Pelnena 2016 Abstract MitoFit Science Camp 2016
|OXPHOS enzyme activity measurements in mitochondria isolated from peripheral blood leukocytes in control group and patients with suspected mitochondrial disease.|
Mitochondrial disorders are a heterogeneous group of diseases usually caused by defects in the mitochondrial oxidative phosphorylation system (OXPHOS). Diagnosis is often challenging and includes both clinical observations and biochemical analysis followed by molecular genetic testing. OXPHOS enzyme activity measurements in muscle biopsies are considered as cornerstone for diagnostic of mitochondrial diseases . As in Latvia diagnosing of mitochondrial diseases has been based so far only on clinical phenotype, metabolite analysis and mtDNA sequencing, we aimed to introduce measurement of OXPHOS enzyme activity in peripheral leukocytes as a routine diagnostic method.
Respiratory Complex I (CI) and citrate synthase (CS) activities in mitochondria isolated from peripheral blood leukocytes were measured. Ten to twenty ml of peripheral blood were collected from 9 healthy persons and 3 patients with suspected mitochondrial diseases. Leukocytes were isolated by centrifugation in RBC lysis buffer in 600 g 10 min 4 °C and after washed with PBS. Isolated leukocytes were disrupted by homogenization with Dounce homogenizer in medium that contained 0.1 mg/ml digitonin as described before . Obtained mitochondria were suspended in buffer with 25 mM KH2PO4 and 5 mM MgCl2, pH 7.2. CI and CS activities were measured spectrophotometrically as previously described  with modifications. The results were expressed as ratio between CI and CS activities in order to compare patients and control group.
Mean CI activity in control group was 25.59 ± 8.25 mU/mg protein, mean CS activity was 196.59 ± 56.57 mU/mg protein and mean CI/CS ratio was 0.14 ± 0.06. One of the examined patients had lower CI activity and all three patients had increased CS activities compared to control group. All three examined patients had decreased CI/CS ratio (Fig 1).
OXPHOS enzyme activity measurements in peripheral leukocytes is fast robust method for routine diagnosis of patients with suspected mitochondrial disease as it is less traumatic than comprehensive biopsy. The limitation of this method is relatively large volume of blood that is essential to measure activities of all OXPHOS enzymes and citrate synthase spectrophotometrically. Therefore, more sensitive and reliable method as high-resolution respirometry would be required for this approach.
Labels: MiParea: mt-Medicine, Patients
Stress:Mitochondrial disease Organism: Human Tissue;cell: Blood cells, Macrophage-derived Preparation: Isolated mitochondria Enzyme: TCA cycle and matrix dehydrogenases
Event: B1, Poster MitoFit Science Camp 2016
1-Latvian Biomedical Research Study Centre, Latvia; 2-Laval Univ; 3-Centre hospitalier univ Québec, Canada; 4-Children's Clin Univ Hosp, Latvia. – [email protected]
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Figure 1. Ratio between complex I and citrate synthase activities of peripheral blood leukocytes in control group and patients.