NAD+ measurement.

Cellular NAD⁺ levels were quantified by means of an enzymatic cycling procedure (90). Briefly, cells grown in a 10-cm plate were treated with 125 μM H₂O₂ for the indicated times. The cells were collected in cold PBS and centrifuged at 5,000 rpm for 10 min at 4°C. The pellet was resuspended in 100 μl of 1 N HClO₄ and neutralized with 50 μl of 1 N KOH. After the addition of 150 μl of 100 mM bicine, pH 8, 100 μl of the cell extract was mixed with 50 μl of the bicine buffer containing 0.114 M bicine (pH 7.8), 0.57 M ethanol, 4.8 mM EDTA-Na₄, 1 mg/ml BSA, 0.48 mM 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium, 1.9 mM phenazine ethosulfate, and 48 μg/ml alcohol dehydrogenase. The mixture was incubated at room temperature for 20 min, and then the A₅₉₀ was measured. A standard curve was used to quantify total NAD⁺.