Article

Evaluation of Methods for the Determination of Mitochondrial Respiratory Chain Enzyme Activities in Human Skeletal Muscle Samples

Otto-von-Guericke-Universität Magdeburg, Magdeburg, Saxony-Anhalt, Germany
Analytical Biochemistry (Impact Factor: 2.22). 04/2000; 279(1):55-60. DOI: 10.1006/abio.1999.4434
Source: PubMed

ABSTRACT

The quantification of mitochondrial enzyme activities in skeletal muscle samples of patients suspected of having mitochondrial myopathies is problematic. Therefore, we have evaluated different methods for the determination of activities cytochrome c oxidase and NADH:CoQ oxidoreductase in human skeletal muscle samples. The measurement of cytochrome c oxidase activity in the presence of 200 microM ferrocytochrome c and the detection of NADH:CoQ oxidoreductase as rotenone-sensitive NADH:CoQ(1) reductase resulted in comparable citrate synthase-normalized respiratory chain enzyme activities of both isolated mitochondria and homogenates from control human skeletal muscle samples. These methods allowed the precise detection of deficiencies of respiratory chain enzymes in skeletal muscle of two patients harboring only 20 and 27% of deleted mitochondrial DNA, respectively. Therefore, citrate synthase-normalized respiratory chain activities can serve as stable reference values for the determination of a putative mitochondrial defect in human skeletal muscle.

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Available from: Wolfram S Kunz, Jan 16, 2015
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    • "The activity of citrate synthase, rotenone-sensitive NADH:CoQ 1 oxidoreductase, and COX was determined by standard methods (Wiedemann et al., 2000). The details on the performed biochemical assays are outlined in the Supplementary material. "
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    • "Enzymatic activities were measured using a dual wavelength spectrophotometer (Aminco DW 2000, SLM Instruments, Rochester, NY), as described before (Wiedemann et al., 2000). "
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    • "Then, the sample was sonicated for 15 s with the ultrasonic processor GEX-600. The activity of citrate synthase was determined by standard methods as described elsewhere (Wiedemann et al, 2000). The activity of rotenone-sensitive NADH : "
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