Measurement of ATP production and respiratory chain enzyme activities in mitochondria isolated from small muscle biopsy samples.
A set of methods suitable for assessment of respiratory chain function in mitochondria isolated from 25mg of muscle is described. This set of methods includes determination of the mitochondrial ATP production rate (MAPR) and the activities of the respiratory chain complexes I, I+III, II+III, and IV and citrate synthase. MAPR is determined with an optimized version of a luminometric method previously described. The optimized method measures 50-220% higher activities than the original method. The highest MAPRs are recorded using the substrate combinations glutamate+succinate and N,N,N(1),N(1)-tetramethyl-1,4-phenyldiamine+ascorbate. The respiratory chain complex activities are determined with standard spectrophotometric methods, adapted to an automated photometer. The sensitivity in the determination of complex I, I+III, and II+III activities was increased considerably by pretreating the samples with saponin. The set of methods was evaluated on double biopsy samples from five healthy volunteers and showed coefficients of variation between 7 and 14% when citrate synthase was used as reference base. All of the various measures of mitochondrial function showed high correlation coefficients to each other (r=0.84-0.98; p<0.01). It is concluded that the set of methods is suitable for diagnosis of mitochondrial disorders in adults and small children.
Wibom
Rolf
R
Centre for Inherited Metabolic Diseases, Department of Medical Laboratory Sciences and Technology, Karolinska Institutet, Huddinge University Hospital, S-141 86 Stockholm, Sweden. rolf.wibom@cmms.hs.sll.se
Hagenfeldt
Lars
L
von Döbeln
Ulrika
U
D000255:Adenosine Triphosphate / Q000032:analysis / Q000096:biosynthesis*; D000328:Adult; D002458:Cell Fractionation; D002950:Citrate (si)-Synthase / Q000378:metabolism; D004796:Clinical Enzyme Tests / Q000379:methods*; D004579:Electron Transport; D042967:Electron Transport Complex I; D042963:Electron Transport Complex II; D014450:Electron Transport Complex III; D005260:Female; D006801:Humans; D008163:Luminescent Measurements; D008297:Male; D008931:Mitochondria, Muscle / Q000201:enzymology*; D028361:Mitochondrial Diseases / Q000175:diagnosis; D024101:Mitochondrial Proteins / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*
D024101:Mitochondrial Proteins; C478189:respiratory complex II; D000255:Adenosine Triphosphate; D042963:Electron Transport Complex II; D009247:NADH, NADPH Oxidoreductases; D002950:Citrate (si)-Synthase; D042967:Electron Transport Complex I; D014450:Electron Transport Complex III