[Densitometric quantitation of platelet activating factor and other phospholipids in human saliva using enzyme...

A sensitive quantitative analysis by thin layer chromatography was developed for the determination of platelet activating factor (PAF) and other phospholipids in human saliva. The saliva sample (0.6 ml) was pretreated by diatomite column extraction with chloroform-methanol (95:5, v/v). The extract (20 microliters) was spotted on a TLC plate. The mobile phase was chloroform-methanol-water (65:35:7, v/v). The development proceeded until the mobile phase front reached 8 cm from the spotted point, this process usually required 30 min. After development, phospholipase C and alkaline phosphatase solutions were sprayed on the TLC plate at 45 degrees C to hydrolyze phospholipids. By spraying a mixture of ammonium molybdate and Malachite Green, the produced phosphate was changed to molybdophosphate-Malachite Green aggregate, which gave a blue green spot. The colored spots were scanned at 620 nm by chromatoscanner. A linear relationship was obtained between peak area and PAF concentration in the range from 2 to 100 pmol/spot with a relative standard deviation of 2% (n = 7). By this procedure, lysophosphatidylcholine, phosphatidylcholine and phosphatidylethanolamine in human saliva were also determined sensitively. PAF levels in the range from 40 to 300 ng/ml were found in normal human salivas. Although differences in the total amounts of phospholipids in saliva were found for each healthy volunteer and sampling time, the composition of phospholipids was proved to be virtually constant.