Pyruvate dehydrogenase (PDH) and lactate dehydrogenase (LDH) activity assay

In vitro PDH activity was measured by coupling to the reduction of NAD⁺ (nicotinamide adenine dinucleotide) to NADH, using a commercially available kit (Abcam, USA). NADH concentration was measured spectrophotometrically at 450 nm at room temperature using an Infinite M200 microplate reader, and the results were expressed as change of absorbance per minute per mg of protein.

In vitro LDH activity was measured spectrophotometrically by quantifying the linear decrease in NADH absorbance at varying pyruvate (sodium salt) concentrations at 339 nm using an Infinite M200 microplate reader. The maximum velocity (Vmax) and the Michaelis-Menten constant (Km) were estimated using the Lineweaver-Burk plot.