Lipid hydroperoxide assay kit

Plasma lipid hydroperoxide levels were determined using a specific kit (Kamiya Biomedical Company, Seattle, WA, USA). Their rationale is based on the reaction of hydroperoxides with a derivative of methylene blue, 10-N-methylcarbamoyl-3,7-dimethylamino-10-H-phenothiazine (MCDP), in a reaction catalyzed by hemoglobin, to give rise to the formation of methylene blue. LPO was quantified colorimetrically by measuring the methylene blue formed. Quantification of LPO in liver homogenate was carried out by means of a lipid hydroperoxide assay kit according to the manufacturer’s instructions (Cayman Chemical, Ann Arbor, MI, USA). It measures the hydroperoxides directly utilizing the redox reactions with ferrous ions. The amount of lipid hydroperoxides was obtained from the linear regression of the standard curve substituting corrected absorbance values for each sample. This procedure eliminates any interference caused by hydrogen peroxide or endogenous ferric ions in the sample and provides a more sensitive and reliable assay for LPO.

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.

Lipid peroxidation was quantified in liver tissues and ER fraction lysates using a lipid hydroperoxide assay kit (Cayman Chemicals, Ann Arbor, MI, USA). In this assay, lipid hydroperoxide was extracted from the samples into chloroform using the extraction buffer provided by the manufacturer. The absorbance of each well was read at 500 nm using a 96 well plate spectrometer (SpectraMax 190, Molecular Devices, Sunnyvale, CA, USA). 13-Hydroperoxy-octadecadienoic acid was used as the standard. The cellular levels of lipid hydroperoxide were calculated as described by the manufacturer.

Lipid hydroperoxides contained in total hepatic extracts from whole liver tissue were quantified using a commercially available lipid hydroperoxide assay kit (Cayman Chemical) according to the manufacturer's instructions. To extract lipids, portions of liver tissue were removed immediately after killing, weighed (average weight, 0.16 g) and immersed in liquid nitrogen for 5 min prior to storage at −80°C. Frozen tissue samples were then placed in glass tubes containing 0.25 mL of water, homogenized for 30 sec using an Omni TH hand held tissue homogenizer, extracted using chloroform/methanol, and assayed for lipid hydroperoxides.
To determine hepatic total lipid content, 0.5 mL of lipid extracts were placed in microfuge tubes and evaporated overnight for complete solvent removal. Lipid weight was determined as the difference in tube weight before addition of extract and after solvent evaporation.

Intracellular lipid peroxidation of aHSCs was fluorescently (ex. 515 nm; em. 553 nm) measured by the determination of the MDA–TBA complex with fluorometeror (Thermo Scientific) using HPLC with LiChrospher column (RP-18, 5μm, Merck), mobile phase of 25 mM Na₂HPO₄–methanol (58/42, v/v) at a flow rate of 1 ml/min. The complex of MDA–TBA was eluted in 4.8 min. A MDA–TBA complex standard curve was constructed for calibration. Additionally, the lipid peroxidation (LPO) assays were performed using a Lipid Hydroperoxide Assay kit (Cayman Chemical). Lipid hydroperoxides were extracted into chloroform and measured by the redox reactions with ferrous ions. Chromogenic reaction was performed at room temperature for 5 min, followed by reading the mixture at 500 nm. The calibration curve was constructed using 13-Hydroperoxy-octadecadienoic acid. Lipid hydroperoxide was expressed as nmol/mg protein.