摘要：Abstract Background Mitochondrial diseases belong to the most severe inherited metabolic disorders affecting pediatric population. Despite detailed knowledge of mtDNA mutations and progress in identification of affected nuclear genes, diagnostics of a substantial part of mitochondrial diseases relies on clinical symptoms and biochemical data from muscle biopsies and cultured fibroblasts. Methods To investigate manifestation of oxidative phosphorylation defects in isolated lymphocytes, digitonin-permeabilized cells from 48 children were analyzed by high resolution respirometry, cytofluorometric detection of mitochondrial membrane potential and immunodetection of respiratory chain proteins with {SDS} and Blue Native electrophoreses. Results Evaluation of individual respiratory complex activities, {ATP} synthesis, kinetic parameters of mitochondrial respiratory chain and the content and subunit composition of respiratory chain complexes enabled detection of inborn defects of respiratory complexes I, {IV} and V within 2 days. Low respiration with NADH-dependent substrates and increased respiration with glycerol-3-phosphate revealed complex I defects; changes in p50 for oxygen and elevated uncoupling control ratio pointed to complex {IV} deficiency due to {SURF1} or {SCO2} mutation; high oligomycin sensitivity of state 3-ADP respiration, upregulated mitochondrial membrane potential and low content of complex V were found in lymphocytes with {ATP} synthase deficiency due to {TMEM70} mutations. Conclusion Based on our results, we propose the best biochemical parameters predictive for defects of respiratory complexes I, {IV} and V manifesting in peripheral blood lymphocytes. General significance The noninvasiveness, reliability and speed of an approach utilizing novel biochemical criteria demonstrate the high potential of isolated lymphocytes for diagnostics of oxidative phosphorylation disorders in pediatric patients.
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