IN VITRO AND IN VIVO EFFICACY COMPARISON OF THREE CYANIDE ANTIDOTE CANDIDATES AND FLUORESCENT METHOD DEVELOPMENT FOR MEASURING THE SELECTED CYANIDE ANTIDOTE DIMETHYL TRISULFIDE AT LOW CONCENTRATIONS
Gaspe Ralalage, Ramesha Dilhani
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Cyanide (CN) is a toxic molecule that inhibits oxygen utilization by cells. Thiosulfate converts CN- into the less toxic thiocyanate (SCN-), a reaction that is catalyzed by rhodanese (Rh). The comparison of sulfur donor (SD) efficacy (in vitro) and antidotal efficacy (in vivo) of different SDs (SD1, SD2 and SD3) is described in the first part. The in vitro SD efficacy was monitored by quantifying the formation of SCN- with and without Rh at physiological pH (7.4) and the optimum pH for Rh (8.6). The in vitro SD efficacy without Rh at pH 7.4 and with Rh at both pH values varies in the order of SD1 > SD2 > SD3. The in vivo antidotal efficacies were expressed and compared using the antidotal potency ratios (APR) (APR= CN LD50 with antidote/CN LD50 without antidote). Mice were injected with CN and SD1 and the APR value was calculated. The antidotal efficacy varies in the order of SD2 > SD1 > SD3. The in vitro SD efficacy values and in vivo antidotal efficacy values were different due to several pharmacokinetic reasons. In the second part, a fluorescent method to detect low concentrations of DMTS (below 1 µg/mL) in blood samples is described. DMTS is a sulfane sulfur type CN antidote. Sulfane sulfur probe (SSP4) is a probe that selectively binds with sulfane sulfurs. Upon the reaction of SSP4 and DMTS, a fluorescent product is expected. The mixture of SSP4 and DMTS was scanned to determine its excitation and emission characteristics using a fluorescence spectrophotometer. The fluorescent product yielded from the reaction between SSP4 and DMTS exhibited peak absorbance at 280 nm and peak emission at 309 nm. A calibration curve was obtained at λex/λem=280/309 nm for fluorescence intensity vs. DMTS concentrations (R2=0.9935). The mixture of SSP4 and DMTS/ACN was then analyzed using an HPLC-fluorescence experiment with λex/λem=280/309 nm. A fluorescent peak with a retention time of 11.1 minutes showed good linear correlation with DTMS concentration in simple aqueous solutions of DMTS (R2=0.993), and in DMTS-spiked blood (R2=0.9995).