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M Yudkoff

Publications and source records attributed to M Yudkoff.

At least 73 records · Page 4Linked to original sources
PubMed1990

Carbon flux through tricarboxylic acid cycle in rat renal tubules.

Our aim was to delineate the effect(s) of chronic metabolic acidosis on renal TCA-cycle metabolism. Renal tubules isolated from control and chronically acidotic rats were incubated at pH 7.4 with either 2 mM [2,3-13C]pyruvate or [2-13C]acetate. GC-MS and/or 13C-NMR were utilized to monitor the flux of 13C through pyruvate dehydrogenase, pyruvate carboxylase and the TCA-cycle. With either, precursor acidosis was associated with significantly decreased formation of 13C-labelled citrate, malate, aspartate and alanine and increased formation of glucose, lactate and acetyl-CoA as compared with the control. The results indicate that adaptation of renal metabolism to chronic metabolic acidosis is associated with diminished flux through citrate synthetase and concomitantly increased flux through pyruvate carboxylase. The data suggest that depletion of TCA-cycle intermediates and enhanced ammoniagenesis in the kidney of chronically acidotic rats may be regulated at the site of mitochondrial citrate-condensing enzyme.

Acidosis↗
PubMed1990

Measurement of (C13)arginine incorporation into apolipoprotein B-100 in very low density lipoproteins and low density lipoproteins in normal subjects using (13C)sodium bicarbonate infusion and isotope ratio mass spectrometry.

We describe a new stable isotope technique for the in vivo study of hepatic plasma protein synthesis in humans. The method involves the infusion of (13C)sodium bicarbonate for 1 h and the measurement of the isotopic enrichment of (13C)arginine in newly synthesized apolipoprotein B of very low density lipoproteins (VLDL-apoB) and low density lipoproteins (LDL-apoB) in blood samples taken over a 5-6 h period from the commencement of the infusion. Isotope ratio mass spectrometry was utilized to measure 13CO2 enrichment following hydrolysis of these proteins and conversion of the guanidinium carbon of arginine in the hydrolysate to carbon dioxide by sequential incubation with arginase and urease. The method is capable of measuring isotopic enrichment as low as 0.001 at.% excess (APE) with a precision of 1.2%. In both subjects studied, the (13C)arginine of VLDL-apoB reached enrichments of 0.2 APE and that of the arginine of LDL-apoB, 0.03 APE. Incorporation of labeled arginine into LDL-apoB was demonstrable at 60-90 min. The new technique is safe and is applicable to the study of the hepatic biosynthesis of a wide range of plasma proteins.

Adult↗
PubMed1990

Precursors of glutamic acid nitrogen in primary neuronal cultures: studies with 15N.

We utilized gas chromatography-mass spectrometry to study the transfer of 15N from [2-15N]glutamine, [15N]leucine, [15N]alanine, or 15NH4Cl to [15N]glutamate and [15N]aspartate in cultured cerebrocortical GABA-ergic neurons from the mouse. Initial rates of 15N appearance (atom % excess) were somewhat higher with 2mM [2-15N]glutamine as a precursor than with 1mM [15N]leucine or 1mM [15N]alanine, but initial net formation (nmol [15N]glutamate/mg protein.min-1) was roughly comparable with all precursors. At steady-state 15N labeling was about two times greater with 2mM [2-15N]glutamine as precursor. The subsequent transfer of 15N from glutamate to aspartate was extremely rapid, the labelling pattern of these two amino acid pools being virtually indistinguishable. We observed little reductive amination of 2-oxo-glutarate to yield [15N]glutamate in the presence of 0.3mM 15NH4Cl. Reductive amination through glutamate dehydrogenase was much more prominent at a concentration of 3.0mM 15NH4Cl. Glutamate formation via reductive amination was unaffected by inclusion of 1mM 2-oxo-glutarate in the incubation medium. These results indicate that glutamate synthesis in cultured GABA-ergic neurons is derived not only from the glutaminase reaction, but also from transamination reactions in which both leucine and alanine are efficient N donors. Reductive amination of 2-oxo-glutarate in the glutamate dehydrogenase pathway plays a relatively minor role at lower concentrations of extracellular ammonia but becomes quite active at 3mM ammonia.

Alanine↗
PubMed1990

Neuronal glutamine utilization: glutamine/glutamate homeostasis in synaptosomes.

The synaptosomal metabolism of glutamine was studied under in vitro conditions that simulate depolarization in vivo. With [2-15N]glutamine as precursor, the [glutamine]i was diminished in the presence of veratridine or 50 mM KCl, but the total amounts of [15N]glutamate and [15N]aspartate formed were either equal to those of control incubations (veratridine) or higher (50 mM [KCl]). This suggests that depolarization decreases glutamine uptake and independently augments glutaminase activity. Omission of sodium from the medium was associated with low internal levels of glutamine which indicates that influx occurs as a charged Na(+)-amino acid complex. It is postulated that a reduction in membrane potential and a collapse of the Na+ gradient decrease the driving forces for glutamine accumulation and thus inhibit its uptake and enhance its release under depolarizing conditions. Inorganic phosphate stimulated glutaminase activity, particularly in the presence of calcium. At 2 mM or lower [phosphate] in the medium, calcium inhibited glutamine utilization and the production of glutamate, aspartate, and ammonia from glutamine. At a high (10 mM) medium [phosphate], calcium stimulated glutamine catabolism. It is suggested that a veratridine-induced increase in intrasynaptosomal inorganic phosphate is responsible for the enhancement of flux through glutaminase; calcium affects glutaminase indirectly by modulating the level of free intramitochondrial [phosphate]. Because phosphate also lowers the Km of glutaminase for glutamine, augmentation of the amino acid breakdown may occur even when depolarization lowers [glutamine]i. Reducing the intrasynaptosomal glutamate to 26 nmol/mg of protein had little effect on glutamine catabolism, but raising the pH to 7.9 markedly increased formation of glutamate and aspartate. It is concluded that phosphate and H+ are the major physiologic regulators of glutaminase activity.

Animals↗
PubMed1990

Glutathione turnover in cultured astrocytes: studies with [15N]glutamate.

The incorporation of [15N]glutamic acid into glutathione was studied in primary cultures of astrocytes. Turnover of the intracellular glutathione pool was rapid, attaining a steady state value of 30.0 atom% excess in 180 min. The intracellular glutathione concentration was high (20-40 nmol/mg protein) and the tripeptide was released rapidly into the incubation medium. Although labeling of glutathione (atom% excess) with [15N]glutamate occurred rapidly, little accumulation of 15N in glutathione was noted during the incubation compared with 15N in aspartate, glutamine, and alanine. Glutathione turnover was stimulated by incubating the astrocytes with diethylmaleate, an electrophile that caused a partial depletion of the glutathione pool(s). Diethylmaleate treatment also was associated with significant reductions of intraastrocytic glutamate, glycine, and cysteine, i.e., the constituents of glutathione. Glutathione synthesis could be stimulated by supplementing the steady-state incubation medium with 0.05 mM L-cysteine, such treatment again partially depleting intraastrocytic glutamate and causing significant reductions of 15N labeling of both alanine and glutamine, suggesting that glutamate had been diverted from the synthesis of these amino acids and toward the formation of glutathione. The current study underscores both the intensity of glutathione turnover in astrocytes and the relationship of this turnover to the metabolism of glutamate and other amino acids.

Amino Acids↗
PubMed1990

Familial hyperuricemia and renal insufficiency.

A kindred is described in which hyperuricemia and renal insufficiency were observed in three generations. The hyperuricemia appeared to precede the renal diseases. Lowering the serum uric acid level to normal did not decrease the progression of renal insufficiency. This suggested that the hyperuricemia was a marker of a familial nephropathy and possibly not the cause.

Adolescent↗
PubMed1989

Effect of 5-amino-4-imidazolecarboxamide riboside (AICA-riboside) on the purine nucleotide synthesis and growth of rat kidney cells in culture: study with [15N]aspartate.

The present investigation evaluates the effect of AICA-Riboside on the synthesis of purine nucleotides and the growth of normal rat kidney cells in culture. Experiments in the presence and absence of various concentrations of AICA-Riboside were conducted with Dulbecco's Modified Eagle's Medium supplemented with either 1 mM [15N]aspartate or [14N]aspartate. Addition of 50 microM AICA-Riboside to the incubation medium significantly stimulated intracellular adenine nucleotide concentrations following incubation for 48 hours. This stimulation was associated with augmented cell growth and DNA concentration. In contrast, with concentrations above 100 microM of AICA-Riboside in the incubation medium, there was a remarkable inhibition of cell growth and a significant depletion of intracellular pools of adenine nucleotides and DNA. Experiments with [15N]aspartate showed that the initial rate (0-24 hours) of [6-15NH2]adenine nucleotide formation from 1 mM [15N]aspartate was 38.8 +/- 9.6, 67.9 +/- 12.5, and 20.1 +/- 3.8 pmol h-1/10(6) cells in the presence of 0 (control), 50 microM and 500 microM AICA-Riboside, respectively. These observations indicate that the main effect of AICA-Riboside is on the formation of AMP from aspartate and IMP via the sequential action of adenylosuccinate synthetase and adenylosuccinate lyase. The current studies suggest that AICA-Riboside could be used as a factor mediating renal cell mitosis in culture. AICA-Riboside has a biphasic effect on the growth of renal epithelial cells in culture and on their intracellular purine nucleotides and DNA concentration.

Adenine Nucleotides↗
PubMed1989

Organic aciduria and butyryl CoA dehydrogenase deficiency in BALB/cByJ mice.

A metabolic screening program of inbred strains of mice has detected a marked organic aciduria in the BALB/cByJ strain. Gas chromatographic and mass spectrometric analysis identified large quantities of n-butyrylglycine plus lesser quantities of ethylmalonic acid. Crosses with the nonexcreting C57BL/6J strain indicate that this condition is inherited as an autosomal recessive trait. Independently from this screening a variant with no detectable enzyme activity of butyryl CoA dehydrogenase (BCD) in liver and kidney of the BALB/cByJ strain but not other BALB/c sublines was discovered. Data from a three-point cross indicated that the null variant maps to the structural locus for the enzyme, Bcd-1, on chromosome 5. The findings indicate that a mutation at or near Bcd-1 in the BALB/cByJ strain resulted in a biochemical abnormality manifest as the BCD deficiency. It is concluded that accumulation of butyryl CoA due to a block in the oxidation of short-chain fatty acids results in an overproduction of organic metabolites leading to the observed organic aciduria. The fact that other BALB/c substrains do not exhibit this abnormality further suggests that this disorder reflects subline divergence within the BALB/c family.

Animals↗
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↗
PubMed1988

Isovaleric acidemia: medical and neurodevelopmental effects of long-term therapy.

Nine patients with isovaleric acidemia were treated with a low-protein diet and supplemental glycine for up to 10 years. Carnitine was added to the therapy in four patients. Overall, the treatment was well tolerated, resulting in no significant side effects other than persistent hyperglycinemia. Normal growth was observed in all patients. Of four patients with the chronic phenotype, three, whose treatment was delayed beyond the first year of life, are mentally retarded. Two of five patients with the acute phenotype are retarded. The outcome in these two was complicated in one by neonatal intraventricular hemorrhage and in the other by therapeutic noncompliance. In our patients, only those who were treated successfully from early infancy and had no complications did not develop mental retardation. After initiation of therapy, there was a significant decrease in ketoacidotic attacks requiring hospitalization. Glycine is indicated for the treatment of acute ketoacidosis in these patients; none of the catastrophically ill newborn who received glycine died. The aim of treatment is to reduce the isovaleric acid burden to a minimum. Therapy consisting of leucine restriction with supplemental glycine and carniline should be started as soon as possible after birth.

Amino Acid Metabolism, Inborn Errors↗
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↗