2.9. NAD+ measurements

NAD⁺ levels were measured as previously described [31]. Briefly, frozen liver or small intestine tissues were homogenized in 1 M perchloric acid and neutralized in 3 M K₂CO₃ on ice. After centrifugation, the supernatant was mixed with Buffer A (50 mM K₂PO₄/KHPO₄, pH 7.0) and loaded on to the column. The HPLC was run at a flow rate of 1 ml/min with 100% Buffer A from 0 to 5 min, a linear gradient to 95% Buffer A/5% Buffer B (100% methanol) from 5 to 6 min, 95% Buffer A/5% Buffer B from 6 to 11 min, a linear gradient to 85% Buffer A/15% Buffer B from 11 to 13 min, 85% Buffer A/15% Buffer B from 13 to 23 min, and a linear gradient to 100% Buffer A from 23 to 24 min. NAD⁺ eluted as a sharp peak at 15 min and was quantitated based on the peak area compared to a standard curve and normalized to tissue weight of frozen liver or small intestine tissues.