Determination of lipid peroxidation in desiccated red blood cells

Lipid peroxidation of biological membranes is one of the most studied indicators of oxidative stress. Very little is known about the extent of oxidative damage that occurs during the desiccation of mammalian cells. The objective of this study was to modify and validate a method for the determination of lipid peroxidation in desiccated red blood cells (RBCs). The measurement of malondialdehydes (MDAs), the decomposition products of oxidized polyunsaturated fatty acids, is commonly used as a method for the quantification of lipid peroxidation. The method is based on the reaction of MDAs with thiobarbituric acid, which forms a thiobarbituric acid reactive substances (TBARS) chromophore. Validation of the method to dried RBC specimens included the optimization of an NIDA standard curve, spiking studies, measurement of TBARS in lyophilized RBCs, and reexamination of the reagents and processes that are associated with this assay. The recovery of MDA standards, which were lyophilized with RBCs was reasonable (72.96% +/- 8.85%), as drying and rehydration are associated with sample loss. Significant TBARS concentrations were obtained during lyophilization, dry storage for up to 72 h, and rehydration of RBCs specimens. The efficacy of the antioxidant tert-butyl hydroperoxide in reducing sample autoxidation during the TBARS assay is questionable due to its poor solubility in aqueous solution. Investigation of the interference caused by the 453-nm peak in the TBARS assay revealed that the origin of the peak is, at least in part, related to hydroperoxides. The data shows that the TBARS assay can be applied to desiccated RBC specimens. It also provides first evidence regarding the relationship between oxidative damage and desiccation. The modification of the MDA assay to dried cells will contribute to understanding the mechanism of damage that occurs during dehydration, dry storage and rehydration of dry biomaterials.