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I Nissim

Publications and source records attributed to I Nissim.

At least 73 records · Page 4Linked to original sources
PubMed1989

Effects of palmitate on astrocyte amino acid contents.

The effects of palmitate on intracellular and extracellular amino acid concentrations of cultured astrocytes was studied. Exposure of astrocytes to either 0.72 mM or 0.36 mM palmitate was associated with a significant reduction in the intracellular pool of glutamine and taurine. In contrast, the intracellular concentration of histidine, glycine, citrulline, isoleucine and leucine were increased in the presence of 0.72 mM palmitate. Comparable changes in the extracellular amino acid pool were not observed. The data suggest that palmitic acid, which accumulates in the brain during periods of anoxia, alters the metabolism of several amino acids in cultured astrocytes. These changes may be of significance in terms of the pathophysiology of a stress such as anoxia.

Amino Acids↗
PubMed1989

Fibronectin turnover in the premature neonate measured with [15N]glycine.

Fibronectin is a large-molecular-weight glycoprotein present on most cell surfaces and in plasma. Plasma fibronectin concentrations in neonates are lower than those in adults and a direct relationship exists between plasma concentration and gestational age. We determined the half-life and fractional synthetic rate (FSR) of plasma fibronectin in the premature infant. Infants and adults received a loading dose of [15N]glycine followed by a constant infusion [15N]glycine incorporation into plasma fibronectin and urine hippurate was determined by gas chromatography-mass spectrometry. The plasma fibronectin FSR in the preterm neonates was 15.5 +/- 9.9%/d(means +/- SD) and the half-life was 5.55 +/- 2.25 d. Birth weight correlated inversely with plasma fibronectin half-life. In the adults the plasma fibronectin FSR ranged from 20 to 87%/d and half-life ranged between 0.79 and 3.47 d. These data suggest that decreased plasma fibronectin levels in preterm infants are due to reduced FSRs rather than to greater turnover of a relatively small plasma pool.

Adult↗
PubMed1989

Neuronal glutamine utilization: pathways of nitrogen transfer studied with [15N]glutamine.

Gas chromatography-mass spectrometry was used to evaluate the metabolism of [15N]glutamine in isolated rat brain synaptosomes. In the presence of 0.5 mM glutamine, synaptosomes accumulated this amino acid to a level of 25-35 nmol/mg protein at an initial rate greater than 9 nmol/min/mg of protein. The metabolism of [15N]glutamine generated 15N-labelled glutamate, aspartate, and gamma-aminobutyric acid (GABA). An efflux of both [15N]glutamate and [15N]aspartate from synaptosomes to the medium was observed. Enrichment of 15N in alanine could not be detected because of a limited pool size. Elimination of glucose from the incubation medium substantially increased the rate and amount of [15N]aspartate formed. It is concluded that: (1) With 0.5 mM external glutamine, the glutaminase reaction, and not glutamine transport, determines the rate of metabolism of this amino acid. (2) The primary route of glutamine catabolism involves aspartate aminotransferase which generates 2-oxoglutarate, a substrate for the tricarboxylic acid cycle. This reaction is greatly accelerated by the omission of glucose. (3) Glutamine has preferred access to a population of synaptosomes or to a synaptosomal compartment that generates GABA. (4) Synaptosomes maintain a constant internal level of glutamate plus aspartate of about 70-80 nmol/mg protein. As these amino acids are produced from glutamine in excess of this value, they are released into the medium. Hence synaptosomal glutamine and glutamate metabolism are tightly regulated in an interrelated manner.

Amino Acids↗
PubMed1989

Ammoniagenesis by cultured human renal cortical epithelial cells: study with 15N.

The metabolic fate of 15N-labeled glutamine and glutamate in cultured human renal cortical epithelial cells was investigated. The main goal was to elucidate the major pathways of ammoniagenesis depending on varying H+ concentration. Incubations at pH 7.4 or 6.8 were conducted with either 1 mM [5-15N]glutamine, [2-15N]glutamine, [15N]glutamate, or L-[2-15N]-gamma-glutamylmethylamide. The results demonstrate that acute acidosis had little effect on total ammonia generation from glutamine. However, 15NH3 formation from [5-15N]glutamine was significantly higher at pH 7.4 compared with pH 6.8. Conversely, at pH 6.8, 15NH3 production from either [2-15N]-glutamine or [15N]glutamate was twofold higher than at pH 7.4. Thus the observations indicate that acute acidosis had little effect on net ammonia production from glutamine due to decreased flux through glutaminase and concomitant increased flux through glutamate dehydrogenase. When L-[2-15N]-gamma-glutamylmethylamide was utilized as the sole substrate, significantly higher amounts of 15NH3 and 15N-labeled amino acids were formed at pH 6.8 compared with pH 7.4. Addition of either 1 mM pyruvate or alpha-ketoglutarate significantly decreased 15NH3 and increased 15N-amino acid formation from either [2-15N]glutamine or [2-15N]-gamma-glutamylmethylamide. The metabolism of either substrate via transamination reaction was significantly stimulated at acidic pH, presumably due to a depleted pool of alpha-ketoglutarate during the course of the incubations. The data indicate that in addition to glutaminase I and glutamate dehydrogenase, the glutamine aminotransferase (glutaminase II) pathway exists in cultured human renal cells. The data suggest that glutamate dehydrogenase flux and/or the alpha-ketoglutarate dehydrogenase reaction may have an important regulatory role in ammoniagenesis from glutamine and/or glutamate in human kidney during acute acidosis.

Ammonia↗
PubMed1988

Astrocyte metabolism of [15N]glutamine: implications for the glutamine-glutamate cycle.

The metabolism of glutamine was studied in cultured astrocytes by incubating these cells with [2-15N]-glutamine and using gas chromatography-mass spectrometry to quantitate the transfer of 15N to other amino acids. We found that astrocytes simultaneously synthesize and consume [2-15N]glutamine, with the respective synthetic and utilization rates being approximately equal (ca. 13.0 nmol min-1 mg protein-1). Considerable 15N was transferred to alanine and a significant amount to the essential amino acids leucine, tyrosine, and phenylalanine, the latter process denoting active reamination of cognate ketoacids. A net export of alanine into the medium was noted. Astrocyte glutamine utilization appeared to be mediated via both the phosphate-activated glutaminase (PAG) pathway and the glutamine aminotransferase pathway, the activity of which was about half that of PAG. The glutamine concentration in the incubation medium determined whether net synthesis or utilization of this amino acid occurred. When glutamine was omitted from the medium, net synthesis occurred. When it was present at a high (5 mM) level, net consumption was observed. At a physiologic (0.5 mM) concentration, neither net synthesis nor consumption was noted, although the 15N data indicated that glutamine was actively metabolized. An implication of this work is that astrocytes clearly are capable of both synthesizing and utilizing glutamine, and current concepts of a glutamate-glutamine cycle functioning stoichiometrically between astrocytes and neurons may be an oversimplification.

Animals↗
PubMed1988

Glucose and synaptosomal glutamate metabolism: studies with [15N]glutamate.

The metabolism of [15N]glutamate was studied with gas chromatography-mass spectrometry in rat brain synaptosomes incubated with and without glucose. [15N]Glutamate was taken up rapidly by the preparation, reaching a steady-state level in less than 5 min. 15N was incorporated predominantly into aspartate and, to a much lesser extent, into gamma-aminobutyrate. The amount of [15N]ammonia formed was very small, and the enrichment of 15N in alanine and glutamine was below the level of detection. Omission of glucose substantially increased the rate and amount of [15N]aspartate generated. It is proposed that in synaptosomes (a) the predominant route of glutamate nitrogen disposal is through the aspartate aminotransferase reaction; (b) the aspartate aminotransferase pathway generates 2-oxoglutarate, which then serves as the metabolic fuel needed to produce ATP; (c) utilization of glutamate via transamination to aspartate is greatly accelerated when flux through the tricarboxylic acid cycle is diminished by the omission of glucose; (d) the metabolism of glutamate via glutamate dehydrogenase in intact synaptosomes is slow, most likely reflecting restriction of enzyme activity by some unknown factor(s), which suggests that the glutamate dehydrogenase reaction may not be near equilibrium in neurons; and (e) the activities of alanine aminotransferase and glutamine synthetase in synaptosomes are very low.

Amino Acids↗
PubMed1987

Metabolic fate of glutamate carbon in rat renal tubules. Studies with 13C nuclear magnetic resonance and gas chromatography-mass spectrometry.

13C-n.m.r. spectroscopy and g.c.-m.s. were used to determine the metabolic fate of glutamate carbon in rat kidney. The main purpose was to characterize the effect of chronic metabolic acidosis on the utilization of glutamate carbon. Renal tubules obtained from normal and chronically acidotic rats were incubated in Krebs buffer, pH 7.4, in the presence of 2.5 mM-[3-13C]glutamate. During the course of incubation the concentrations of total glucose and NH3 were significantly (P less than 0.05) higher in tissue from acidotic rats. The levels of some tricarboxylic-acid-cycle intermediates were higher (P less than 0.05) in control tissue. In control tissue, 13C-n.m.r. spectra demonstrated a significantly higher rate of 13C appearance of aspartate, glutamine and [2,4-13C]glutamate. However, in acidosis the resonances of [13C]glucose carbon atoms were significantly higher. In the control, approx. 15% of glutamate carbon was accounted for by [13C]glucose formation as against 30% in chronic acidosis. However, in control tissue, 44% of glutamate carbon utilization was accounted for by recycling to glutamate and formation of aspartate, glutamine and GABA. In acidosis, only 11% was so recovered. Analysis of 15NH3 formation during the course of incubation with 2.5 mM-[15N]glutamate demonstrated a positive association between the appearance of [13C]glucose and 15NH3 both in the control and in acidosis. The data suggest that the control of gluconeogenesis and ammoniagenesis in acidosis is, in part, referable to a diminution in the rate of the reductive amination of alpha-oxoglutarate, that of the transamination reaction and that of glutamine synthesis.

Acidosis, Renal Tubular↗
PubMed1987

[15N]aspartate metabolism in cultured astrocytes. Studies with gas chromatography-mass spectrometry.

The metabolism of 2.5 mM-[15N]aspartate in cultured astrocytes was studied with gas chromatography-mass spectrometry. Three primary metabolic pathways of aspartate nitrogen disposition were identified: transamination with 2-oxoglutarate to form [15N]glutamate, the nitrogen of which subsequently was transferred to glutamine, alanine, serine and ornithine; condensation with IMP in the first step of the purine nucleotide cycle, the aspartate nitrogen appearing as [6-amino-15N]adenine nucleotides; condensation with citrulline to form argininosuccinate, which is cleaved to yield [15N]arginine. Of these three pathways, the formation of arginine was quantitatively the most important, and net nitrogen flux to arginine was greater than flux to other amino acids, including glutamine. Notwithstanding the large amount of [15N]arginine produced, essentially no [15N]urea was measured. Addition of NaH13CO3 to the astrocyte culture medium was associated with the formation of [13C]citrulline, thus confirming that these cells are capable of citrulline synthesis de novo. When astrocytes were incubated with a lower (0.05 mM) concentration of [15N]aspartate, most 15N was recovered in alanine, glutamine and arginine. Formation of [6-amino-15N]adenine nucleotides was diminished markedly compared with results obtained in the presence of 2.5 mM-[15N]aspartate.

Adenine Nucleotides↗
PubMed1987

Characterization of amino acid metabolism by cultured rat kidney cells: study with 15N.

The present study evaluates the metabolism of glutamine and glutamate by normal rat kidney (NRK) cells. The major aim was to evaluate the effect of acute acidosis on the metabolism of amino acid and ammonia formation by cultured NRK cells. Experiments at either pH 7.0 or 7.4 were conducted with phosphate-buffered saline supplemented with either 1 mM [5-15N]glutamine, [2-15N]glutamine, or [15N]glutamate. Incubation with either glutamine or glutamate as a precursor showed that production of ammonia and glucose was increased significantly at pH 7.0 vs. 7.4. The disappearance [corrected] of glutamine and glutamate was linear during a 60-min incubation at either pH. In experiments with [5-15N]glutamine, we found that approximately 57 and 43% of ammonia N was derived from 5-N of glutamine at pH 7.4 and 7.0, respectively. Experiments with [2-15N]glutamine or [15N]glutamate indicated that approximately 43 and 47% of 2-N glutamine and glutamate N utilization, respectively, was accounted for by ammonia production at pH 7.0. Similarly, 28 and 29% of NH3 was derived from 2-N of glutamine or glutamate N by activity of glutamate dehydrogenase at pH 7.4. In addition to 15NH3 formation, three major metabolic pathways of [2-15N]glutamine or [15N]glutamate disposal were identified: 1) transamination reactions involving the pH-independent formation of [15N] aspartate and [15N]alanine; 2) the synthesis of [6-15NH2]adenine nucleotide, a process more active at pH 7.4 vs. 7.0; and 3) glutamine synthesis from [15N]glutamate, especially at pH 7.4. The data indicate that NRK cells in culture consume glutamine and glutamate and generate ammonia and various amino acids, depending on the H+ concentration in the media. The studies suggest that these cell lines may provide a useful model for studying various aspects of the effect of pH on rat renal ammoniagenesis.

Amino Acids↗
PubMed1987

Albumin synthesis in premature infants: determination of turnover with [15N]glycine.

Albumin turnover was studied in seven small premature infants who received a constant infusion of [15N]glycine for 60-72 h. Gas chromatography-mass spectrometry was used to measure the rate of appearance of [15N]glycine in albumin isolated from the blood. By comparing the linear increment of [15N]glycine in blood albumin with plateau labelling of urinary [15N]hippurate, which was assumed to reflect intrahepatic isotopic abundance in [15N]glycine, the fractional synthetic rate for albumin was found to be 0.09-0.177 day-1 (mean +/- SD = 0.122 +/- 0.041 day-1). The absolute synthesis rate for albumin was 0.3 +/- 0.099 g/dl plasma X day-1 and the total plasma synthetic rate was 117.6 +/- 37.0 mg/kg X day-1. The glycine flux was 326.0-927.7 mumol/kg X h-1 (mean +/- SD 516.7 +/- 218.4 mumol/kg hr-1). The percentage of the glycine flux incorporated into albumin in the total vasculature was 0.425 +/- 0.344. The fractional synthetic rate and the absolute synthetic rate for albumin in these small premature infants are much higher than values obtained in healthy young adults studied with a similar methodology. The relatively low serum albumin concentrations typical of premature infants appear to be referable to more rapid turnover of a small plasma pool rather than a diminution in the rate of albumin synthesis.

Gas Chromatography-Mass Spectrometry↗
PubMed1986

Effect of 5-amino-4-imidazolecarboxamide riboside on renal ammoniagenesis. Study with [15N]aspartate.

[15N]Aspartate and 5-amino-4-imidazolecarboxamide riboside (AICAriboside) were used to evaluate the contribution of the purine nucleotide cycle to ammonia production in renal tubules isolated from control and chronically acidotic rats. Addition of 1 mM AICAriboside to incubation medium containing 2.5 mM [15N] aspartate significantly stimulated production of 15NH3 and 15N in the 6-amino group of adenine nucleotides during a 30-min incubation. In tubules from both control and acidotic animals, the levels of ATP, AMP, and NH3 were increased. In contrast, 5 mM AICAriboside inhibited 15NH3 production and reduced the total purine nucleotide content. In tubules from acidotic rats, enrichment in 15NH3 exceeded that in the 6-amino group of the adenine nucleotides, indicating that no precursor-product relationship existed between the purine nucleotide cycle and ammonia. Conversely, in tubules from control rats, 15N enrichment in the 6-amino group of the adenine nucleotides exceeded that in NH3. This relationship obtained whether or not AICAriboside was included in the incubation mixture. The current investigations show that the metabolism of aspartate through the purine nucleotide cycle is lower in renal tubules obtained from chronically acidotic rats than in control tubules. The observations indicate that AICAriboside has a biphasic effect on renal ammoniagenesis and adenine nucleotide synthesis, and suggest a possible clinical use of AICAriboside in cases of impaired ammonia formation in renal failure.

Adenosine Diphosphate↗
PubMed1986

Utilization of [15N]glutamate by cultured astrocytes.

The metabolism of 0.25 mM-[15N]glutamic acid in cultured astrocytes was studied with gas chromatography-mass spectrometry. Almost all 15N was found as [2-15N]glutamine, [2-15N]glutamine, [5-15N]glutamine and [15N]alanine after 210 min of incubation. Some incorporation of 15N into aspartate and the 6-amino position of the adenine nucleotides also was observed, the latter reflecting activity of the purine nucleotide cycle. After the addition of [15N]glutamate the ammonia concentration in the medium declined, but the intracellular ATP concentration was unchanged despite concomitant ATP consumption in the glutamine synthetase reaction. Some potential sources of glutamate nitrogen were identified by incubating the astrocytes for 24 h with [5-15N]glutamine, [2-15N]glutamine or [15N]alanine. Significant labelling of glutamate was noted with addition of glutamine labelled on either the amino or the amide moiety, reflecting both glutaminase activity and reductive amination of 2-oxoglutarate in the glutamate dehydrogenase reaction. Alanine nitrogen also is an important source of glutamate nitrogen in this system.

Adenosine Triphosphate↗
PubMed1986

Dynamic aspects of amino acid metabolism in alloxan-induced diabetes and insulin-treated rabbits: in vivo studies with 15N and gas chromatography-mass spectrometry.

The present study was designed to determine the effect of alloxan-induced diabetes in rabbits on L-[15N]alanine and [15N]glycine kinetic parameters. This process was measured by single-dose administration of 15N-labeled amino acids to postabsorptive control rabbits and alloxan-induced diabetics and insulin-treated diabetic rabbits. Gas chromatography-mass spectrometry was used to determine the 15N enrichment of plasma glycine and alanine. Glycine and alanine pools and turnover rate constants were estimated from isotope enrichment time decay curves. The data from the present study indicate that plasma glycine and alanine turnover rate constants increased by 25-50% after alloxan administration but pool sizes showed only little changes, resulting in highly significant increases in fluxes and metabolic clearance rates of both alanine and glycine following alloxan administration; single-dose crystalline insulin or protamine zinc insulin treatment failed to restore the turnover rate constants of glycine or alanine toward control values and caused a depletion of 50% in glycine pool size; 7 days prolonged treatment with protamine zinc insulin restored alanine and glycine fluxes and metabolic clearance rates towards control postabsorptive values; and the reduction in flux values following insulin treatment is consistent with the reduction in the plasma glucose levels in rabbits. The data suggest that the regulatory mechanisms for uptake and metabolism of circulating glycogenic amino acids no longer are operative as a consequence of insulin deficiency following alloxan administration. Exogenous insulin restored the activity of the regulatory mechanism toward the postabsorptive control state.

Alanine↗
PubMed1986

Nitrogen sources for renal ammoniagenesis: study with 15N amino acid.

The contribution of amino acids other than glutamine to renal ammoniagenesis was investigated in renal tubules obtained from control rats and rats with metabolic acidosis by incubating 2.5 mM [5-15N]glutamine, [2-15N]glutamine, [15N]glutamate, [15N]aspartate, [15N]alanine, or [15N]glycine, either as the sole N source in Krebs bicarbonate buffer or as a labeled substrate in a medium containing a mixture of unlabeled amino acids. With control tissue in Krebs buffer, approximately 75% of total ammonia was derived from 5-N of glutamine, whereas 2-N of glutamine, glutamate, aspartate, alanine, and glycine supplied 1, 10, 13, 4, and 18%, respectively. In the acidotic state, these values were 51, 30, 30, 30, 10, and 15% of the total ammonia produced, respectively. The ammonia that could not be accounted for by 15N analysis was derived from endogenous sources. Studies with tubules incubated in Krebs medium alone indicated that, in both control and acidosis, the calculated fraction of ammonia derived from endogenous sources was significantly decreased by addition of either 0.7 or 2.5 mM glutamine. However, ammonia production from endogenous sources was similar whether 0.7 or 2.5 mM glutamine was used as a sole exogenous substrate. Incubations of control tissue in buffer supplemented with an amino acid mixture revealed a significant decrease in ammonia production from [5-15N]glutamine compared with incubation in Krebs buffer alone. In chronic acidosis, no significant difference was found in total ammonia formation from [5-15N]glutamine compared with Krebs buffer alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Acidosis↗
PubMed1986

Methods for determining the protein requirement of infants.

Although there is no clearly preferred method to determine the precise protein requirement of infants, a variety of techniques have been employed to provide a reasonable approximation of this variable. These approaches include anthropometric measurements, the determination of nitrogen balance, the estimation of rate of whole body protein synthesis and catabolism by the administration of amino acids labeled with stable isotopes, and the determination of serum concentrations of certain proteins and amino acids.

Amino Acids↗
PubMed1985

Metabolism of glutamine and glutamate by rat renal tubules. Study with 15N and gas chromatography-mass spectrometry.

Gas chromatography-mass spectrometry was utilized to study the metabolism of [15N]glutamate, [2-15N]glutamine, and [5-15N]glutamine in isolated renal tubules prepared from control and chronically acidotic rats. The main purpose was to determine the nitrogen sources utilized by the kidney in various acid-base states for ammoniagenesis. Incubations were performed in the presence of 2.5 mM 15N-labeled glutamine or glutamate. Experiments with [5-15N]glutamine showed that in control animals approximately 90% of ammonia nitrogen was derived from 5-N of glutamine versus 60% in renal tubules from acidotic rats. Experiments with [2-15N]glutamine or [15N]glutamate indicated that in chronic acidosis approximately 30% of ammonia nitrogen was derived either from 2-N of glutamine or glutamate-N by the activity of glutamate dehydrogenase. Flux through glutamate dehydrogenase was 6-fold higher in chronic acidosis versus control. No 15NH3 could be detected in renal tubules from control rats when [2-15N]glutamine was the substrate. The rates of 15N transfer to other amino acids and to the 6-amino groups of the adenine nucleotides were significantly higher in normal renal tubules versus those from chronically acidotic rats. In tubules from chronically acidotic rats, 15N abundance in 15NH3 and the rate of 15NH3 appearance were significantly higher than that of either the 6-amino group of adenine nucleotides or the 15N-amino acids studied. The data indicate that glutamate dehydrogenase activity rather than glutamate transamination is primarily responsible for augmented ammoniagenesis in chronic acidosis. The contribution of the purine nucleotide cycle to ammonia formation appears to be unimportant in renal tubules from chronically acidotic rats.

Acid-Base Equilibrium↗