Received on February 23, 2005
Accepted on July 28, 2005

Automation and Analytical Techniques

Automated Spectrophotometric Analysis of Mitochondrial Respiratory Chain Complex Enzyme Activities in Cultured Skin Fibroblasts

¹ Biochemical Genetics Laboratory, Department of Laboratory Medicine and Pathology, Mayo Clinic College of Medicine, Rochester, MN
² Biochemical Genetics Laboratory, Departments of Laboratory Medicine and Pathology, Medical Genetics, and Pediatric and Adolescent Medicine, Mayo Clinic College of Medicine, Rochester, MN
³ Metabolism Programme, Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada

^* To whom correspondence should be addressed. E-mail: hahn.sihoun{at}mayo.edu.

Background: Mitochondrial respiratory chain complex (RCC) disorders may occur as commonly as 1 in 8500 individuals. Because of the great variability of phenotypic presentations, measurement of individual RCC enzyme activities is a crucial diagnostic process. Current assay methods are time-consuming and labor-intensive and thus constitute a major impediment to clinical practice. A method with a faster turnaround time would therefore be beneficial.

Method: We developed an automated spectrophotometric method for measuring the respiratory chain enzyme activities of complex I, complex II + III, and complex IV with the Hitachi 912, an automated spectrophotometer. Mitochondrial citrate synthase was also determined for normalization of the RCC activities.

Results: A blinded method comparison with samples from an external testing center yielded a 91% concordance of interpretations. Mean intraassay imprecision (as CV; n = 20) in a single batch analysis of each RC complex was 5.9%. Interassay imprecision, evaluated on 2 samples harvested and analyzed 3 times each, gave mean CVs of 10%-18%.

Conclusions: With this automated method, a panel of RCC enzyme activities can be determined in <2 h. In addition, an immunoblot assay using monoclonal antibodies against specific subunits of RCC enzyme complexes can be informative in cases of borderline enzyme activity. Our results suggest that in vitro diagnosis of RCC enzyme deficiencies in skin fibroblasts is an effective alternative to invasive muscle biopsy.