Postprandial exchange of urea and ammonia nitrogen between the digestive tract and portal blood in the conscious pig after ingestion of a diet with or without urea

The concentrations of urea and ammonia were measured in the portal and arterial blood simultaneously to the blood flow rate in the portal vein during the postprandial period (8 hrs.) after ingestion of a normal protein diet with 3% urea (10 meals) or without urea (12 meals) in conscious pigs (mean body weight: 55.5 +/- 2.3 kg). When no urea was present in the diet, there was a slight and permanent uptake of blood urea by the gut (570 mg/h, i.e. 9,5 mmoles/h) as well as a permanent appearance of ammonia in the portal vein (258 mg/h i.e. 15.2 mmoles/h), increasing with time (P less than 0.05). The absorbed ammonia nitrogen represented a maximum of 70% of urea nitrogen taken up. 2. Addition of urea to the diet brought about a large absorption of that substance (73% of the ingested amount) followed by a rather large excretion (960 mg/h, i.e. 16 mmoles/h), 5-6 hrs. after the meal and led to an increase (P less than 0.05) in the absorption of ammonia.     

The potentiation of ammonia toxicity by sodium benzoate is prevented by L-carnitine

Sodium benzoate has been recommended and even been used for the treatment of hyperammonemia in humans. More recently, a note of caution was raised since it has been shown that in experimental animals, sodium benzoate potentiates ammonia toxicity and inhibits urea synthesis in vitro. This has been further confirmed in the work presented here and the mechanism by which benzoate increases mortality and the levels of blood ammonia in mice given ammonium acetate have also been studied. In hyperammonemia, urea production and N-acetylglutamate levels were decreased by sodium benzoate. Pretreatment of mice with L-carnitine suppressed mortality following ammonium acetate plus sodium benzoate administration. Under these conditions L-carnitine lowered blood ammonia and increased urea production and N-acetylglutamate levels.     

Protective effect of ethanol on acute ammonia intoxication in mice

A single i.p injection of 12 mmoles ammonium acetate/kg produced 100% mortality in mice. Ethanol in doses of 11 to 75 mmoles/kg administered along with the ammonium acetate decreased dramatically the mortality, the maximum protective effect being at 75 mmoles/kg. Blood and brain ammonia levels were also significantly reduced, while blood ethanol was higher in animals injected with ammonia and ethanol. Methanol and butanol also had some protective effect.     

Amino acids in the rat liver and plasma and some metabolites in the liver after sodium benzoate treatment

The effect of sodium benzoate administration on amino acids in the liver and plasma and various metabolites in the liver was studied. Changes in glutamine and ornithine were noted only at a higher dose (10 mmol/kg body wt) of benzoate, whereas even a lower dose caused a significant decrease in glycine, serine, and alanine levels of plasma and liver. A dose- and time-dependent decrease in glycine levels was studied. A decrease of up to 50% in the glycine concentration may limit its own transport into mitochondria and availability for the formation of hippurate. A decrease in alanine may have resulted from stimulation of gluconeogenesis from alanine, by increased ammonia. Among the metabolites studied, ATP and acetyl-CoA decreased and ammonia increased significantly even at a lower dose (5 mmol/kg body wt) of benzoate. The compounds that require ATP for their synthesis such as N-acetylglutamate and glutamine decreased significantly only at the higher dose of benzoate, whereas urea and glutathione levels were unaffected under our experimental conditions.     

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.     

Prevention of ammonia toxicity byl-carnitine: metabolic changes in brain

l-Carnitine when injected in mice 30 min before an LD₁₀₀ of ammonium acetate (12 mmol/kg body weight, intraperitoneal) reduced mortality (100% survival with 16 mmoll-carnitine/kg) and prevented the appearance of symptoms of ammonia toxicity. Brain ammonia decreased in the animals givenl-carnitine. Ammonia decreased the levels of glutamate in brain; they were partially restored byl-carnitine, which also reduced the increase in brain glutamine in animals given only ammonia. The redox state of the brain was altered following ammonia intoxication. The ratio of lactate to pyruvate in the cytosol increased while that of glutamate to -ketoglutarate in the mitochondria decreased. These ratios were partially restored byl-carnitine. The implications of these findings are discussed relative to the mechanism of ammonia toxicity.     

Antileishmanial effect of cisplatin against murine visceral leishmaniasis

Drug development in visceral leishmaniasis is extremely vital as the existing therapeutic modalities are plagued by the unwanted twosome of toxicity and drug resistance. Antineoplastic drugs have in the past been effective against the parasitic infections, for example, miltefosine. Cisplatin is a first-generation platinum-containing drug, used in the treatment of various solid tumors. Its in vitro antileishmanial effect has already been demonstrated. In the present study, the leishmanicidal potential of two doses (0.5 mg/kg body weight and 1 mg/kg body weight) of the drug was studied in BALB/c mice. The antileishmanial effect of the drug was revealed by significant reduction in the parasite burden. The infected and treated animals were also found to exhibit increased DTH responses. An initial transient and reversible increase in levels of SGOT, SGPT, BUN, blood urea, creatinine and phosphorus was observed in infected animals treated with both doses of the drug. The reduction in parasite load, increase in DTH response and various biochemical parameters were more pronounced in animals treated with 1 mg/kg body weight of cisplatin as compared to those treated with 0.5 mg/kg body weight of the drug. Though some histopathological changes were observed in the kidneys of animals treated with 1 mg/kg body weight of cisplatin, no such change was observed in mice treated with the lower dose. Thus, we have for the first time characterized the in vivo effect of cisplatin in murine experimental visceral leishmaniasis.     

Antioxidant effects of Emblica officinalis and Zingiber officinalis on arsenic and lead induced toxicity on Albino rats

It is of interest to document the effect of Emblica officinalis (E. officinalis) and Zingiber officinalae (Z. officinalae) leaf extract on reactive oxygen species, antioxidant potential changes in arsenic and lead-induced toxicity in male rats. We used 8 groups of adult male Wistar rats with 1 control group for this study. The animals were divided into Group I: Control and Group II: Lead and sodium arsenite induced rats (animals were induced for metal toxicity by the combined administration of arsenic (13.8 mg/ kg body weight) and lead (116.4 mg/kg body weight). These doses were administered by gastric intubation during 14 consecutive days using known standard procedures. Arsenic and lead induced rats treated with ethanolic extract of Emblica officinalis (60 mg/kg body weight/day, orally for 45 days) are group III rats. Group IV animals are arsenic and lead induced rats treated orally with ethanolic extracts of E. officinalis (120 mg/kg body weight/day for 45 days). Group V animals are arsenic and lead induced rats treated orally with ethanolic extracts of Z. officinalae (60 mg/kg body weight/day for 45 days). Group VI animals are arsenic and lead induced rats orally treated with ethanolic extracts of Zingiber officinalis (120 mg/kg body weight/day for 45 days). Group VII animals are arsenic and lead induced rats treated orally with ethanolic extracts of E. officinalis and Z. officinalae (60 + 60 mg/kg body weight/day for 45 days). Group VIII animals are arsenic and lead induced rats treated orally with ethanolic extracts of E. officinalis and Z. officinalae (120 + 120 mg/kg body weight/day, orally for 45 days). Normal Control animals were treated orally with ethanolic extracts of E. officinalis (120mg/kg body weight) + Z. officinalae (120mg/kg body weight) for 45 days. The control and experimental animals were then subjected to analysis for oxidative stress markers such as H2O2, *OH, and lipid peroxidation (LPO), antioxidant enzymes in addition to liver and kidney function markers. Results: Arsenic and lead induced rats showed a significant increase in the levels of reactive oxygen species (H2O2, OH* and LPO) with concomitant alterations in the renal and liver tissues. However, enzymic and non-enzymic antioxidant levels were decreased. Nevertheless, an oral effective dose of E. officinalis and Z. officinalae (120 + 120 mg/kg body weight/day increased the antioxidant enzymes and retrieved the altered levels of ROS and LPO that were induced by arsenic and lead. Thus, we show that E. officinalis and Z. officinalae leaf extract exhibits nephroprotective and hepatoprotective role through the restoration of reactive oxygen species and antioxidant enzymes in the kidney and liver tissue of Arsenic and Lead-induced nephrotoxicity and hepatotoxicity in rats. Hence, E. officinalis and Z. officinalae leaf extract are potential therapeutic options for the treatment of metal toxicity-induced kidney and liver diseases.     

The Effect of Ouabain and External Potassium on the Ion Transport of Rabbit Red Cells

Na efflux of rabbit RBC is approximately 10 mmoles/kg wet weight. hr. One-half of this consists of a ouabain-insensitive exchange diffusion component. Ouabain inhibits 2.5 mmoles/kg.hr of Na efflux. K influx is 3.0 mmoles/kg.hr; 2.2 mmoles/kg.hr are inhibited by ouabain. In contrast with human RBC, ouabain inhibition of Na efflux and K influx of rabbit RBC is easily reversible. After 2 hr, ouabain inhibition of Na efflux is completely compensated for by increased internal Na concentration and Na efflux returns to initial levels. Removal of ouabain at this stage results in stimulation of the efflux by 4.3 mmoles/kg.hr. Na influx is initially not affected by ouabain but is increased by 2.4 mmoles/kg.hr after 2 hr incubation with the drug. Removal of K from normal Ringer does not affect Na efflux and increases Na influx by 1.6 mmoles/kg.hr. Addition of ouabain to K-free Ringer inhibits Na efflux and influx to the same extent (1.6 mmoles/kg.hr). Removal of Na from K-free Ringer has an inhibitory effect on efflux similar to that of ouabain. These findings suggest that the fraction of Na efflux inhibited by removal of external K is completely replaced by a new, ouabain-sensitive exchange diffusion of Na ions.     

Effects of a slow-release urea source on absorption of ammonia and endogenous production of urea by cattle

Three experiments were conducted with Angus or Holstein steers to evaluate effects of dietary urea–calcium (a slow rumen-release urea source) on absorption of ammonia N from the gut and urea N production in the liver. Steers were fed a high-grain diet (Experiment 1) or an all-forage diet (Experiments 2 and 3). Urea or urea–calcium (0.25 g/kg body weight) was dosed into the esophagus (Experiments 1 and 2) or rumen (Experiment 3), and blood samples were serially collected for 180 min. Blood concentrations of ammonia N and urea N were measured in all experiments, and net flux of metabolites across splanchnic tissues was measured in Experiment 3. Compared to urea, urea–calcium reduced (P<0.05) plasma concentrations of ammonia N in steers fed all-forage diets, and tended (P<0.06) to reduce arterial glucose concentrations in Experiment 3. Plasma concentrations of urea N were not affected by treatment in any experiment. Treatment and time post-dosing interactions (P<0.05) in Experiment 3 were due to increased ruminal fluid concentrations of ammonia N, net release of ammonia N by portal-drained viscera and total splanchnic tissues with urea versus urea–calcium treatment shortly after dosing. Similar interactions (P<0.05) indicated that urea caused higher hepatic glucose release and increased l-lactate release by total splanchnic tissues after dosing than urea–calcium. Urea–calcium was effective in mitigating rapid ammonia release in the rumen and subsequent effects on glucose and lactate metabolism.     

The effect of protein content of the diet on the rate of urea formation in sheep liver

1. In the livers of six sheep given a high-protein diet, the concentrations of certain urea-cycle enzymes [ornithine transcarbamoylase, arginine synthetase (combined activity of argininosuccinate synthetase and argininosuccinase) and arginase] were significantly greater than when the sheep were given a low-protein diet. Alkaline phosphatase activity/mg. of liver protein was not significantly affected by diet. 2. Three sheep previously given the high-protein diet showed no significant rise in the concentration of ammonia in the blood after the administration of urea (0·5g./kg. body wt.). The concentration of ammonia in the blood of the three sheep given the low-protein diet rose exponentially with time after dosing with urea and all sheep died. 3. It is suggested that tolerance to ammonia toxicity in the sheep is at least partly a function of the activity of the urea-cycle enzymes in the liver.     

Benzoate stimulates glutamate release from perfused rat liver.

In isolated perfused rat liver, benzoate addition to the influent perfusate led to a dose-dependent, rapid and reversible stimulation of glutamate output from the liver. This was accompanied by a decrease in glutamate and 2-oxoglutarate tissue levels and a net K+ release from the liver; withdrawal of benzoate was followed by re-uptake of K+. Benzoate-induced glutamate efflux from the liver was not dependent on the concentration (0-1 mM) of ammonia (NH3 + NH4+) in the influent perfusate, but was significantly increased after inhibition of glutamine synthetase by methionine sulphoximine or during the metabolism of added glutamine (5 mM). Maximal rates of benzoate-stimulated glutamate efflux were 0.8-0.9 mumol/min per g, and the effect of benzoate was half-maximal (K0.5) at 0.8 mM. Similar Vmax. values of glutamate efflux were obtained with 4-methyl-2-oxopentanoate, ketomethionine (4-methylthio-2-oxobutyrate) and phenylpyruvate; their respective K0.5 values were 1.2 mM, 3.0 mM and 3.8 mM. Benzoate decreased hepatic net ammonia uptake and synthesis of both urea and glutamine from added NH4Cl. Accordingly, the benzoate-induced shift of detoxication from urea and glutamine synthesis to glutamate formation and release was accompanied by a decreased hepatic ammonia uptake. The data show that benzoate exerts profound effects on hepatic glutamate and ammonia metabolism, providing a new insight into benzoate action in the treatment of hyperammonaemic syndromes.     

Ammonia absorption from the rumen to the systemic circulation with urea poisoning in goats.

To confirm the transfer of ammonia leaking from the rumen content via the liver to the perid by laparotomy. When ammonia leakage from the hepatic vein occurred, it was followed by an increase in ammonia concentration in the jugular vein. There were increases of ammonia concentration in the intestinal vein and in the thoracic duct after urea drenching. These increases suggested neither trapping ammonia in the peritoneal fluid nor responsibility for increases of ammonia in the systemic circulation, respectively. At times when respiration ceased due to urea poisoning, the peritoneal fluids wee in the fluid. The hypothesis of the peritoneal cavity-thoracic duct route of ammonia absorption, presented by some of previous workers on urea toxicity, was not supported in the present study.     

Sodium benzoate inhibits fatty acid oxidation in rat liver: effect on ammonia levels

Sodium benzoate inhibited PC and octanoic acid-mediated State 3 respiration rates by 39 and 29%, respectively, at 0.5 mM in isolated rat liver mitochondria. At 2 mM, benzoate did not affect State 3 respiration rates with either succinate or malate plus glutamate, indicating that it did not act as an uncoupler. The oxidation of palmitate and octanoate was inhibited by 39 and 54% at 2 mM benzoate in liver homogenates. Benzoate, at 10 mmol/kg caused significant decreases in the levels of hepatic ATP, CoA, and acetyl-CoA. Administration of sodium benzoate to rats caused a dose-dependent increase in hepatic ammonia levels. However, the inhibitory effect of benzoate on fatty acid oxidation is not mediated through ammonia since ammonium chloride, at 1 mM, did not inhibit PC or octanoate oxidation in mitochondria or their oxidation in liver homogenate. Our results warrant a reevaluation of the use of sodium benzoate in the treatment of hyperammonemia.     

Developmental toxicity evaluation of emodin in rats and mice

BACKGROUND: Emodin, a widely available herbal remedy, was evaluated for potential effects on pregnancy outcome. METHODS: Emodin was administered in feed to timed-mated Sprague-Dawley (CD) rats (0, 425, 850, and 1700 ppm; gestational day [GD] 6-20), and Swiss Albino (CD-1) mice (0, 600, 2500 or 6000 ppm; GD 6-17). Ingested dose was 0, 31, 57, and approximately 80-144 mg emodin/kg/day (rats) and 0, 94, 391, and 1005 mg emodin/kg/day (mice). Timed-mated animals (23-25/group) were monitored for body weight, feed/water consumption, and clinical signs. At termination (rats: GD 20; mice: GD 17), confirmed pregnant dams (21-25/group) were evaluated for clinical signs: body, liver, kidney, and gravid uterine weights, uterine contents, and number of corpora lutea. Fetuses were weighed, sexed, and examined for external, visceral, and skeletal malformations/variations. RESULTS: There were no maternal deaths. In rats, maternal body weight, weight gain during treatment, and corrected weight gain exhibited a decreasing trend. Maternal body weight gain during treatment was significantly reduced at the high dose. In mice, maternal body weight and weight gain was decreased at the high dose. CONCLUSIONS: Prenatal mortality, live litter size, fetal sex ratio, and morphological development were unaffected in both rats and mice. At the high dose, rat average fetal body weight per litter was unaffected, but was significantly reduced in mice. The rat maternal lowest observed adverse effect level (LOAEL) was 1700 ppm; the no observed adverse effect level (NOAEL) was 850 ppm. The rat developmental toxicity NOAEL was > or =1700 ppm. A LOAEL was not established. In mice, the maternal toxicity LOAEL was 6000 ppm and the NOAEL was 2500 ppm. The developmental toxicity LOAEL was 6000 ppm (reduced fetal body weight) and the NOAEL was 2500 ppm.     

The effects of prenatal tertiary butanol administration in CBA/J and C57BL/6J mice

Pregnant mice of the CBA/J and C57BL/6J strains were given either tertiary butanol (10.5 mmoles/kg, p.o.) or an equivalent volume of tap water twice daily from day 6 through day 18 of gestation. Examination on day 18 revealed significantly more resorptions per litter in the t-butanol-treated animals but no interstrain difference. Tertiary butanol did not significantly affect the body weight of the survivors nor produce significant abnormalities in either strain. Subsequent blood concentration profiles in female C57BL/6J mice indicated that the treatment regimen produced blood levels equivalent to teratogenic ethanol treatment. Mice receiving 3 days of t-butanol treatment did not eliminate the drug more rapidly than control animals, indicating that tolerance was not a factor in the treatment regimen. Since t-butanol shares membrane disordering effects with ethanol but is not metabolized by the same pathway, a role for acetaldehyde or the process of ethanol metabolism is suggested in ethanol teratogenicity.     

Maternal and developmental toxicity of low doses of cytosine arabinoside in mice

1-beta-D-Arabinofuranosylcytosine (Ara-C), an effective drug for the treatment of leukemia and breast cancer, was evaluated for developmental toxicity in pregnant Swiss mice. Ara-C was administered by intraperitoneal injection on gestational days 6-15 at doses of 0, 0.5, 2, and 8 mg/kg/day. Maternal observations included clinical signs, body weight change, food consumption, and gross evaluation of organs and uterine contents at necropsy (day 18). Live fetuses were examined for external, visceral, and skeletal alterations. Maternal toxicity was observed at 2 and 8 mg/kg/day, as evidenced by a significant decrease in body weight gain and food consumption during the treatment period. Significantly increased early and late resorptions and reduced number of live fetuses per liter as well as decreased fetal body weight were observed at 8 mg/kg/day. At 2 mg/kg/day, the incidence of cleft palate, renoureteral agenesis or hypoplasia, and poly- or oligodactyly was significantly increased, whereas fetal weight was reduced at 0.5 mg/kg/day. Thus, the developmental no-observed-adverse-effect-level (NOAEL) of Ara-C in the pregnant mouse is lower than 0.5 mg/kg/day, while the NOAEL for maternal toxicity is 0.5 mg/kg/day. We believe that exposure to this agent ought to be avoided during organogenesis.     

Preliminary studies on acute and subacute toxicity of an antidiabetic herbal preparation, Dianex

Dianex, a polyherbal formulation intended to use for diabetic patients, has been screened for toxic effects. For acute toxicity studies, Dianex was administered orally in graded doses of 0.75-10 g/kg to the mice. For subacute toxicity studies, different doses of Dianex (1.0, 1.5 and 2.5 g/kg) were administered orally to the rats once daily for 30 days. Animals were observed for physiological and behavioural responses, mortality, food and water intake and body weight changes. Hematological evaluation was performed weekly. All the animals were sacrificed on 31st day and changes in organ weights and histology were examined. Biochemical studies were done in liver and serum. No mortality was observed up to 10 g/kg of Dianex in acute toxicity study. Daily administration of as high as 2.5 g/kg dose of Dianex did not result in any mortality or changes in gross behaviour, body weight, weight and histology of different organs or serum and liver biochemistry. However, significant increase in RBC count and hemoglobin level was observed in the treated animals at all doses. Other peripheral blood constituents were in the normal range. The dose of Dianex to produce significant antidiabetic activity in mouse, 0.25-0.5 g/kg, is much lower than the doses used in the present study. Therefore such doses may be safe for daily administration without causing any serious side effects.     

The effectiveness of steers and heifers treated with oestrogen or testosterone to detect oestrus in cattle

Two experiments were carried out to determine the effectiveness of steers and heifers, treated with oestrogen or testosterone, in the detection of oestrus in cattle.In the first experiment 17 steers castrated at birth and 16 castrated at 6 months of age were randomly allocated to three groups and received an 800 mg subcutaneous implant of testosterone, subcutaneous injections of 10 mg oestradiol benzoate per week for 16 weeks or no hormone (controls). In addition, six heifers were injected subcutaneously with 10 mg oestradiol benzoate per week for 16 weeks while six untreated heifers served as controls. Animals were observed in a standardised libido test 2, 4, 8, 16, 20 and 24 weeks after treatment commenced. The time to first mount and the number of mounts per animal responding in the presence of oestrous heifers were recorded. Both steers and heifers treated with oestradiol benzoate were superior at detection of oestrus in cattle than animals treated with testosterone or those receiving no hormone. Oestrogen-treated animals generally detected heifers in oestrus in less than 3 min after introduction and mounted these animals between 20 and 30 times in one hour. This response was consistent throughout the duration of the experiment. There was no effect of age at castration of steers on development of male behaviour.The second experiment determined the rate and degree of development of male behaviour in steers in response to weekly subcutaneous injections of 0, 2, 4, 8 or 16 mg oestradiol benzoate per 250 kg body weight, 250 mg testosterone or 150 mg dihydrotestosterone for a period of 15 weeks. Steers treated with oestradiol benzoate again proved to be more successful than untreated or testosterone-treated steers at consistently detecting and mounting oestrous heifers. The best response was obtained from steers treated with 8 mg/250 kg body weight per week. The practical application of this work is discussed.     

Failure of sodium benzoate to alleviate plasma and liver ammonia in rats

The intraperitoneal administration of L-norvaline and L-methionine-SR-sulfoximine to rats caused an increase in the concentration of ammonia in plasma as well as in liver. These compounds interfere with urea and glutamine formation, respectively. Subsequent injection of sodium benzoate failed to alleviate ammonia levels, and on the contrary, caused a further increase. Sodium benzoate itself, when administered, resulted in higher levels of ammonia in plasma and liver of the rats. Administration of glycine to rats treated with benzoate did not lower ammonia levels indicating that other factors besides glycine may also be necessary for the removal of sodium benzoate.