IADR Abstract Archives
Fluoride and MFP Penetration into Bacterial Cell Using F-19 and P-31 NMR
Objectives: Fluoride (F) and Monofluorophosphate (MFP) are known anti-caries agent. Questions remain on the degree and form of penetration of these ions into bacterial cell. F-19 and P-31 NMR allow for selective detection of both of these agents and their possible interaction within the cell.
Methods: E. coli and Streptococcus mutans were grown in an enriched medium, harvested, washed twice and re-suspended in buffered artificial saliva containing either no treatment, MFP or F. Afterwards exposed cells were washed and centrifuged 3-times in buffer solution to remove surface fluorides and then sonicated and centrifuged at ultra-high speeds. The resulting pellets were re-suspended in buffered/D2O media to transfer into NMR tubes.
Results: At modest concentrations of agents, distinct peaks could be detected in samples for both free Fluoride 500 cps (120 ppm) and doublet MFP 500 cps (60 ppm). Further, two much larger F-19 peaks 500 cps (170; 200 ppm) were identified, likely indicating bound fluoride to the intracellular matrix. Further using P-31 NMR, MFP has a distinct doublet, which bracketed the phosphate peak at 500 cps (4.2 ppm). Incorporation of both agents into the cells was found to be much lower with Streptococcus mutans than E-coli.
Conclusions: Although these studies are largely limited to modest concentrations and presently focused on the easier bacterial system of E. coli, on-going experimentation with Streptococcus mutans at lower agent concentrations is underway in order to both quantify and determine the form of binding that has been observed.
Methods: E. coli and Streptococcus mutans were grown in an enriched medium, harvested, washed twice and re-suspended in buffered artificial saliva containing either no treatment, MFP or F. Afterwards exposed cells were washed and centrifuged 3-times in buffer solution to remove surface fluorides and then sonicated and centrifuged at ultra-high speeds. The resulting pellets were re-suspended in buffered/D2O media to transfer into NMR tubes.
Results: At modest concentrations of agents, distinct peaks could be detected in samples for both free Fluoride 500 cps (120 ppm) and doublet MFP 500 cps (60 ppm). Further, two much larger F-19 peaks 500 cps (170; 200 ppm) were identified, likely indicating bound fluoride to the intracellular matrix. Further using P-31 NMR, MFP has a distinct doublet, which bracketed the phosphate peak at 500 cps (4.2 ppm). Incorporation of both agents into the cells was found to be much lower with Streptococcus mutans than E-coli.
Conclusions: Although these studies are largely limited to modest concentrations and presently focused on the easier bacterial system of E. coli, on-going experimentation with Streptococcus mutans at lower agent concentrations is underway in order to both quantify and determine the form of binding that has been observed.
