Subject(s)
Alcohol Oxidoreductases/deficiency , Cerebral Palsy/enzymology , Cerebral Palsy/genetics , Metabolism, Inborn Errors/drug therapy , Metabolism, Inborn Errors/genetics , Alcohol Oxidoreductases/cerebrospinal fluid , Brain/pathology , Cerebral Palsy/epidemiology , Child , Chromosomes, Human, Pair 2/genetics , Cognition Disorders/diagnosis , Cognition Disorders/epidemiology , Disorders of Excessive Somnolence/diagnosis , Disorders of Excessive Somnolence/epidemiology , Electroencephalography , Female , Humans , Infant , Magnetic Resonance Imaging , Male , Metabolism, Inborn Errors/epidemiology , Neuropsychological Tests , Severity of Illness Index , Siblings , Videotape RecordingABSTRACT
L-2-hydroxyglutaric aciduria (L-2-HGA) is a rare inherited autosomal recessive neurometabolic disorder caused by mutations in the gene encoding L-2-hydroxyglutarate dehydrogenase. An assay to evaluate L-2-hydroxyglutarate dehydrogenase (L-2-HGDH) activity in fibroblast, lymphoblast and/or lymphocyte lysates has hitherto been unavailable. We developed an L-2-HGDH enzyme assay in cell lysates based on the conversion of stable-isotope-labelled L-2-hydroxyglutarate to 2-ketoglutarate, which is converted into L-glutamate in situ. The formation of stable isotope labelled L-glutamate is therefore a direct measure of L-2-HGDH activity, and this product is detected by liquid chromatography-tandem mass spectrometry. A deficiency of L-2-HGDH activity was detected in cell lysates from 15 out of 15 L-2-HGA patients. Therefore, this specific assay confirmed the diagnosis unambiguously affirming the relationship between molecular and biochemical observations. Residual activity was detected in cells derived from one L-2-HGA patient. The L-2-HGDH assay will be valuable for examining in vitro riboflavin/FAD therapy to rescue L-2-HGDH activity.
Subject(s)
Alcohol Oxidoreductases/analysis , Alcohol Oxidoreductases/deficiency , Brain Diseases, Metabolic, Inborn/diagnosis , Cell Extracts/chemistry , Enzyme Assays/methods , Alcohol Oxidoreductases/cerebrospinal fluid , Animals , Brain Diseases, Metabolic, Inborn/cerebrospinal fluid , Brain Diseases, Metabolic, Inborn/pathology , Calibration , Cell Extracts/analysis , Cells, Cultured , Chromatography, High Pressure Liquid , Chromatography, Liquid/methods , Enzyme Assays/standards , Fibroblasts/chemistry , Fibroblasts/enzymology , Humans , Lymphocytes/chemistry , Lymphocytes/enzymology , Models, Biological , Models, Molecular , Rats , Research Design , Tandem Mass Spectrometry/methodsABSTRACT
BACKGROUND: Sepiapterin reductase (SR) deficiency is a rare inherited disorder of neurotransmitter metabolism; less than 25 cases have been described in the literature so far. METHODS: We describe the clinical history and extensive cerebrospinal fluid (CSF) and urine examination of two Greek siblings with the diagnosis of SR deficiency. The diagnosis was confirmed by enzyme activity measurement in cultured fibroblasts and by mutation analysis. RESULTS: Both patients suffered from a progressive and complex L-dopa responsive movement disorder. Very low concentrations of the neurotransmitter metabolites homovanillic acid (HVA), 5-hydroxyindolacetic acid (5-HIAA) and 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG) were observed in CSF. CSF neopterin and biopterin concentrations were abnormal in one case only, whereas in both cases sepiapterin concentrations were abnormally high and 5-hydroxytryptophan was undetectable. Urine concentrations of HVA, 5-HIAA and vanillyl mandelic acid (VMA) were decreased in both cases. Both patients had no detectable SR enzyme activity in primary dermal fibroblasts, and upon analysis of genomic DNA revealed the same homozygous point mutation introducing a premature stop codon into the reading frame of the SPR gene (mutant allele K251X). CONCLUSIONS: Our cases illustrate that, apart from HVA and 5-HIAA analysis, the specific quantification of sepiapterin in CSF, rather than neopterin and biopterin alone, is crucial to the final diagnosis of SR deficiency. In addition, urinary concentrations of neurotransmitter metabolites may be abnormal in SR deficiency and may provide an initial indication of SR deficiency before CSF analysis is performed. The known, impressive beneficial response of SR deficient patients to treatment with L-dopa, is illustrated again in our cases.
Subject(s)
Alcohol Oxidoreductases/genetics , Metabolism, Inborn Errors/enzymology , Alcohol Oxidoreductases/cerebrospinal fluid , Alcohol Oxidoreductases/metabolism , Alcohol Oxidoreductases/urine , Biosynthetic Pathways , Child , Female , Fibroblasts/enzymology , Greece , Homovanillic Acid/cerebrospinal fluid , Homovanillic Acid/urine , Humans , Hydroxyindoleacetic Acid/cerebrospinal fluid , Hydroxyindoleacetic Acid/urine , Metabolism, Inborn Errors/cerebrospinal fluid , Metabolism, Inborn Errors/genetics , Metabolism, Inborn Errors/urine , Mutation , Neurotransmitter Agents/cerebrospinal fluid , Neurotransmitter Agents/urine , Pterins/cerebrospinal fluid , Pterins/urine , SiblingsABSTRACT
The activities of several glycolytic enzymes (hexokinase, phosphofructokinase, pyruvate kinase, lactate dehydrogenase) as well as glycerol-1-phosphate dehydrogenase and (Mg2+)ATPase in normal cerebrospinal fluid (CSF) and blood plasma samples, from 12 healthy infants, aged 2-18 months, and in supernatants from brain tissue slices, taken during neurosurgical operations from infants of the same range of age were estimated. The values obtained confirm the high activity of the above enzymes found in animal brains, and indicate an independence of these activities in blood plasma and CSF. The origin of the activities of the investigated enzymes in CSF seems to be mainly, if not, exclusively, from brain tissue. This might be useful for detection of brain tissue damage as was earlier proven with LDH activity in CSF.
