Minimal ureagenesis is necessary for survival in the murine model of hyperargininemia treated by AAV-based gene therapy.

Hyperammonemia is less severe in arginase 1 deficiency compared with other urea cycle defects. Affected patients manifest hyperargininemia and infrequent episodes of hyperammonemia. Patients typically suffer from neurological impairment with cortical and pyramidal tract deterioration, spasticity, loss of ambulation, seizures and intellectual disability; death is less common than with other urea cycle disorders. In a mouse model of arginase I deficiency, the onset of symptoms begins with weight loss and gait instability, which progresses toward development of tail tremor with seizure-like activity; death typically occurs at about 2 weeks of life. Adeno-associated viral vector gene replacement strategies result in long-term survival of mice with this disorder. With neonatal administration of vector, the viral copy number in the liver greatly declines with hepatocyte proliferation in the first 5 weeks of life. Although the animals do survive, it is not known from a functional standpoint how well the urea cycle is functioning in the adult animals that receive adeno-associated virus. In these studies, we administered [1-13C] acetate to both littermate controls and adeno-associated virus-treated arginase 1 knockout animals and examined flux through the urea cycle. Circulating ammonia levels were mildly elevated in treated animals. Arginine and glutamine also had perturbations. Assessment 30 min after acetate administration demonstrated that ureagenesis was present in the treated knockout liver at levels as low at 3.3% of control animals. These studies demonstrate that only minimal levels of hepatic arginase activity are necessary for survival and ureagenesis in arginase-deficient mice and that this level of activity results in control of circulating ammonia. These results may have implications for potential therapy in humans with arginase deficiency.

Selective inhibition of NAALADase, which converts NAAG to glutamate, reduces ischemic brain injury.

We describe here a new strategy for the treatment of stroke, through the inhibition of NAALADase (N-acetylated-alpha-linked-acidic dipeptidase), an enzyme responsible for the hydrolysis of the neuropeptide NAAG (N-acetyl-aspartyl-glutamate) to N-acetyl-aspartate and glutamate. We demonstrate that the newly described NAALADase inhibitor 2-PMPA (2-(phosphonomethyl)pentanedioic acid) robustly protects against ischemic injury in a neuronal culture model of stroke and in rats after transient middle cerebral artery occlusion. Consistent with inhibition of NAALADase, we show that 2-PMPA increases NAAG and attenuates the ischemia-induced rise in glutamate. Both effects could contribute to neuroprotection. These data indicate that NAALADase inhibition may have use in neurological disorders in which excessive excitatory amino acid transmission is pathogenic.

Metabolic pathway profiling of mitochondrial respiratory chain mutants in C. elegans.

Caenorhabditis elegans affords a model of primary mitochondrial dysfunction that provides insight into cellular adaptations which accompany mutations in nuclear genes that encode mitochondrial proteins. To this end, we characterized genome-wide expression profiles of C. elegans strains with mutations in nuclear-encoded subunits of respiratory chain complexes. Our goal was to detect concordant changes among clusters of genes that comprise defined metabolic pathways. Results indicate that respiratory chain mutants significantly upregulate a variety of basic cellular metabolic pathways involved in carbohydrate, amino acid, and fatty acid metabolism, as well as cellular defense pathways such as the metabolism of P450 and glutathione. To further confirm and extend expression analysis findings, quantitation of whole worm free amino acid levels was performed in C. elegans mitochondrial mutants for subunits of complexes I, II, and III. Significant differences were seen for 13 of 16 amino acid levels in complex I mutants compared with controls, as well as overarching similarities among profiles of complex I, II, and III mutants compared with controls. The specific pattern of amino acid alterations observed provides novel evidence to suggest that an increase in glutamate-linked transamination reactions caused by the failure of NAD(+)-dependent ketoacid oxidation occurs in primary mitochondrial respiratory chain mutants. Recognition of consistent alterations both among patterns of nuclear gene expression for multiple biochemical pathways and in quantitative amino acid profiles in a translational genetic model of mitochondrial dysfunction allows insight into the complex pathogenesis underlying primary mitochondrial disease. Such knowledge may enable the development of a metabolomic profiling diagnostic tool applicable to human mitochondrial disease.

In vivo nitrogen metabolism in ornithine transcarbamylase deficiency.

We developed a new technique that monitors metabolic competency in female heterozygotes for ornithine transcarbamylase deficiency (OTCD). The method uses mass spectrometry to measure conversion of (15)NH4Cl to [15N]urea and [5-(15)N]glutamine following an oral load of (15)NH4Cl. We found that heterozygotes converted significantly less NH3 nitrogen to urea, with this difference being particularly obvious for symptomatic carriers, in whom the blood [15N]urea concentration (mM) was significantly less than control values at most time points. The blood concentration of [5-(15)N]-glutamine (microM) was significantly higher in both asymptomatic and symptomatic heterozygotes than it was in the control subjects. The administration of a test dose of sodium phenylbutyrate to the control group did not affect the rate of [15N]urea formation. We conclude: (a) This test effectively monitors in vivo N metabolism and might obviate the need for liver biopsy to measure enzyme activity in OTCD; (b) Asymptomatic OTCD carriers form urea at a normal rate, indicating that ureagenesis can be competent even though enzyme activity is below normal; (c) Although ostensibly asymptomatic OTCD carriers form urea at a normal rate, their nitrogen metabolism is still abnormal, as reflected in their increased production of [5-(15)N]glutamine; and (d) This new test may be important for monitoring the efficacy of novel treatments for OTCD, e.g., liver transplantation and gene therapy.

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.

Pathologic Variants of the Mitochondrial Phosphate Carrier SLC25A3: Two New Patients and Expansion of the Cardiomyopathy/Skeletal Myopathy Phenotype With and Without Lactic Acidosis.

Variants in the SLC25A3 gene, which codes for the mitochondrial phosphate transporter (PiC), lead to a failure of inorganic phosphate (Pi) transport across the mitochondrial membrane, which is required in the final step of oxidative phosphorylation. The literature described two affected sibships with variants in SLC25A3; all cases had skeletal myopathy and cardiomyopathy (OMIM 610773). We report here two new patients who had neonatal cardiomyopathy; one of whom did not have skeletal myopathy nor elevated lactate. Patient 1 had a homozygous splice site variant, c.158-9A>G, which has been previously reported in a Turkish family. Patient 2 was found to be a compound heterozygote for two novel variants, c.599T>G (p.Leu200Trp) and c. 886_898delGGTAGCAGTGCTTinsCAGATAC (p.Gly296_Ser300delinsGlnIlePro). Protein structure analysis indicated that both variants are likely to be pathogenic. Sequencing of SLC25A3 should be considered in patients with isolated cardiomyopathy, even those without generalized skeletal myopathy or lactic acidosis.

Phenylalanine-tyrosine deficiency syndrome as a complication of the management of hereditary tyrosinemia.

A male infant with type I hereditary tyrosinemia developed a phenylalanine-tyrosine deficiency syndrome after receiving a synthetic diet which was low in these amino acids. The syndrome was characterized by growth failure, anorexia, lethargy, and hypotonia. Hypophenylalaninemia and hypotyrosinemia were discovered. The blood concentration of most other amino acids were increased. Supplementation of the patient's diet with phenylalanine and tyrosine resulted in a prompt and dramatic reversal of both clinical and biochemical abnormalities. Dietary therapy had no effect on the child's hepatic cirrhosis.

On the clinical significance of the plasma alpha-amino-n-butyric acid:leucine ratio.

The ratio of plasma alpha-aminobutyric acid to plasma leucine that has been proposed as a specific biochemical marker of alcoholic liver injury, was measured in normal children an children with a variety of metablic disorders. The ratio was found to be lower in children that in adults. It was elevated in children with Reye's syndrome, tyrosinemia, homocystinuria, nonketotic hyperglycinemia, and ornithine transcarbamylase deficiency. The results indicate that an elevated ratio is not specific for adults with alcoholic liver injury and that the ratio may be high even without the presence of clinically apparent hepatic disease. An altered ratio may reflect the presence of an inherent metabolic defect.

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.

The pancytopenia of isovaleric acidemia.

Severe pancytopenia developed in two infants with isovaleric acidemia. Previous reports indicate these hematologic abnormalities are a leading cause of death in affected infants. Our findings suggest that the pancytopenia may be due to arrested maturation of hematopoietic precursors. Prompt transfusion of appropriate blood components prevented complications due to the hematologic abnormalities.

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.

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.

A model for determination of total body protein synthesis based upon compartmental analysis of the plasma [15N] glycine decay curve.

A model for whole body glycine nitrogen flux based on the compartmental analysis of plasma [15N] glycine decay curves is described and used for the measurements of (1) total body glycine nitrogen flux and the components of this flux in three healthy young adults and (2) total body protein synthesis based on the conversion of 15N to excretory products, ie, the sum of urinary [15N] urea and 15NH3 and the amount of labeled urea remaining in the body at five hours following administration of [15N] glycine. The mean glycine nitrogen flux was 3.93 +/- 0.42 mg N X kg-1 X h-1 (SEM). The major components of this flux are de novo synthesis of glycine, which accounts for 18% to 27%, and release from protein breakdown, which accounts for 62% to 73%. The outward pathways of glycine from the total body free glycine pool are conversion to other amino acids and oxidation to excretory end products (30% to 42%) and incorporation into protein, which accounts for 45% to 61% of glycine N loss from the metabolic pool. The mean rate of total body protein synthesis as determined by compartmental analysis was 3.56 g protein X kg-1 X day-1. The results that were obtained for protein synthesis and whole body glycine kinetics accord well with previous studies in normal adults, using the stochastic model.

Cysteamine inhibition of [15N]-glycine turnover in cystinosis and of glycine cleavage system in vitro.

In order to clarify the hyperglycinemic effect of cysteamine treatment in children with nephropathic cystinosis, we measured [15N]-glycine turnover in three affected patients. Administration of cysteamine lowered the glycine flux and the glycine metabolic clearance rate but did not alter the glycine pool size. Formation of [15N]-serine from [15N]-glycine was lower in untreated patients than in control subjects and was reduced still further by cysteamine. Studies in vitro with isolated rat liver mitochondria and acetone extracts of mitochondria indicated that even low cysteamine concentrations (0.1 mM) inhibited the glycine cleavage system in both the direction of glycine oxidation and glycine synthesis. Cysteamine was a more potent inhibitor of the glycine cleavage system than any other sulfhydryl containing compound. Although no ill effects of cysteamine treatment were immediately apparent, patients receiving cysteamine should be monitored carefully for the appearance of any neurologic symptoms which might be referable to inhibition of the glycine cleavage system.

Expression of glutamic acid decarboxylase during human neuronal differentiation: studies using the NTera-2 culture system.

Human NTera-2N neurons, but not the parental NTera-2 teratocarcinoma line, decarboxylate [2-(15)N]glutamine to form gamma-[15N]aminobutyric acid (GABA). The reverse transcriptase-polymerase chain reaction (RT-PCR) followed by Southern blotting showed that NTera-2N neurons transcribe the glutamic acid decarboxylase p67 (GAD67) gene, and also demonstrated that there is developmentally regulated alternative splicing of GAD67 mRNA in NTera-2N neurons. As in rat central nervous system (CNS), this mRNA processing generates two RNA transcripts, owing to the inclusion or exclusion of an approximately 80 bp coding region insert. In embryonic day 16 (E16) rat brain, the larger of the two GAD67 mRNAs, which encodes a truncated, inactive apoenzyme, reaches a concentration almost equal to that of the smaller transcript, which encodes functional GAD67. In developing NTera-2N neurons, however, the larger transcript is barely detectable by RT-PCR. RT-PCR also revealed that rat CNS of all ages examined contains GAD65 mRNA, and that GAD65 mRNA is below the detectable range in NTera-2N neurons.

Determination of [amide-15N]glutamine in plasma with gas chromatography-mass spectrometry.

A gas chromatographic-mass spectrometric method is described for determination of isotopic enrichment in [amide-15N]glutamine. The method involves the isolation of glutamine with an anionic exchange resin and the enzymatic hydrolysis of glutamine with glutaminase. The ammonia derived from the amide group is converted enzymatically to glutamic acid and isotopic enrichment in the latter compound is determined following formation of the N-TFA-glutaminate derivative. The method gave a linear result for [amide-15N]glutamine in the 0.5--21.5 atom % excess range with a mean coefficient of variance of 7.8%. Determination of plasma glutamine by isotope dilution showed a mean recovery of 94%. The appearance of plasma [amide-15N]glutamine was measured in a healthy adult following the intravenous administration of 15NH4Cl (350 mumol). A peak enrichment of 12 atom % excess was observed by 3.5 min.

Unusual pattern of metabolites in the urine of child with tyrosinemia: glyceraldehyde.

An unusual metabolite has been found in the acidic fraction of the urine of a child with tyrosinemia. This compound has been identified as glyceraldehyde, in addition to phenylacetic acid and p-hydroxybenzaldehyde, on the basis of gas chromatograph-mass spectral data and comparison with authentic samples.

Rapid gas chromatographic-mass spectrometric analysis of [15N]urea: application to human metabolic studies.

A rapid gas chromatographic-mass spectrometric method for [15N]urea analysis is described which is based on the formation of a TFA-urea derivative. The method is extremely sensitive, being capable of detecting less than 0.2 atom % excess [15N]urea in as little as 50 microliter of plasma and 10 microliter of urine with a coefficient of variation of 11%. The method was used to measure in vivo the disappearance of [15N]urea from plasma and the incorporation of [15N]ammonium acetate into [15N]urea. The values obtained for urea pool size and flux are 4.5 +/- 0.30 g nitrogen/total body weight and 16.2 +/- 1.34 g nitrogen per 24 h per 1.73 m2. Following the ingestion of [15N]ammonium acetate nearly 90% of the 15N label was incorporated into [15N]urea by 30 min.

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.