Isolation and identification of hydroxyl–platelet-activating factor from natural sources

Abstract

Platelet-activating factor (PAF), a potent inflammatory mediator that has previously been detected in elevated levels in inflamed gingival tissues, in gingival crevicular fluid (GCF) and in saliva, is implicated in periodontal disease. The biologically active phospholipid detected in gingival crevicular fluid is a hydroxyl–PAF analogue. In a preliminary study this bioactive molecule was detected for the first time in human blood derived from volunteers with chronic periodontitis as well as from periodontally healthy volunteers. Compounds isolated from natural sources as well as synthetic ones have been reported as biologically active lipids with physiological importance based on the fact that they induce platelet aggregation with EC 50 values ranging from 100 to 0.01 μM through interaction with G-protein-coupled receptors like the PAF receptor, leading to altered signal transduction. In this study, the existence of hydroxyl–PAF analogue in human blood was further studied as well as its distribution in plasma and in blood components. The existence of hydroxyl–PAF analogue was also investigated in samples from rabbit blood hen's egg yolk. The hydroxyl–PAF analogue was purified by high-performance liquid chromatography, detected by biological assays and identified by electrospray MS analysis. Quantitative determination of PAF and hydroxyl–PAF analogue (expressed as PAF-like activity) showed a statistically significant increase in the ratio of plasma hydroxyl–PAF analogue levels to plasma PAF levels in volunteers with periodontitis. Moreover, hydroxyl–PAF analogue was also detected in rabbit blood and hen's egg yolk samples. These data support that this bioactive lipid may play a role in oral inflammation and suggest PAF as a member of a lipid molecule family with different structures and from different sources which share the same or similar biological activities, apparently with different physiological roles in human and animals.