Measurement of the energy-generating capacity of human muscle mitochondria: diagnostic procedure and application to human pathology.
BACKGROUND
Diagnosis of mitochondrial disorders usually requires a muscle biopsy to examine mitochondrial function. We describe our diagnostic procedure and results for 29 patients with mitochondrial disorders.


METHODS
Muscle biopsies were from 43 healthy individuals and 29 patients with defects in one of the oxidative phosphorylation (OXPHOS) complexes, the pyruvate dehydrogenase complex (PDHc), or the adenine nucleotide translocator (ANT). Homogenized muscle samples were used to determine the oxidation rates of radiolabeled pyruvate, malate, and succinate in the absence or presence of various acetyl Co-A donors and acceptors, as well as specific inhibitors of tricarboxylic acid cycle or OXPHOS enzymes. We determined the rate of ATP production from oxidation of pyruvate.


RESULTS
Each defect in the energy-generating system produced a specific combination of substrate oxidation impairments. PDHc deficiencies decreased substrate oxidation reactions containing pyruvate. Defects in complexes I, III, and IV decreased oxidation of pyruvate plus malate, with normal to mildly diminished oxidation of pyruvate plus carnitine. In complex V defects, pyruvate oxidation improved by addition of carbonyl cyanide 3-chlorophenyl hydrazone, whereas other oxidation rates were decreased. In most patients, ATP production was decreased.


CONCLUSIONS
The proposed method can be successfully applied to the diagnosis of defects in PDHc, OXPHOS complexes, and ANT.
Janssen
Antoon J M
AJ
Department of Pediatrics and Laboratory of Pediatrics and Neurology, the Nijmegen Centre for Mitochondrial Disorders (NCMD), Nijmegen, The Netherlands.
Trijbels
Frans J M
FJ
Sengers
Rob C A
RC
Wintjes
Liesbeth T M
LT
Ruitenbeek
Wim
W
Smeitink
Jan A M
JA
Morava
Eva
E
van Engelen
Baziel G M
BG
van den Heuvel
Lambert P
LP
Rodenburg
Richard J T
RJ
D000255:Adenosine Triphosphate / Q000096:biosynthesis; D000293:Adolescent; D000328:Adult; D001706:Biopsy; D002250:Carbon Radioisotopes; D002331:Carnitine / Q000378:metabolism; D002648:Child; D002675:Child, Preschool; D004734:Energy Metabolism; D005260:Female; D006801:Humans; D008293:Malates / Q000378:metabolism; D008297:Male; D008314:Malonates / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism*; D000226:Mitochondrial ADP, ATP Translocases / Q000172:deficiency; D028361:Mitochondrial Diseases / Q000175:diagnosis* / Q000378:metabolism; D018482:Muscle, Skeletal / Q000378:metabolism*; D010084:Oxidation-Reduction; D010085:Oxidative Phosphorylation; D011768:Pyruvate Dehydrogenase Complex / Q000378:metabolism; D019289:Pyruvic Acid / Q000378:metabolism; D019802:Succinic Acid / Q000378:metabolism
D002250:Carbon Radioisotopes; D008293:Malates; D008314:Malonates; D011768:Pyruvate Dehydrogenase Complex; C030298:malic acid; D019289:Pyruvic Acid; D000255:Adenosine Triphosphate; D000226:Mitochondrial ADP, ATP Translocases; C030290:malonic acid; D019802:Succinic Acid; D002331:Carnitine