title: High-throughput assay to measure oxygen consumption in digitonin-permeabilized cells of patients with mitochondrial disorders.
abstract: BACKGROUND
  Muscle biopsy analysis is regarded as the gold standard in diagnostic workups of patients with suspected mitochondrial disorders. Analysis of cultured fibroblasts can provide important additional diagnostic information. The measurement of individual OXPHOS complexes does not always provide sufficient information about the functional state of the complete mitochondrial energy-generating system. Thus, we optimized a high-throughput fluorescence-based methodology for oxygen consumption analysis in patient-derived cells.
  
  
  METHODS
  We analyzed mitochondrial respiration in digitonin-permeabilized cells in the presence of a substrate mix containing pyruvate and malate, using a phosphorescent probe, 96-well plates, and a fluorescence plate reader.
  
  
  RESULTS
  In control fibroblasts, we observed clear stimulation by ADP of the pyruvate + malate-driven respiration. Known inhibitors of the OXPHOS system and the Krebs cycle significantly reduced respiration. In patient fibroblasts with different OXPHOS deficiencies, ADP-stimulated respiratory activity was decreased in comparison to control cells. In several patients with reduced ATP production rate in muscle tissue but with normal OXPHOS enzyme activities, the fibroblasts displayed reduced respiratory activity. Finally, we observed a clear difference between control and complex I-deficient transmitochondrial cybrid cells.
  
  
  CONCLUSIONS
  These results confirm the validity of the assay as a high-throughput screening method for mitochondrial function in digitonin-permeabilized cells. The assay allows primary and secondary mitochondrial abnormalities in muscle to be differentiated, which is of great importance with respect to counseling, and also will facilitate the search for new genetic defects that lead to mitochondrial disease.
authors.affiliation: Laboratory of Pediatrics and Neurology, Department of Pediatrics, Nijmegen Center for Mitochondrial Disorders, Radboud University Nijmegen Medical Center, Nijmegen, the Netherlands.
authors.forename: An I
authors.initials: AI
authors.lastname: Jonckheere
authors.forename: Merei
authors.initials: M
authors.lastname: Huigsloot
authors.forename: Antoon J M
authors.initials: AJ
authors.lastname: Janssen
authors.forename: Antonia J H
authors.initials: AJ
authors.lastname: Kappen
authors.forename: Jan A M
authors.initials: JA
authors.lastname: Smeitink
authors.forename: Richard J T
authors.initials: RJ
authors.lastname: Rodenburg
mesh_terms: D002463:Cell Membrane Permeability; D019069:Cell Respiration; D002478:Cells, Cultured; D004072:Digitonin; D005347:Fibroblasts / Q000378:metabolism / Q000473:pathology; D005453:Fluorescence; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D008293:Malates / Q000378:metabolism; D008931:Mitochondria, Muscle / Q000378:metabolism / Q000473:pathology; D028361:Mitochondrial Diseases / Q000175:diagnosis / Q000378:metabolism*; D010101:Oxygen Consumption; D019289:Pyruvic Acid / Q000378:metabolism
chemical_list: D008293:Malates; C030298:malic acid; D019289:Pyruvic Acid; D004072:Digitonin