The reevaluation of the ferric thiocyanate assay for lipid hydroperoxides with special considerations of the mechanistic aspects of the response

The mechanistic aspects of the spectrophotometric method of analysis of lipid hydroperoxides (LOOH) based on the oxidation of ferrous to ferric ion and subsequent complexation of the latter by thiocyanate are considered. The method of analysis, as revised by us, was carried out in the same: solvent that had been used far the extraction of lipids from the sample, a deoxygenated chloroform:methanol or a dichloromethane:methanol (2:1, v/v) mixture, and used a single solution containing both reagents, Fe2+ and SCN-, for developing the response. In that solvent, total lipids up to 5 mg/ml did not interfere, and linear increase of the absorbance of ferric thiocyanate complex was obtained up to 2 . 10(-5) M LOOH. Molar absorptivity of the ferric thiocyanate complex expressed per mol of LOOH was determined as 58,440 M(-1) cm(-1), based on the average of four ferric ions produced by each LOOH molecule. The estimated lowest detectable limit was about 170 pmol LOOH/ml of analyzed solution, which corresponded to about 50 mu mol LOOH/kg lipid in complex natural mixtures. In addition to good sensitivity, and in contrast to some other more popular spectrophotometric assays for LOOH, the method is responsive also to hydroperoxides of mono- and di-unsaturated fatty acids. The method, thus, provides an easy, rapid, sensitive, and complete measure of hydroperoxidation of lipids.