The aim of the present investigation is to purify, characterize, and determine the chemical and biological properties of Porphyromonas gingivalis free dihydroceramides (fDHC). Effects were tested by examination of human gingival fibroblast morphologic changes in addition to their synthesis of prostaglandin E2 (PGE2) and prostaglandin F2α (PGF2) produced in vitro. Free dihydroceramides as well as phosphoglycerol dihydroceramide (PGDHC), and de-esterified phosphoglycerol dihydroceramide (PGDHC-C15:0) were obtained by total lipid extraction of P. gingivalis followed by high performance liquid chromatography (HPLC). Confirmation and structural analysis was obtained by electrospray mass spectrometry (ES-/+MS) and nuclear magnetic resonance spectroscopy (NMR). A synthetic free dihydroceramide (A) was obtained, and its purification and structural analysis was similar. Ethanol solvent served as a negative control, and PGDHC served as a positive control. HGF were obtained from tissue samples of two periodontally healthy volunteers during clinical crown lengthening procedures, and cell cultures were introduced to 35 mm wells previously coated with ethanol or 20g of each lipid/well. Additional experimentation was carried out to investigate fibroblast response to different concentrations of P. gingivalis fDHC. These experiments were carried out in a similar manner except P. gingivalis fDHC were studied at 0.2, 2, and 20 g/35 mm well. After one hour, 30 ng of interleukin-1 (IL-1) was added to each sample. After 24 hours, supernatants were harvested and 100 ng of both deuterated prostaglandin E2 (DPGE2) and prostaglandin F2α (DPGF2) were added. Analyses were performed by derivatization of samples followed by gas chromatograph mass spectrometry (GC-MS). HGF synthesis of PGF2α was unaffected for all lipids tested. IL-1 mediated HGF synthesis of PGE2 was greater for all treatment groups compared to control; however, statistical significance was only achieved for PGDHC and P. gingivalis fDHC (p < .05). P. gingivalis PGDHC and fDHC treated HGF resulted in similar PGE2 synthesis approximately 1.8 fold compared to ethanol vehicle. Morphologic changes suggestive of HGF atrophy and apoptosis were evident only for PGDHC, and this was noted for the majority of PGDHC experiments. These findings suggest that IL-1 mediated HGF secretion of PGE2 can occur even in the absence of a phosphate linkage and 15-carbon isobranched methyl ester, but these structural elements seem to play an important role in the induction of cellular morphological changes observed in phase contrast microscopy.
Chapokas, Andrew Robert, "Characterization of Chemical and Biological Properties of Purified Porphyromonas gingivalis Free Dihydroceramides" (2008). SoDM Masters Theses. 161.