Hepatic Lipid and Antioxidant Assessment

Hepatic TG, cholesterol, and FFAs were determined enzymatically using commercial kits (FUJIFILM Wako Diagnostics, Richmond, VA) as previously described (20 (link), 21 (link)). Hepatic collagen content was determined using a commercial kit (QuickZyme Biosciences; catalog no.: QZBTOTCOL1), following instructions with minor modifications. In brief, about 80 mg of snap-frozen liver was homogenized in 1 ml water, and an equal volume of 12 N HCl added to the homogenate and hydrolyzed at 95^oC for 20 h. Collagen content in the mixture was measured spectrometrically and normalized to protein content. Hepatic lipid hydroperoxide and glutathione levels were measured on fresh liver samples after homogenization using a commercial kit (catalog Nos.: 705002 and 705003; lipid hydroperoxide assay kit [catalog no.: 703002]; glutathione assay kit [Cayman Chemical, Ann Arbor, MI]). Hepatic thiobarbituric acid reactive substances (TBARS) were determined by Parameter™ TBARS kit (R&D Systems; catalog no.: KGE013) following the manufacturer's instructions with modifications for use on lipid-rich samples. In brief, liver lipids were extracted by chloroform:methanol (2:1). The organic phase was dried under nitrogen and redissolved in 1% Triton. TBARS in the mixture were determined spectrometrically following the manufacturer's instructions.