At the end of the experiments, blood samples and tissue specimens were collected. Lactate dehydrogenase in plasma was measured using a colorimetric assay kit according to the manufacturer’s instructions. α-Ketoglutarate dehydrogenase activity in heart tissue was determined by the rate of reduction of NAD+ in the presence of α-ketoglutarate (33 (link)), while cytochrome c oxidase activity was measured by the rate of enzymatic oxidation of reduced cytochrome c (34 (link)).
Measurement of ATP content was performed according to the manufacturer’s instructions. Briefly, 20 mg of left ventricular tissue was homogenized in 5% trichloroacetic acid and centrifuged at 12,000 rpm for 10 min at 4°C. The same amount of the supernatant for each group was neutralized with K2CO3 and then incubated with the assay buffer. The relative light units were determined with a luminometer (VICTOR X, PerkinElmer, Waltham, MA, USA), and the ATP content was calculated by a standard curve. The data are presented as nmol ATP/mg of wet tissue weight (35 (link)).
Measurement of ATP content was performed according to the manufacturer’s instructions. Briefly, 20 mg of left ventricular tissue was homogenized in 5% trichloroacetic acid and centrifuged at 12,000 rpm for 10 min at 4°C. The same amount of the supernatant for each group was neutralized with K2CO3 and then incubated with the assay buffer. The relative light units were determined with a luminometer (VICTOR X, PerkinElmer, Waltham, MA, USA), and the ATP content was calculated by a standard curve. The data are presented as nmol ATP/mg of wet tissue weight (35 (link)).
