Interference caused by acid extraction in the study of diacylglycerol in platelets

A neutral mixture of chloroform and methanol was compared to an acidic mixture of these solvents for the extraction of diacylglycerol from platelets labelled with 3H-arachidonic acid. Using a neutral solvent we found that thrombin caused a rapid increase in the radioactivity of diacylglycerol. With an acidic solvent there was 10 times more background radioactive diacylglycerol, but no increase was detected after stimulation with thrombin. Acidic extraction, but not neutral extraction, caused a small percentage of phosphatidylinositol and phosphatidylcholine to hydrolyse and form diacylglycerol. The extent of hydrolysis accounted for the greater amount of radioactive diacylglycerol found after acidic extraction of radiolabelled platelets. In addition, when platelets were extracted by the acidic solvent a modified form of hydroxy-heptadecatrienoic acid appeared, and thin-layer chromatography in two dimensions was required to separate it from diacylglycerol. It is therefore important to use a neutral extraction method when studying diacylglycerol in platelets.