Effect of polyethylene glycol on lipid peroxidation in cold-stored rat hepatocytes

Abstract

A mechanism suggested to cause injury to preserved organs is the generation of oxygen free radicals either during the cold-storage period or after transplantation (reperfusion). Oxygen free radicals can cause peroxidation of lipids and alter the structural and functional properties of the cell membranes. Methods to suppress generation of oxygen free radicals of suppression of lipid peroxidation may lead to improved methods of organ preservation. In this study we determined how cold storage of rat hepatocytes affected lipid peroxidation by measuring thiobarbituric acid reactive products (malondialdehyde, MDA). Hepatocytes were stored in the UW solution ± glutathione (GSH) or ± polyethylene glycol (PEG) for up to 96 h and rewarmed (resuspended in a physiologically balanced saline solution and incubated at 37 °C under an atmosphere of oxygen) after each day of storage. Hepatocytes rewarmed after storage in the UW solution not containing PEG or GSH showed a nearly linear increase in MDA production with time of storage and contained 1.618 ± 0.731 nmol MDA/mg protein after 96 h. When the storage solution contained PEG and GSH there was no significant increase in MDA production after up to 72 h of storage and at 96 h MDA was 0.827 ± 0.564 nmol/mg protein. When freshly isolated hepatocytes were incubated (37 °C) in the presence of iron (160 μM) MDA formation was maximally stimulated (3.314 ± 0.941 nmol/mg protein). When hepatocytes were stored in the presence of PEG there was a decrease in the capability of iron to maximally stimulate lipid peroxidation. The decrease in iron-stimulated MDA production was dependent upon the time of storage in PEG (1.773 nmol/mg protein at 24 h and 0.752 nmol/mg protein at 48 h). In the absence of PEG, iron-stimulated MDA formation was nearly maximal at all times of storage. These results show that lipid peroxidation is stimulated by cold storage of hepatocytes. Inclusion of PEG in the storage medium suppressed lipid peroxidation suggesting that PEG is accumulated, in a time-dependent manner, by hepatocytes (either into the plasma membrane or into the cell cytosol) and either scavenges oxygen free radicals or alters the availability of lipids to these radicals. PEG may be a useful additive to organ preservation solutions.