Lactate Oxidation for the Detection of Mitochondrial Dysfunction in Human Skin Fibroblasts

To screen fibroblasts for defects in lactate/pyruvate oxidation, cells were grown to confluence in 25-cm² flasks, rinsed, and incubated in glucose-free media containing 25 μM L-lactate and 0.1 μCi [D,L-1-¹⁴C]lactate. Lactate oxidation was measured as the amount of lactate oxidized in nmol of ¹⁴CO₂ generated /mg protein/min. Fibroblasts from patients with mitochondrial or peroxisomal disorders had decreased lactate oxidation compared to the control (CON): CON, 1.9 ± 0.13 nmol/mg/min; neonatal adrenoleukodystrophy (NALD), 0.45 ± 0.01 (P < 0.001); rhizomelic chondrodysplasia punctata (RCDP), 0.13 ± 0.002 (P < 0.001); mitochondrial defect of unknown etiology (MIT), 0.77 ± 0.003 (P <0.001); pyruvate dehydrogenase (PDH) deficiency, 0.98 ± 0.02 (P < 0.001). This method is useful for screening fibroblasts for defects in lactate oxidation in patients with mitochondrial or peroxisomal disorders. Confirmation of the site of the defect may then be investigated with specific assays, e.g., PDH, in cellular homogenates: CON, 0.93 ± 0.02 nmol/mg/min; NALD, 0.55 ± 0.02; RCDP, 0.44 ± 0.02; MIT, 0.53 ± 0.03; PDH deficiency, 0.19 ± 0.02.