Assay of mitochondrial respiratory chain complex I in human lymphocytes and cultured skin fibroblasts

doi:10.1016/S0006-291X(02)03016-4

Biochemical and Biophysical Research Communications

Volume 301, Issue 1, 31 January 2003, Pages 222-224

https://doi.org/10.1016/S0006-291X(02)03016-4 Get rights and content

Abstract

Respiratory chain complex I (NADH:ubiquinone oxidoreductase) deficiency is one of the most frequent causes of mitochondrial disease in humans. The activity of this complex can be confidently measured in most tissue samples, but not in cultured skin fibroblasts or circulating lymphocytes. Highly contaminating non-mitochondrial NADH–quinone oxidoreductase activity in fibroblasts and the limited access of substrates to complex I in lymphocytes hinder its measurement in permeabilized cells. Complex I assay in these cells requires the isolation of mitochondria, which in turn necessitates large quantities of cells and is not feasible when studying circulating lymphocytes. Here we report a simple method to measure complex I activity in a minute amount of either cell type. The procedure strongly reduces contaminating NADH:quinone oxidoreductase activity and permits measuring high rates of rotenone-sensitive complex I activity thanks to effective cell permeabilization.

Section snippets

Materials and methods

Patient. Cultured skin fibroblasts were obtained from healthy controls and one patient born to non-consanguineous healthy parents. He presented with Leigh-like syndrome. Its clinical presentation has been previously reported [6].

Cells. Cultured skin fibroblasts were grown under standard conditions and circulating lymphocytes were isolated on a Ficoll cushion as previously described [3]. A small aliquot of the cell pellet (fewer than 1 million cells; 0.5–0.7 mg protein) was deep-frozen in 50 μl

Results and discussion

A hypotonic shock has been previously found to both selectively destabilize the contaminating NADH:ubiquinone oxidoreductase and to break open the mitochondria to NADH in skeletal muscle and in most human tissue homogenates allowing measurement of complex I activity [11]. However, we found hypotonic shock insufficient to decrease the contaminating activity in freeze-thaw permeabilized fibroblasts or to allow easy access of the NADH and/or decylubiquinone to mitochondrial CI in freeze-thaw

Acknowledgements

This work was supported in part by the Association Frančaise contre les Myopathies (AFM), by INSERM, and by the European Community (contract QLRT-CT-1999-00584).

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Assay of mitochondrial respiratory chain complex I in human lymphocytes and cultured skin fibroblasts

Abstract

Section snippets

Materials and methods

Results and discussion

Acknowledgements

Am. J. Hum. Genet.

Clin. Chim. Acta

Am. J. Hum. Genet.

Methods

Anal. Biochem.

Respiratory chain deficiency

Am. J. Hum. Genet.