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Biomedical subjects

I Nissim

Publications and source records attributed to I Nissim.

At least 37 records · Page 2Linked to original sources
PubMed1996

A mass isotopomer study of urea and glutamine synthesis from 15N-labeled ammonia in the perfused rat liver.

This study examines the incorporation of 15N from 15NH4Cl into urea and glutamine, predicts the pattern of isotopomers produced as a function of the 15N enrichment of the relevant precursor pools, and presents a means of determining the isotopic enrichment of these pools. Rat livers were perfused, in the nonrecirculating mode, with 0.3 mM 15NH4Cl, and the isotopomers of urea and of glutamine produced were determined by gas chromatography-mass spectrometry methodology. Three different nitrogen mass isotopomers of urea were found, containing no, one, or two atoms of 15N. Four glutamine isotopomers were found, containing no 15N, one atom of 15N in either the amino or amide position, or two 15N atoms. A mathematical relationship was deduced that predicts that the relative proportions of the urea isotopomers depends not only on the relative enrichment of 15N in the two precursor pools of urea nitrogen (mitochondrial ammonia and cytoplasmic aspartate) but on their absolute enrichment. This relationship was validated in experiments in which the isotopic enrichment of the substrate, 15NH4Cl, was varied. The proportions of the urea isotopomers produced can be predicted if one knows the 15N enrichment in the two precursor pools. We found that when the 15N enrichment of citrulline and aspartate in the perfusate were used as proxies for that in the mitochondrial ammonia and cytoplasmic aspartate pools we could accurately predict the relative proportion of the three isotopomers. The production of the four nitrogen isotopomers of glutamine could be used to determine the 15N enrichment in the two precursor pools of glutamine nitrogen, the cytoplasmic ammonia and glutamate pools of the perivenous hepatocytes.

Ammonia↗
PubMed1996

Glycine attenuates Fanconi syndrome induced by maleate or ifosfamide in rats.

It has become widely recognized that glycine (Gly) depletion predisposes isolated proximal tubules (PT) to necrotic cell damage induced by diverse insults and that Gly replacement in vitro is highly cytoprotective. However, the effectiveness of supplementation with Gly in vivo, where blood and tissue Gly normally are maintained at high levels, is incompletely defined. Our aim was to assess whether: (a) supplementation of Gly in drinking water of rats would attenuate the proximal tubule damage and the Fanconi syndrome (FS) induced by maleate (Mal), a classical proximal tubule toxin, or ifosfamide (IFO), an antineoplastic drug; and (b) to explore the mechanisms responsible for such effects, since Gly supplementation might be especially beneficial in treating the FS, where the kidney tends to waste amino acids. Rats received daily injection of Mal (2 mmol/kg) for two days without or with oral supplementation of 2% Gly. IFO, 50 mg/kg, was injected daily for five days without or with oral Gly. Control rats were injected with saline, without or with oral Gly. The results demonstrated that both Mal and IFO induced a FS characterized by wasting of amino and organic acids, glucose, and electrolytes, along with elevated plasma creatinine (Crn) and BUN, and decreased Crn clearance rate. Light microscopy revealed a necrotic lesion in the proximal tubules of the Mal group, but no necrosis after IFO. Gly strongly ameliorated the severity of renal necrosis and/or dysfunction induced by Mal or IFO, with significant decreases in total and fractional excretion of Na+, K+, PO4(3-) and glucose, decreased plasma BUN and Crn, and increased Crn clearance. Analysis of freeze-clamped cortical tissue showed substantial depletion of [Gly], [ATP] and [GSH] along with increased GSSG in Mal or IFO groups and correction of [Gly] and [ATP] with Gly supplementation, but no improvement with Gly of reduced gluthatione [GSH] or the ratio of reduced to oxidized gluthatione (GSH/GSSG). 31P-NMR analysis of the renal cortex indicated a decrease in Pi and various membrane phospholipids in Mal and IFO rats and prevention of this damage with Gly. These observations demonstrate that oral supplementation of Gly can provide protection against Mal or IFO-induced renal tubular cell dysfunction and structural damage. The lack of effect on glutathione oxidation and depletion suggests an action distal to toxin uptake and intracellular interactions, which is similar to the characteristics of Gly cytoprotection against diverse insults in vitro. The results also suggest modification by Gly of the primary toxicity of the agents and effects on phospholipid synthesis that could contribute to repair.

Administration, Oral↗
PubMed1996

Astrocyte leucine metabolism: significance of branched-chain amino acid transamination.

We studied astrocytic metabolism of leucine, which in brain is a major donor of nitrogen for the synthesis of glutamate and glutamine. The uptake of leucine into glia was rapid, with a Vmax of 53.6 +/- 3.2 nmol/mg of protein/min and a Km of 449.2 +/- 94.9 microM. Virtually all leucine transport was found to be Na+ independent. Astrocytic accumulation of leucine was much greater (3x) in the presence of alpha-aminooxyacetic acid (5 mM), an inhibitor of transamination reactions, suggesting that the glia rapidly transaminate leucine to alpha-ketoisocaproic acid (KIC), which they then release into the extracellular fluid. This inference was confirmed by the direct measurement of KIC release to the medium when astrocytes were incubated with leucine. Approximately 70% of the leucine that the glia cleared from the medium was released as the keto acid. The apparent Km for leucine conversion to extracellular KIC was a medium [leucine] of 58 microM with a Vmax of approximately 2.0 nmol/mg of protein/min. The transamination of leucine is bidirectional (leucine+alpha-ketoglutarate<==>KIC+glutamate) in astrocytes, but flux from leucine-->glutamate is more active than that from glutamate-->leucine. These data underscore the significance of leucine handling to overall brain nitrogen metabolism. The release of KIC from glia to the extracellular fluid may afford a mechanism for the "buffering" of glutamate in neurons, which would consume this neurotransmitter in the course of reaminating KIC to leucine.

Amination↗
PubMed1996

Neuronal metabolism of branched-chain amino acids: flux through the aminotransferase pathway in synaptosomes.

The metabolism of branched-chain amino acids (BCAAs) was studied in cortical synaptosomes. With [15N]leucine (1 mM) as precursor, the cumulative appearance of 15N in [15N]glutamate and [15N]aspartate was 0.2 nmol/min/mg of protein without supplemental alpha-ketoglutarate and 0.3 nmol/min/mg of protein in the presence of alpha-ketoglutarate (0.5 mM). The BCAA amino-transferase reaction also proceeded in the "reverse" direction [alpha-ketoisocaproate (KIC) + glutamate-->leucine + alpha-ketoglutarate]. This was documented by incubating synaptosomes with [15N]glutamate and measuring the formation of [15N]leucine. Without KIC in the medium, the rate of [15N]leucine production was 0.13 nmol/min/mg of protein. In the presence of 25 microM KIC the rate was 0.79 nmol/min/mg of protein and even greater (1.0 nmol/ min/mg of protein) in the presence of 500 microM KIC. The reamination of KIC was two- to threefold faster with [2-15N]glutamine as precursor compared with [15N]-glutamate. The ketoacid of valine, alpha-ketoisovalerate (KIV), was reaminated to [15N]valine at a rate comparable to that observed with respect to KIC. The BCAA transaminase mediated not only the bidrectional transfer of amino groups between leucine or valine and glutamate, but also the direct transfer of nitrogen between leucine and valine. This was ascertained in studies in which the incubation medium was supplemented with either [15N]leucine and KIV or [15N]valine and KIC (amino acids at 1 mM and ketoacids at 25 or 500 microM). The rate was faster in the direction of leucine formation at both the lower (6.1-fold) and higher (1.7-fold) KIC concentration. It is suggested that in synaptosomes the BCAA transaminase (a) functions predominantly in the direction of leucine formation and (b) maintains a constant ratio of BCAAs and ketoacids to one other.

Amino Acids, Branched-Chain↗
PubMed1995

Endogenous synthesis of galactose in normal men and patients with hereditary galactosaemia.

Despite restricted ingestion of lactose, patients with galactose-1-phosphate uridyltransferase deficiency have raised concentrations of galactose metabolites in blood and urine. Endogenous production of galactose may underlie this phenomenon. Using isotopically labelled galactose in a continuous intravenous infusion, we employed the steady-state flux method to calculate endogenous galactose production rate in three normal men and three patients with classic galactosaemia. We found that galactosaemic patients and normal subjects synthesise gram quantities of galactose per day. The rate of synthesis ranged from 0.53-1.05 mg/kg per h. Endogenous production of galactose may be an important factor in the pathogenesis of the complications of the brain and ovary, and could explain the persistent elevation of galactose metabolites in patients despite dietary restriction of galactose.

Adult↗
PubMed1995

In vivo oxidation of [13C]galactose in patients with galactose-1-phosphate uridyltransferase deficiency.

We developed an intravenous and oral [13C]galactose breath test for the in vivo study of galactose metabolism. Following an intravenous bolus of 7 mg/kg of [1-13C]galactose in the fasting state, normal children and adults eliminated 3-6% and 21-47% of the bolus as 13CO2 in expired air collected over 1 and 5 h, respectively. Comparable fractional elimination was seen when the dose was given orally. Patients with galactosemia who have barely detectable or absent galactose-1-phosphate uridyltransferase (GALT) activity in erythrocytes and are homoallelic for the Q188R gene mutation, when given a 7 mg/kg intravenous bolus had barely detectable 13CO2 in air samples in the first hour, but eventually eliminated as much as 3.6% of the dose in 5 h. A galactosemia/Duarte (Q188R/N314D) compound heterozygote and a homozygous Duarte subject, as well as a subject with one normal allele and one Q188R allele, showed normal in vivo oxidation. An assessment of whole body galactose metabolism can be made with this procedure. Further use of this in vivo modality in patients with different genetic backgrounds should increase our understanding of genotype-phenotype relationships in hereditary galactosemia.

Adolescent↗
PubMed1995

Hormonal regulation of glutamine metabolism by OK cells.

The precise mechanism(s) of action of PTH, insulin or glucagon in the regulation of renal glutamine and ammonia metabolism is unknown. Our aim was to delineate the effects and the site(s) of action of these hormones on renal glutamine metabolism. Experiments were carried out using OK cells as a model system. Cell cultures were incubated for three hours in a bicarbonate buffer of pH 7.4 supplemented with either 1 mM [2-15N] or [5-15N] glutamine and 10(-7) M PTH, insulin or glucagon. Comparative studies were performed at pH 6.8, 7.4 or 7.6 without hormone. PTH and acute acidosis significantly stimulated glutamine metabolism via both the phosphate-dependent glutaminase (PDG) and glutamate dehydrogenase (GLDH) pathways. The opposite was observed at pH 7.6. Insulin augmented flux via PDG with little effect on the GLDH pathway. Glucagon had insignificant effects on either PDG or GLDH pathways. Intracellular [15N] glutamate formed from [2-15N] glutamine was removed partially by transamination to alanine, aspartate and serine and partially by translocation to an extracellular compartment. Acidosis, PTH and insulin enhanced the formation of [15N] alanine with little effect on [15N] aspartate. PTH, insulin and glucagon significantly stimulated the production of [15N]serine, whereas acidosis had little effect. The translocation of intracellular glutamate was significantly increased by acidosis, PTH and insulin and decreased by acute alkalosis. The data indicate that: (a) PTH mimicks the effect of acute acidosis on renal glutamine metabolism, that is, augmented glutamine metabolism through both PDG and GLDH pathways and stimulated the output of intracellular glutamate. This effect might be mediated via decreased activity of the Na(+)-H+ exchanger associated with cellular acidification and/or through a second messenger; (b) insulin, but not glucagon, increased glutamine uptake and metabolism, and simultaneously enhanced output of intracellular glutamate sufficiently to stimulate the PDG pathway; and (c) overall, glucagon had little effect on glutamine metabolism by OK cells compared with either PTH or insulin.

Adenosine Triphosphate↗
PubMed1995

Prostaglandin F2 alpha- and 12-O-tetradecanoylphorbol-13-acetate-induced alterations in the pathways of renal ammoniagenesis.

The mechanisms whereby prostaglandin F2 alpha (PGF2 alpha) and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibit ammoniagenesis and the reason why they behave differently at pH 7.4, were examined with (15N)glutamine to assess the metabolic pathways and 2'-7'-bis(2-carboxyethyl)-5-(and-6)-carboxylfluorescein, acetoxymethylester (BCECF-AM) to evaluate Na+/H+ antiporter activity. LLC-PK1 cultures were incubated for 1 h in a Krebs-Hensleit bicarbonate buffer of pH 7.4 and pH 6.8 supplemented either with 5-15N- or 2-15N-labeled glutamine, followed by the assessment of (15N)ammonia and (15N)amino acid formation. Exposure of cells to either PGF2 alpha or TPA completely inhibited the low pH-induced increases in (15N)ammonia formation from incubations with 5-15N, reflecting reduced flux through the mitochondrial phosphate-dependent glutaminase, and from (2-15N)glutamine, reflecting reduced flux through the mitochondrial glutamate dehydrogenase pathway. They also qualitatively reversed the acute acidosis-induced changes in (15N)alanine formation and (15N)glutamate accumulation in the media. By contrast only TPA, but not PGF2 alpha, modified glutamine metabolism at pH 7.4. Na+/H+ antiporter activity was assessed under both acidified and basal (pH 7.4) conditions by measuring changes in intracellular pH in cells loaded with BCECF. TPA and PGF2 alpha both stimulated Na+/H+ antiporter activity comparably under acidified conditions. When cells were studied at pH 7.4, TPA but not PGF2 alpha stimulated the Na+/H+ antiporter and increased steady-state intracellular pH.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1995

Metabolic studies of rat renal tubule cells loaded with cystine: the cystine dimethylester model of cystinosis.

The cause of Fanconi syndrome in cystinosis is enigmatic. It has previously been shown that renal tubules could be loaded with cystine by incubating them with cystine dimethylester (CDE), mimicking the biochemical hallmark of cystinosis. Such tubules have impaired transport, decreased whole-cell O2 consumption, and substrate utilization. In this study, the metabolic disturbances in cystine-loaded renal tubule cells were further characterized. Isolated rat renal tubules were loaded with cystine by incubating them with 2 mM CDE for 10 min. This had no significant effect on total ATPase, Na(+)-K(+)-ATPase, or the ouabain-insensitive ATPase activity of renal tissue homogenates from these cystine-loaded tubules. Intracellular K was significantly lower in the cystine-loaded tubules (37 +/- 2 versus 47 +/- 3 nEq/mg; P < 0.008). Intracellular ATP was reduced by 39% in the cystine-loaded tubules (23.7 +/- 2.4 versus 38.1 +/- 3.3 nmol/mg of protein; P < 0.0025). CDE (2 mM) reduced isolated mitochondrial O2 consumption with glutamate as the substrate by 66% (4.7 +/- 0.7 versus 13.9 +/- 0.8 nm/min per mg of protein, P < 0.001) but had no effect on mitochondrial O2 consumption with succinate as the substrate. It was speculated that the impaired transport from cystine loading with CDE is secondary to a decrease in energy generation.

Adenosine Triphosphate↗
PubMed1995

Stable isotopes as metabolic probes for nutritional studies in neonates.

Metabolic and nutritional studies with stable isotope tracers have proved valuable in the human newborn. This technique is acceptable ethically, because it does not expose the infant to radioactivity, and it is sensitive enough for the analysis of very small samples. The stable isotope method has been especially applied to studies of neonatal carbohydrate, protein, and energy metabolism.

Dietary Carbohydrates↗
PubMed1994

The intensity of acidosis differentially alters the pathways of ammoniagenesis in LLC-PK1 cells.

Utilizing [5-15N] and [2-15N]-labeled glutamine and gas chromatography mass spectrometry methodology, we examined the pathways of ammoniagenesis under basal and acute acidotic conditions of pH 7.0 and pH 6.8, respectively. LLC-PK1 cultures were incubated for one hour with gentle rocking in a bicarbonate buffer of pH 7.4, pH 7.0, or pH 6.8 supplemented either with [5-15N] or [2-15N] glutamine at 37 degrees C in a 5% CO2/95% air incubator atmosphere. Incubation of cultures with [5-15N] glutamine at pH 7.4 resulted in a substantial amount of 15N ammonia formation which was not significantly altered by incubations at pH 7.0. By contrast, exposure to pH 6.8 significantly increased 15N ammonia formation in comparison with its production at pH 7.0 or 7.4. However, 15N ammonia production from [2-15N] glutamine was significantly stimulated at pH 7.0 and was further increased at pH 6.8. Incubation of the cells with [2-15N] glutamine resulted in a substantially lower amounts of 15N ammonia formation than produced with [5-15N] glutamine. Alanine formation from [2-15N] glutamine increased significantly at pH 7.0; but in contrast to 15N ammonia formation, pH 6.8 had no additional stimulatory effect on 15N alanine formation. Cells incubated with [2-15N] glutamine resulted in a significant decrement in 15N glutamate production at both pH 7.0 and 6.8 when compared with pH 7.4. 15N aspartate formation was unaltered by the changes in media pH.(ABSTRACT TRUNCATED AT 250 WORDS)

Acidosis↗
PubMed1994

Interrelationships of leucine and glutamate metabolism in cultured astrocytes.

The aim was to study the extent to which leucine furnishes alpha-NH2 groups for glutamate synthesis via branched-chain amino acid aminotransferase. The transfer of N from leucine to glutamate was determined by incubating astrocytes in a medium containing [15N]leucine and 15 unlabeled amino acids; isotopic abundance was measured with gas chromatography-mass spectrometry. The ratio of labeling in both [15N]glutamate/[15N]leucine and [2-15N]glutamine/[15N]leucine suggested that at least one-fifth of all glutamate N had been derived from leucine nitrogen. At the same time, enrichment in [15N]leucine declined, reflecting dilution of the 15N label by the unlabeled amino acids that were in the medium. Isotopic abundance in [15N]isoleucine increased very quickly, suggesting the rapidity of transamination between these amino acids. The appearance of 15N in valine was more gradual. Measurement of branched-chain amino acid transaminase showed that the reaction from leucine to glutamate was approximately six times more active than from glutamate to leucine (8.72 vs. 1.46 nmol/min/mg of protein). However, when the medium was supplemented with alpha-ketoisocaproate (1 mM), the ketoacid of leucine, the reaction readily ran in the "reverse" direction and intraastrocytic [glutamate] was reduced by approximately 50% in only 5 min. Extracellular concentrations of alpha-ketoisocaproate as low as 0.05 mM significantly lowered intracellular [glutamate]. The relative efficiency of branched-chain amino acid transamination was studied by incubating astrocytes with 15 unlabeled amino acids (0.1 mM each) and [15N]glutamate. After 45 min, the most highly labeled amino acid was [15N]alanine, which was closely followed by [15N]leucine and [15N]isoleucine. Relatively little 15N was detected in any other amino acids, except for [15N]serine. The transamination of leucine was approximately 17 times greater than the rate of [1-14C]leucine oxidation. These data indicate that leucine is a major source of glutamate nitrogen. Conversely, reamination of alpha-ketoisocaproate, the ketoacid of leucine, affords a mechanism for the temporary "buffering" of intracellular glutamate.

Amination↗
PubMed1994

Cerebral alanine transport and alanine aminotransferase reaction: alanine as a source of neuronal glutamate.

Alanine transport and the role of alanine amino-transferase in the synthesis and consumption of glutamate were investigated in the preparation of rat brain synaptosomes. Alanine was accumulated rapidly via both the high- and low-affinity uptake systems. The high-affinity transport was dependent on the sodium concentration gradient and membrane electrical potential, which suggests a cotransport with Na+. Rapid accumulation of the Na(+)-alanine complex by synaptosomes stimulated activity of the Na+/K+ pump and increased energy utilization; this, in turn, activated the ATP-producing pathways, glycolysis and oxidative phosphorylation. Accumulation of Na+ also caused a small depolarization of the plasma membrane, a rise in [Ca2+]i, and a release of glutamate. Intra-synaptosomal metabolism of alanine via alanine amino-transferase, as estimated from measurements of N fluxes from labeled precursors, was much slower than the rate of alanine uptake, even in the presence of added oxoacids. The velocity of [15N]alanine formation from [15N]glutamine was seven to eight times higher than the rate of [15N]-glutamate generation from [15N]alanine. It is concluded that (a) overloading of nerve endings with alanine could be deleterious to neuronal function because it increases release of glutamate; (b) the activity of synaptosomal alanine aminotransferase is much slower than that of glutaminase and hence unlikely to play a major role in maintaining [glutamate] during neuronal activity; and (c) alanine amino-transferase might serve as a source of glutamate during recovery from ischemia/hypoxia when the alanine concentration rises and that of glutamate falls.

Alanine↗
PubMed1994

Inhibition of astrocyte glutamine production by alpha-ketoisocaproic acid.

We have evaluated the effect of alpha-ketoisocaproic acid (KIC), the ketoacid of leucine, on the production of glutamine by cultured astrocytes. We used 15NH4Cl as a metabolic tracer to measure the production of both [5-15N]glutamine, reflecting amidation of glutamate via glutamine synthetase, and [2-15N]glutamine, representing the reductive amination of 2-oxoglutarate via glutamate dehydrogenase and subsequent conversion of [15N]glutamate to [2-15N]glutamine. Addition of KIC (1 mM) to the medium diminished the production of [5-15N]glutamine and stimulated the formation of [2-15N]glutamine with the overall result being a significant inhibition of net glutamine synthesis. An external KIC concentration as low as 0.06 mM inhibited synthesis of [5-15N]glutamine and a level as low as 0.13 mM enhanced labeling (atom% excess) of [2-15N]glutamine. Higher concentrations of KIC in the medium had correspondingly larger effects. The presence of KIC in the medium did not affect flux through glutaminase, which was measured using [2-15N]glutamine as a tracer. Nor did KIC inhibit the activity of glutamine synthetase that was purified from sheep brain. Addition of KIC to the medium caused no increased release of lactate dehydrogenase from the astrocytes, suggesting that the ketoacid was not toxic to the cells. KIC treatment was associated with an approximately twofold increase in the formation of 14CO2 from [U-14C]glutamate, indicating that transamination of glutamate with KIC increases intraastrocytic alpha-ketoglutarate, which is oxidized in the tricarboxylic acid cycle. KIC inhibited glutamine synthesis more than any other ketoacid tested, with the exception of hydroxypyruvate.(ABSTRACT TRUNCATED AT 250 WORDS)

Ammonium Chloride↗
PubMed1993

Effect of glutamine on heat-shock-induced mRNA and stress proteins.

Our aim was to delineate the effect of glutamine on the level of heat shock-inducible mRNA and synthesis of stress protein(s) in cultured kidney cells. Experiments were carried out using opossum kidney (OK) cells. The induction of HSP70 mRNA as well as the synthesis of 72,73 kDa stress proteins was evaluated in cell monolayers exposed to 45 degrees C for 15 minutes followed by a recovery period at 37 degrees C for 3 hours. Incubations were performed in Krebs buffer supplemented with 0, 2, 5, or 10 mM glutamine. A separate series of experiments was performed in the presence of glutamine metabolites, such as NH4Cl, glutamate, or aspartate. Glutamine without preincubation at 37 degrees C remarkably increased the steady-state level of HSP70 mRNA as well as the production of 72,73 kDa stress proteins in a dose-dependent manner. The production of stress protein(s) in the presence of glutamine was associated with decreased percent LDH efflux, suggesting cytoprotective action of glutamine in cultured kidney cells. However, when OK cells were preincubated for 1 hour at 37 degrees C with 10 mM glutamine, there was an approximately fourfold decline in level of HSP70 mRNA compared with experiments in the presence of 10 mM glutamine without preincubation. In addition, metabolites of glutamine, i.e., ammonia and glutamate decreased the level of heat-inducible HSP70 mRNA. Furthermore, aspartate or NH4Cl had little effect on LDH release compared with heat shock experiments, without addition of amino acids. These observations suggest that metabolites of glutamine may blunt the steady-state level of glutamate or HSP70 mRNA. The decreased level of HSP70 mRNA in the presence of NH4Cl may explain the role of ammonia in renal injury and brain toxicity, as well as glutamate excitotoxicity.

Ammonium Chloride↗