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Adaptation of a Mitochondrial Complex III Assay for Automation: Examination of Reproducibility and Precision

¹ Department of Paediatrics and Child Health, University of Sydney, Sydney, 2006 Australia;
² Western Sydney Genetics Program, Children’s Hospital, Sydney, 2145 Australia;
³ Department of Clinical Immunology, Royal Prince Alfred Hospital, Sydney, 2050 Australia;

^aaddress correspondence to this author at: Western Sydney Genetics Program, Royal Alexandra Hospital for Children, Locked Bag 4001, Westmead, NSW 2145 Australia; fax 612-9845-1864, e-mail Johnc@chw.edu.au

The first 20% of the full text of this article appears below.

Complex III (ubiquinol:ferricytochrome c reductase; EC 1.10.2.2) catalyzes the reduction of cytochrome c by ubiquinol (1)(2). The assay measures the reduction of cytochrome c as catalyzed by complex III at 550 nm (3) in the presence of reduced decylubiquinone (DBH₂) (2).

Despite numerous in vivo methods to screen for disorders of ATP production, biochemical assays for the respiratory chain (RC) remain the best way to identify and characterize this group of disorders (4). Although investigators have documented various methodologies to measure complex III in a variety of tissue types (3)(5)(6)(7), these are based on manual methods that are unsuitable for screening large numbers of samples. We recently automated mitochondrial and RC-specific enzyme (8) and protein (9) assays on a random access analyzer (Roche Mira S). The benefits of automation include enhanced speed and simplicity; savings in costs, including labor and reagents, and in the amount of sample needed; and improvements in precision. We adapted and automated the complex III assay (2)(6) on the Roche Mira S random access automated analyzer and compared methods of isolation, performed stability studies, and examined the precision of this new automated method.

Skin fibroblasts from healthy human controls and patients were grown from skin explants. Fibroblasts were cultured in DMEM (high glucose) supplemented with 100 mL/L fetal bovine serum and 225 µmol/L uridine (10).

Two procedures were used to isolate mitochondria from cultured fibroblasts, with all sample preparation being carried out at 4 °C. The first method used a glass-on-Teflon homogenizer, based on the long method of Pitkänen et al. (11), except that four . . . [Full Text of this Article]

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				K. A. Kramer, D. Oglesbee, S. J. Hartman, J. Huey, B. Anderson, M. J. Magera, D. Matern, P. Rinaldo, B. H. Robinson, J. M. Cameron, et al. Automated Spectrophotometric Analysis of Mitochondrial Respiratory Chain Complex Enzyme Activities in Cultured Skin Fibroblasts Clin. Chem., November 1, 2005; 51(11): 2110 - 2116. [Abstract] [Full Text] [PDF]