Measuring oxidative phosphorylation in human skin fibroblasts

An approach has been developed to quantitate oxidative phosphorylation in harvested human skin fibroblasts that have been permeabilized with digitonin. In protocol 1, state 3 rates are measured with complex I and II substrates, followed by uncoupled maximal oxidative capacity measured in the presence of these combined substrates as well as through complex IV. In protocol 2, state 3 rates are measured using palmitoylcarnitine to monitor fatty acid oxidation and duroquinol to assess the flux through complex III; uncoupled duroquinol oxidation measures maximal oxidative capacity through complex III. The activity of citrate synthase is determined in every experiment as a marker of the amount of mitochondria per chamber. Data are expressed on the basis of cell count (per million fibroblasts), of protein, or of citrate synthase activity. Cell growth conditions are optimized, and it is necessary to keep cultured cells from reaching confluency. Cultures in passages 3 to 10 show reproducible oxidative phosphorylation data. Based on the data from the 15 normal human skin fibroblast lines, we are evaluating the use of this approach to diagnose systemic mitochondrial disease and avoid issues associated with open skeletal muscle biopsy.