NAD metabolite extraction from liver, mitochondria, and hepatocytes.

NAD was extracted from 50 mg freeze-clamped liver, 50–100 μg isolated mitochondria, or 1 × 10⁶ primary hepatocytes in 500 μL ice-cold 0.6 M perchloric acid. Tissues were homogenized at 20 Hz for 2 minutes at 4°C using a tissue lyser (Qiagen). The insoluble materials were precipitated by centrifugation at 15,000g for 10 minutes at 4°C, and the clear supernatant was diluted to 1:100 in ice-cold 100 mM sodium phosphate buffer, pH 8. NAD was measured by an enzymatic cycling assay in a 96-well format modified from the procedure of Graeff and Lee (44) as previously described. Briefly, 5 μL NAD standards or diluted tissue extracts were freshly prepared and added to 95 μL cycling mix consisting of 2% ethanol, 100 μL/mL alcohol dehydrogenase, 10 μL/mL diaphorase, 20 mM resazurin, 10 mM flavin mononucleotide, 10 mM nicotinamide, and 0.1% BSA in 100 mM phosphate buffer, pH 8. Alcohol dehydrogenase reduces NAD to NADH, which in the presence of diaphorase, quantitatively reduces resazurin to the fluorescent molecule resorufin with regeneration of NAD. The cycling reaction was incubated for 30 minutes at room temperature, and the concentration of NAD was determined based on the rate of resorufin accumulation as measured by fluorescence with excitation at 544 nm and emission at 590 nm.

NADH was similarly extracted from freeze-clamped liver tissue, mitochondria, and hepatocytes in a prechilled extraction buffer (25 mM NH₄Ac, 25 mM NaOH, 50% [v/v] acetonitrile) bubbled with nitrogen gas. Alkaline extracts were incubated at 55°C for 10 minutes to degrade any residual NAD, cooled. and centrifuged. The supernatants were diluted 1:50 in ice-cold 100 mM phosphate buffer, pH 8, for NADH measurement by cycling assay. Mitochondria and hepatocytes were separately vortexed vigorously for 1 minute and then subjected to centrifugation for 10 minutes (15,000g, 4°C), the clear supernatant was removed and either diluted to 1:10 or 1:5 in ice-cold 100 mM sodium phosphate buffer, pH 8, for NAD and NADH measurement.