PHOSPHOLIPID METABOLISM IN THE HYPOTHERMIC LIVER

Abstract

39 The activity of phospholipase A2 (PLA2) on membrane-bound phospholipids (PLs) during cold storage (CS) is a cause of liver injury due to: 1)loss of membrane structural and functional integrity, 2)generation of cytotoxic level of free fatty acids, and 3)formation of detergent-like, lysoPL compounds. In this study we show that not only are PLA2 active during CS of the liver, but also fatty acid acylation of PLs occurs rapidly. The continued activity of enzymes involved in PL metabolism may be a major cause of membrane alterations in the CS liver that leads to organ failure on reperfusion (transplantation). Rat hepatocytes (Hc) were CS (4°C) for up to 48 hours and rate of incorporation of radiolabelled arachadonic acid (AA) into specific PLs measured. PLs were extracted and separated by conventional thin layer chromatography (TLC) methods. The isolated and identified PL was used for determination of amount of radiolabelled AA. The results shown are for at least three separate Hc preparations. (PC = phosphatidylcholine, PE = phosphatidylethanolamine) Table The rate of incorporation of AA into specific PL species was not decreased by hypothermia. Also, there was no significant loss of PL species as judged by phosphorus analysis of the TLC spots. The rate of incorporation into PC, PE, PS (not shown) and PI (not shown) was nearly complete within one hour of incubation at both 37°C and 4°C in both L-15 tissue culture medium or UW solution. The incorporation was ATP dependent (blocked by KCN and iodoacetate) indicating a role for the ATP-dependent fatty acid acyltransferase in the incorporation of AA into PLs. Also, the incorporation was blocked by PLA2 inhibitor (trifluoroperazine) showing the role of PLA2 in the reaction. Thus, the acylation-deacylation of fatty acids that occurs at normothermia, occurs at a similar rate at hypothermia (4°C). The implications of this for liver preservation are: 1)acylation-deacylation is an important energy consuming reaction that can contribute to the energy deficit in the cold stored liver, 2)acylation-deacylation will lead to a restructuring of cellular and organelle membranes, contributing to abnormal cellular transport functions, and 3)PLA2 activity is not significantly suppressed by hypothermia. We will discuss how the effects of hypothermic preservation on liver PL metabolism can lead to methods to improve organ preservation.Table