Detection of mitochondrial respiratory dysfunction in circulating lymphocytes using resazurin.
BACKGROUND
Laboratory methods currently available for detecting mitochondrial respiratory dysfunction are labor-intensive, require large amounts of isolated mitochondrial protein, invasive (require a skeletal muscle biopsy), and usually produce conflicting results.


OBJECTIVE
To develop a rapid, reliable, and noninvasive method for detecting oxidative phosphorylation activity without the need to isolate mitochondrial fractions.


METHODS
Lymphocytes from 6 patients with mitochondrial disorders (3 with mitochondrial myopathy and 3 with Leber hereditary optic neuropathy) and 51 normal control subjects were incubated with 6 microM resazurin without and with mitochondrial inhibition by amiodarone (200 microM), and the fluorescence intensity resulting from resazurin reduction was monitored spectrofluorometrically over time. Mitochondrial respiratory activity was calculated as the difference between uninhibited and inhibited measurements.


RESULTS
Mitochondrial respiratory activity was established for 51 normal control subjects and was decreased in all 6 patients with mitochondrial syndromes. Mitochondrial respiratory activity values for patients 1 through 6 compared to the control group after 240 minutes' incubation with resazurin were 55%, 71%, 49%, 61%, 68%, and 59%, respectively (mean mitochondrial respiratory activity of patients, 13.6% or 60.5% of control mean; P < .001).


CONCLUSIONS
This resazurin-based technique proved to be a fast and reproducible method for quantifying mitochondrial activity and identifying respiratory functional defects in patients with mitochondrial disorders.
Abu-Amero
Khaled K
KK
Department of Genetics, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia. kamero@kfshrc.edu.sa
Bosley
Thomas M
TM
D000293:Adolescent; D000328:Adult; D000638:Amiodarone / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D006801:Humans; D007202:Indicators and Reagents / Q000378:metabolism*; D008214:Lymphocytes / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D008297:Male; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D028361:Mitochondrial Diseases / Q000378:metabolism* / Q000473:pathology; D010078:Oxazines / Q000378:metabolism*; D010085:Oxidative Phosphorylation; D014966:Xanthenes / Q000378:metabolism*
D004791:Enzyme Inhibitors; D007202:Indicators and Reagents; D010078:Oxazines; D014966:Xanthenes; C005843:resazurin; D000638:Amiodarone