title: Automated spectrophotometric analysis of mitochondrial respiratory chain complex enzyme activities in cultured skin fibroblasts.
abstract: 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.
  
  
  METHODS
  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 RCC 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.
authors.affiliation: Biochemical Genetics Laboratory, Department of Laboratory Medicine and Pathology, Mayo Clinic College of Medicine, Rochester, MN 55905, USA.
authors.forename: Karen A
authors.initials: KA
authors.lastname: Kramer
authors.forename: Devin
authors.initials: D
authors.lastname: Oglesbee
authors.forename: Stacy J
authors.initials: SJ
authors.lastname: Hartman
authors.forename: Joe
authors.initials: J
authors.lastname: Huey
authors.forename: Bambi
authors.initials: B
authors.lastname: Anderson
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Magera
authors.forename: Dietrich
authors.initials: D
authors.lastname: Matern
authors.forename: Piero
authors.initials: P
authors.lastname: Rinaldo
authors.forename: Brian H
authors.initials: BH
authors.lastname: Robinson
authors.forename: Jessie M
authors.initials: JM
authors.lastname: Cameron
authors.forename: Si Houn
authors.initials: SH
authors.lastname: Hahn
mesh_terms: D001322:Autoanalysis; D015153:Blotting, Western; D002478:Cells, Cultured; D042967:Electron Transport Complex I / Q000378:metabolism*; D042963:Electron Transport Complex II / Q000378:metabolism*; D014450:Electron Transport Complex III / Q000378:metabolism*; D003576:Electron Transport Complex IV / Q000378:metabolism*; D005347:Fibroblasts / Q000201:enzymology*; D006801:Humans; D012016:Reference Values; D012867:Skin / Q000166:cytology / Q000201:enzymology*; D013053:Spectrophotometry
chemical_list: C478189:respiratory complex II; D042963:Electron Transport Complex II; D003576:Electron Transport Complex IV; D042967:Electron Transport Complex I; D014450:Electron Transport Complex III