title: Noninvasive diagnostics of mitochondrial disorders in isolated lymphocytes with high resolution respirometry.
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 p 50 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.
  
  
  CONCLUSIONS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Bioenergetics, Institute of Physiology Academy of Sciences of the Czech Republic v.v.i., 142 20 Prague 4, Czech Republic.
authors.forename: Petr
authors.initials: P
authors.lastname: Pecina
authors.affiliation: Department of Pediatrics and Adolescent Medicine, Thomayer Hospital, 140 00 Prague 4, Czech Republic.
authors.forename: Hana
authors.initials: H
authors.lastname: Houšťková
authors.affiliation: Department of Bioenergetics, Institute of Physiology Academy of Sciences of the Czech Republic v.v.i., 142 20 Prague 4, Czech Republic.
authors.forename: Tomáš
authors.initials: T
authors.lastname: Mráček
authors.affiliation: Department of Bioenergetics, Institute of Physiology Academy of Sciences of the Czech Republic v.v.i., 142 20 Prague 4, Czech Republic.
authors.forename: Alena
authors.initials: A
authors.lastname: Pecinová
authors.affiliation: Department of Bioenergetics, Institute of Physiology Academy of Sciences of the Czech Republic v.v.i., 142 20 Prague 4, Czech Republic.
authors.forename: Hana
authors.initials: H
authors.lastname: Nůsková
authors.affiliation: Clinic of Pediatrics and Adolescent Medicine, General University Hospital in Prague, 128 08 Prague 2, Czech Republic.
authors.forename: Markéta
authors.initials: M
authors.lastname: Tesařová
authors.affiliation: Clinic of Pediatrics and Adolescent Medicine, General University Hospital in Prague, 128 08 Prague 2, Czech Republic.
authors.forename: Hana
authors.initials: H
authors.lastname: Hansíková
authors.affiliation: Department of Neonatology, Thomayer Hospital, 140 00 Prague 4, Czech Republic.
authors.forename: Jan
authors.initials: J
authors.lastname: Janota
authors.affiliation: Clinic of Pediatrics and Adolescent Medicine, General University Hospital in Prague, 128 08 Prague 2, Czech Republic.
authors.forename: Jiří
authors.initials: J
authors.lastname: Zeman
authors.affiliation: Department of Bioenergetics, Institute of Physiology Academy of Sciences of the Czech Republic v.v.i., 142 20 Prague 4, Czech Republic.
authors.forename: Josef
authors.initials: J
authors.lastname: Houštěk
keywords: AA, antimycin A; BNE, Blue Native PAGE; COX, cytochrome c oxidase; Diagnostics; FCCP, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; GP, glycerol-3-phosphate; GPDH, mitochondrial FAD-dependent glycerophosphate dehydrogenase; Lymphocytes; Mitochondrial diseases; OXPHOS, oxidative phosphorylation; Oxidative phosphorylation; PAGE, polyacrylamide gel electrophoresis; Respirometry; TMPD, tetramethylphenylenediamine; TMRM, tetramethylrhodamine methyl ester; cI–cV, respiratory chain complexes I–V; s3, state 3-ADP; s3u, state 3-uncoupled; s4o, state 4-oligomycin; ΔΨm, mitochondrial membrane potential