Ultraviolet and Infrared Spectroscopy for Microdetermination of Oxidized and Unoxidized Fatty Acyl Esters in Cells

Abstract

New methods based on ultraviolet and infrared spectroscopy were developed to quantify oxidized and unoxidized fatty acyl esters (FAE) in cells. For this study, rat hepatocyte cultures (2.5 × 10 6 cells) were submitted to an oxidative stress by a 5-h incubation with iron(III) chelated with nitrilotriacetic acid (100 μM). Control hepatocytes were incubated under the same conditions except in the absence of iron. After cell lipid extraction, oxidized FAE were evaluated by the second derivative of the conjugated-diene (CD) spectrum, which exhibited minima at 233 and 242 nm ascribed to trans,trans (t,t) and cis,trans (c,t) CD isomers, respectively. These minima were quantified in arbitrary units as d 2 A/ dλ 2 hydroperoxide concentration was determined using a linear regression curve obtained from autoxidized linoleic acid micelles. Total (oxidized and unoxidized) FAE were measured by Fourier transform infrared spectroscopy using the absorption band at 1740 cm −1. A highly significant correlation coefficient ( r = 0.992) was found for the standard curve performed with glycerol trioleate expressed as nanomoles fatty acid equivalents. The extent of lipid oxidation could be estimated by the sum of minima at 233 and 242 nm which allowed the calculation of hydroperoxide concentrations. The amount of oxidized FAE was related to the amount of total FAE in the same sample. The ratio of minima at 242 nm (c,t isomers) and 233 nm (t,t isomers) could provide an evaluation of cell antioxidant capacity. A decrease of this ratio would indicate a large depletion of radical termination antioxidants.