The clinically and biochemically heterogeneous group of diseases characterized by Idiopathic Lactic Acidosis are a major therapeutic and diagnostic challenge in the pediatric patient. They often present in infancy leading to major morbidity and death. Patients are frequently severely neurologically damaged as a consequence of both the ongoing metabolic defect and episodic deterioration. These diseases are very probably genetically determined, but in most cases the biochemical nature of the defect remains unknown and adequate treatment is not available. The usual approach to the investigation of these patients involves fasting and dietary challenges along with the assay in blood or fibroblasts of a few of the enzymes concerned with pyruvate metabolism. Occasionally other specific enzyme assays are carried out on a """"""""hit and miss"""""""" basis. Over 80% of these patients remain undiagnosed. Citric acid cycle or cytochrome chain defects are the likely errors in many of these children. The propsed studies will provide an overview of these areas of metabolism indicating sites of specific metabolic defect. Location of the exact defect provides the best chance of designing effective therapy for the patient.
The specific aims of this proposal are the development and application of three different techniques for studying the citric acid cycle and oxidative phosphorylation in tissue samples and cultured cell lines. Control samples will be used to establish normal patterns and cells and muscle tissue from patients with lactic acidosis will be studied. These techniques are: 1) High pressure liquid chromatography (HPLC) assay of citric acid cycle intermediates in fibroblasts and muscle tissue. The extracts will also be analyzed for nucleotides, ketones, and pyruvic and lactic acid. 2) Mitochondria isolated from fibroblass wil be polarographically assayed utilizing a microchamber developed by this investigator. These mitochondria will also undergo HPLC analysis. 3) Fibroblast mitochondria will be used to measure the protonmotive force and proton conductance across the inner mitochondrial membrane. This assay provides information on the integrity of the inner mitochondrial membrane and along with the polarographic assay provides an excellent overview of oxidative phosphorylation and the cytochrome chain. The HPLC techniques will be used to investigate the metabolic effects of thiamine deficiency in the rat pyrithiamine treated/thiamine deficient model of lactic acidosis.
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