Hyperammonaemia in V1a vasopressin receptor knockout mice caused by the promoted proteolysis and reduced intrahepatic blood volume.

An analysis of arginine-vasopressin (AVP) V1a receptor-deficient (V1aR-/-) mice revealed that glucose homeostasis and lipid metabolism were altered in the mutant mice. Here, we used V1aR-/- mice to investigate whether the deficiency of the V1a receptor, which led to altered insulin sensitivity, affected protein metabolism. The serum 3-methylhistidine levels were increased in V1aR-/- mice under feeding conditions, indicating that proteolysis was enhanced in muscle tissue from V1aR-/- mice. Furthermore, serum amino acid profiling revealed that the amino acid levels, including glycogenic and branched-chain amino acids, were reduced in V1aR-/- mice. In addition, an alanine-loading test showed that gluconeogenesis was enhanced in V1aR-/- mice. Blood ammonia, which is a by-product of amino acid catabolism, was two times higher in V1aR-/- mice without hepatopathy under the feeding and fasting conditions than in wild-type mice. Amino acid profiling also revealed that the amino acid pattern was not typical of a urea-cycle enzymatic disorder. An ammonia tolerance test and an indocyanine green elimination test showed that V1aR-/- mice had lower ammonia clearance due to a decreased intrahepatic circulating blood volume. Metabolic acidosis, including lactic- and keto-acidosis, was not observed in V1aR-/- mice. These results provide evidence that proteolysis promotes the production of glucose in the muscles of V1aR-/- mice and that hyperammonaemia is caused by promoted protein catabolism and reduced intrahepatic blood volume. Thus, our study with V1aR-/- mice indicates that AVP plays a physiological role via the V1a receptor in regulating both protein catabolism and glucose homeostasis.

Method for the determination of 15NH3 enrichment in biological samples by gas chromatography/electron impact ionization mass spectrometry.

An alternative method for the determination of [15N]ammonia enrichment in biological fluids was developed. It is based on the use of glutamate dehydrogenase of bovine liver (EC 1.4.1.2.) with 2-oxopentanoic acid as substrate, to convert the ammonia present in the sample into norvaline, the enrichment of which can be measured by gas chromatography/mass spectrometry as its tertiary butyldimethylsilyl (TBDMS) derivative under electron impact selective ion recording (SIR) conditions. The principal advantage of the present approach is that it is simpler and quicker than the previously described methods, because the synthetic product, norvaline, is not present in biological fluids and pre-processing of the sample is unnecessary. The procedure includes a pre-incubation stage which allows removal of contaminant ammonia present in the reagents used for the enzyme reaction. The contributions of other sources of nitrogen to norvaline production have been checked and quantified: these may provide limitations of the technique when samples for analysis are low in ammonia (e.g. arterial or hepatic venous blood). To reduce these contributions, short times of incubation are proposed. The results from two experiments in vivo in which two sheep were infused with [15N]ammonium chloride in the mesenteric vein are presented and the biological implications which arise from the results are discussed. The validity of the procedures was demonstrated by the quantitative recovery from the mesenteric and portal veins of [15N]ammonia infused.

Use of peritoneal dialysis, continuous arteriovenous hemofiltration, and continuous arteriovenous hemodiafiltration for removal of ammonium chloride and glutamine in rabbits.

OBJECTIVE: We compared the ability of peritoneal dialysis, hemofiltration, and continuous hemodiafiltration to remove infused ammonium chloride. STUDY DESIGN: Anesthetized adult rabbits received an intravenous infusion of ammonium chloride. Two methods of removal of ammonium chloride were performed in each animal and compared. In group 1 (n = 6), peritoneal dialysis (dialysate = 75 ml.kg-1) and continuous arteriovenous hemofiltration (CAVH) with a polysulfone 800 cm2 hemofilter (Minifilter Plus; Amicon Division, W. R. Grace & Co., Danvers, Mass.) were simultaneously performed for 40 minutes. In group 2 (n = 6), peritoneal dialysis and continuous arteriovenous hemodiafiltration (CAVHD) (dialysate flow = 1000 ml.hr-1) were simultaneous performed for 40 minutes. In group 3 (n = 6), CAVH and CAVHD were performed successively in random order for 30 minutes each. RESULTS: Animals had high and stable ammonium chloride and glutamine plasma levels during the experimental procedure. No significant difference in ammonium chloride clearance was observed between PD and CAVH (group 1). In comparison with PD or CAVH, CAVHD resulted in significantly higher clearances of ammonium chloride (40% +/- 10% vs 96% +/- 34%, respectively) and of glutamine (195% +/- 17% vs 77% +/- 25%, respectively). CONCLUSION: The overall results indicate that CAVHD should be considered for hyperammonemia when peritoneal dialysis is indicated but unfeasible or inefficient.

The influence of pH and various anions on the distribution of NH4+ in human blood.

The pH-dependent distribution of ammonia between blood cells and plasma was investigated with oxygenated blood samples from healthy subjects at 37 degrees C. Blood pH was varied between 6.95 and 7.65 by equilibration with different CO2 mixtures. Plasma ammonia concentrations were measured directly with a specific enzymatic method. Ammonia concentrations within blood cells were calculated from a) the concentration changes of ammonia in plasma after addition of 87.7 mumol/l NH4Cl to whole blood and b) the pH-dependent haematocrit. The pH-dependency of the distribution ratio(ammonia) = P-ammonia/cell ammonia (substance concentrations in water spaces) is described by the equation distribution ratio(ammonia) = 3.095 - 0.342 x pHplasma (r = 0.928, n = 36) in good agreement with available literature data on the distribution of H+. A quantitative figure to describe the actual NH4+ concentration in oxygenated whole blood at defined values of P-NH4+, P-pH and haematocrit is given. Values of distribution ratio(ammonia) at pH 7.4 (0.57 or 0.75, ammonia concentrations corrected/not corrected for water content) are higher than those assumed so far in the literature. Addition of non-permeating anions (citrate, EDTA) to whole blood results in a shift of NH4+ from the intra- to the extracellular compartment. In contrast, chaotropic anions like iodide or thiocyanate lower distribution ratio(ammonia). To avoid medically important bias in the measurement of plasma ammonia concentration, the changes in pH or in the ionic composition of the blood sample following pretreatment with anticoagulants or preservatives should not exceed certain limits. Citrate is not a suitable anticoagulant.

Effect of muscular exercise by bicycle ergometer on erythrocyte purine nucleotides.

The effect of muscular exercise by bicycle ergometer on erythrocyte purine nucleotides was investigated in 6 athletes. Muscular exercise increased the concentration of inosine monophosphate from 5.9 +/- 1.1 to 7.3 +/- 1.3 nmol/ml in venous erythrocytes and from 5.7 +/- 1.0 to 6.8 +/- 1.4 nmol/ml in arterial erythrocytes, respectively, while it decreased the concentrations of adenosine diphosphate and adenosine monophosphate from 189.3 +/- 42.7 to 141.2 +/- 26.9 and from 26.0 +/- 7.8 to 15.7 +/- 4.3 nmol/ml in venous erythrocytes and also decreased their concentrations from 195.1 +/- 51.0 to 141 +/- 29.2 and from 26.5 +/- 9.6 to 14.8 +/- 3.0 nmol/ml in arterial erythrocytes, respectively. The muscular exercise also increased the concentration of inorganic phosphate in venous plasma from 1.12 +/- 0.12 to 1.46 +/- 0.22 mmol/l, that of NH3 in blood from 41.90 +/- 6.91 to 150.22 +/- 50.80 mumol/l, that of lactic acid in blood from 7.90 +/- 1.71 to 61.03 +/- 18.43 mg/dl and that of hypoxanthine in venous plasma from 1.32 +/- 0.36 to 18.14 +/- 4.87 mumol/l, respectively. Therefore, in vitro study was performed to investigate whether inorganic phosphate, NH4Cl, lactic acid or hypoxanthine affects nucleotides in erythrocytes. After 2 hour-incubation, 2 mM inorganic phosphate increased the erythrocyte concentration of inosine monophosphate 1.6 fold but decreased the erythrocyte concentrations of adenosine monophosphate and adenosine diphosphate 0.72 and 0.89 fold, respectively, in the suspension (pH 7.35), as compared with 1 mM inorganic phosphate. However NH4Cl, lactic acid or hypoxanthine did not affect erythrocyte purine nucleotides.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute zinc chloride ingestion in a child: local and systemic effects.

A 16-month-old boy ingested liquid zinc chloride/ammonium chloride soldering flux. He developed severe local burns, metabolic acidosis, hepatic damage, hyperamylasemia, lethargy, and hypertension. Peak measured plasma zinc was 1,199 micrograms/dL. Because of persistent signs of systemic toxicity, he was chelated with dimercaprol (BAL) and EDTA. Although clinical improvement was noted coincident with the initiation of chelation, there was no apparent increase in urinary zinc excretion. Scarring in the gastric antrum necessitated an antrectomy. The child recovered without other apparent complications.

Studies in red blood cell preservation. 5. Determining the limiting concentrations of NH4Cl and Na2HPO4 needed to maintain red blood cell ATP during storage.

The purpose of the present study was to define the lowest concentrations of ammonium (NH4+) and phosphate (Pi) in an experimental additive solution (EAS) that would support suitable red blood cell (RBC) ATP levels and other in vitro characteristics for at least 84 days. It was determined that ATP maintenance was dependent upon both NH4+ and Pi concentrations. RBCs stored for 84 days in additive solutions containing 10 mM NH4+ and 0, 15, 25 and 40 mM Pi had ATP values averaging 1.87, 2.49, 2.70 and 2.65 mumol/g Hb, respectively. The shedding of exocytic hemoglobin-containing vesicles and percent hemolysis were significantly (p less than 0.001) elevated in the preservative containing 40 mM Pi. These data suggest that an EAS containing 10 mM NH4+ and 15 mM Pi would be optimal for storing RBCs up to 84 days. The extended storage would be particularly advantageous for autologous transfusion programs.

Brain phenylalanine and tyrosine levels and hepatic encephalopathy induced by CCl4 in rats.

The correlation between the levels of brain aromatic amino acids and hepatic encephalopathy induced by CCl4 was investigated in rats. CCl4 (1.0 ml/kg three times per week for over 10 weeks) caused hyperammonemia and hepatic encephalopathy in rats. The brain levels of aromatic amino acids, especially tyrosine (Tyr) and phenylalanine (Phe) in rats with hepatic encephalopathy were increased by 605% and 255% respectively from that of the corresponding controls. Furthermore, a single intraperitoneal injection of 2,4-dinitrophenol (DNP; 30 mg/kg) in CCl4-treated rats (1.0 ml/kg three times per week for 7 weeks) showed hyperammonemia and hepatic encephalopathy (loss of consciousness) elicited a marked increase of Tyr and Phe levels in the brain. In addition, the blood levels of Tyr and Phe in all rats with hepatic encephalopathy were greatly elevated as compared to controls. On the other hand, continuous injection of ammonium chloride (20 mg N/ml) into the jugular vein for 1 h caused severe hyperammonemia without loss of consciousness. The brain levels of Tyr and Phe showed no change from the corresponding controls. These results suggest that the increase of aromatic amino acids, such as Tyr and Phe, in brain produced by hyperammonemia and high blood levels of Tyr and Phe may be a critical event to the development of hepatic encephalopathy.

[A comparative study of the oral and rectal ammonium overload test in liver cirrhosis]. / Estudio comparativo del test de sobrecarga amónica por vía oral y rectal en la cirrosis hepática.

The ammonium loading test has been realized in 66 patients, 8 of them without clinical or laboratory data of hepatic disease and 58 diagnosed of hepatic cirrhosis (HC). In 40 patients with HC and 8 patients without liver disease the ammonium was administered by rectum and in the remaining 18 patients with HC it was administrated orally. In each case, non stagnant venous blood was drawn at 0, 30, 45, 60 and 75 minutes after the administration of ammonium and plasmatic levels were measured. The results show that in patients with HC there are no significant differences between rectal and oral administration although the rectal way presented less secondary effects and is better tolerated. The test is discriminatory when comparing patients with HC and patients without liver disease as well as between patients with HC and portal hypertension and those without clinical signs of portal hypertension.

Urine electrolytes in the assessment of extracellular fluid volume contraction.

The purpose of this study was to determine which urine electrolytes should be measured to confirm that the extracellular fluid (ECF) volume is depleted. ECF volume contraction was induced by furosemide administration to rats consuming an electrolyte-free diet. An external potassium balance was achieved by replacing potassium losses with KHCO3 and KCl so that the sodium and chloride deficits were comparable (equivalent to a 30% reduction in ECF volume). As expected, the urine sodium and chloride concentrations fell to 2 +/- 0.3 mmol/l and 3 +/- 0.3 mmol/l, respectively. Rats were then randomized to receive 50-75% of their sodium or chloride deficit as either: NaCl (control group), NH4Cl or NaHCO3 to mimic clinical situations associated with ECF volume contraction. In the NaCl group, the urine sodium and chloride concentrations remained low (6 +/- 2 mmol/l and 7 +/- 2 mmol/l), consistent with persistent ECF volume contraction. Although the NH4Cl group continued to have a low urine sodium concentration (2 +/- 0.2 mmol/l), there was now a marked increase in the urine chloride concentration (51 +/- 7 mmol/l; p less than 0.01 vs. NaCl group). In contrast, although the NaHCO3 group continued to have a low urine chloride concentration (2 +/- 1 mmol/l), there was a significant increase in the urine sodium concentration (19 +/- 3 mmol/l; p less than 0.01 vs. NaCl group). We conclude that the clinical assessment of ECF volume by urine electrolytes requires an evaluation of both the urine sodium and chloride concentrations.

Effect of acetazolamide on renal metabolism and ammoniagenesis in the dog.

The effect of acetazolamide (ACZ) on renal metabolism and ammoniagenesis was studied in the dog in vivo and in vitro. ACZ was administered to 10 dogs with normal acid-base status and five with chronic metabolic acidosis induced by NH4Cl. In both groups of dogs, the acute administration of ACZ markedly reduced the urinary excretion of ammonium (from 33 to 10 in normal dogs and from 100 to 23 mumoles/100 ml GFR in acidotic dogs) whereas its release into the renal vein was increased in a reciprocal fashion (from 69 to 95 in normal dogs and from 91 to 152 mumoles/100 ml GFR in acidotic dogs). ACZ did not change the total ammonium production nor the renal glutamine utilization. The renal utilization or production of glutamate, alphaketoglutarate, alanine and citrate also remained unchanged. Despite a marked urinary alkalinization, citraturia remained minimal. However, the renal cortical concentrations of glutamine, glutamate, succinate, fumarate, malate, aspartate and phosphoenolpyruvate fell following ACZ administration, especially in acidotic dogs showing rapid renal utilization of glutamine. ACZ had no effect on the same metabolites in the kidney of normal dogs even when lactate utilization was enhanced by lactate infusion. This study demonstrates that an accelerated ammoniagenic flux can proceed in the dog kidney without the renal cortical changes produced by metabolic acidosis in this species. In vitro, using dog tubules, a selective effect of ACZ on glutamine metabolism as compared to lactate was observed. ACZ reduce the rate of the reactions catalyzed by alphaketoglutarate dehydrogenase and by succinyl CoA synthetase. Other enzymes of the ammoniagenic and gluconeogenic pathways (glutaminase, GLDH, malic enzyme, PEPCK) were not changed by ACZ. The metabolic effects of ACZ observed in the intact kidney in vivo or with tubules in vitro may be in part related to the effect of ACZ on these enzymes critical for the ammoniagenic process.

Determination of factor XIII activity and of factor XIII inhibitors using an ammonium-sensitive electrode.

An assay for rapid factor XIII activity measurement has been developed based on the determination of the ammonium released during fibrin stabilization. Factor XIII was activated by thrombin and calcium. Ammonium was measured by an ammonium-sensitive electrode. It was demonstrated that the assay procedure yields accurate and precise results and that factor XIII-catalyzed fibrin stabilization can be measured kinetically. The amount of ammonium released during the first 90 min of fibrin stabilization was found to be 7.8 +/- 0.5 moles per mole fibrinogen, which is in agreement with the findings of other authors. In 15 normal subjects and in 15 patients suffering from diseases with suspected factor XIII deficiency there was a satisfactory correlation between the results obtained by the "ammonium-release-method', Bohn's method, and the immunological assay (r1 = 0.65; r2 = 0.70; p less than 0.01). In 3 of 5 patients with paraproteinemias the values of factor XIII activity determined by the ammonium-release-method were markedly lower than those estimated by the other methods. It could be shown that inhibitor mechanisms were responsible for these discrepancies.

In vitro adsorption of possible aetiological factors of hepatic encephalopathy.

Four different adsorbents (activated charcoal, XAD-4, a strong base anion and a strong acid cation-exchange resin) were tested in vitro for their capacity to remove substances that may be important in the development of hepatic encephalopathy. Separate columns packed with one of these adsorbents were perfused for three hours with a reconstituted plasma solution containing simultaneously high concentrations of amino-acids, ammoniumchloride, short-chain fatty acids, octopamine and bile salts. Effective removal of all these substances was only obtained when either activated charcoal, or XAD-4, were combined with the cation-exchange resin. Possible implications for the treatment of hepatic coma are discussed.

Polycystic disease and hepatic fibrosis in children. Renal function studies.

Renal function studies were done in five children with infantile polycystic disease (IPCD)of kidneys and liver and in four with congenital hepatic fibrosis (CHF). Glomerular filtration rate was reduced in all IPCD patients and in two of four CHF patients. Urinary concentrating ability following water deprivation and vasopressin administration was impaired in all IPCD patients and in three of four CHF patients. During control period, all patients had asymptomatic metabolic acidosis with total carbon dioxide content less than or equal to 20.5 millimols/liter, and net acid excretion (NAE) was reduced in all but one. Ammonium chloride was administered to seven patients; NAE increased in all, but the increments were subnormal in four. The inability to excrete maximally concentrated urine and an adequate amount of net acid may best be explained by abnormal tubular structure or alterations in medullary architecture secondary to progressive scarring, or both.

Arterialization of the liver in combination with a portacaval shunt in the dog.

A dog preparation has been developed combining an end-to-side portacaval shunt with arterialization of the hepatic portion of the portal vein through an anastomosis between the inferior branch of the splenic artery and the stump of the portal vein. In this dog preparation, total hepatic blood flow, perfusion of the intrahepatic portal vein, and sinusoidal pressure remained within the preoperative range in the majority of the dogs. The data presented indicate that arterialization of the liver, under those conditions, resulted in no histologic damage or atrophy of the parenchyma of the liver and was effective in achieving significant prolongation of life and prevention of most of the adverse metabolic sequelae that follow a portacaval shunt in dogs. The indocyanine green extraction was restored to normal. The extraction of ammonia was impaired in all dogs with shunts; however, a significantly better ammonia extraction was seen in dogs with arterialization of the liver. It is being postulated that the dual perfusion of the liver through the hepatic artery and the portal vein is essential for the maintenance of the normal morphologic and functional states of the liver in dogs. The avoidance of hyperfusion of the sinusoidal bed with arterial blood is the most critical factor in preventing morphologic damage of the liver if arterialization of the portal vein is used.