A multi-center comparison of diagnostic methods for the biochemical evaluation of suspected mitochondrial disorders

Abstract

A multicenter comparison of mitochondrial respiratory chain and complex V enzyme activity tests was performed. The average reproducibility of the enzyme assays is 16% in human muscle samples. In a blinded diagnostic accuracy test in patient fibroblasts and SURF1 knock-out mouse muscle, each lab made the correct diagnosis except for two complex I results. We recommend that enzyme activities be evaluated based on ratios, e.g. with complex IV or citrate synthase activity. In spite of large variations in observed enzyme activities, we show that inter-laboratory comparison of patient sample test results is possible by using normalization against a control sample.

Fig. 1

Reproducibility of complex and citrate synthase activity measurements in control muscle. The results obtained with biopsies (1A) or muscle homogenates (1B), and comparison of enzyme activities measured in muscle biopsies and muscle homogenates (1C) are shown. The %CVs were calculated as the SD / mean activity of the biopsy or muscle homogenate. Activities were expressed as mU/U citrate synthase for the OXPHOS complex activities and mU/mg protein for citrate synthase. The median of the %CV for each enzyme assay is indicated by a line in A and B. The ratios given in C were calculated by dividing the activities measured in the muscle homogenate by those measured in the corresponding muscle biopsy. A and B: Diamond: lab 1; triangle: lab 3; cross: lab 4; asterisk: lab 5. C: gray circle: complex I, black circle: complex II, triangle: complex III, cross: complex IV, asterisk: complex V, and white circle: citrate synthase.

Fig. 2

Results of the analysis of wild type and SURF1 knock-out mouse muscle analysis. Two wild type (WT) and two SURF1 knock-out (KO) samples were analyzed in a blinded manner and results were expressed on citrate synthase base (A). The relative activities were calculated by dividing the activities measured in individual samples by the average of activities measured in wild type mouse muscle (B). Diamond: lab 1; square: lab 2; triangle: lab 3; cross: lab 4; and asterisk: lab 5.

Fig. 3

Effect of mitochondrial extract preparation on the outcome of the enzyme activity measurements in fibroblasts. The extraction procedures were according to the protocols of the participating labs, the enzyme activity measurements were according to the protocols of lab 4. For this purpose, a control cell line was used. Fig. 3A: activities are expressed as mU/mg protein; Fig. 3B: mU/U citrate synthase; Fig. 3C: mU/U complex IV. Gray bars: lab 1; black bars: lab 3; and white bars: lab 4.

Fig. 4

Effect of the buffer in which mitochondrial extracts were resuspended on the outcome of the enzyme activity measurements in fibroblasts. The extraction procedure was according to the protocol of lab 4, and the final mitochondrial pellet was resuspended in the final buffer according to the protocols of the different labs. Enzyme activities were determined using the methods described in Methods section 2.2. The activities are given as mU/U CS. Gray bars: lab 1; black bars: lab 3; and white bars: lab 4.