Lucigenin

A lucigenin assay was performed as described previously.³³ Snap-frozen livers were homogenized on ice in sucrose buffer (0.3 mol/L sucrose, 10 mmol/L HEPES, 10 mmol/L KCl, 0.1 mmol/L EDTA, 0.1 mmol/L ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, 0.5 mmol/L spermidine, and 1 mmol/L dithiothreitol, pH 7.4) with protease inhibitor, and centrifuged at 1000 × g for 5 minutes. The supernatant was centrifuged further at 100,000 × g for 1 hour at 4°C to obtain membrane-enriched fractions. The pellet was suspended in Krebs buffer (100 mmol/L NaCl, 5 mmol/L KCl, 2 mmol/L CaCl₂, 1.2 mmol/L MgSO₄, 1.0 mmol/L K₂HPO₄, 25 mmol/L NaHCO₃, 20 mmol/L Na-HEPES, and 0.2% glucose, pH 7.4) with protease inhibitor and incubated at 37°C for 30 minutes. Membrane fractions were incubated with 10 μmol/L lucigenin (L6868; Invitrogen) at room temperature for 15 minutes. Then, 100 μmol/L NADPH (Sigma-Aldrich) was added and the chemiluminescence intensity was read with a Pharmingen Monolight 3010 luminometer (BD Biosciences) every minute, for up to 10 counts. Data were normalized to the protein concentration.

NADPH oxidase activity was determined by measuring NADPH‐induced chemiluminescence in the presence of lucigenin (5 μmol/L, Invitrogen) and NADPH (100 mol/L, ICN Biomedicals),19 following manufacturer's instructions. The specificity of the NADPH‐dependent superoxide anion production was verified by treatment with diphenylene iodinium (Sigma).