Kenji Toshima

Enzymologic Studies and Therapy of Leigh's Disease Associated with Pyruvate Decarboxylase Deficiency

Summary: An 11-month-old boy with muscle hypotonia and neurologic deteriorations had lactic acidosis, pyruvic acidemia and alaninemia due to deficiency of the pyruvate dehydrogenase complex in his platelets and of pyruvate decarboxylase in his muscle. When placed on a low carbohydrate-high fat diet for 6 months, his blood levels of lactate and pyruvate became nearly normal, but his cerebrospinal fluid levels of lactate and pyruvate remained high. Despite this dietary therapy, neurologic deterioration progressed slowly. He died of pneumonia after artificial respiration for 3 wk. At autopsy, extensive symmetric necrotic lesions were found in the brain including proliferation of capillaries and gliosis in the brain stem and diffuse demyelination in the white matter. These lesions were consistent with those observed in Leighs disease. The activities of the pyruvate dehydrogenase complex and pyruvate decarboxylase in various tissues obtained at autopsy were less than 10% of control values; however, the activities of pyruvate carboxylase and α-ketoglutarate decarboxylase were within the normal limits. This patient with Leighs disease had an isolated deficiency of pyruvate decarboxylase in various tissues.Speculation: Deficiency of pyruvate decarboxylase in brain may be one cause of clinical and pathologic features of Leighs disease. The absence of pyruvate decarboxylase in the cerebellum, cerebrum, kidney, liver, muscle and platelets of the patient supports the hypothesis that there is only one form of pyruvate decarboxylase in humans.

Treatment of chronic congenital lactic acidosis by oral administration of dichloroacetate

Sodium dichloroacetate (DCA) was administered orally at a dose of 50 mg per kg body weight twice or three times per day to a newborn infant with lactic acidosis of unknown cause (patient 1) and to a 15-year-old boy with mitochondrial encephalomyopathy associated with lactic acidosis (patient 2). In patient 1, during treatment with DCA, DCA accumulated in the blood judging from the findings that the urinary excretion of DCA increased cumulatively and the blood lactate level rapidly decreased to the normal range. In patient 2, the blood DCA level gradually increased during treatment to a concentration of 250 µgml−1 and the blood lactate level decreased and was maintained within the normal range. DCA was detected in the brain (25 µg g tissue−1) and the liver, kidney and muscle (33.8, 33.8 and 26.3 µg g tissue−1, respectively) obtained at autopsy of patient 1, and in the cerebrospinal fluid of patient 2 at a concentration of 125 µg ml−1 when the blood concentration was 250 µg ml−1. The lactate levels in the cerebrospinal fluid decreased from 7 and 4mmoll−1 to 2.4 and 2.6 mmoll−1 in patients 1 and 2, respectively. Thus DCA may be useful in clinical treatment of chronic congenital lactic acidosis because it seems to cross the blood-brain barrier. However, it must be given at non-toxic doses, determined by monitoring the concentrations of lactate and DCA in the blood, because orally administered DCA tends to accumulate in tissues.

Effects of Dichloroacetate on Pyruvate Metabolism in Rat Brain in Vivo

Summary: The effects of dichloroacetate (DCA) on the activity of the pyruvate dehydrogenase (PDH) complex and associated changes in the lactate and glucose levels in rat brain were investigated in vivo. The average activities of the active form of the PDH complex in the brain, liver and muscle of starved rats were respectively 0.40 ± 0.04, 0.07 ± 0.04, and 0.17 ± 0.11 μmol/min/g tissue, and amounted to 21, 11, and 16% of the total activity of the complex. Intraperitoneal injection of DCA (125 mg/kg) increased the percentage of the active form of the PDH complex in the brain, liver, and muscle to 107, 40, and 84%, respectively. DCA significantly lowered the lactate and glucose concentrations of the brain and blood. A lower dose of DCA (12.5 mg/kg) also caused significant increase in activity of the PDH complex in the brain, but did not significantly change the lactate or glucose concentration of the brain. These results suggest that DCA crosses the blood-brain barrier reasonably well.

Rapid diagnosis of vitamin D-dependent rickets type II by use of phytohemagglutinin-stimulated lymphocytes

The interactions of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] with phytohemagglutinin (PHA)-stimulated lymphocytes from normal subjects and three patients with vitamin D-dependent rickets (DDR) type II were investigated. Impaired nuclear uptake and normal cytosol binding of [3H]1,25-(OH)2D3 were observed with PHA-stimulated lymphocytes of these patients as with their cultured skin fibroblasts. Furthermore, the incorporation of [14C]thymidine into PHA-stimulated lymphocytes of the patients was not reduced by 1,25-(OH)2D3, which is known to inhibit proliferation of various cells. These findings suggest that 1,25-(OH)2D3 receptors are reduced or absent in patients with DDR type II. Thus, the capacities of cytosol binding and nuclear uptake of 1,25-(OH)2D3 in PHA-stimulated lymphocytes seem to reflect those of endo-organs such as the intestine and bone. These findings show that a test of the effect of 1,25-(OH)2D3 on thymidine incorporation into PHA-stimulated lymphocytes is useful for rapid diagnosis of DDR type II.

Effect of Long-term Administration of Sodium Benzoate to a Patient with Partial Ornithine Carbamoyl Transferase Deficiency

An 8-year-old girl with partial ornithine carbamoyl transferase deficiency was treated with sodium benzoate (200 mg/kg/day) for 13 months. Before administration of sodium benzoate, her protein intake was reduced to 1.0 to 1.5 g/kg/day and her caloric intake fluctuated. Hy perammonemic attacks were frequently observed in winter. After the start of administration of sodium benzoate, the severity and frequency of these attacks decreased, although her protein intake was increased to 1.5 to 2.0 g/kg/day. No adverse effect of sodium benzoate were detected by clinical and laboratory examinations. It is concluded that long-term oral administration of sodium benzoate was effective in reducing the frequency and severity of hyperammonemic attacks in this patient. Sodium benzoate therapy in combination with dietary manipulation may improve the growth and development of these patients by allowing reduced dietary protein restriction.

Effect of sodium dichloroacetate on human pyruvate metabolism

Sodium dichloroacetate (DCA) was administered orally at doses of 12.5 to 50 mg/kg body weight twice or three times per day to a patient with mitochondrial encephalomyopathy associated with congenital lactic acidemia. During therapy, the rates of decarboxylation of (1-14C) pyruvate and (3-14C) pyruvate, which represent the activity of the pyruvate dehydrogenase (PDH) complex and the function of the TCA cycle, respectively, were markedly increased in the platelets and increases in the lactate levels in the blood and urine during exercise were markedly reduced. These results suggest that oral administration of DCA causes significant increases in the activities of the PDH complex and TCA cycle not only in the platelets but also in various tissues of humans, which is important as a pathway for production of energy, resulting in decreases in the lactate and pyruvate levels in the blood and cerebrospinal fluid.

Hereditary renal hypouricemia in children

The renal handling of urate was investigated in four children with hereditary renal hypouricemia and in their parents. The urate/creatinine clearance ratios in the four patients were 1.02 +/- 0.28, 0.93 +/- 0.11, 1.03 +/- 0.24, and 1.46 +/- 0.26, markedly higher than those in control subjects. Except for a partial response to pyrazinamide (change in clearance ratio from 1.46 to 1.07) in one patient, pyrazinamide and benzbromarone did not affect the clearance ratios in our patients. In the parents, the urate/creatinine clearance ratios were intermediate between those of the patients and control subjects, but responses to pyrazinamide and benzbromarone were normal. These data indicate that our patients have a combined defect in renal urate reabsorption, and that one of them might be subclassified as having the hypersecretion of defect. Results also show that heterozygotes can be identified by testing their urate/creatinine clearance ratio.

Diagnosis of partial deficiency of the pyruvate dehydrogenase complex in biopsied muscle.

We have measured the total activity of pyruvate dehydrogenase (PDH) complex, by in vitro activation with a broad specificity protein phosphatase, and the basal activity, supposed to be present in vivo, in biopsied muscles from three patients with PDH complex deficiency and 11 patients with lactic acidemia. Results showed that the total PDH complex activity must be determined in biopsied muscles for the diagnosis, because the basal activities of two of three patients with PDH complex deficiency overlapped those of two patients with lactic acidemia whose total activities were within normal range.

Activation of branched-chain α-ketoacid dehydrogenase complex by α-chloroisocaproate in normal and enzyme-deficient fibroblasts

Abstract A method has been developed for the activation of the branched-chain α-ketoacid dehydrogenase complex by α-chloroisocaproate, an inhibitor of branched-chain α-ketoacid dehydrogenase kinase in human cultured skin fibroblasts. The enzyme could be activated by pretreating the cells with α-chloroisocaproate before they were disrupted for measurement of the activity. After this treatment, the activity was 2- to 3-fold that of untreated cells (24.8–81.4 pmol/min per mg protein). The enzyme activity in fibroblasts from a patient with maple syrup urine disease was measured by this procedure. After activation by α-chloroisocaproate, the activity of fibroblasts from the patient was only 10–14 pmol/min per mg protein (10% of that of controls), and was almost the same as that of the untreated cells from this patient. These results show that it is important to consider the activation state of branched-chain α-ketoacid dehydrogenase complex when assaying it in disrupted cells.

Muscle involvement in pyruvate dehydrogenase complex (PDHC) deficiency

Muscle biopsies from a 13-month-old female infant with a delay in developmental milestones, lactic acidosis and visual disturbance, and a 6 year-old female with frequent epileptic fits are described. Biochemical studies of biopsied muscles and skin fibroblasts demonstrated markedly decreased pyruvate dehydrogenase complex (PDHC) activity to about 16% of normal value. Muscle histochemistry in both patients showed disorganized intermyofibrillar networks containing large diformazan granules on NADH-TR, small angulated fibers with high nonspecific esterase (NSE) activity and basophilic fibers. Ragged-red fibers and increased lipid droplet accumulation were absent. Patient 1 had increased numbers of type 2C fibers (11.3%) and mild fiber type grouping. On electron microscopy, most mitochondria were nearly normal. There were focal aggregates of mildly enlarged mitochondria in the subsarcolemmal areas in both patients. Morphometric study showed that the mean mitochondrial size and the mitochondrial percentage of fiber volume were not significantly different amongst patients and normal controls.