Devendra R. Deshmukh

Adenosine triphosphate and adenosine increase the pulmonary blood flow to postnatal levels in fetal lambs

ABSTRACT: We investigated the hypothesis that purine nucleotides may mediate the pulmonary vasodilation that occurs at bulb in fetal lambs. We studied nine fetal lambs 3 d after placement of intravascular catheters, a flow transducer around the left pulmonary artery, and an inflatable vascular occluder around the ductus arteriosus. The pressure-flow relationship of left lung during a brief occlusion of the ductus arteriosus was studied as an index of pulmonary vascular resistance. We investigated the pulmonary vascular effects of adenosine, ATP, or saline (control) in doses of 0.01–2.50 μmol/kg/min infused into the right atrial line, and measured blood adenosine and ATP levels in samples from the pulmonary artery and left atrium. We also investigated the mechanism of pulmonary vascular effects of adenosine and ATP. Adenosine and ATP caused significant decreases in pulmonary vascular resistance and increases in pulmonary blood flow in doses of 0.08–2.5 μmol/kg/min. The pulmonary blood flow increased to levels seen in postnatal lambs at doses of 1.2 and 2.5 μmol/kg/min of adenosine and ATP. The baseline blood adenosine and ATP levels in fetus were 8 and 70% of levels in postnatal lambs. ATP concentrations increased to postnatal levels and adenosine levels increased to 20% of postnatal levels at infusion rates of 1.2 and 23 μmol/ kg/rain. The pulmonary vasodilation caused by adenosine and ATP was attenuated by 8-phenyltheophylline and ci-bacron blue, respectively, but not by indomethacin. We conclude that adenosine and ATP are pulmonary vasodilators and increase the fetal pulmonary flow to postnatal levels in doses that increase their blood concentrations to ≤ postnatal levels. The effects of adenosine and ATP are mediated by stimulation of P1 and P2 pnrinergk receptors and are independent of prostacyclin synthesis. Purine nucleotides may be important mediators of pulmonary vasodilation that occurs in the perinatal Iamb.

Immobilization of D-xylose (D-glucose) isomerase from a Chainia species.

D-Xylose isomerase is a heat-stable enzyme which isomerizes D-xylose into D-xylulose. D-Xylose isomerase from various species also isomerizes D-glucose into D-fructose. This enzyme is used in industry for the production of high-fructose corn syrup. The enzyme is specific for both, xylose and glucose. In most species xylose isomerase is localized intracellularly. However, in a rare actinomycete, Chainia sp. (NCL 82-5-1), xylose isomerase is present in both intracellular and extracellular compartments. We have previously purified and characterized intracellular enzyme from Chainia sp. In the present paper, we describe a procedure for immobilization of intracellular xylose isomerase on INDION 48-R by ionic binding. This method is inexpensive, does not require cross-linking agents and results in firm binding of the enzyme with the resin. The properties of immobilized enzyme such as pH optimum, substrate specificity, Km and inhibition by various metabolites are described and compared with those of purified, nonimmobilized enzyme.

Effect of arginine-free diet on plasma and tissue amino acids in young and adult ferrets

Abstract A single meal of arginine-free diet caused severe hyperammonemia in young (2-month-old) ferrets, whereas adult (18-month-old) ferrets did not develop hyperammonemia after identical treatment. To explore the mechanism of hyperammonemia induced by arginine-free diet, we tested the effect of a single meal of arginine-free diet on the amino acid levels in plasma, liver, and kidney of young and adult ferrets. Plasma and tissues were obtained three hours after feeding the diets. Arginine-containing diet caused a rapid increase in plasma arginine and ornithine (compared to fasting levels) only in young ferrets, indicating that these amino acids were absorbed more rapidly in young ferrets than in adult ferrets. Young ferrets fed arginine-free diet had lower levels of plasma arginine than those fed arginine-containing diet, whereas in adult ferrets no differences were observed in plasma arginine levels between arginine-free and arginine-containing diet groups. Both young and adult ferrets had low levels of hepatic ornithine. However, only young ferrets became hyperammonemic after ingesting an arginine-free diet. A decreased synthesis or an increased catabolism of arginine in young ferrets may explain the rapid development of hyperammonemia resulting from ingestion of an arginine-free diet.

Purification and properties of α-aminoadipate aminotransferase from rat liver and kidney mitochondria

Recently we reported an affinity chromatography method to purify alpha-aminoadipate aminotransferase (AadAT) activity from rat kidney supernatant fraction. Using the same affinity column, we purified AadAT activities from rat kidney and liver mitochondria. The physical and kinetic properties such as pH optima, Km for substrates, molecular weight, subunit structure, isoelectric pH, electrophoretic mobility and inhibition by dicarboxylic acids of mitochondrial AadAT were similar to those of the AadAT from rat kidney supernatant fraction. These results indicate that AadAT from different subcellular fractions is structurally and immunologically identical.

Purification and properties of α-aminoadipate aminotransferase from rat kidney

Abstract Previous studies with rat kidney preparations indicated that α-aminoadipate aminotransferase (AadAT) and kynurenine aminotransferase (KAT) activities are associated with a single protein. However, recent studies from our laboratory demonstrated that AadAT and KAT activities belong to two different proteins. AadAT from rat kidney supernatant fraction was purified by affinity chromatography to electrophoretic homogeneity. This rapid and efficient procedure improved the yield and the degree of purification over previously published methods and separated AadAT from KAT. The molecular weight of the enzyme was estimated to be 89,000 by Sephadex G-200 gel filtration chromatography. SDS-PAGE indicated that the enzyme is composed of two apparently identical subunits. Absorption spectra and the kinetic properties of AadAT are reported.

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.

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.

Hepatic polyamine metabolism in children with Reye's syndrome

Acute mitochondrial insult has been suggested as a primary reason for the clinical, histopathological and biochemical abnormalities seen in Reyes syndrome. However, the etiology of mitochondrial dysfunction has not been identified. Polyamines have been known to alter the mitochondrial structure and function. Influenza infection may cause an increase in ornithine decarboxylase activity and thereby channel ornithine for polyamine biosynthesis, leading to mitochondrial dysfunction in Reyes syndrome. To test this hypothesis, the hepatic concentrations of polyamines, polyamine-metabolizing enzymes and urea cycle enzyme activities in Reyes syndrome patients were determined and compared with patients who died from illnesses other than Reyes syndrome. The hepatic concentration of putrescine, spermidine and spermine were increased in Reyes syndrome patients. The activity of ornithine decarboxylase was elevated but, due to the small number of samples, these values did not reach statistical significance. Ornithine carbamoyltransferase activity was decreased in the liver of Reyes syndrome patients. Our results suggest that increased synthesis of polyamines from ornithine may initiate mitochondrial injury in Reyes syndrome.

PROTECTION AGAINST INTESTINAL ISCHEMIA/REPERFUSION INJURY IN TRANSGENIC MICE OVEREXPRESSING SUPEROXIDE DISMUTASE. • 691

We investigated the effect of intestinal ischemia, with and without reperfusion, in transgenic mice overexpressing the human superoxide dismutase(SOD) gene. Ischemia was induced by occluding the superior mesenteric artery in 2-4 month old (young) and 8 month old (adult) transgenic mice and in age-matched nontransgenic controls. In young animals, 60 min of ischemia without reperfusion was associated with a 55% (12/22) mortality in nontransgenic mice and 60% (6/10) mortality in transgenic mice. There were no deaths after 60 min of ischemia in adult, nontransgenic or transgenic mice. Fifty percent (5/10) of young nontransgenic mice died during the 4 hr reperfusion period, whereas no deaths were observed in transgenic mice in the same age group. All adult nontransgenic mice (4/4) died during the reperfusion period whereas all the transgenic mice in the same age group survived. In another experiment, the nontransgenic and transgenic mice survived 45 min ischemia followed by 4 hr reperfusion. After reperfusion, the young as well as adult nontransgenic mice exhibited a significant increase in lung myeloperoxidase activity whereas transgenic mice did not show this increase. These results indicate that older mice are able to tolerate a longer duration of intestinal ischemia than young animals. The overexpression of SOD in transgenic mice did not offer protection against mortality induced by ischemia without reperfusion. However, overexpression of SOD in transgenic mice provided protection against mortality induced by reperfusion injury. Protection appears to be due to the elimination of free radicals by SOD and due to decreased neutrophil sequestration in the lungs.

Effect of hyperammonemia on brain amino acids in young and adult ferrets

SummaryEffects of arginine deficiency and hyperammonemia on the brain concentrations of amino acids and urea cycle enzyme activities in young and adult ferrets were investigated. Only young ferrets developed hyperammonemia and encephalopathy immediately after consuming the arginine-free diet. Brain ornithine and citrulline concentrations in young ferrets fed arginine containing diet were significantly lower than those in adult ferrets. Compared to rats and other animals, young and adult ferrets had lower concentrations of brain glutamic acid and glutamine. Unlike in other species, brain glutamine was not elevated in young, hyperammonemic ferrets. Brain arginase and glutamate dehydrogenase activities were significantly increased in young ferrets fed arginine-free diet. Young ferrets provide a useful animal model for investigating the neurotoxicity of acute hyperammonemia.