Sensitive densitometry for the determination of platelet-activating factor and other phospholipids in human tears

Abstract

A sensitive TLC method is reported for the determination of the platelet-activating factor (PAF) and other phospholipids in human tears. Tear samples absorbed on filter-paper were subjected to diatomite column extraction with chloroform–methanol. The eluent containing phospholipids was spotted on a silica gel plate. After removing lipids other than phospholipids by pre-development with hexane–diethyl ether (4 + 1 v/v), individual phospholipid separation was carried out by development with chloroform–methanol–water (65 + 35 + 7 v/v). Phospholipase C and then alkaline phosphatase solutions were sprayed on the TLC plate at 45 °C to liberate phosphate from each phospholipid. By spray application of a mixture of ammonium molybdate and Malachite Green, the liberated phosphates appeared as blue–green spots of molybdophosphate–Malachite Green aggregate on a yellow–brown background. The absorbance of each spot on the plate was measured at 620 nm with a densitometer. The peak area was found to be linearly related to PAF content in the range 2–100 pmol per spot, the RSD being 2%(n= 7). Approximately 86% of standard PAF added to tears was recovered. By this method, lysophosphatidylcholine (lysoPC), phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in human tears could also be detected with high sensitivity. Each phospholipid in healthy human tears showed a nearly constant content; PAF, lysoPC, PC and PE were present at levels of 26.2, 42.3, 10.0 and 19.7%, respectively.