Comparison of disruption methods to optimize the evaluation of mitochondrial enzymatic activities in human fibroblasts

Abstract

Mitochondriopathies often lead to chronic degenerative pathology. Muscle biopsy is considered the gold standard for diagnosis; however, its invasiveness restricts the amount of tissue obtained, and issue that is limiting for prospective, longitudinal, and pharmacological studies. Primary human fibroblasts (HF) lines are a valid alternative to biochemically characterize mitochondrial syndromes. Cell disruption is essential for measuring mitochondrial enzyme activity, and yet, no systematic comparative study has evaluated the different existing methods. Using 17 different cell lysis procedures, we evaluated the activity preservation of two mitochondrial enzymes: complex IV or cytochrome c oxidase (CIV) and citrate synthase (CS), in normal HF samples from pediatric patients. Procedures were categorized as chemical, mechanical, physical, and enzymatic. Only the enzymatic disruption using Pronase leads to high activity values for both CIV and CS activities, exhibiting intra- and inter-sample consistency. We then measured the activities of all mitochondrial complexes (CI, CI+CIII, CII, CII+CIII) and CV (ATP hydrolysis) in Pronase-disrupted cells, finding proper reproducibility and values comparable to those in the literature. We propose Pronase cell disruption is an adequate method for evaluating mitochondrial activities in HF samples.

Keywords: cell disruption; citrate synthase; cytochrome c oxidase; enzymatic activities; human fibroblasts.