Optimization And Application Of Method For Measuring Hydroxide Free Radical By Colorimetry

[Objective] To confirm the optimization conditions for measuring hydroxide free radicals by colorimetry in vitro and the actual application significance in order to provide a measure method for filtrating antioxidants in vitro.[Method] Adopting classical response system composed of ferrous sulfate(FeSO₄) and hydrogen peroxide (H₂O₂) and using dimethyl sulfoxide(DMSO) as a trapping reagent of hydroxide free radicals, we improved original assays on a certain extent. Primary influencing factors and suitable range was ascertained by many tries. Influence of three levels FeSO₄, H₂O₂ and DMSO on detecting system was analyzed and optimization arrange in pairs or groups was ascertained by orthogonal experimental design. Feasibility and actual value was validated by measure anti-oxidation ability of thiourea, reduced glutathione(GSH), strawberry extract and metallothionein (MT).[Result] The stability of color was increased when fast blue BB salt was replaced by fast blue B salt and it was not necessary for pyridine in this assay. In this system suitable content of hydrochloric acid was 7.5-10mmol/L, suitable response time was from 10 to 40min, suitable concentration for FeSO₄, H₂O₂ and DMSO were 3.0-4.0mmol/L, 16- 32mmol/L and 40-60mmol/L respectively. The range of FeSO₄, H₂O₂ and DMSO were 0.066, 0.026and 020 respectively, then it was considered that effects of FeSO₄ is most, next is H₂O₂, DMSO is least by orthogonal experimental design. Most reasonable arrangement for FeSO₄, H₂O₂ and DMSO were 3.6mmol/L, 24mmol/L and 60mmol/L in this system. Results was stable and repeated during 3 hours, coefficient of variance (CV) was less than 6 percent. GSH and thiourea had ability of clear hydroxide free radicals, the clear rate of GSH and thiourea were 43.2 and 77.5 percent respectively when concentration was arrived at same 40mmol/L and the clear ability for hydroxide free radicals, thiourea was much more than GSH. strawberry extract also had ability for clear hydroxide free radicals, clear rate can reached 33.8 percent when volume of strawberry extract was 2ml which was corresponded to 10g strawberry. When different hepatic homogenate was added to detecting system there was a obvious difference in ability of clear hydroxide free radicals between two groups which MT content was different. The clear rate was24.27% for Zinc-induced group which MT content was 76.5μg/ml and it was 7.85%.for control group which MT content was 28.5μg/ml.[Conclusion] The suitable experimental condition is 10mmol/L for hydrochloric acid,3.6mmol/L for FeSO₄, 24mmol/L for H₂O₂ and 60mmol/L for DMSO, response timeis 10 minute in this detecting system which can be used .to filtrate antioxidants invitro and to evaluate the anti-oxidation ability for some biological materials.