Establishment Of Methods For Measuring Antioxidant Capacity In Serum Based On Oxidation Reduction Potential

Objective:The present study was meant to establish some kinds of accurate, sensitive method with good precision to measure the antioxidant capacity of human serum.Three methods based on the principle of Redox Reaction and Oxidation Reduction Potential(ORP)were established. In the meanwhile,the methods established in this study were used to determine the antioxidant capacity of patients with type 2 diabetes mellitus(T2DM), and AS-PCR was used to detect the CETP TaqIB polymorphism of patients with T2 DM.Goal of the present study was to figure out the association of CETP TaqIB polymorphism and the antioxidant capacity of human serum with T2 DM.Methods:1. Method 1 for measuring antioxidant capacity was established based on the linear relationship between logarithm of concentration ratio of redox couple(I2/KI) and ORP.The value of ORP of electrical couple solution will be changed when serum reacts with a known fixed concentration ratio of I2/KI. In order to measure the accuracy of the method, both serum and vitamin C were applied to react with electrical couple solution of I2/KI. The degree of precision was measured by document of EP5 from NCCLS. The antioxidant capacity of patients with T2 DM was determined by Method 1 to certify thepotential clinical application value of this method.2. Method 2 was established based on the linear relationship between logarithm of concentration ratio of redox couple(Fe3+/Fe2+) and ORP. The value of ORP of electrical couple solution will be changed when serum reacts with a known fixed concentration ratio of Fe3+/Fe2+. In order to measure the accuracy of method 2, both serum and vitamin C were applied to react with electrical couple solution of Fe3+/Fe2+. The multifactor linear regression analysis was used to evaluate the effect of each factor on this method. The antioxidant capacity of patients with T2 DM was determined by Method 2 to certify the potential clinical application value of this method.3. Method 3 was established based on the linear relationship between logarithm of concentration ratio of oxidant hydrogen peroxide and ORP. The value of ORP of oxidant hydrogen peroxide solution will be changed when serum reacts with a known fixed concentration ratio of H2O2. In order to measure the accuracy of Method 3, both serum and vitamin C were applied to react with electrical couple solution of H2O2. The multifactor linear regression analysis was used to evaluate the effect of each factor on this method. The antioxidant capacity of patients with T2 DM was determined by Method 3 to certify the potential clinical application value of this method.4. Total 30 cases of serum from patients with T2 DM and the healthy were collected separately and the antioxidant capacity of the two group were measured by three established methods.CETP TaqIB polymorphism of the two group were detected using AS-PCR.Results:1. There was a linear relationship between logarithm of concentration ratio of redox couple(I2/KI) and ORP(R2=0.9970). On the condition of redox couple solution, there was a linear relationship between serum and ORP(R2=0.9970) and a linear relationship between vitamin C and ORP(R2=0.9980).The precision results showed that the intra-,inter- and days coefficient of variation(CV) of 100% serum was 0.20%, 0.21% and0.43%, and the intra-, inter- and days CV of 50% serum was 0.11%, 0.12% and 0.39%,respectively. The ORP value of T2 DM group was 385.40±5.93 mv and the controlgroup was 380.72±5.54 mv. There was a statistical significance(P<0.05).2. There was a linear relationship between logarithm of concentration ratio of redox couple(Fe3+/Fe2+) and ORP(R2=0.9987). On the condition of redox couple solution,there was a linear relationship between serum and ORP(R2=0.9700) and a linear relationship between vitamin C and ORP(R2=0.9960). The multifactor linear regression analysis was used to measure the effect of serum, Fe3+and Fe2+on ORP and the results were statistically significant(P<0.05) among which, Fe3+ was positively associated with ORP and did the most contribution(β=0.7890), while both serum and Fe2+ were inverse correlation with ORP. The ORP of T2 DM group was 399.17±10.99 mv and the control group was 392.60±8.36 mv. There was a statistical significance(P<0.01).3. There was a linear relationship between logarithm of concentration ratio of H2O2 and ORP(R2=0.9991). On the condition of H2O2, there was a linear relationship between serum and ORP(R2=0.9890) and a linear relationship between vitamin C and ORP(R2=0.975). The multifactor linear regression analysis was used to measure the effect of serum, H2O2 and repeat count on ORP. The contribution of serum and H2O2 to ORP were statistically significant(P<0.05) while repeat count was not statistically significant(P=0.0610). Serum which did the most contribution was inverse correlation with ORP(β=-0.757) and H2O2 was positively associated with ORP. The ORP of T2 DM group was 384.35±11.23 mv and the control group was 380.67±8.98 mv. There was a statistical significance(P=0.015).4. The antioxidant capacity of human serum from 30 T2 DM patients measured by the three methods was significantly lower than that of the healthy and the difference was statistically significant(P<0.05). The TaqIB locus was detected in B1 Bl, BlB2 and B2B2 three genotypes in both the healthy controls and the T2 DM patients. Analysis of the polymorphism showed that the frequency of B1 and B2 alleles and three genotypes was not significantly different in patients and controls(P>0.05). The binary logistic regression analysis showed that no significant association was found between CETP gene TaqIB polymorphism and T2 DM, but the antioxidant capacity of human serum increased the risk of T2 DM significantly(OR=1.029, 95% CI: 1.011~1.048).Conclusions:1. The present study primarily established three methods which through the application of redox couple solution or H2O2 in combination with ORP to measure the antioxidant capacity of human serum. The methods were adapted to measure the antioxidant capacity of human serum and had clinical applications value.2. The antioxidant capacity of human serum from T2 DM patients was significantly lower than that of the healthy which suggested that T2 DM was associated with the antioxidant capacity of human.