Porphyromonas gingivalis Dihydroceramide Lipids Recovered in Carortid Atheroma Samples

Objective: Pophyromonas gingivalis produces unusual phosphorylated dihydroceramide lipids that mediate biological effects through engagement of Toll-like receptor 2 (TLR2).  Because TLR2 is expressed in developing atherosclerotic lesions, the present study examined the recovery of these lipids in carotid atheroma samples.  

Method: Carotid endarterectomy samples were collected in compliance with the UCHC approved IRB protocol.  After thawing, each carotid endarterectomy sample was sectioned to provide a patent common carotid segment and a carotid sinus atheroma segment.  Each control or atheroma sample was extracted for phospholipids according to the procedure of Bligh and Dyer.  An aliquot of each lipid extract (100 ug) was dissolved in HPLC solvent (hexane:isopropanol:water, 6:8:0.75, v/v/v).  Each lipid sample was then eluted over a 3 cm x 4 mm silica gel column interfaced to an AB Sciex 4000 Qtrap instrument.  Samples were eluted at 350 ul/min with a flow splitter interface for mass spectrometric analysis.   Phosphorylated dihyroceramide lipids were quantified using multiple reaction monitoring.  Instrument parameters were optimized using highly enriched preparations of phosphorylated dihydroceramide lipids. 

Result: Five carotid atheroma samples provided paired common carotid and atheroma lipid extracts.  High and low mass phosphorylated dihydroceramide lipids were recovered in all common carotid and paired atheroma samples.  However, the bacterial lipid levels in the control artery lipid extracts were at least tenfold greater than that recovered in paired atheroma lipid extracts. Only the high mass phosphoethanolamine dihydroceramide lipid class was significantly elevated in control artery segments (two way ANOVA with pairwise comparisons). 

Conclusion: P gingivalis phosphorylated dihydroceramide lipids are recovered in carotid atheroma samples but the levels are considerably higher in proximal artery samples without  grossly apparent atheroma formation. However, the patent artery segments exhibited mineral deposition in the vessel wall when sectioned.  We therefore propose that bacterial lipid deposition contributes to the mineralization process in diseased artery walls.