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Biomedical subjects

M Yudkoff

Publications and source records attributed to M Yudkoff.

At least 19 recordsLinked to original sources
PubMed2004

Metabolic and neuropsychological phenotype in women heterozygous for ornithine transcarbamylase deficiency.

We compared neurocognitive indices with clinical status, mutation analysis, and urea synthetic capacity in 19 women heterozygous for ornithine transcarbamylase deficiency. Although as a group, these women had average IQ scores, they displayed a specific neuropsychological phenotype with significant strengths in verbal intelligence, verbal learning, verbal memory, and reading, and significant weaknesses in fine motor dexterity/speed and nonsignificant weaknesses in nonverbal intelligence, visual memory, attention/executive skills, and math. This suggests selective vulnerability of white matter and better preservation of gray matter. When the group was divided into symptomatic and asymptomatic subgroups, based on either clinical history or residual urea synthetic capacity, the asymptomatic subgroup outperformed the symptomatic subgroup on all tested domains of neuropsychological functioning. Furthermore, the amount of residual urea synthetic capacity was predictive of several end point cognitive measures. There was no correlation between neonatal versus late-onset mutation or between normal or abnormal allopurinol challenge and neuropsychological outcome. In sum, we identified a specific metabolic and neurocognitive phenotype in women heterozygous for ornithine transcarbamylase deficiency. The findings support the importance of maintaining meticulous metabolic control in children with urea cycle disorders, because even mildly symptomatic subjects demonstrate cognitive deficits.

Adult↗
PubMed2001

Ketogenic diet, amino acid metabolism, and seizure control.

The ketogenic diet has been utilized for many years as an adjunctive therapy in the management of epilepsy, especially in those children for whom antiepileptic drugs have not permitted complete relief. The biochemical basis of the dietary effect is unclear. One possibility is that the diet leads to alterations in the metabolism of brain amino acids, most importantly glutamic acid, the major excitatory neurotransmitter. In this review, we explore the theme. We present evidence that ketosis can lead to the following: 1) a diminution in the rate of glutamate transamination to aspartate that occurs because of reduced availability of oxaloacetate, the ketoacid precursor to aspartate; 2) enhanced conversion of glutamate to GABA; and 3) increased uptake of neutral amino acids into the brain. Transport of these compounds involves an uptake system that exchanges the neutral amino acid for glutamine. The result is increased release from the brain of glutamate, particularly glutamate that had been resident in the synaptic space, in the form of glutamine. These putative adaptations of amino acid metabolism occur as the system evolves from a glucose-based fuel economy to one that utilizes ketone bodies as metabolic substrates. We consider mechanisms by which such changes might lead to the antiepileptic effect.

Amino Acids↗
PubMed2001

Brain amino acid metabolism and ketosis.

The relationship between ketosis and brain amino acid metabolism was studied in mice that consumed a ketogenic diet (>90% of calories as lipid). After 3 days on the diet the blood concentration of 3-OH-butyrate was approximately 5 mmol/l (control = 0.06-0.1 mmol/l). In forebrain and cerebellum the concentration of 3-OH-butyrate was approximately 10-fold higher than control. Brain [citrate] and [lactate] were greater in the ketotic animals. The concentration of whole brain free coenzyme A was lower in ketotic mice. Brain [aspartate] was reduced in forebrain and cerebellum, but [glutamate] and [glutamine] were unchanged. When [(15)N]leucine was administered to follow N metabolism, this labeled amino acid accumulated to a greater extent in the blood and brain of ketotic mice. Total brain aspartate ((14)N + (15)N) was reduced in the ketotic group. The [(15)N]aspartate/[(15)N]glutamate ratio was lower in ketotic animals, consistent with a shift in the equilibrium of the aspartate aminotransferase reaction away from aspartate. Label in [(15)N]GABA and total [(15)N]GABA was increased in ketotic animals. When the ketotic animals were injected with glucose, there was a partial blunting of ketoacidemia within 40 min as well as an increase of brain [aspartate], which was similar to control. When [U-(13)C(6)]glucose was injected, the (13)C label appeared rapidly in brain lactate and in amino acids. Label in brain [U-(13)C(3)]lactate was greater in the ketotic group. The ratio of brain (13)C-amino acid/(13)C-lactate, which reflects the fraction of amino acid carbon that is derived from glucose, was much lower in ketosis, indicating that another carbon source, i.e., ketone bodies, were precursor to aspartate, glutamate, glutamine and GABA.

Amino Acids↗
PubMed2001

Dynamic regulation of metabolism and respiration by endogenously produced nitric oxide protects against oxidative stress.

One of the many biological functions of nitric oxide is the ability to protect cells from oxidative stress. To investigate the potential contribution of low steady state levels of nitric oxide generated by endothelial nitric oxide synthase (eNOS) and the mechanisms of protection against H(2)O(2), spontaneously transformed human ECV304 cells, which normally do not express eNOS, were stably transfected with a green fluorescent-tagged eNOS cDNA. The eNOS-transfected cells were found to be resistant to injury and delayed death following a 2-h exposure to H(2)O(2) (50-150 microM). Inhibition of nitric oxide synthesis abolished the protective effect against H(2)O(2) exposure. The ability of nitric oxide to protect cells depended on the presence of respiring mitochondria as ECV304+eNOS cells with diminished mitochondria respiration (rho(-)) are injured to the same extent as nontransfected ECV304 cells and recovery of mitochondrial respiration restores the ability of nitric oxide to protect against H(2)O(2)-induced death. Nitric oxide also found to have a profound effect in cell metabolism, because ECV304+eNOS cells had lower steady state levels of ATP and higher utilization of glucose via the glycolytic pathway than ECV304 cells. However, the protective effect of nitric oxide against H(2)O(2) exposure is not reproduced in ECV304 cells after treatment with azide and oligomycin suggesting that the dynamic regulation of respiration by nitric oxide represent a critical and unrecognized primary line of defense against oxidative stress.

Adenosine Triphosphate↗
PubMed2001

Alanine metabolism in the perfused rat liver. Studies with (15)N.

We have utilized [(15)N]alanine or (15)NH(3) as metabolic tracers in order to identify sources of nitrogen for hepatic ureagenesis in a liver perfusion system. Studies were done in the presence and absence of physiologic concentrations of portal venous ammonia in order to test the hypothesis that, when the NH(4)(+):aspartate ratio is >1, increased hepatic proteolysis provides cytoplasmic aspartate in order to support ureagenesis. When 1 mm [(15)N]alanine was the sole nitrogen source, the amino group was incorporated into both nitrogens of urea and both nitrogens of glutamine. However, when studies were done with 1 mm alanine and 0.3 mm NH(4)Cl, alanine failed to provide aspartate at a rate that would have detoxified all administered ammonia. Under these circumstances, the presence of ammonia at a physiologic concentration stimulated hepatic proteolysis. In perfusions with alanine alone, approximately 400 nmol of nitrogen/min/g liver was needed to satisfy the balance between nitrogen intake and nitrogen output. When the model included alanine and NH(4)Cl, 1000 nmol of nitrogen/min/g liver were formed from an intra-hepatic source, presumably proteolysis. In this manner, the internal pool provided the cytoplasmic aspartate that allowed the liver to dispose of mitochondrial carbamyl phosphate that was rapidly produced from external ammonia. This information may be relevant to those clinical situations (renal failure, cirrhosis, starvation, low protein diet, and malignancy) when portal venous NH(4)(+) greatly exceeds the concentration of aspartate. Under these circumstances, the liver must summon internal pools of protein in order to accommodate the ammonia burden.

Alanine↗
PubMed2001

N(epsilon)-(gamma-L-glutamyl)-L-lysine (GGEL) is increased in cerebrospinal fluid of patients with Huntington's disease.

Pathological-length polyglutamine (Q(n)) expansions, such as those that occur in the huntingtin protein (htt) in Huntington's disease (HD), are excellent substrates for tissue transglutaminase in vitro, and transglutaminase activity is increased in post-mortem HD brain. However, direct evidence for the participation of tissue transglutaminase (or other transglutaminases) in HD patients in vivo is scarce. We now report that levels of N(epsilon)-(gamma-L-glutamyl)-L-lysine (GGEL)--a 'marker' isodipeptide produced by the transglutaminase reaction--are elevated in the CSF of HD patients (708 +/- 41 pmol/mL, SEM, n = 36) vs. control CSF (228 +/- 36, n = 27); p < 0.0001. These data support the hypothesis that transglutaminase activity is increased in HD brain in vivo.

Adult↗
PubMed2001

The glutamine/glutamate couplet and cellular function.

All cells require glutamine as a nitrogen donor as well as an energy source for cell-specific functions. Understanding how glutamine utilization is metered to these demands is fundamental to basic cell processes as well as to therapeutic manipulation of regulatory mechanisms. The regulatory role of the glutamine/glutamate couplet in cellular function is illustrated for acid-base homeostasis and for production of the extracellular matrix.

Acidosis↗
PubMed2000

Is there a relationship between 3-hydroxy-3-methylglutaryl coenzyme a reductase activity and forebrain pathology in the PKU mouse?

Previous reports have suggested that elevated levels of phenylalanine inhibit cholesterol synthesis. The goals of this study were to investigate if perturbations in cholesterol synthesis exist in the PAH(enu2) genetic mouse model for phenylketonuria (PKU), and if so, initiate studies determining if they might underlie the white matter pathology that exists in PKU forebrain. Gross sections and electron microscopy showed that select tracts were hypomyelinated in adult PKU mouse forebrain but not hindbrain. The activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), the rate controlling enzyme in the cholesterol biosynthetic pathway, was examined in isolated microsomes from forebrain, hindbrain, and liver to assess if perturbations in cholesterol biosynthesis were occurring. HMGR activity was normal in unaffected PKU hindbrain and was increased 2-4-fold in PKU liver compared to control. HMGR activity in the forebrain, however, was decreased by 30%. Because normal numbers of MBP-expressing glia (oligodendrocytes) were present, but the number of glia expressing HMGR was reduced by 40% in the hypomyelinated tracts, the decreased HMGR activity seemed to result from a down-regulation of HMGR expression in affected oligodendrocytes. Exposure of an oligodendrocyte-like glioma cell line to physiologically relevant elevated levels of Phe resulted in a 30% decrease in cholesterol synthesis, a 28% decrease in microsomal HMGR activity, and a 28% decrease in HMGR protein levels. Measurement of HMGR activity after addition of exogenous Phe to control brain microsomes revealed that Phe is a noncompetitive inhibitor of HMGR; physiologically relevant elevated levels of exogenous Phe inhibited HMGR activity by 30%. Taken together, these data suggest that HMGR is moderately inhibited in the PKU mouse. Unlike other cell types in the body, a subset of oligodendrocytes in the forebrain seems to be unable to overcome this inhibition. We speculate that this may be the cause of the observed pathology in PKU brain.

Alkyl and Aryl Transferases↗
PubMed2000

Proteolytic degradation of tyrosine nitrated proteins.

Tyrosine nitration is a covalent posttranslational protein modification that has been detected under several pathological conditions. This study reports that nitrated proteins are degraded by chymotrypsin and that protein nitration enhances susceptibility to degradation by the proteasome. Chymotrypsin cleaved the peptide bond between nitrated-tyrosine 108 and serine 109 in bovine Cu,Zn superoxide dismutase. However, the rate of chymotryptic cleavage of nitrated peptides was considerably slower than control. In contrast, nitrated bovine Cu,Zn superoxide dismutase was degraded at a rate 1. 8-fold faster than that of control by a gradient-purified 20S/26S proteasome fraction from bovine retina. Exposure of PC12 cells to a nitrating agent resulted in the nitration of tyrosine hydroxylase and a 58 +/- 12.5% decline in the steady-state levels of the protein 4 h after nitration. The steady-state levels of tyrosine hydroxylase were restored by selective inhibition of the proteasome activity with lactacystin. These data indicate that nitration of tyrosine residue(s) in proteins is sufficient to induce an accelerated degradation of the modified proteins by the proteasome and that the proteasome may be critical for the removal of nitrated proteins in vivo.

Animals↗
PubMed2000

Genotype spectrum of ornithine transcarbamylase deficiency: correlation with the clinical and biochemical phenotype.

Ornithine transcarbamylase (OTC) deficiency, a partially dominant X-linked disorder, is the most common inherited defect of the urea cycle. Previous reports suggested a variable phenotypic spectrum, and several studies documented different "private" mutations in the OTC genes of patients. Our laboratory identified disease-causing mutations in 157 families with OTC deficiency, 100 of which came to medical attention through a hemizygous propositus and in 57 the index case was a heterozygous female. We correlated the genotype with age of onset, liver OTC activity, incorporation of nitrogen into urea, and peak plasma ammonia levels. The "neonatal onset" group has a homogeneous clinical and biochemical phenotype, whereas the "late onset" group shows an extremely wide phenotype; 60% of the mutations are associated exclusively with acute neonatal hyperammonemic coma. The remaining mutations caused a nonuniform phenotype ranging from severe disease to no symptoms; 31% of the mutations in the OTC gene occur in CpG dinucleotides (methylation-mediated deamination), and none of them accounted for more than 4% of the total. Eighty-six percent of the mutations represented single-base substitutions and 68% of the substitutions were transitions. G-to-A and C-to-T transitions were the most frequent substitutions (34 and 21%, respectively) whereas C-to-A, A-to-C, C-to-G, and T-to-A transversions were the least common (1.5-3%). Twenty percent of propositi and 77% of propositae carried new mutations. Forty percent of female germinal mutations were in CpG dinucleotides whereas this number appears much smaller in male germinal mutations. These data allow classification of patients with OTC deficiency into at least two groups who have discordant disease course and prognoses. In addition, they improve our understanding on the origin of mutations in the OTC gene and allow better counseling of affected families.

Adolescent↗
PubMed2000

Rapid method for determining the rate of DNA synthesis and cellular proliferation.

A new method has been developed for determination of DNA synthesis during cell proliferation. The method is based on the metabolism of [U-(13)C(6)]glucose to deoxyribose (DR) and then incorporation of [U-(13)C(5)]DR into newly synthesized DNA. Extracted cellular DNA is subjected to HCl hydrolysis (2 h at 100 degrees C), which converts DR into levulinic acid. The (13)C enrichment in DR is determined in the trimethylsilyl derivative of levulinate using gas chromatography-mass spectrometry. The method is rapid and sensitive. It can precisely determine (13)C enrichment below 1 at.% excess in as little as 4 ng DNA. We have used this method to determine the rate of cell proliferation in vitro and the level of DR in a given amount of DNA. The current approach has significant advantages over previously described methods and overcomes several difficulties related to the determination of DNA synthesis both in vivo and in vitro.

Biological Assay↗
PubMed2000

The molecular basis of ornithine transcarbamylase deficiency.

UNLABELLED: The ornithine transcarbamylase (OTC) gene is located on the short arm of the X-chromosome and encodes the second enzyme of the urea cycle. OTC deficiency is an X-linked disorder that causes hyperammonemia leading to brain damage, mental retardation and death. The clinical and biochemical phenotype is extremely variable and can only partially be explained by the genotype. We identified mutations in the OTC gene of more than 150 patients with OTC deficiency. The "neonatal onset" group of patients has mutations that abolish enzyme activity, whereas the "late onset group" shows partial enzyme deficiency to variable degree. Of the mutations, 60% are associated exclusively with acute neonatal hyperammonemic coma while the remaining cause "late onset" disease. Several symptomatic and asymptomatic adults have now been identified to have deleterious mutations in the OTC gene leading to predisposition to hyperammonemia. CONCLUSION: The enlarging clinical, biochemical and molecular spectrum observed in patients with ornithine transcarbamylase deficiency suggests that this disorder behaves like a single gene disorder at one end of the spectrum and as a multi-factorial disease at the other.

Adolescent↗
PubMed2000

Acidosis and astrocyte amino acid metabolism.

The relationship between acidosis and the metabolism of glutamine and glutamate was studied in cultured astrocytes. Acidification of the incubation medium was associated with an increased formation of aspartate from glutamate and glutamine. The rise of the intracellular content of aspartate was accompanied by a significant decline in the extracellular concentration of both lactate and citrate. Studies with either [2-(15)N]glutamine or [15N]glutamate indicated that there occurred in acidosis an increased transamination of glutamate to aspartate. Studies with L-[2,3,3,4,4-(2)H5]glutamine indicated that in acidosis glutamate carbon was more rapidly converted to aspartate via the tricarboxylic acid cycle. Acidosis appears to result in increased availability of oxaloacetate to the aspartate aminotransferase reaction and, consequently, increased transamination of glutamate. The expansion of the available pool of oxaloacetate probably reflects a combination of: (a) Restricted flux through glycolysis and less production from pyruvate of acetyl-CoA, which condenses with oxaloacetate in the citrate synthetase reaction; and (b) Increased oxidation of glutamate and glutamine through a portion of the tricarboxylic acid cycle and enhanced production of oxaloacetate from glutamate and glutamine carbon. The data point to the interplay of the metabolism of glucose and that of glutamate in these cells.

Acidosis↗
PubMed2000

Compartmentation of brain glutamate metabolism in neurons and glia.

Intrasynaptic [glutamate] must be kept low in order to maximize the signal-to-noise ratio after the release of transmitter glutamate. This is accomplished by rapid uptake of glutamate into astrocytes, which convert glutamate into glutamine. The latter then is released to neurons, which, via mitochondrial glutaminase, form the glutamate that is used for neurotransmission. This pattern of metabolic compartmentation is the "glutamate-glutamine cycle." This model is subject to the following two important qualifications: 1) brain avidly oxidizes glutamate via aspartate aminotransferase; and 2) because almost no glutamate crosses from blood to brain, it must be synthesized in the central nervous system (CNS). The primary source of glutamate carbon is glucose, and a major source of glutamate nitrogen is the branched-chain amino acids, which are transported rapidly into the CNS. This arrangement accomplishes the following: 1) maintenance of low external [glutamate], thereby maximizing signal-to-noise ratio upon depolarization; 2) the replenishing of the neuronal glutamate pool; 3) the "trafficking" of glutamate through the extracellular fluid in a nonneuroactive form (glutamine); 4) the importation of amino groups from blood, thus maintaining brain nitrogen homeostasis; and 5) the oxidation of glutamate/glutamine, a process that confers an additional level of control in terms of the regulation of brain glutamate, aspartate and gamma-aminobutyric acid.

Animals↗
PubMed1999

Factors determining the selectivity of protein tyrosine nitration.

Tyrosine nitration is a covalent posttranslational protein modification derived from the reaction of proteins with nitrating agents. Protein nitration appears to be a selective process since not all tyrosine residues in proteins or all proteins are nitrated in vivo. To investigate factors that may determine the biological selectivity of protein tyrosine nitration, we developed an in vitro model consisting of three proteins with similar size but different three-dimensional structure and tyrosine content. Exposure of ribonuclease A to putative in vivo nitrating agents revealed preferential nitration of tyrosine residue Y(115). Tyrosine residue Y(23) and to a lesser extent residue Y(20) were preferentially nitrated in lysozyme, whereas tyrosine Y(102) was the only residue modified by nitration in phospholipase A(2). Tyrosine Y(115) was the residue modified by nitration after exposure of ribonuclease A to different nitrating agents: chemically synthesized peroxynitrite, nitric oxide, and superoxide generated by SIN-1 or myeloperoxidase (MPO)/H(2)O(2) plus nitrite (NO(-2)) in the presence of bicarbonate/CO(2). The nature of the nitrating agent determined in part the protein that would be predominantly modified by nitration in a mixture of all three proteins. Ribonuclease A was preferentially nitrated upon exposure to MPO/H(2)O(2)/NO(-2), whereas phospholipase A(2) was the primary target for nitration upon exposure to peroxynitrite. The data also suggest that the exposure of the aromatic ring to the surface of the protein, the location of the tyrosine on a loop structure, and its association with a neighboring negative charge are some of the factors determining the selectivity of tyrosine nitration in proteins.

Amino Acid Sequence↗
PubMed1999

Studies of hepatic glutamine metabolism in the perfused rat liver with (15)N-labeled glutamine.

This study examines the role of glucagon and insulin in the incorporation of (15)N derived from (15)N-labeled glutamine into aspartate, citrulline and, thereby, [(15)N]urea isotopomers. Rat livers were perfused, in the nonrecirculating mode, with 0.3 mM NH(4)Cl and either 2-(15)N- or 5-(15)N-labeled glutamine (1 mM). The isotopic enrichment of the two nitrogenous precursor pools (ammonia and aspartate) involved in urea synthesis as well as the production of [(15)N]urea isotopomers were determined using gas chromatography-mass spectrometry. This information was used to examine the hypothesis that 5-N of glutamine is directly channeled to carbamyl phosphate (CP) synthesis. The results indicate that the predominant metabolic fate of [2-(15)N] and [5-(15)N]glutamine is incorporation into urea. Glucagon significantly stimulated the uptake of (15)N-labeled glutamine and its metabolism via phosphate-dependent glutaminase (PDG) to form U(m+1) and U(m+2) (urea containing one or two atoms of (15)N). However, insulin had little effect compared with control. The [5-(15)N]glutamine primarily entered into urea via ammonia incorporation into CP, whereas the [2-(15)N]glutamine was predominantly incorporated via aspartate. This is evident from the relative enrichments of aspartate and of citrulline generated from each substrate. Furthermore, the data indicate that the (15)NH(3) that was generated in the mitochondria by either PDG (from 5-(15)N) or glutamate dehydrogenase (from 2-(15)N) enjoys the same partition between incorporation into CP or exit from the mitochondria. Thus, there is no evidence for preferential access for ammonia that arises by the action of PDG to carbamyl-phosphate synthetase. To the contrary, we provide strong evidence that such ammonia is metabolized without any such metabolic channeling. The glucagon-induced increase in [(15)N]urea synthesis was associated with a significant elevation in hepatic N-acetylglutamate concentration. Therefore, the hormonal regulation of [(15)N]urea isotopomer production depends upon the coordinate action of the mitochondrial PDG pathway and the synthesis of N-acetylglutamate (an obligatory activator of CP). The current study may provide the theoretical and methodological foundations for in vivo investigations of the relationship between the hepatic urea cycle enzyme activities, the flux of (15)N-labeled glutamine into the urea cycle, and the production of urea isotopomers.

Ammonia↗
PubMed1999

Blood levels of ammonia and nitrogen scavenging amino acids in patients with inherited hyperammonemia.

Plasma levels of glutamine (456 determinations), alanine (434 determinations), and asparagine (431 determinations) and corresponding ammonia levels (260 determinations) were retrospectively analyzed in 30 patients with hyperammonemia secondary to urea cycle disorders (including 3 patients with amino acid transport defects) and 5 patients with propionic acidemia (PA). All patients had elevated glutamine levels on one or more testing except for 2 patients with severe PA and 1 patient with a mild urea cycle disorder. All but 4 patients with urea cycle disorders showed a maximal glutamine level higher than 100 micromol/dl, and 3 patients had a maximal glutamine level of higher than 200 micromol/dl. The only exceptions were 2 asymptomatic ornithine transcarbamylase (OTC)-deficient females, 1 male with mild OTC deficiency, and 1 patient with citrullinemia (CIT) whose plasma glutamine levels were never above 100 micromol/L. Patients with CIT and argininosuccinic aciduria (ASA) showed statistically significant lower levels of glutamine than patients with other urea cycle disorders. However, the maximal glutamine level did not directly correlate with severity of the disorder and within disorders correlated inversely with severity of outcome. Patients with PA showed statistically significant lower glutamine, alanine, and asparagine levels than patients with urea cycle disorders and the severity of this disorder correlated inversely with plasma glutamine levels. Plasma ammonia levels showed a positive correlation with glutamine in patients with carbamyl phosphate synthetase I and OTC deficiency and a negative correlation in patients with PA. Although, most patients also showed elevated levels of alanine and asparagine, their levels generally did not show a good correlation with glutamine (R2 = 0.25 and 0.34, respectively).

Adolescent↗