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Notes: Abstract— D-β-hydroxybutyrate (β-OHB) was compared to glucose as a precursor for brain amino acids during rat development. In the first study [3-14C]β-OHB or [2-14C]glucose was injected subcu-taneously (01 μCi/g body wt) into suckling rats shortly after birth and at 6. 11, 13, 15 and 21 days of age. Blood and brain tissue were obtained 20 min later after decapitation. The specific activity of the labelled precursor in the blood and in the brain tissue was essentially the same for each respective age suggesting that the labelled precursor had equilibrated between the blood and brain pools before decapitation. [3-14C]β-OHB rapidly labelled brain amino acids at all ages whereas [2-14C]glucose did not prior to 15 days of age. These observations are consistent with a maturational delay in the flux of metabolites through glycolysis and into the tricarboxylic acid cycle. Brain glutamate, glutamine, asparate and GABA were more heavily labelled by [3-14C]β-OHB from birth-15 days of age whereas brain alanine was more heavily labelled by [2-14C]glucose at all ages of development. The relative specific activity of brain glutamine/glutamate was less than one at all ages for both labelled precursors suggesting that β-OHB and glucose are entering the‘large’glutamate compartment throughout development. In a second study, 6 and 15 day old rats were decapitated at 5 min intervals after injection of the labelled precursors to evaluate the flux of the [14C]label into brain metabolites. At 6 days of age, most of the brain acid soluble radioactivity was recovered in the glucose fraction of the [2-,4C]glucose injected rats with 72, 74, 65 and 63% after 5, 10, 15 and 20 min. In contrast, the 6 day old rats injected with [3-14C]β-OHB accumulated much of the brain acid soluble radioactivity in the amino acid fraction with 22, 47, 57 and 54% after 5, 10, 15 and 20 min. At 15 days of age the transfer of the [14C]label from [2-14C]glucose into the brain amino acid fraction was more rapid with 29, 40, 45, 61 and 73% of the brain acid soluble radioactivity recovered in the amino acid fraction after 5, 10, 15, 20 and 30 min. There was almost quantitative transfer of [14C]label into the brain amino acids of the 15-day-old [3-14C]β-OHB injected rats with 66, 89, 89, 89 and 90% of the brain acid soluble radioactivity recovered in the amino acid fraction after 5, 10, 15, 20 and 30 min. The calculated half life for /?-OHB at 6 days was 19 8 min and at 15 days was 12-2 min. Surprisingly, the relative specific activity of brain GABA/glutamate was lower at 15 days of age in the [3-14C]β-OHB injected rats compared to the [2-14C]glucose injected rats despite a heavier labelling of brain glutamate in the [3-14C]β-OHB injected group. We interpreted these data to mean that β-OHB is a less effective precursor for the brain glutamate ‘subcompartment’ which is involved in the synthesis of GABA.

Notes: Summary Two patients with atypical manifestations of aberrant peroxisomal biogenesis are described. Contrary to previous studies, which had shown that Zellweger syndrome patients usually have normal levels of urinary oxalate excretion, the patients in the present study had evidence of abnormal oxalate metabolism in the form of hyperoxaluria and, in one of the patients, calcium oxalate urolithiasis. Activity of the liver-specific peroxisomal enzyme alanine:glyoxylate aminotransferase (AGT), which is a major determinant of the level of endogenous oxalate synthesis in humans, was normal in one patient and markedly supranormal in the other. Using the technique of post-embedding protein A-colloidal gold immunoelectron microscopy, AGT was found to be mainly cytosolic in the livers of both patients, with significant amounts also localized in the nuclei. In a small minority of the hepatocytes of one patient, who was homozygous for the more common (major) AGT allele, large numbers of unidentified fibrillar arrays were found in the cytosol, which labelled heavily for immunoreactive AGT. The background cytosolic AGT labelling was markedly reduced in such cells when compared to the majority of cells that did not contain fibrils. In the other patient, who was heterozygous for the major and minor AGT alleles, there appeared to be low levels of mitochondrial AGT labelling. In the light of these data, the possible metabolic function of cytosolic AGT in the livers of panperoxisomal disease patients is discussed.

Notes: Abstract We performed comparative biochemical studies in cultured fibroblast mitochondria from patients with the T8993G or the T8993C point mutations in the ATPase 6 gene of mitochondrial DNA. We found that ATP production was much more severely decreased in cells from patients with the T8993G mutation than in those from patients with the T8993C mutation. Kinetic studies suggest that both mutations affect only the F0 sector of the mitochondrial ATPase complex. We conclude that these two mutations, which result in the substitution of different amino acids at the same site of the ATPase, result in an enzyme with different biochemical characteristics.

Notes: Abstract The mitochondrial myopathies or encephalomyopathies with known biochemical defects can be divided into 5 groups: (1) defects of mitochondrial transport, such as CPT deficiency or carnitine deficiencies; (2) defects of substrate utilization, such as PDHC deficiency or defects of β-oxidation; (3) defects of the Krebs cycle, such as fumarase deficiency; (4) defects of oxidation-phosphorylation coupling, such as Luft disease, and (5) defects of the respiratory chain. These disorders are reviewed, with particular emphasis on the defects of the respiratory chain. Defects of complex I, III and IV show remarkable clinical and biochemical heterogeneity. All 3 complexes contain some subunits encoded by mtDNA and others encoded by nuclear DNA. At least some of the cytoplasmically made subunits appear to be tissue specific and may be developmentally regulated, thus explaining the genetic heterogeneity of these disorders.

Notes: A quantitative assay for 4-hydroxybutyric acid was developed using D6-4-hydroxybutyric acid as an internal standard. 4-Hydroxybutyric acid was isolated by liquid chromatography and the amount quantified by selected ion monitoring, ammonia chemical ionization gas chromatography/mass spectrometry of the trimethylsilyl derivatives. The concentrations of 4-hydroxybutyric in control physiological fluids were: 2.64 ± 3.46 mmol mol-1 creatinine in urine, 1.09 ± 2.87 μmol l-1 in plasma, 0.98 ± 1.17 μmol l-1 in cerebrospinal fluid and 1.28 ± 0.47 μmol l-1 in amniotic fluid. The concentration of 4-hydroxybutyric acid in the amniotic fluid from a pregnancy at risk for 4-hydroxybutyric aciduria was 2.30 μmol l-1, indicating an unaffected fetus. The stable isotope dilution assay of 4-hydroxybutyric acid in physiological fluid samples is a rapid, sensitive and accurate method for quantification, as well as a valuable technique for the prenatal diagnosis of 4-hydroxybutyric aciduria.