Tbars parameter kit

The lipid peroxidation product Malondialdehyde (MDA) was quantified as Thiobarbituric Acid Reactive Substances (TBARs). Cells, seeded in 6-well plates and treated with the materials for 24h, were lysed and samples acidified before being tested using the TBARs Parameter kit (R&D System, Abingdon, UK) according to manufacturer’s instructions. Results were calculated according to absorbance (530–540 nm) readings obtained with standards and expressed as TBARs (μM).

Thiobarbituric acid reactive substances (TBARS) formation was assessed, using the TBARS Parameter Kit (R&D systems, Minneapolis, MN, USA), following the manufacturer’s instructions. Two hundred micrograms of mitochondria were lysed, the proteins were precipitated using trichloroacetic acid (TCA) and pelleted at 12,000 g, room temperature, 4 min. The supernatants were transferred to fresh tubes. Samples (75 µL) were incubated with 37.5 µL of thiobarbituric acid in a 96-well plate for 3 h at 50 °C. Absorbances were read at 532 nm on a SpectraMax 190 microplate reader (Molecular Devices, Sunnyvale, CA, USA) and TBARS quantification was performed using a standard curve provided in the kit. Oxidized samples were compared to control mitochondria that were incubated under the same conditions in PBS devoid of TBHP.

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.