Hideko Morishita

Two cases of NADH-coenzyme Q reductase deficiency: Relationship to MELAS syndrome

Muscle biopsy specimens from two patients with MELAS syndrome (mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes) were studied biochemically. 14CO2 production rates from (1-14C)pyruvate, (U-14C)malate, and (1-14C)2-ketoglutarate were all decreased in intact mitochondria in both patients. Rotenone-sensitive NADH cytochrome c reductase activities were decreased to 8% (patient 1) and 6% (patient 2) of control values; succinate cytochrome c reductase and cytochrome c oxidase values were within normal limits. These results indicate that both patients have a defect of NADH-CoQ reductase of the respiratory chain and that MELAS can be brought about by a defect of NADH-CoQ reductase.

Mitochondrial Myopathy, Encephalopathy, Lactic Acidosis and Stroke-like Episodes Syndrome and NADH-CoQ Reductase Deficiency

M. KOBAYASHI 1, H. MORISHITA 1, N. SUGIYAMA 1, K. YOKOCHI 1, M. NAKANO 1, Y. WADA 1, Y. HOTTA 2, A. TERAUCHI 3 and I. NONAKA 4 1Department of Paediatrics and ~First Department of Anatomy, Medical School, Nagoya City University, Kawasumi-cho, Mizuho-ku, Nagoya 467, Japan 3Higashimatsumoto National Sanatorium, Matsumoto 399-65, Nagano, Japan 4National Centre for Nervous, Mental and Muscular Disorders, Kodaira 187, Tokyo, Japan

Molecular characterization of histidinemia: identification of four missense mutations in the histidase gene

Histidinemia (MIM235800) is characterized by elevated histidine in body fluids and decreased urocanic acid in blood and skin and results from histidase (histidine ammonia lyase, EC 4.3.1.3) deficiency. It is the most frequent inborn metabolic error in Japan. Although the original description included mental retardation and speech impairment, neonatal screening programs have identified the majority of histidinemic patients with normal intelligence. Molecular characteristics of histidase in histidinemia have not been determined, and cytogenetically visible deletions of 12q22-24.1 in which histidase gene resides have not been identified in histidinemic patients. In order to investigate whether individuals with this disorder have small deletions, additions, or point mutations in the histidase gene, we screened genomic DNA isolated from 50 histidinemic individuals who were discovered by the neonatal screening program. The methods employed included polymerase chain reaction (PCR) amplification of exons 1–21 of the histidase gene, followed by mutation detection enhancement gel electrophoresis and sequencing of the PCR products displaying heteroduplex bands. Four missense mutations (R322P, P259L, R206T, and R208L), two exonic polymorphisms (T141T c.423A→T and P259P c.777A→G), and two intronic polymorphisms (IVS6−5T→C and IVS9+25A→G) were identified. The frequencies of each polymorphism estimated either by dot blot allele-specific oligonucleotide hybridization, restriction enzyme digestion, or direct sequencing of the PCR products amplified from 50 unrelated normal individuals were 0.28, 0.30, 0.40, and less than 0.01, respectively. Mutation analysis of one family demonstrated that the patient inherited R322P from the mother and P259L from the father. This report describes the first mutations occurring in the coding region of the histidase structural gene in patients with histidinemia.

Different ketogenic response to medium-chain triglycerides and to long-chain triglycerides in a case of muscular carnitine palmitoyltransferase deficiency

CASE REPORT The patient, a 12-year-old Japanese boy, was born uneventfully at term but for breech extraction. Family history is unremarkable. He had muscular torticollis but was healthy throughout childhood. At the age of 10 years, he was operated on for the torticollis under general anaesthesiawith GOF and succinylcholine. Soon after the introduction of anaesthesia, malignant hyperthermia developed and lasted for about 12h. At the age of 11 years he had an episode of muscular pain with swelling and gait disturbance of the lower legs. In marathon races held in school, he was slow. Physical examination was almost normal except for moderate obesity. Laboratory data were as follows: serum triglyceride, 220 mg/100 ml; insulin, 31.8 laU/ml; pyruvate, 1.40 mg/100 ml. Other studies including blood glucose, lactate, glutamic oxalacetic transaminase, glutamic pyruvic transaminase, lactic dehydrogenase, creatine phosphokinase, free-fatty acids and cholesterol were normal. Amino acids and organic acids were all within the normal range both in serum and urine. Muscle biopsy specimens were obtained from the right biceps brachii and the left gastrocnemius one year after the episode of malignant hyperthermia. Routine histological examinations and histochemical staining of the section revealed that muscle fibres were mildly varied in size and a slight increase in fat-droplet content was seen in type I fibres. The ATPase stain at pH 4.2 showed an increase of type IIC fibres, suggesting that the patient was still in the subclinical stage of myopathy.

Pyroglutamic Aciduria in Propionyl CoA Carboxylase Deficiency

Propionyl CoA carboxylase (PCC; EC 6.4.1.3) deficiency (McKusick 23200) results in accumulation of propionyl CoA in the bodyfluid. Clinically, the disorder leads to severe metabolic disturbance, which often appears in the neonatal period, and which secondarily produces hyperammonaemia, hyperglycinaemia and hyperlactataemia. Large amounts of abnormal metabolites of propionyl CoA, methylcitrate, 3-hydroxypropionate and others, are excreted in the urine. The urinary excretion of pyroglutamic acid has not been reported anywhere in the literature. In this first report, pyroglutamic aciduria was detected in a neonatal case of propionic acidaemia, and the possible causes of excessive production of pyroglutamic acid are discussed.

Biochemical evidence of carnitine effect on propionate elimination

In propionic acidaemia (McKusick 23200), an inborn error of organic acid metabolism, the primary lesion is accumulation of propionyl CoA due to a defect of its carboxylation (Rosenberg, 1983). As propionyl CoA is known to be a substrate for carnitine acetyltransferase (CAT) (EC 2.3.1.7) (Bohmer and Bremer, 1968), we postulated that carnitine might counteract the accumulation of propionyl CoA. However, according to our literature survey there has been only one report (Roe and Bohan, 1982) on carnitine supplementation in propionic acidaemia, and the effect was still uncertain. The purpose of this study, therefore, is to examine the effect of carnitine on this condition. A patient in whom propionyl CoA carboxylase (EC 6.4.1.3) deficiency was diagnosed using cultured skin fibroblasts (Gompertz et al., 1975), was given carnitine with the permission of his family. Urinary propionylcarnitine and propionate concentrations were estimated thereafter. Additionally we measured the carnitine contents of the specially prepared formulae for the dietary treatment of this disorder. These results will be reported below.

Glutaric aciduria type II: autopsy study of a case with electron-transferring flavoprotein dehydrogenase deficiency.

An autopsy study of glutaric aciduria type II in a 62-day-old Japanese boy is presented. The diagnosis was made by analysis of organic acids in the urine. Immunoblot analysis of liver homogenate confirmed the diagnosis, revealing absence of electron-transferring flavoprotein dehydrogenase. The major findings were fatty changes of variable degree in many organs and tissues, the most severe being found in cardiac myocytes, hepatocytes, renal tubular epithelium, and skeletal muscle fibers. Other pertinent findings included multicystic and dysplastic kidney, pulmonary alveolar proteinosis, and spongiosis and gliosis of the spinal cord. The thymus was markedly depleted, and lymphocytes in the lymph nodes were mainly B cells. Although some of these changes may have been secondary to the sepsis and immunosuppression complicating 2 months of intensive care, the abnormal organic acid metabolism with severe acidosis may have been a significant contributing factor.

Identification of glutarylcarnitine in glutaric aciduria type 1 by carboxylic acid analyzer with an ODS reverse-phase column

A technique for the identification of glutarylcarnitine in urine from a patient with glutaric aciduria type 1 is described. The patients urine sample was partially purified using an anion exchange column and analyzed by a carboxylic acid analyzer fitted with an ODS reverse-phase column. The chromatogram of the patients urine sample revealed 3 different peaks, which corresponded respectively to those of carnitine with amino acids, acetylcarnitine and glutarylcarnitine. Following hydrolysis of the sample, the chromatogram had no peaks of acetylcarnitine and glutarylcarnitine but had remarkably amplified peaks of carnitine, acetic acid and glutaric acid. The eluent fraction of glutarylcarnitine from the non-hydrolyzed sample was hydrolyzed and analyzed again. It no longer had the glutarylcarnitine peak on the chromatogram, but had only two separate peaks of carnitine and glutaric acid. This technique simplifies the identification of glutarylcarnitine, in that it requires only removal of organic acids for preparation of samples, and does not require radioisotope or mass spectrometry.

Effect of hypocalcemia on muscle in disorders of calcium metabolism

We reported on three hypocalcemic patients with various serum creatine kinase (CK) levels and Ca metabolic disorders. Two patients with moderate hypocalcemia had increased CK levels (hyper‐CK‐emia), which normalized during treatment for the hypocalcemia; a negative correlation between the Ca and CK levels was observed in both patients. The remaining patient with mild hypocalcemia had a normal CK level. We discuss the effect of hypocalcemia on muscle in our patients as well as previously reported patients. Muscle may respond to hypocalcemia in three stages, namely homeostatic, asymptomatic hyper‐CK‐emic and myopathic stages.

Urinary acylcarnitines in a patient with neonatal multiple acyl-CoA dehydrogenation deficiency, quantified by a carboxylic acid analyzer with a reversed-phase column

A quantitative analysis for urinary acylcarnitines in a patient with neonatal multiple acyl-CoA dehydrogenation deficiency is described. This method (liquid chromatography) can quantify twelve acylcarnitines including glutarylcarnitine and 3 isomeric acylcarnitines (butyryl-1, valeryl- and octanoylisomer) in urine. Before and up to the 15th hour of DL-carnitine therapy, isovalerylcarnitine was the largest single component existing in urinary acylcarnitines. Its excretion increased approximately 10 times within 1 day of DL-carnitine therapy. However, the acetyl-, the isobutyryl- and the butyrylcarnitine values increased gradually. From the 8th day of the therapy, the isobutyrylcarnitine value exceeded the isovalerylcarnitine. The patients dominant urinary specific acylcarnitine derived from amino acids oxidation deficiency was changed from isovalerylcarnitine(leucine) to isobutyrylcarnitine(valine) during the early period of DL-carnitine therapy. Glutarylcarnitine was a minor component in the urine. Its degree of increase was as small as that of octanoylcarnitine. 2-Methylbutyrylcarnitine and propionylcarnitine were not detected.