Ornithine aminotransferase activity, tissue ornithine concentrations and polyamine metabolism

1. Inactivation of L-ornithine:2-oxoacid aminotransferase (OAT) by 5-fluoromethylornithine (5FMOrn), a specific inactivator of OAT, causes a great elevation of tissue ornithine (Orn) concentrations. 2. Inhibition of L-ornithine decarboxylase (ODC) by 2-difluoromethylornithine (DFMO) had no effect on Orn concentrations. 3. The combined administration of 5FMOrn and DFMO produced a 2- to 3-fold greater enhancement of tissue Orn concentrations than treatment with 5FMOrn alone. 4. The increase of tissue Orn concentrations had a long-lasting enhancing effect on polyamine metabolism. 5. In the brain this could be demonstrated by the elevation of putrescine and spermidine concentrations and the increase of spermidine turnover rate. 6. In visceral organs polyamine concentrations were not elevated because polyamines can be eliminated by transport. 7. In line with this notion is the fact that urinary polyamine excretion was increased for several days, even after a single dose of 5FMOrn. 8. Inhibitors of 4-aminobutyric acid:2-oxoglutarate aminotransferase which are also inactivators of OAT had the same effect on polyamine excretion as 5FMOrn.     

DL-canaline and 5-fluoromethylornithine. Comparison of two inactivators of ornithine aminotransferase.

5-Fluoromethylornithine (5FMOrn) is an enzyme-activated irreversible inhibitor or ornithine aminotransferase (L-ornithine:2-oxo-acid 5-aminotransferase, OAT). For purified rat liver OAT, Ki(app.) was found to be 30 microM. and tau 1/2 = 4 min. Of the four stereomers of 5FMOrn only one reacts with OAT. The formation of a chromophore with an absorption maximum at 458 nm after inactivation of OAT by 5FMOrn suggests the formation of an enamine intermediate, which is slowly hydrolysed to release an unsaturated ketone. L-Canaline [(S)-2-amino-4-amino-oxybutyric acid] is a well-known irreversible inhibitor of OAT. Not only the natural L-enantiomer but also the D-enantiomer reacts by oxime formation with pyridoxal 5'-phosphate in the active site of the enzyme, although considerably more slowly. This demonstrates that the stereochemistry at C-2 of ornithine is not absolutely stringent. In vitro, canaline reacted faster than 5FMOrn with OAT. In vivo, however, only incomplete OAT inhibition was observed with canaline. Whereas intraperitoneal administration of 10 mg of 5FMOrn/kg body wt. to mice was sufficient to inactivate OAT in brain and liver by 90% for 24 h, 500 mg of DL-canaline/kg body wt. only produced a transient inhibition of 65-70%. The accumulation of ornithine in these tissues was considerably slower and the maximum concentrations lower than were achieved with 5FMOrn. It appears that DL-canaline, in contrast with 5FMOrn, is not useful as a tool in studies of biological consequences of OAT inhibition.     

Effects of inhibition of ornithine aminotransferase on thioacetamide-induced hepatogenic encephalopathy

Repeated administration of thioacetamide (TAA) to CD1 mice produced hepatic failure and biochemical and behavioral effects characteristic of hepatogenic encephalopathy (HE). The symptoms in mice resembled those previously observed in rats after similar treatments. It is, howeve, obvious that both in rats and mice the severity of symptoms depends not only on dose and dosing schedule of TAA, but also on strain and body weight (age). Administration of 5-fluoromethylornithine (5FMOrn), a selective inactivator of ornithine aminotransferase (OAT), significantly reduced mortality, and it ameliorated most of the TAA-induced pathologic symptoms, such as hypothermia, decreased locomotor and exploratory behavior, pathologic liver function and amino acid patterns. The most prominent biochemical consequence of 5FMOrn administration is the elevation of ornithine concentrations in tissues, including the brain, and in body fluids. Elevated ornithine concentrations are, therefore, the most likely basis for the therapeutic effects of 5FMOrn. In agreement with this notion is the enhancement of citrulline and urea formation. These findings and the observation that administration of ornithine in combination with a branched-chain 2-oxoacid ameliorated the pathologic symptoms of portal-systemic encephalopathy suggest inhibition of OAT in the treatment of this disease. The liver protective effect of 5FMOrn is not yet understood; the enhancement of regenerative processes is a likely explanation.Abbreviations GABA 4-aminobutyrate - GABA-T 4-aminobutyrate aminotransferase - GOT plasma glutamate oxaloacetate transaminase - HE hepatogenic encephalopathy - LDH plasma lactate dehydrogenase - MAO monoamine oxidase - OAT ornithine aminotransferase - TAA thioacetamide - 5FMOrn 5-fluoromethylornithine Special issue dedicated to Dr. Claude Baxter.     

5-Fluoromethylornithine, an irreversible and specific inhibitor of L-ornithine:2-oxo-acid aminotransferase.

5-Fluoromethylornithine (5-FMOrn) is the first specific irreversible inhibitor of L-ornithine:2-oxoacid aminotransferase (OAT) found. Single doses (greater than 10 mg/kg) of this compound inactivate OAT to a residual OAT-like activity. This activity (10-20% of total activity) is resistant to further inactivation by higher or repeated doses of 5-FMOrn, or incubation with the inactivator in vitro. Ornithine concentrations are greatly enhanced in various tissues, and urinary ornithine is dramatically increased, but no other amino acid is affected after acute treatment with 5-FMOrn. Repeated administration decreases carnosine and homocarnosine concentrations in brain. Toxic effects were not observed. The new inactivator is considered as a tool in the establishment of functions of OAT under physiological and pathological conditions.     

A New Synaptosomal Biosynthetic Pathway of Glutamate and GABA from Ornithine and Its Negative Feedback Inhibition by GABA

In sonicates of mouse brain synaptosomes, we demonstrated that gamma-aminobutyric acid (GABA) can be formed when L-ornithine (Orn) through L-glutamic acid (Glu), but not through putrescine (Put). Incubation of these sonicates with [3H]ORN yielded not only [3H]Glu and [3H]L-proline (Pro) but also produced [3H]GABA from the [3H]Glu. Formation of each of these three major amino acids from [3H]Orn was strongly inhibited by the addition of GABA (1-5 mM). The likely enzymatic site of this negative feedback inhibition by GABA appeared to be ornithine delta-aminotransferase (OAT). A radiometric procedure was employed to study the effects of the three amino acids cited above and of others found in the free form in brain on the activity of a 30-fold-purified OAT from rat brain. Enzyme activity was measured in the presence of low concentrations of Orn, such as might occur in vivo. OAT was inhibited by GABA to a considerably greater extent than by Glu, L-glutamine, or Put; no inhibition was found with Pro, glycine, aspartarte, taurine, or beta-alanine. The inhibition of GABA was competitive with Orn. These results clearly show that one of the molecular mechanisms underlying the negative feedback inhibition of synaptosomal GABA biosynthesis from Orn is a competitive inhibition by GABA of the brain OAT activity that is responsible for the formation of L-glutamic-gamma-semialdehyde in equilibrium with L-delta 1-pyrroline-5-carboxylic acid from Orn. Thus, the results suggest that GABA may play an important role in restricting the metabolic flow from Orn to Glu and thence to GABA. It is confirmed that L-canaline (delta-aminooxy-L-alpha-aminobutyric acid) is a potent and specific inhibitor of brain OAT whereas much weaker inhibition was observed with two other carbonyl-trapping agents, aminooxyacetic acid and hydrazine.     

Interrelationships between ornithine,glutamate and GABA—I. Feed-back inhibition of ornithine aminotransferase by elevated brain GABA levels

In this work new methods for the determination of ornithine (Orn) and l-ornithine:2-oxoacid aminotransferase (OAT) activity are described. These methods were used to demonstrate linear interrelationships between brain GABA and Orn concentrations. Brain GABA levels were modulated by administration of vigabatrin (4-aminohex-5-enoic acid), a specific inactivator of GABA-T, which is not an inhibitor of OAT. The results suggest feed-back inhibition of OAT by GABA, a mechanism which is compatible with the assumption that Orn may serve in certain neurons as a precursor of glutamate and GABA.     

Interrelationships between ornithine,glutamate, and GABA. II. Consequences of inhibition of GABA-T and ornithine aminotransferase in brain

The objective of the present study was to compare the effects of elevation of GABA concentration and those of inactivation ofl-ornithine: 2-oxoacid aminotransferase (OAT) on the in vivo metabolism ofl-ornithine (Orn) in brain. Vigabatrin (4-aminohex-5-enoic acid) and gabaculine (5-amino-1,3-cyclohexadienyl carboxylic acid), two well known inactivators of GABA-T, were used to elevate brain GABA concentrations. The latter inactivates OAT also. Transamination of Orn is, from a quantitative point of view, a significant reaction in mouse brain. GABA is a feed-back regulator of OAT. Within GABAergic neurons Orn concentration may be regulated by endogenous GABA. Extensive inactivation of OAT causes a considerable increase of Orn concentration, both in synaptosomes and in non-synaptosomal compartments. The results are compatible with a role of Orn as precursor of glutamate and/or GABA in certain neurons.     

Differential sensitivity of cytotoxic T lymphocytes and lymphokine-activated killer cells to inhibition by L-ornithine

The selective inhibition of murine cytotoxic T lymphocyte (CTL) differentiation in C57B1/6 (B6) anti-DBA/2 mixed leukocyte cultures (MLC) by the amino acid L-ornithine (Orn) could not be reversed by addition of up to 1000 U/ml IL-2. Analysis of the effects of Orn on induction of lymphokine-activated killer (LAK cells), using dosages of IL-2 from 10-1000 U/ml and measuring cytolytic activity against two tumor targets (P815 and YAC-1) over the course of 5 days, indicated that LAK cells were not suppressed by Orn. LAK precursors and effector cells were CD8- and ASGM1+, indicating that they were derived from natural killer (NK) cells. We also found that the growth and maintenance of cloned CTL lines were not sensitive to inhibition by Orn; nor was their acquisition of nonspecific cytolytic activity in the presence of high lymphokine concentrations. Thus, induction of naive CTL shows differential susceptibility to Orn inhibition relative to LAK and LAK-like activities by NK and cloned CTL lines in response to IL-2.     

Effects of L-carnitine on urea synthesis following acute ammonia intoxication in mice

L-Carnitine protects mice against acute ammonia intoxication. The effect of L-carnitine on in vivo incorporation of [14C] bicarbonate into urea has been investigated in mice given large doses of ammonium acetate. The hepatic content of N-acetylglutamate has been measured. Following ammonia administration the animals given L-carnitine have much higher production of urea than the unprotected mice. The marked protective effect of L-carnitine on ammonium acetate-induced hyperammonemia and on the increase in urea synthesis is not due primarily to activation of N-acetylglutamate synthetase.     

Interrelationships between ornithine,glutamate and GABA-III. an ornithine aminotransferase activity that is resistant to inactivation by 5-fluoromethylornithine

5-Fluoromethylornithine (5-FMOrn) is a specific inactivator of l-ornithine:2-oxoacid aminotransferase (OAT). However, a certain proportion of the OAT activity in mouse brain, liver and kidney is not inactivated by this compound. In the present work, the occurrence, distribution and subcellular localization of this 5-FMOrn-resistant OAT is reported. It was shown that the 5-FMOrn-resistant brain enzyme is kinetically different from the corresponding liver enzyme, and it also differs from the 5-FMOrn-sensitive OAT. The most conspicuous difference between the 5-FMOrn-resistant OAT of liver and brain is the sensitivity of the latter against excessive concentrations of its substrate 2-oxoglutarate.5-FMOrn and GABA are reversible inhibitors of the 5-FMOrn-resistant enzyme. Both compounds compete with Orn for the enzymes active site. A number of known inactivators of GABA-T which are at the same time inactivators of OAT, and canaline, a natural inhibitor of OAT, inactivate both the 5-FMOrn-sensitive and the 5-FMOrn-resistant enzyme. Gabaculine is the most potent inhibitor of the 5-FMOrn-resistant enzyme that is presently known. Our results are compatible with the suggestion that the 5-FMOrn-resistant OAT is an isoenzyme. From the fact that this form of OAT prevails in the brain, and its occurrence in the nerve ending fraction of brain homogenates supports the view that 5-FMOrn-resistant OAT may be involved in the intraneuronal generation of neurotransmitter glutamate and/or GABA from Orn as precursor. Further support in favour of this notion are previous findings which suggest feedback inhibition of OAT by GABA in GABAergic nerve endings.     

Prophylactic and Therapeutic Potential of Acetyl‐l‐carnitine against Acetaminophen‐Induced Hepatotoxicity in Mice

Prophylactic and therapeutic effects of acetylcarnitine against acetaminophen‐induced hepatotoxicity were studied in mice. To evaluate the prophylactic effects of acetylcarnitine, mice were supplemented with acetylcarnitine (2 mmol/kg/day per oral (p.o.) for 5 days) before a single dose of acetaminophen (350 mg/kg intraperitoneal (i.p.)). Animals were sacrificed 6 h after acetaminophen injection. Acetaminophen significantly increased the markers of liver injury, hepatic reactive oxygen species, and nitrate/nitrite, and decreased hepatic glutathione (GSH) and the antioxidant enzymes. Acetylcarnitine supplementation resulted in reversal of all biochemical parameters toward the control values. To explore the therapeutic effects of acetylcarnitine, mice were given a single dose of acetylcarnitine (0.5, 1, and 2 mmol/kg p.o.) 1.5 h after acetaminophen. Animals were sacrificed 6 h after acetaminophen. Acetylcarnitine administration resulted in partial reversal of liver injury only at 2 mmol/kg p.o. At equimolar doses, N‐acetylcystiene was superior as therapeutic agent to acetylcarnitine. However, acetylcarnitine potentiated the effect of N‐acetylcystiene in the treatment of acetaminophen toxicity.     

alpha-Tocopherol protects against alpha-naphthylisothiocyanate-induced hepatotoxicity in rats less effectively than melatonin

The protective effect of alpha-tocopherol (alpha-Toc), which exerts antioxidant and anti-inflammatory actions, against alpha-naphthylisothiocyanate (ANIT)-induced hepatotoxicity in rats was compared with that of melatonin because orally administered melatonin is known to protect against ANIT-induced hepatotoxicity in rats through its antioxidant and anti-inflammatory actions. Rats intoxicated once with ANIT (75 mg/kg, intraperitoneal (i.p.)) showed liver cell damage and biliary cell damage with cholestasis at 24 h, but not 12 h, after intoxication. ANIT-intoxicated rats received alpha-Toc (100 or 250 mg/kg) or melatonin (100 mg/kg) orally at 12 h after intoxication. The alpha-Toc administration protected against liver cell damage in ANIT-intoxicated rats, while the melatonin administration protected against both liver cell damage and biliary cell damage with cholestasis. ANIT-intoxicated rats had increased hepatic lipid peroxide concentration and myeloperoxidase activity at 12 and 24 h after intoxication. ANIT-intoxicated rats also had increased serum alpha-Toc and non-esterified fatty acid (NEFA) concentrations at 12 and 24 h after intoxication and increased serum triglyceride and total cholesterol concentrations at 24h. The administration of alpha-Toc to ANIT-intoxicated rats increased the hepatic alpha-Toc concentration with further increase in the serum alpha-Toc concentration and attenuated the increased hepatic lipid peroxide concentration and myeloperoxidase activity and serum NEFA concentration at 24 h after intoxication. The melatonin administration did not affect the hepatic alpha-Toc concentration but attenuated the increased hepatic lipid peroxide concentration and myeloperoxidase activity and serum alpha-Toc, NEFA, triglyceride, and total cholesterol concentrations at 24 h after ANIT intoxication. These results indicate that orally administered alpha-Toc protects against ANIT-induced hepatotoxicity in rats possibly through its antioxidant and anti-inflammatory actions less effectively than orally administered melatonin.     

Effect of a cysteine prodrug, L-2-oxothiazolidine-4-carboxylic acid, on the metabolism and toxicity of bromobenzene: an acute study

The effect of a cysteine prodrug, L-2-oxothiazolidine-4-carboxylic acid (OTCA), on certain aspects of the metabolism and toxicity of bromobenzene administered acutely to mice was investigated by (i) characterizing the influence of OTCA on the metabolic profile of low and high bromobenzene dose at 0-6, 6-12, and 12-24 h, (ii) determining the effective doses range and administration time for OTCA, as well as the optimum period for urine sampling; and (iii) measuring the efficacy of OTCA for protection against bromobenzene induced toxicity. Coadministration of OTCA and bromobenzene enhanced the urinary excretion of mercapturic acid and phenolic metabolites, during 6-12 h, by approximately 152 and 193%, respectively. Maximum efficacy was observed when OTCA (16.0 mmol/kg) was administered concomitantly with bromobenzene (4.0 mmol/kg). Finally, OTCA administration was found to afford substantial protection against elevation of plasma transaminases used as indices of bromobenzene-induced hepatotoxicity. N-acetylcysteine, another cysteine prodrug, had essentially similar effects on the metabolism and toxicity of bromobenzene. Thus, administration of cysteine prodrugs enhances the urinary excretion of several metabolites of bromobenzene and affords protection against bromobenzene-induced hepatotoxicity.     

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.     

Changes in urea cycle-related metabolites in the mouse after combined administration of valproic acid and an amino acid load

Increased blood ammonia was induced in fasting mice by ip administration of 200 mg/kg Na-valproate followed 1 h later by 13 and 4 mmol/kg alanine and ornithine, respectively. When valproate was not used blood or liver ammonia was not increased, but increases were observed in liver glutamate (5-fold), glutamine (2-fold), aspartate (5-fold), acetylglutamate (15-fold), citrulline (35-fold), argininosuccinate (11-fold), arginine (11-fold), and urea (3-fold). The level of carbamoyl phosphate (less than 2 nmol/g) was, by far, the lowest of all urea cycle intermediates. The large increase in citrulline indicates that argininosuccinate synthesis was limiting, and that the increase in acetylglutamate induced a considerable activation of carbamoyl phosphate synthetase, which agrees with theoretical expectations, irrespective of the actual KD value for acetylglutamate. Pretreatment with valproate resulted in lower hepatic levels of glutamate, glutamine, aspartate, acetyl-CoA, and acetylglutamate. At the level found of acetylglutamate the activation of carbamoyl phosphate synthetase would be expected to be similar to that without valproate. Indeed, the levels of citrulline were similar with or without valproate. Argininosuccinate, arginine, and urea levels exhibited little if any change. Although the model used may not replicate exactly the situation in patients, from our results it appears that changes in citrullinogenesis or in other steps of the urea cycle do not account for the increase in blood ammonia induced by valproate, and it is proposed that valproate may alter glutamine metabolism.     

Enhanced cell-mediated protection against fatal Escherichia coli septicemia induced by treatment with recombinant IL-2

Administration of rIL-2 to BALB/c mice induces a rapid, cell-mediated response that is sufficient to protect mice from a lethal i.p. dose of Escherichia coli. Mice were protected from septic death if IL-2 was administered i.p. within 1 h after the bacterial challenge. Optimal protection was provided by treating the lethally challenged mice with rIL-2 at 1 and 5 h or 1, 5, and 10 h after the bacterial challenge and was dose-dependent (greater than or equal to 5.0 x 10(5) U/kg). Furthermore, treatment of mice with anti-IL-2R antibody abolished the protective effect induced by rIL-2 administration. These data suggest that the rIL-2-induced protection against septic death in mice is mediated by a cell type expressing a functional IL-2R. One potentially important therapeutic application of rIL-2 may be to modulate the course of sepsis once the host has been exposed to potentially lethal microbial pathogens.     

Postprandial intestinal and whole body nitrogen kinetics and distribution in piglets fed a single meal

Our aim was to characterize the postprandial total and dietary N fluxes in the portal drained viscera (PDV) and whole body after administration of a single meal in young pigs. Seven 4-wk-old piglets, implanted with a portal flow probe and portal, arterial and venous catheters, received a primed constant [(18)O]urea intravenous infusion and were studied for 8 h after a bolus mixed meal ingestion (46 mmol N/kg body wt) intrinsically labeled with (15)N to trace dietary N fluxes. The real cecal digestibility of the formula was 94.3% (SD 1.8). PDV output of dietary N was found principally in the pool of circulating protein (51% of the measured dietary N PDV output), in the free alpha-amino N pool (44%), and to a lesser extent in ammonia (5%). Dietary N release in alpha-amino N and ammonia mainly occurred during the first 3 h. Total and exogenous postprandial urea productions were 5.8 and 2.0 mmol N/kg body wt, respectively. At the end of the postprandial period, losses of dietary N amounted to 10.3% of the dose: 5.7% through ileal losses and 4.6% by deamination and transfer to urea. Net postprandial retention of dietary N was 90.4% (SD 1.3), of which 20% was found in splanchnic zone (small intestine 10%, liver 5%, and plasma protein 3%) and 42% in peripheral zone (muscle 31%, skin 6%). In conclusion, our results show a high efficiency of dietary N utilization for muscular uptake and anabolic utilization. However, the results obtained point out the necessity to further explore the form of dietary N released into the portal blood.     

Characterization of N-methyl-D-aspartate receptor subunits involved in acute ammonia toxicity

Rapid administration of large doses of ammonia leads to death of animals, which is largely prevented by pretreatment with N-methyl-D-aspartate (NMDA) receptor antagonists. The present study focuses on a subunit(s) of NMDA receptor involved in ammonia-induced death by use of NMDA receptor GluRepsilon subunit-deficient (GluRepsilon(-/-)) mice and the selective GluRepsilon2 antagonist CP-101,606. Acute ammonia intoxication was induced in mice (eight per group) by a single intraperitoneal (i.p.) injection of ammonium chloride. Appearance of neurological deteriorations depended on the doses of ammonium chloride injected. While wild-type, GluRepsilon1(-/-), GluRepsilon4(-/-), and GluRepsilon1(-/-)/epsilon4(-/-) mice all died by ammonium chloride at 12 mmol/kg during the first tonic convulsions, two of eight GluRepsilon3(-/-) mice survived. Pretreatment of wild-type mice with CP-101,606 prevented two mice from ammonia-induced death. Pretreatment of GluRepsilon3(-/-) mice with CP-101,606 prevented the death of three mice and prolonged the time of death of non-survivors. Similarly, the neuronal form of nitric oxide synthase (NOS) inhibitor 7-nitroindazole (7-NI) as well as the nonselective NOS inhibitor L-NMMA, but not the inducible NOS inhibitor 1400W, partially prevented the death of mice and prolonged the period of death. Furthermore, ammonium chloride prolonged the increase in intracellular free Ca2+ concentration ([Ca2+]i) and subsequent NO production induced by NMDA in the cerebellum. These results suggest that activation of NMDA receptor containing GluRepsilon2 and GluRepsilon3 subunits and following activation of neuronal NOS are involved in acute ammonia intoxication which leads to death of animals.     

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.