Mitochondrial Physiology Network 10.9: 67 (2005) - download pdf

Assessment of mitochondrial function and respiratory complex function using mitochondrial containing blood cells following membrane permeabilization.

Joseph F Clark, TJ DeGrauw, GJ Pyne-Geithman

Dept. Neurology, University of Cincinnati, Medical Center, 231 Bethesda Ave, Cincinnati, OH 45267-0525, USA. - clarkjf@email.uc.edu

    Assessing mitochondrial function for the diagnosis of metabolic disorders and inborn errors of metabolism is generally performed using a muscle biopsy. In the pediatric population this generally means an invasive surgical procedure with substantial emotional stress and concomitant morbidity. What is needed is a method for assessing mitochondrial function that is less invasive and usable on functional (coupled) mitochondria.

    From normal healthy adult volunteers, we obtained 50 ml of blood into EDTA containing vacutainer tubes. The red blood cells were separated using the Lymphoprep method from Granier^®. Membrane permeabilization was accomplished in the oxygraph chamber using 40 µg/ml saponin. The mitochondrial containing cells were then assessed for oxidative phosphorylation by measuring oxygen consumption in the presence of specific inhibitors or substrates. Cells were aliquoted and frozen in the presence of 20 % DMSO, quickly frozen on dry ice slurry, and stored for a minimum of 24 hours, at –70 °C, and tested in the oxygraph.

    Coupled respiration was 1.16 + 0.35 µM∙min^-1∙g^-1 protein for the freshly isolated cells and 0.95 + 0.14 µM∙min^-1∙g^-1 protein for the frozen cells. Rotenone (complex I inhibitor) produced a 29 % decrease in respiration and atractyloside (adenine nucleotide translocase inhibitor) produced a 52 % decrease in respiration. Frozen cells retained their ability to be inhibited by rotenone and atractyliside (43 and 38 % respectively).

    We conclude from this study that the isolation of mitochondrial containing blood cells and saponin permeabilization of these cells results in coupled respiration of the mitochondria and provides an avenue for assessing the mitochondrial complex activity. Following freeze/thaw of these cells there is still coupled respiration and an opportunity for measuring coupled respiration and oxidative complexes.