Inositol phospholipid levels of rat forebrain obtained by freeze-blowing method

Four different techniques of handling rat brain prior to lipid extraction and assay were tested to investigate the levels of inositol phospholipids in the brain. In these four techniques, the rat forebrains were either (1) freeze-blown followed by being preserved in liquid N2, (2) subjected to microwave irradiation prior to decapitation, (3) removed and frozen in liquid N2, or (4) removed at room temperature and subjected to lipid extraction as rapidly as possible. There was little change in phosphatidylinositol levels under any of these conditions; however, higher levels of phosphatidylinositol 4-phosphate were observed in freeze-blown and microwave-irradiated samples compared to the other samples. Even more striking differences were seen in phosphatidylinositol 4,5-bisphosphate fractions. The highest level of this lipid, 763 +/- 39 nmol/g tissue, which was obtained from freeze-blown samples, was more than 2-fold higher than that of the lowest values which were obtained by extraction without prior inactivation. These results indicate that the values of phosphatidylinositol 4,5-bisphosphate in brain in situ are higher than those generally reported, and that the freeze-blowing method has an advantage for further investigation of inositol phospholipid metabolism in brain due to the rapid breakdown of these compounds.