Citrate synthase activity assay kit

Myocardial ATP content was detected using an ATP Assay Kit (Beyotime, China) according to the manufacturer's instructions. Approximately 20 mg LV myocardial tissues were lysed and homogenized, followed by centrifugation at 12000g for 5 minutes. Supernatant was mixed with diluted luciferase reagent. The emitted light was measured by a microplate luminometer (Thermo Scientific, USA) and ATP content was calculated from a standard curve. Finally ATP level was normalized to protein concentration, which was determined with a BCA protein assay kit (Beyotime, China). CS activity was measured using an Citrate Synthase Activity Assay Kit (Sigma-Aldrich, USA). Briefly, 10 mg tissue samples were homogenized in CS assay buffer and centrifuged at 10000g for 5 minutes. Supernatants were diluted appropriately and mixed with reaction mixes. Both the initial and final absorbance at 412 nm were measured for each sample for calculating the changes in absorbance (ΔA₄₁₂). Sample blank ΔA₄₁₂ value was subtracted from the sample ΔA₄₁₂ reading to obtain the corrected measurement. Sample values were then read off the standard curve and expressed as nmole per minute per milligram of total protein.

Citrate synthase (CS) activity of liver samples under the conditions indicated was measured using the Citrate Synthase Activity Assay Kit from Sigma (MAK193), following manufacturer’s instructions. CS activity was determined using a coupled enzyme reaction, resulting in a colorimetric (412 nm) product proportional to the enzymatic activity. The reaction was measured every 5 min for 40 min at 25°, considering the penultimate reading the final result. CS activity was represented as a percentage. To measure the liver cytosolic SOD activity, a commercially available kit (STA-340, Cell Biolabs, INC.) was used according to the manufacturer’s protocol. Absorbance was measured at 490 nm after 1 h incubation at 37 °C. SOD activity was represented as a function of inhibition percentage.