Diphosphoinositide and triphosphoinositide in animal tissues. Extraction, estimation and changes post mortem

Abstract

A method is presented for the determination of the di- and tri-phosphoinositide in animal tissues. The polyphosphoinositides are quantitatively extracted into chloroform-methanol-hydrochloric acid solvent after a preliminary chloroform-methanol (11, v/v) extraction to remove the bulk of the other phospholipids. On washing this extract with [image]-hydrochloric acid the polyphosphoinositides pass completely into the lower chloroform-rich phase. Their concentrations in the lower phase are determined by chromatography on formaldehyde-treated paper or chromatography and ionophoresis of the acid hydrolysis products. When guinea-pig brain is extracted by the method of Folch (1942), considerable hydrolysis of the triphosphoinositide and accumulation of diphosphoinositide occurs during the initial acetone extraction. The tri- and di-phosphoinositide contents of rat and guinea-pig brain decline substantially within a few minutes after death. The concentrations of tri- and di-phosphoinositide in rat brain are not changed by insulin-hypoglycaemia or electrical stimulation. Examination of frozen rat tissues showed that the brain contained the highest concentrations of polyphosphoinositides. Much smaller amounts are present in kidney, and only trace quantities in liver and lung. None could be detected in spleen, heart and skeletal muscle.