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Platelet-activating factor is the term used to denote a class of extremely potent lipid mediators that consist predominantly of 1-O-alkyl- and 1-O-acyl-2-acetyl-sn-glycero-3-phosphocholines. A method has been devised for rapid isolation of these acetylated phospholipids by solid-phase extraction prior to direct derivatization with pentafluorobenzoic anhydride and analysis by gas chromatography (GC)/electron-capture mass spectrometry. Recovery through the entire method (lipid isolation, derivatization, and purification) typically ranged from 70% to 85%. Using the direct derivatization procedure described here, the practical limit of detection for each of the standard alkyl- and acyl-platelet-activating factor homologs was 1 fmol injected into the GC. Results from the application of the method to the analysis of alkyl and acyl homologs of platelet-activating factor isolated from stimulated human umbilical vein endothelial cells are presented, exhibiting excellent accuracy and precision for a wide range of tissue levels of this class of potent autacoids.

Alkyl-PAF induced two components of polymorphonuclear leukocyte (PMN) priming for enhanced fMLP- and rC5a-induced superoxide anion (O2-) production. Component A priming had a shallow, linear alkyl-PAF concentration-response slope (10 pM-1 nM), and component B priming had a significantly steeper concentration-response slope (1-100 nM alkyl-PAF). Whereas the extent of component B priming decayed significantly within 5-10 min after pretreatment of PMNs with alkyl-PAF, component A priming was completely stable. WEB 2086, a specific and potent PAF receptor antagonist, abolished component A priming when PMNs were simultaneously stimulated with alkyl-PAF and either fMLP or rC5a but only partially reduced component B priming. However, whereas WEB 2086 also obliterated component A priming when PMNs were pretreated with alkyl-PAF for 2.5, 5, or 10 min prior to fMLP stimulation, WEB 2086 had little or no inhibitory effect on component B priming. Paradoxically, WEB 2086 significantly augmented alkyl-PAF-induced component B priming for enhanced rC5a-induced PMN O2. production yet concomitantly obliterated component A priming. PMN priming by acyl-PAF (1 nm-1 micron) had characteristics identical to those of alkyl-PAF-induced component A priming. These studies suggest that there are at least two effector pathways modulating alkyl-PAF-induced PMN respiratory burst priming. They are also consistent with the notion that component A priming is initiated via high-affinity PAF receptors and component B priming is mediated through low-affinity PAF receptors; and whereas alkyl-PAF interacts with both high- and low-affinity PAF receptors, both acyl-PAF and WEB 2086 preferentially bind to the high-affinity PAF receptors.

Platelet-activating factor (PAF), a family of phospholipid autacoids with potent pro-inflammatory activities, is present in saliva. The current study has quantitated various species of PAF isolated from normal human mixed saliva. Choline-containing, sn-2 acetylated phospholipids with sn-1 ether- or ester-linked fatty alcohol/acid moieties (alkyl-PAF or acyl-PAF, respectively) were evaluated after direct derivatization with pentafluorobenzoic (PFB) anhydride. Individual species of PFB-derivatized PAF were separated by gas chromatography prior to mass spectral analysis; quantitative estimates of six different species of PAF in saliva were made by comparison to corresponding authentic, synthetic PAF standards. In each saliva sample, all six species of PAF were readily detected by this facile procedure. The predominant PAF was 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine or 16:0-alkyl-PAF (0.75 +/- 0.09 pmol/ml saliva; mean +/- S.E.; n = 5) which represented only 30.4 +/- 1.5% of the total PAF. Substantial amounts of 18:1- and 18:0-alkyl-PAF and 16:0-acyl-PAF were also identified (0.52 +/- 0.07, 0.35 +/- 0.06, and 0.35 +/- 0.02 pmol/ml saliva, respectively). In summary, mass spectrometric analysis of PAF after direct derivatization with PFB anhydride has revealed that at least six different species of PAF are present in normal human mixed saliva. This structural diversity may represent an important aspect of homeostasis in the healthy oral cavity.

Platelet-activating factor (PAF), a potent phospholipid inflammatory mediator, is increased in the mixed saliva of subjects with periodontal disease and correlates with the extent of oral inflammation. The present study was designed to provide a longitudinal evaluation of the effect of initial periodontal therapy (home care instruction, prophylaxis, and scaling/root planing) on salivary PAF levels in chronic adult periodontitis patients (n = 15). Mixed saliva was collected prior to, during, and after initial therapy and was utilized to assess PAF levels after lipid extraction and fractionation as well as to histologically assess the number of polymorphonuclear leukocytes (PMN). PAF activity was determined in bioassay relative to authentic PAF (1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine; 16:0-alkyl-PAF). Initial salivary PAF levels (12.1 +/- 2.8 pmole equivalents of 16:0-alkyl-PAF/ml saliva; mean +/- SE) decreased following supragingival plaque control (9.6 +/- 2.4) and were further reduced following scaling and root planing (5.7 +/- 1.4). In parallel, salivary PMN levels were significantly reduced and clinical estimates of periodontal disease were significantly improved; i.e., there was a decrease in the percentage of sites with both bleeding on probing (from 46.1 +/- 4.6% of sites at pretreatment to 25.9 +/- 2.6% after scaling and root planing) and probing depths > or = 4 mm (from 16.7 +/- 1.9% of sites to 10.3 +/- 1.2%). Thus, initial periodontal therapy reduced salivary PAF levels in concert with improvements in clinical estimates of marginal and submarginal periodontal inflammation suggesting that PAF may participate in inflammatory events during periodontal tissue injury and disease.

Platelet-activating factor (PAF), a potent phospholipid mediator of inflammation, has been previously identified in inflamed gingival tissues and gingival crevicular fluid. However, the role of PAF in oral pathobiology remains unknown. The purpose of the present study was to evaluate the relationship between salivary PAF levels and the severity of periodontal inflammation. PAF activity in lipid extracts of whole (mixed) saliva collected from 69 untreated subjects immediately prior to routine oral evaluation was determined by platelet bioassay. Significant positive correlations were observed between the level of PAF in saliva and measures of periodontal inflammation, i.e., the percentage of periodontal probing depths greater than 4 mm, the number of periodontal bleeding sites, and the number of histologically identified polymorphonuclear neutrophilic leukocytes (PMN) in saliva. Moreover, when subjects were subdivided into groups on the basis of periodontal probing depths, a significant correlation was observed between salivary PAF levels and the extent of periodontal disease, i.e., PAF levels in saliva progressively increased from the healthiest group to the most severely affected group. Thus, salivary PAF levels correlate with the severity of periodontal inflammation. These results support the hypothesis that this pro-inflammatory phospholipid autacoid may participate in the pathogenesis of periodontal tissue injury and disease.

The physiologic responses during prolonged exposure to platelet activating factor (PAF) are largely unexplored. Thus, the cardiopulmonary and intravascular effects of a sustained infusion of 1-O-hexadecyl-2-acetyl-sn-glycero-3- phosphocholine (C16:0-AGEPC; 0.159 nmole/kg/min for 120 min) were characterized in anesthetized rabbits. Within minutes, a transient period of tachypnea developed and was followed by 30 min of stable ventilation. Subsequently, both respiratory rate and tidal volume irreversibly increased. The latter pulmonary ventilatory alterations were preceded by lung mechanical changes, i.e., dynamic lung compliance (CLdyn) decreased 21.6 +/- 3.4% (mean +/- SE) and total pulmonary resistance (Rpulm) increased 25.1 +/- 8.5%. In contrast to CLdyn which partially recovered during infusion, Rpulm remained elevated throughout the study, Concurrent with these pulmonary alterations, significant cardiovascular changes also occurred. Right ventricular hypertension developed immediately and was maximal within 7.9 +/- 0.9 min; this hypertension persisted throughout the infusion period but rapidly reversed thereafter. Mean arterial pressure (MAP) decreased 37.1 +/- 5.8% within 31.4 +/- 2.5 min and then remained at this level. The patterns and extent of alterations in left ventricular pressure, +dP/dtmax, and -dP/dtmax paralleled the changes in MAP and were accompanied by a progressive decrease in left ventricular end-diastolic pressure. These cardiopulmonary effects of C16:0-AGEPC developed in association with prolonged thrombocytopenia and intravascular platelet activation. In contrast, C16:0-AGEPC-induced neutropenia at 5 min was reversed within 60 min and was followed by sustained neutrophilia. In combination, these data suggest that the continuous biosynthesis and release of PAF in vivo could modulate significant, persistent pathophysiological alterations.(ABSTRACT TRUNCATED AT 250 WORDS)