result of hemolysis, favored intestinal absorption of Fe. This situation gave rise to an increase in plasma Fe levels, which in turn favored Fe uptake and storage by different organs, especially the liver and spleen. However, despite the increased Fe content seen in the tissues of rats fed the Mg-deficient diet, these animals were unable to compensate for the hemolysis caused by this nutritional deficiency.     

Formation of taurine and amino acid pools in rat tissues upon stimulation of NAD synthesis

A single intraperitoneal injection of nicotinamide (500 mg/kg) to mongrel albino rats causes a 6-hour increase in the 2-oxoglutarate level and the free NAD+/NADH ratio in liver mitochondria. The levels of taurine and taurocholates as well the activity of cysteine oxidase in liver tissues remains thereby unchanged, whereas the cysteine transaminase activity diminishes. In the heart and brain of experimental animals the activity of both enzymes is decreased. In the liver, blood plasma and heart of experimental animals, the Ala and Ser levels are low, whereas the taurine content is elevated both in blood plasma and brain. Nicotinamide administration eliminates positive correlations between the levels of taurine, its precursors and metabolically bound amino acids. In the liver the negative correlations between the activities of cysteine oxidase and cysteine transaminase observed in the control group disappear in the experimental group. Apparently, one of regulatory mechanisms of the taurine pool formation in the liver is the ratio of activities of the both enzymes as well as their competition at the substrate level. This emphasizes the importance of the transamination reactions in the metabolism of sulphur-containing amino acids.     

Influence of dietary hexachlorocyclohexane isomers on lipid metabolism in albino rats

Dietary intake of beta- and gamma-hexachlorocyclohexane (beta- and gamma-HCH) by albino rats for two weeks (at 800 ppm level) resulted in impairment of lipid metabolism, viz. hyperlipemia and fatty metamorphosis of liver. Liver fat content was increased by both beta- and gamma-HCH. Significant increases were observed in triglyceride and phospholipid fractions of blood in these experimental animals. The incorporation of [14C]acetate and palmitate into liver and blood lipids was higher in HCH pretreated animals, suggesting a higher rate of fat synthesis in liver and of secretion as well.     

Age-related changes of lipid spectrum, level of lipid peroxidation, and antioxidant defence in the liver and blood of rats

It has been shown that the lowest level of total lipids, cholesterol, triacylglycerols and products of lipid peroxidation of blood and liver, as a rule, is specific to adult rats. These characteristics are significantly higher for old and young animals. At the same time, the level of glutathione and alphatocopherols in adults' liver is much higher than in young and old rats. It suggests the lower level of processes of lipid peroxidation in adult mature rats. The relative high level of products of lipid peroxidation and low content of alpha-tocopherols in old rats' liver (against the background of higher activity and glutamineperoxidase, and glutathionereductase than in adults) suggests tocopherol deficiency in old animals. High content of total lipids, cholesterol and cholesterol entering into the composition of lipoprotein of different density, triacylglycerols, diene conjugates and malonic dialdehide, activity of glutathione-dependent enzymes of antioxidant defence in young animals as compared with these levels in adult rats seems to be associated with agerelated hypercholesterolemia and intensive plastic changes of a growing organism.     

The relationship between homozygosity level and animal physiology: Iron content of plasma and whole blood as well as total iron binding capacity by transferrin (TIBC) in rats of various inbreeding coefficient

Male and female wild Norway rats (Rattus norvegicus Erxleben) and male and female albino outbred rats (Ipf:RIZ) were crossbred. These animals were the control, noninbred group (0% inbred). By systematic full-sib mating, two experimental groups (50 and 91% of inbred) were raised. Half of each group (both males and females) was exposed to physical stress (3 days of starvation and 3 hr of swimming). The other half of each group was ether anesthetized to collect blood. The iron content of plasma and whole blood, as well as the total iron binding capacity, was determined by the Atom-Spec method. A significant decrease in the iron content of plasma and whole blood as well as the TIBC was observed by an increase in the inbreeding coefficient. Stress significantly influenced the iron content of plasma and whole blood as well as the TIBC, whereas the sex of the rats affected the whole-blood iron concentration and TIBC. Moreover, some double interactions had an impact on the iron content and TIBC. The interactions were as follows: plasma—inbreeding level and stress; whole blood—sex and stress; and TIBC—inbreeding level and sex.     

Targeted activation of transcription in vivo through hairpin-triplex forming oligonucleotide in Saccharomyces cerevisiae

The aims of the present study were to analyse the effects of an oral daily dose (10 mg/kg) of the dietary flavonoid quercetin for five weeks in two-kidney, one-clip (2K1C) Goldblatt (GB) hypertensive rats. The evolution of systolic blood pressure was followed by weekly measurements, and morphological variables, proteinuria, plasma nitrates plus nitrites (NO_x) and thiobarbituric acid reactive substances (TBARS), liver oxidative stress markers and endothelial function were determined at the end of the experimental period. Quercetin treatment reduced systolic blood pressure of GB rats, producing no effect in control animals. It also reduced cardiac hypertrophy and proteinuria developed in GB hypertensive rats. Decreased endothelium-dependent relaxation to acetylcholine of aortic rings from GB rats was improved by chronic quercetin treatment, as well as increased endothelium-dependent vasoconstrictor response to acetylcholine and overproduction of TXB₂ by aortic vessels of GB rats, being without effect in normotensive animals. Increased plasma NO_x and TBARS, and decreased liver total glutathione (GSH) levels and glutathione peroxidase (GPX) activity were observed in GB hypertensive rats compared to the control animals. Normalisation of plasma NO_x and TBARS concentrations and improvement of the antioxidant defences system in liver accompanied the antihypertensive effect of quercetin. We conclude that chronic oral treatment with quercetin shows both antihypertensive and antioxidant effects in this model of renovascular hypertension. (Mol Cell Biochem 270: 147–155, 2005)M.F. García-Saura and M. Galisteo are equal contributors to this work     

Norepinephrine turnover in the heart and blood vessels of rats subjected to bilateral renal infarction

The total norepinephrine (NE) content, the uptake of [3H]NE, the turnover rate and the synthesis rate of the neurotransmitter at the heart and blood vessels have been studied during the development of hypertension in rats subjected to bilateral renal infarction. Normal and sham-operated rats were used as controls. Fifty percent of the rats with renal infarction became hypertensive. The weight of the hearts and blood vessels of the experimental animals was significantly increased 15 days after renal infarction. Changes were greater in hypertensive animals. NE concentration in the heart was slightly decreased without achieving statistical significance, while total NE content was unchanged. In the artery wall NE concentration was significantly decreased in normotensive and hypertensive operated rats. [3H]NE uptake in the heart and blood vessels was similar in experimental and control animals. In relation to NE turnover, in both the heart and blood vessels, normal and sham-operated animals behaved as one population while normotensive and hypertensive rats behaved as another population. The rate constant of NE turnover was increased in both tissues of operated experimental animals without achieving statistical significance in the case of the heart. NE synthesis rate was unchanged in the cardiac muscle but was significantly increased in the blood vessels of operated animals. Present data indicate that results describing NE dynamics in the heart cannot be extrapolated for the blood vessels level; on the other hand changes in the neurotransmitter do not seem to be related to the development of high blood pressure after renal infarction in the rat.     

Metabolic cooperation of ascorbic acid and glutathione in normal and vitamin C-deficient ODS rats.

Although the coordination of various antioxidants is important for the protection of organisms from oxidative stress, dynamic aspects of the interaction of endogenous antioxidants in vivo remain to be elucidated. We studied the metabolic coordination of two naturally occurring water-soluble antioxidants, ascorbic acid (AA) and reduced glutathione (GSH), in liver, kidney and plasma of control and scurvy-prone osteogenic disorder Shionogi (ODS) rats that hereditarily lack the ability to synthesize AA. When supplemented with AA, its levels in liver and kidney of ODS rats increased to similar levels of those in control rats. Hepato-renal levels of glutathione were similar with the two animal groups except for the slight increase in its hepatic levels in AA-supplemented ODS rats. Administration of L-buthionine sulfoximine (BSO), a specific inhibitor of GSH synthesis, rapidly decreased the hepato-renal levels of glutathione in a biphasic manner, a rapid phase followed by a slower phase. Kinetic analysis revealed that glutathione turnover was enhanced significantly in liver mitochondria and renal cytosol of ODS rats. Administration of BSO significantly increased AA levels in the liver and kidney of control rats but decreased them in AA-supplemented ODS rats. Kinetic analysis revealed that AA is synthesized by control rat liver by some BSO-enhanced mechanism and the de novo synthesized AA is transferred to the kidney. Such a coordination of the metabolism of GSH and AA in liver and kidney is suppressed in AA-deficient ODS rats. These and other results suggest that the metabolism of AA and GSH forms a compensatory network by which oxidative stress can be decreased.