Free lipid A isolated from Porphyromonas gingivalis lipopolysaccharide is contaminated with phosphorylated dihydroceramide lipids: recovery in diseased dental samples. Infect. Immun. 80:860

Recent reports indicate that Porphyromonas gingivalismediates alveolar bone loss or osteoclast modulation through engage-ment of Toll-like receptor 2 (TLR2), though the factors responsible for TLR2 engagement have yet to be determined. Lipopoly-saccharide (LPS) and lipid A, lipoprotein, fimbriae, and phosphorylated dihydroceramides of P. gingivalis have been reported to activate host cell responses through engagement of TLR2. LPS and lipid A are the most controversial in this regard because con-flicting evidence has been reported concerning the capacity of P. gingivalis LPS or lipid A to engage TLR2 versus TLR4. In the present study, we first prepared P. gingivalis LPS by the Tri-Reagent method and evaluated this isolate for contamination with phosphorylated dihydroceramide lipids. Next, the lipid A prepared from this LPS was evaluated for the presence of phosphory-lated dihydroceramide lipids. Finally, we characterized the lipid A by the matrix-assisted laser desorption ionizationmass spec-trometry (MALDI-MS) and electrospray-MSmethods in order to quantify recovery of lipid A in lipid extracts from diseased teeth or subgingival plaque samples. Our results demonstrate that both the LPS and lipid A derived from P. gingivalis are con-taminated with phosphorylated dihydroceramide lipids. Furthermore, the lipid extracts derived from diseased teeth or subgingi-val plaque do not contain free lipid A constituents of P. gingivalis but contain substantial amounts of phosphorylated dihydro-ceramide lipids. Therefore, the free lipid A of P. gingivalis is not present in measurable levels at periodontal disease sites. Our results also suggest that the TLR2 activation of host tissues attributed to LPS and lipid A of P. gingivalis could actually be medi-ated by phosphorylated dihydroceramides