Figure 8.

Evaluation of enzymatic assay for quantitation of NADPH/NADP⁺. (a) Consistent with the principle of the enzymatic assay, basic pH selectively degrades NADP⁺. 10 µM NADPH and 10 µM NADP in aqueous buffer containing 20mM nicotinamide, 20mM NaHCO₃, and 100mM Na₂CO₃ were heated at 60°C for the indicated times, and their concentrations were compared to the unheated samples by liquid chromatography-mass spectrometry (LC-MS). Note that modest degradation of NADPH is sufficient to alter NADP⁺ measurements, which are determined by subtraction. (b) Aqueous extraction without detergent using typical enzymatic assay conditions results in a dramatic loss of NADPH and a gain in NADP⁺, undermining utility of the enzyme assay. NADPH and NADP⁺ were measured from cultured HEK293T cells and mouse liver using two extraction methods: the suggested conditions of the enzymatic assay kit (20mM nicotinamide, 20mM NaHCO₃, 100 mM Na₂CO₃ in water) or our suggested extraction for LC-MS analysis (40:40:20 acetontrile:methanol:water + 0.1 M formic acid, followed by neutralization with NH₄HCO₃). Results were normalized to the LC-MS extraction buffer.