Phospholipid metabolism of hypothermically stored rat hepatocytes

Isolated rat hepatocytes were suspended and stored in either Liebovitz-15 m edium (37 degrees C or 4 degrees C) or University of Wisconsin (UW) solutio n (4 degrees C) containing [H-3] arachidonic acid (AA), At varying times, m embrane phospholipids were separated by thin layer chromatography. AA label ed phospholipids similarly at both 4 degrees C and 37 degrees C. Analysis o f the ratios of [H-3] AA and [C-14] glycerol incorporated into phosphatidic acid or other phospholipids in dual-labeled cells indicated that the deacy lation/reacylation cycle was the major route of AA incorporation at hypothe rmia. This was supported by showing that blocking phospholipase A(2) (PLA(2 )) activity by trifluoperazine suppressed AA incorporation into phospholipi ds. PLA(2) activity, measured by determining the release of AA, was slow du ring 48-hour cold storage, but increased significantly when ATP was deplete d by inhibition of mitochondria and glycolysis. In the whole rat liver, the re was no significant loss of phospholipids during 48-hour storage (total p hospholipids [mu mol phosphorus/L/mg] : 0.197 +/- .001 at 0 hours) unless e nergy blockers were used (0.155 +/- .005 at 48 hours) or glycogen depleted by fasting the rat (0.167 +/- .001 at 48 hours). This study shows that a ne t PLA(2) stimulated hydrolysis of phospholipids is seen only when ATP is de pleted and its generation from anaerobic glycolysis inhibited. Thus, PLA(2) hydrolysis of phospholipids is not a significant cause of liver cell injur y during cold storage when livers are obtained in optimal condition. Howeve r, conditions affecting the generation of ATP during cold storage could alt er PLA(2) leading to membrane damage.