Development of a new assay for complex I of the respiratory chain

Background: Measurement of complex I activity has been hampered by the larg e amounts of tissue required and the resulting turbidity of the assay solut ion, which makes spectrophotometric analysis difficult. We have developed a new assay for measuring the activity of complex I in isolated mitochondria that is also applicable to skeletal muscle homogenate in patients with sus pected mitochondrial diseases. Methods: The method was a radioenzymatic assay based on the preferential ox idation of the 4B hydrogen of NADH by complex I. We prepared tritiated isof orms of NADH for both the respective 4A-H-3 and 4B-H-3 positions. Enzyme in the form of purified mitochondria or homogenate was prepared from rat or h uman skeletal muscle and incubated with the respective radioisotopes. The p roduct ((H2O)-H-3) was collected after charcoal adsorption of unreacted NAD H and taken as an indicator of NADH oxidation. Sensitivity to rotenone was used as a measure of complex I specific activity. Results: The assay was linear with time and protein for isolated mitochondr ia and tissue homogenates from rats and humans. The V-max and K-m values ob tained for 4B-NADH with isolated rat skeletal muscle mitochondria were 35 m u mol/L and 90 mu mol . min(-1) . mg protein(-1) respectively. The assay wa s reproducible and useable for routine measurements in human skeletal muscl e. The sensitivity was >10-fold higher than the sensitivities of spectropho tometric techniques. Conclusions: The results of our studies demonstrate the successful developm ent of a new assay for complex I that is rapid, easy to perform, and that e nables the processing of multiple samples at one time. (C) 2000 American Association for Clinical Chemistry.