The characteristics of nitrogen metabolism in the tissues of rabbits with ammonium toxicosis]

Disturbances of nitrogen metabolism under acute ammonium toxicosis have been studied in tissues of rabbit. A sharp increase of the ammonium content in the blood and tissues of the liver and kidneys is accompanied by an increase in the glutamine and glutamate level in all tissues. The level of urea nitrogen in the blood of rabbits increases. The activity of phosphate-independent and phosphate-activated glutaminase also increases in tissues of the liver and kidneys, while arginase activity decreases as compared with the control, which is connected with fall of the ATP level under hyperammonemia. A nomograph method of representation of the redox state has been used.     

Selective regional distribution of tubulin induced in cerebrum by hyperammonemia

Ingestion of ammonium induces hyperammonemia which increases tubulin content in cerebrum but not in cerebellum. We have dissected 11 discrete areas of cerebrum and quantified the tubulin content in control and hyperammonemic rats. An heterogeneity in the induction of tubulin is shown. The areas more affected are ventral hippocampus, dorsal hippocampus, hypothalamus, septum, reticular formation and frontal cortex, in which tubulin content increased by 63%, 27%, 32%, 48%, 45%, and 25%, respectively, after two months of feeding the ammonium diet.     

Long-term ingestion of ammonium increases acetylglutamate and urea levels without affecting the amount of carbamoyl-phosphate synthase

Rats were fed the following diets: standard (20% protein), high-protein (80%), protein-free, standard plus ammonium and protein-free plus ammonium for six weeks. The standard plus ammonium diet was prepared to contain ammonia equivalent to that supplied by the high-protein diet. Addition of ammonium acetate (20% by mass) to the 20% protein or protein-free diets results in 2.3- and 10-fold increases of urea excretion respectively, without increase of carbamoyl-phosphate synthase. Supplementation of the standard diet with ammonium increases the mitochondrial content of acetylglutamate from 830 to 1590 pmol/mg protein, and of the protein-free diet from 130 to 1040 pmol/mg. However, ingestion of ammonium did not increase the activity of acetylglutamate synthase. Therefore the efflux of acetylglutamate from mitochondria was determined. After 30 min at 37 degrees C liver mitochondria from rats on standard diet released 61% of the initial acetylglutamate while mitochondria from animals on standard plus ammonium diet released only 20%. These results indicate that ingestion of ammonium increases the content of acetylglutamate in rat liver by decreasing its efflux from mitochondria. This effect is similar to that produced in mice by a high protein diet [Morita et al. (1982) J. Biochem. (Tokyo) 91, 563-569]. However, while the high-protein diet increases carbamoylphosphate synthase content, the ammonium diet does not.     

Guanidino compound metabolism in arginine-free diet induced hyperammonemia.

Guanidino compounds, intermediates of arginine metabolism, are altered in many pathological conditions especially those involving the urea cycle. Arginine and creatine play an important role in nitrogen metabolism whereas other guanidino compounds such as guanidinosuccinic acid and N-acetylarginine are toxins. Our objective was to investigate the relationship between guanidino compounds and hyperammonemia. Young and adult ferrets were fed a single meal of either an arginine-containing diet (ACD) or an arginine-free diet (AFD). Guanidino compounds were determined by HPLC in the plasma, liver, kidney and brain 3 h after feeding the specified diet. Only young ferrets fed AFD developed hyperammonemia. Plasma and kidney arginine was decreased whereas guanidinosuccinic acid was increased in young ferrets fed AFD. Hepatic creatine and kidney and brain guanidinoacetic acid were significantly decreased in this group. These results indicate that AFD-induced hyperammonemia produced decreased methylation activity in the liver and transamidination activity in kidney. Elevated guanidinosuccinate levels coupled with deficient hepatic creatine synthesis may play a role in the pathophysiology of hyperammonemia.     

Lipid changes of goat sperm plasma membrane during epididymal maturation

Highly purified plasma membranes of maturing goat caput-, corpus- and cauda-epididymal spermatozoa were isolated by aqueous two-phase polymer methods and their lipid constituents were analysed. Phospholipid (approx. 75% w/w), neutral lipid (approx. 15% w/w) and glycolipid (approx. 10% w/w) were the major sperm membrane lipids. There was a significant decrease in the total lipids (approx. 25% w/w), phospholipid (approx. 30% w/w) and glycolipid (approx. 80% w/w) contents of sperm membrane during epididymal maturation. On the contrary, the mature cauda-sperm membrane showed greater (approx. 50% w/w) neutral lipid content than that of the immature caput sperm. Phosphatidylcholine (PC), phosphatidylethanolamine (PE) and sphingomyelin were the phospholipids of the sperm membrane, the former two being the major lipids. Both PC and PE fractions consisted of three species--diacyl, alkylacyl and alkenylacyl forms, the last one being the dominant species in both PC and PE. Of all the phospholipids, diacyl PE decreased most strikingly (approx. 65% w/w) during sperm maturation. The neutral lipid fraction contained sterols, wax esters, 1-O-alkyl-2,3-diacylglycerol, triacylglycerol and fatty acids. Sterols represented nearly 75% w/w of the neutral lipids and cholesterol was the major component (approx. 95% w/w) of the sterol fraction. The sperm maturity was associated with marked increase of sterol (approx. 60% w/w) and steryl ester (approx. 200% w/w) and decrease (approx. 50-65% w/w) of the other membrane-bound neutral lipids. The glycolipid was identified as monogalactosyldiacylglycerol. The fatty acid profile of the various membrane lipids underwent marked alteration during the epididymal transit of the male gametes. Cholesterol/phospholipid and saturated/unsaturated fatty acid ratios increased greatly in the maturing sperm membrane. The altered lipid profile of the mature sperm membrane leads to changes in its fluidity that play an important role in determining the structure and functions of the biomembrane.     

Metabolic responses of folic acid and related compounds to thyroxine in rats

This study deals with the effects of thyroidectomy and feeding thyroid powder on histidine and folic acid metabolism. Normal rats maintained on a soy protein diet, low in methionine but supplemented with vitamin B-12, oxidize approx. 10% of an injected dose of [2-¹⁴C]histidine in 3 h and excrete low levels of formiminoglutamic acid. Addition of methionine increases histidine oxidation to approx. 20%. The feeding of thyroid powder or the injection of high levels of thyroxine decreases histidine oxidation and increases formiminoglutamic acid excretion. Surgical thyroidectomy at weaning increases histidine oxidation to approx. 45% and, thus, resembles the effect of methionine in promoting histidine oxidation and decreasing formiminoglutamic acid excretion. The feeding of methionine to the thyroidectomized animal further increases histidine oxidation to 65%. The distribution of folate forms in the liver was determined by column chromatography following administration of a dose of tritiated folic acid. In the normal animal, tetrahydrofolate accounts for 38% of the total folate present. The feeding of methionine increases this to 48%, which is consistent with the observed increase in histidine metabolism. Thyroidectomy increases the percentage of tetrahydrofolate to 63% and the feeding of methionine further increases it to 68%. The percentage of tetrahydrofolate relative to total folate is in proportion to the observed rate of histidine metabolism. The action of thyroidectomy in increasing histidine oxidation may be accounted for by its effect in increasing the proportion of tetrahydrofolate.     

Long-term ingestion of ammonium inhibits lysosomal proteolysis in rat liver

A standard diet was supplemented with ammonium acetate (20%, w/w). The effect on liver protein degradation of oral administration of the ammonium diet to rats for 6 weeks has been studied. It is shown that lysosomal proteolysis is markedly decreased (by 62%) while non-lysosomal proteolysis is inhibited by 11%. This is the first report showing that ammonium ingestion inhibits liver proteolysis.     

Time-restricted feeding mice a high-fat diet induces a unique lipidomic profile

Time-restricted feeding (TRF) can reduce adiposity and lessen the co-morbidities of obesity. Mice consuming obesogenic high-fat (HF) diets develop insulin resistance and hepatic steatosis, but have elevated indices of long-chain polyunsaturated fatty acids (LCPUFA) that may be beneficial. While TRF impacts lipid metabolism, scant data exist regarding the impact of TRF upon lipidomic composition of tissues. We (1) tested the hypothesis that TRF of a HF diet elevates LCPUFA indices while preventing insulin resistance and hepatic steatosis and (2) determined the impact of TRF upon the lipidome in plasma, liver, and adipose tissue. For 12 weeks, male, adult mice were fed a control diet ad libitum, a HF diet ad libitum (HF-AL), or a HF diet with TRF, 12 hours during the dark phase (HF-TRF). HF-TRF prevented insulin resistance and hepatic steatosis resulting from by HF-AL treatment. TRF-blocked plasma increases in LCPUFA induced by HF-AL treatment but elevated concentrations of triacylglycerols and non-esterified saturated fatty acids. Analysis of the hepatic lipidome demonstrated that TRF did not elevate LCPUFA while reducing steatosis. However, TRF created (1) a separate hepatic lipid signature for triacylglycerols, phosphatidylcholine, and phosphatidylethanolamine species and (2) modified gene and protein expression consistent with reduced fatty acid synthesis and restoration of diurnal gene signaling. TRF increased the saturated fatty acid content in visceral adipose tissue. In summary, TRF of a HF diet alters the lipidomic profile of plasma, liver, and adipose tissue, creating a third distinct lipid metabolic state indicative of positive metabolic adaptations following HF intake.     

Effect of ammonium acetate-induced hyperammonemia on metabolism of guanidino compounds.

Guanidino compounds are synthesized from arginine in various tissues such as liver, kidney, brain, and skeletal muscle. Guanidino compounds such as arginine and creatine play an important role in nitrogen metabolism, whereas other guanidino compounds such as guanidinosuccinic acid and alpha-N-acetylarginine are known toxins. In order to understand the changes in the metabolism of guanidino compounds during ammonia toxicity, we investigated the effect of hyperammonemia induced by an ammonium acetate injection on the levels of guanidino compounds in plasma, liver, kidney, and brain of rats. Control animals were injected with an equal volume of saline. Blood and tissues were removed 1 h following ammonium acetate or saline injection and guanidino compounds were analyzed by high-performance liquid chromatography. Plasma and kidney levels of guanidinosuccinic acid were significantly elevated in rats challenged with ammonium acetate. Brain alpha-N-acetylarginine levels were also significantly higher in rats injected with ammonium acetate as compared to those in controls. Our results suggest that guanidinosuccinic acid and alpha-N-acetylarginine may play an important role in hyperammonemia.     

Inhibition of lipid synthesis by beta beta'-tetramethyl-substituted, C14-C22, alpha, omega-dicarboxylic acids in the rat in vivo

beta beta'-Methyl-substituted alpha, omega-dicarboxylic acids (MEDICA) of C14-C18 chain length were found to inhibit liver lipid synthesis in the rat in vivo. Maximum inhibition was observed with MEDICA 16 amounting to a 50% decrease in fatty acid and cholesterol biosynthesis in the presence of 0.07 and 0.015% (w/w) of the drug in the diet, respectively. Inhibition of lipid biosynthesis by MEDICA 16 involved a reduction in cytosolic acetyl-CoA content, while the carbon flux from glucose to glycogen, protein, and carbon dioxide remained unaffected. Inhibition of lipogenesis by MEDICA 16 resulted in a 50% decrease in liver and carcass (but not brain) neutral lipid ester content at 0.25% (w/w) of the drug in the diet, as well as in a dose-dependent hypotriglyceridemic effect, with an up to 3-fold reduction in serum triacylglycerols. Inhibition of cholesterogenesis by MEDICA 16 resulted in a hypocholesterolemic effect, with 60 and 45% reductions in (very low density + low density lipoprotein) cholesterol and high density lipoprotein cholesterol, respectively.     

Lipid peroxidation and glutathione system in hyperlipemic rabbits: influence of olive oil administration

We studied the effect of supplementation (10% w/w) of a hyperlipemic diet (1% cholesterol) with olive oil (OLIV) for 6 weeks in four groups of 10 rabbits each. At the end of this period, we determined lipid peroxidation, glutathione content, and glutathione peroxidase, reductase and transferase activities in liver, brain, heart, aorta and platelets. The atherogenic diet increased tissue lipid peroxidation and decreased the protective antioxidant effect of glutathione. Dietary supplementation with olive oil reduced tissue lipid peroxidation by 71.6% in liver, 20.3% in brain, 84.5% in heart, 63.6% in aorta, 72% in platelets. The ratios total/oxidized glutathione were increased in all tissues (49% in liver, 48% in brain, 45% in heart, 83% in aorta, 70% in platelets). Olive oil increased glutathione peroxidase and transferase activities in all tissues. We conclude that in rabbits made hyperlipemic with a diet rich in saturated fatty acids, olive oil decreased tissue oxidative stress.     

Ammonium ingestion prevents depletion of hepatic energy metabolites induced by acute ammonium intoxication

Ingestion of an ammonium containing diet produces hyperammonemia and protects rats against acute ammonium intoxication. Acute ammonium toxicity has been attributed to the depletion of energy metabolite intermediates. We show here that hyperammonemia affords considerable protection against depletion of hepatic energy metabolites evoked by ammonium acetate injection. In control rats there were marked decreases in the content of acetoacetate, beta-hydroxybutyrate, ATP, 2-oxoglutarate, lactate, and pyruvate while phosphoenolpyruvate increased markedly. In hyperammonemic rats beta-hydroxybutyrate, ATP, 2-oxoglutarate, and lactate were not significantly affected while pyruvate increased markedly and phosphoenolpyruvate slightly. These results suggest that in controls the activity of pyruvate kinase is inhibited after ammonium injection while in hyperammonemic rats it is not inhibited. The content of alanine (an inhibitor of pyruvate kinase) reached 2.8 mumol/g in controls and 1.6 mumol/g in hyperammonemic rats, 15 min after ammonium injection. This could explain the different effects of ammonium injection on control and hyperammonemic rats.     

Morin a flavonoid exerts antioxidant potential in chronic hyperammonemic rats: a biochemical and histopathological study

Plant flavonoids are emerging as potent therapeutic drugs effective against a wide range of free radical-mediated diseases. Morin (3,5,7,2′,4′-pentahydroxyflavone), a member of flavonols, is an important bioactive compound by interacting with nucleic acids, enzymes and protein. In this study, we found that morin (30 mg/kg body weight) by oral administration offers protection against hyperammonemia by means of reducing blood ammonia, oxidative stress and enhancing antioxidant status in ammonium chloride-induced (100 mg/kg body weight; i.p) hyperammonemic rats. Enhanced blood ammonia, plasma urea, lipid peroxidation in circulation and tissues (liver and brain) of ammonium chloride-treated rats was accompanied by a significant decrease in the tissues levels of superoxide dismutase (SOD), catalase, reduced glutathione (GSH) and glutathione peroxidase (GPx). Morin administered rats showed a significant reduction in ammonia, urea, lipid peroxidation with a simultaneous elevation in antioxidant levels. Cotreatment with morin prevented the elevation of liver marker enzymes induced by ammonium chloride. The body weight of the animals decreased significantly on ammonium chloride administration when compared with control group. However, cotreatment with morin significantly prevented the decrease of the body weight caused by ammonium chloride. Hyperammonemic rats show liver fibrosis, steatosis, sinusoidal dilatation, etc., along with necrosis, microcystic degeneration in brain. All these changes were reduced in hyperammonemic rats treated with Morin, which too correlated with the biochemical observations. In conclusion, these findings indicate that morin exert antioxidant potential and offer protection against ammonium chloride-induced hyperammonemia. But the exact underlying mechanism needs to be elucidated.     

Increased activity of stearoyl-CoA desaturation in liver from rat fed clofibric acid

Male rats were fed a diet containing 0.5% (w/w) p-chlorophenoxyisobutyric acid (clofibric acid), a hypolipidemic drug. Activities of stearoyl-CoA desaturation in hepatic microsomes were increased approx. 4 times following the administration of clofibric acid for 7 days. An increase in the activity of desaturation of stearic acid was also observed in the liver of clofibric acid-fed rats in vivo. The increase in the activity of microsomal stearoyl-CoA desaturation by clofibric acid-feeding was due to the increase in the activity of terminal desaturase as measured by the rate constant for cytochrome b5 reoxidation, but not due to the changes in cytochrome b5 content and NADH-cytochrome b5 reductase activity. Increases in the activity of stearoyl-CoA desaturation by clofibric acid-feeding were also observed in rats of hormonally altered state, such as diabetic rats, hyperthyroid rats and hypothyroid rats. Percentages of octadecenoic acid in total fatty acid of hepatic lipid were increased with the increase in the activity of stearoyl-CoA desaturation.     

Enhancement of rat liver catalase activity dietary cholesterol

The effects of a fat-supplemented diet and clofibrate (ethylchlorophenoxyisobutirate) upon serum lipids and liver catalase activity were studied in male rats. A butter-supplemented diet produced a striking increase of serum triglycerides but did not affect the liver catalase activity. Cholesterol (1%, w/w), added to the butter supplemented diet markedly increased liver catalase activity. This diet produced a hypercholesterolemic state higher than that induced by a butter-supplemented diet only, although the hypertriglyceridemic effect was less pronounced. Clofibrate given a butter-supplemented diet produced a marked increase of liver catalase activity (about four-fold). When clofibrate is administered with the cholesterol-supplemented diet, the increment observed in the liver catalase activity was the same as that induced with the cholesterol supplemented diet alone. Clofibrate, in either lipid-rich diet, failed to induce a hypocholesterolemic response, although a clear hypotrigliceridemic effect was evident. This effect appears to be potentiated with clofibrate and the cholesterol supplemented diet. Thus the increment in liver catalase activity induced by dietary cholesterol and clofibrate seems to be related to a hypotriglyceridemic effect which gives support to a role of liver peroxisomes in lipid metabolism. The role that liver catalase would play, in this regard, remains unclear from these results.     

Effect of the Supplementation of Sulfur Amino Acids to a Diet Containing Soy Protein Isolate on Lipid Metabolism in Rats

The effect of the supplementation of sulfur amino acids to a low casein or soy protein isolate diet on tissue lipid metabolism was investigated. Supplementation of methionine to a 8% casein diet produced a fatty liver in rats, however, supplementation of methionine to a 8.8% soy protein diet (corresponding to a 8% casein diet as to sulfur amino acids content) did not produce a fatty liver. At the level of 8% or less of soy protein in the diet, the accumulation of liver lipids and serum triglyceride was observed. An amino acid mixture simulating the composition of soy protein isolate caused significant accumulation of liver lipids, but serum triglyceride was not changed. Serum cholesterol in rats fed the soy protein diet was lower than that in rats fed the casein diet, but on feeding the amino acid mixtures simulating these protein diets, there was no difference between the two groups. The small differences between soy protein isolate and casein as to lipid metabolism might be due to the small differences in the contents of sulfur amino acids or the specific nature of the soy protein or casein. The supplemental effect of methionine and cystine was also studied. About 60% of total sulfur amino acids could be substituted by cystine for maximum growth.     

In vivo model for dyslipidemia with diabetes mellitus in hamster

Due to similarities in lipid metabolism to those in humans, hamster is considered as a good model for the study of regulatory mechanisms of plasma lipoproteins in response to cholesterol or fatty acid-enriched diet. This model of hyperlipidemia has been modified to produce dyslipidedmia with diabetes complexities by feeding with high fat diet added with 9% (w/w) fructose. Feeding this diet to hamster for 10 days markedly increases plasma levels of triglyceride, cholesterol, fatty acids followed by a significant increase in glycerol, beta lipoproteins, high density lipoprotein, glucose and glycosylated proteins. This model is being used for research and development of lipid lowering drugs with hypoglycemic activity in collaboration with Novo Nordisk, Denmark. The modified high fat diet formulation has now been prepared (Research diet D.99122211) and supplied by Research Diets Inc, Burnswick USA.     

Effects of saturated and unsaturated fats given with and without dietary cholesterol on hepatic cholesterol synthesis and hepatic lipid metabolism

Hepatic cholesterol synthesis was studied in rats after consuming diets of varying neutral lipid and cholesterol content. Cholesterol synthesis was evaluated by measuring 3-hydroxy-3-methylglutaryl-CoA reductase and by determining the rate of 3H-labeled sterol production from [3H]mevalonate. Results were correlated with sterol balance data and hepatic lipid content. Hepatic cholesterol synthesis was relatively great when cholesterol was excluded from the diet. The source of neutral dietary lipids, saturated vs. unsaturated, produced no change in hepatic sterol synthesis. Values for fecal sterol outputs and hepatic cholesterol levels were also similar in rats consuming either saturated or unsaturated fats. When 1% cholesterol was added to the diet, hepatic cholesterol synthesis was suppressed but the degree of suppression was greater in rats consuming unsaturated vs. saturated fats. This was associated with greater accumulation of cholesterol in livers from rats consuming unsaturates and a reduction in fecal neutral sterol output in this group as opposed to results from rats on saturated fats. Cholesterol consumption also altered the fatty acid composition of hepatic phospholipids producing decreases in the percentages of essential polyunsaturated fatty acids. It is concluded that dietary cholesterol alters cholesterol and fatty acid metabolism in the liver and that this effect is enhanced by dietary unsaturated fats.     

Dietary fat saturation produces lipid modifications in peritoneal macrophages of mouse

We investigated the effects of a saturated fat diet on mice lipid metabolism in resident peritoneal macrophages. Male C57BL/6 mice were weaned at 21 days of age and assigned to either the experimental diet, containing coconut oil (COCO diet), or the control diet, containing soybean oil as fat source. Fat content of each diet was 15% (w/w). Mice were fed for 6 weeks until sacrifice. In plasma of mice fed the COCO diet, the concentration of triglyceride, total cholesterol, HLD- and (LDL+VLDL)-cholesterol, and thiobarbituric acid-reactive substances (TBARS) increased, without changes in phospholipid concentration, compared with the controls. In macrophages of COCO-fed mice, the concentration of total (TC), free and esterified cholesterol, triglyceride, phospholipid (P) and TBARS increased, while the TC/P ratio did not change. The phospholipid compositions showed an increase of phosphatidylcholine and phosphatidylserine + phosphadytilinositol, a decrease of phosphatidylethanolamine, and no change in phosphatidylglycerol. (3)H(2)O incorporation into triglyceride and phospholipid fractions of macrophages increased, while its incorporation into free cholesterol decreased. Incorporation of [(3)H]cholesterol into macrophages of COCO-fed mice and the fraction of [(3)H]cholesterol ester increased. COCO diet produced an increase in myrystic, palmitic and palmitoleic acids proportion, a decrease in linoleic and arachidonic acids and no changes in stearic and oleic acids, compared with the control. Also, a higher relative percentage of saturated fatty acid and a decrease in unsaturation index (p <0.001) were observed in macrophages of COCO-fed mice. These results indicate that the COCO-diet, high in saturated fatty acids, alters the lipid metabolism and fatty acid composition of macrophages and produces a significant degree of oxidative stress.     

Effects of Cricket Extract on Lipid Metabolism and Body Fat Content in High- Fat Diet Fed Rats

The effect of cricket extract on high‐fat diet fed rat was observed. It was shown that cricket extract prevented the increment of body weight by high‐fat diet. The extract also decreased the value of AST in liver. The most significant effect of the extract was shown on lipid metabolism. The contents of total lipid and total cholesterol in liver and feces were reduced by the extract on dose‐dependent. These statistically significant results were clear in 3% extract treated group (HFD3) while were slight in 1% extract treated group (HFD1). The same result was also shown in body fat content.