Abstract
We employed a microphotometric approach to examine whether a defect in the mitochondrial respiratory complex I expected in Leber hereditary optic neuropathy (LHON) as the consequence of a mtDNA (11778G>A) mutation in the ND4 gene coding for a subunit of the respiratory complex I can be detected at the single-cell level. Genetically stable fibroblast cell lines were established from skin biopsies of two members of a Chinese Indonesian family with LHON. The fibroblasts were homoplasmic for the 11778G>A mutation. The activity of the respiratory complex I was examined histochemically by staining for NADH-tetrazolium reductase. The histochemical staining showed a typical pattern with an apparent concentration of the activity around the nucleus, suggested as the reflection of the gradient in the thickness of the unsectioned fibroblast cells. Microphotometric quantification of the staining intensity showed that the activity is linear for at least 60min. The activity shows a discontinuity in its Arrhenius kinetics with a break point at 13.0–13.5°C (activation energy at 50–58 J/mol and 209–238 J/mol above and below the break temperature, respectively), indicating the membrane association of the NADH-tetrazolium reductase activity. Both patients showed lower fibroblast NADH-tetrazolium reductase activity, with a reduction of ∼30%. Our results demonstrate the utility of microphotometric analysis in the study of biochemical defects associated with mutations in the mtDNA.
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Malik, S., Sudoyo, H. & Marzuki, S. Microphotometric analysis of NADH-tetrazolium reductase deficiency in fibroblasts of patients with Leber hereditary optic neuropathy. J Inherit Metab Dis 23, 730–744 (2000). https://doi.org/10.1023/A:1005687031531
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DOI: https://doi.org/10.1023/A:1005687031531