Measurement Of Antioxidant Ability In Serum Based On Redox Titration And Analysis Of Relationship Between Polymorphism Of HLA-DQB1and Antioxidant Ability

Objective：In order to measure the ability of antioxidant in serum and provide a good indexfor diagnosing and treating diseases which were associated with lower oxidationresistance, four methods to measure the antioxidant ability in serum based on redoxtitration were established. Aim at researching the relationship between polymorphism ofHLA-DQB1and antioxidant ability, Antioxidant ability and the polymorphism ofHLA-DQB1by PCR-SSP in58individuals were detected. Also we tested the levels ofnatural antibodies and routine biochemical parameters in these individuals.Methods:1. Based on the reaction principle of routine iodimetry analysis, two assays tomeasure the antioxidant capacity of substances reacting with iodine in serum wererespectively developed using the microtitration method and agar-gel diffusion method.Besides, the accuracy and the stability of these methods were tested by dilution test andreproducibility test. At last these assays were employed by measuring the antioxidantability in older group and younger group.2. On the basis of the classical potassium permanganate titration, two assays tomeasure the antioxidant ability were developed using the microtitration method andagar-gel diffusion method.Also, we tested the accuracy and the stability of thesemethods by dilution test and reproducibility test. At last these assays were employed bymeasuring the antioxidant ability in older group and younger group.3. Specific amounts of the six proteins including bovine serum albumin(BSA), r-globin, protamine, Casein Enzymatic Hydrolysate, human serum albumin(HAS) andlysozyme were dissolved in0.9%normal saline. Concentrations were determined with TOSHIBA120FR using the kit. Serum samples with equivalent concentrations ofprotein were selected. The antioxidant capacities of these specimens were determinedwith these four assays, with a view to identifying the specific components of theantioxidant system such as the organic and inorganic parts that can be measured usingthese assays.4. Blood samples were collected from the First Affiliated Hospital of DalianMedical University. Overall, individuals were divided into two groups according to ageand gender parameters. The younger group was comprised with15males and15females, with an average age of29years. The older group included15males and15females, with an average age of82years. Blood were analyzed using these four assaysimmediately after sampling. At the same time the level of natural antibody and routinebiochemical parameters(ALT, r-GT, AST, LDH, TP, ALB, Glob, A/G, Urea, Crea, TG,Chol, HDL, LDL and glucose) in these individuals were detected in order to analysisthe correlation among the antioxidant ability、natural antibody and these routinebiochemical parameters.5. The allelomorphic genes of HLA-DQB1were determined by polymerase chainreaction (PCR) between the older and the younger group, in order to analyze thecorrelation between the antioxidant ability and the polymorphism of HLA-DQB1.Results:1. A method of measuring the antioxidant ability in serum using the microtitrationmethod based on the permanganate titration was established. The dilution test revealed alinear relationship between serum volume and assay results. The intra-and inter-assayaverage coefficients of variation (CV) were0.929%and0.692%, respectively. Data onthe antioxidative capacities in the older group measured with this method revealedsignificantly lower antioxidant capacity than in the younger group (p<0.05).2. A method of measuring the antioxidant ability in serum using the agar platediffusion method based on the permanganate titration was established. Also, the dilutiontest revealed a linear relationship between serum volume and assay results. The intra-and inter-assay average coefficients of variation (CV) were2.246%%and5.350%,respectively. Data on the antioxidative capacities in the older group measured with thismethod revealed significantly lower antioxidant capacity than in the younger group(p<0.05).3. An assay to measure the antioxidant capacity of substances reacting with iodinein serum was developed using the microtitration method. The dilution test revealed a linear relationship between serum volume and iodine assay results. The intra-andinter-assay average coefficients of variation (CV) were3.329%and7.219%,respectively. Data on the antioxidative capacities in the older group measured with thismethod revealed significantly lower antioxidant capacity than in the younger group(p<0.05).4. An assay to measure the antioxidant capacity of substances reacting with iodinein serum based on the iodometric and agar diffusion method was established. Thedilution test revealed a linear relationship between serum volume and assay results. Theintra-and inter-assay average coefficients of variation (CV) were3.942%and22.390%,respectively. Data on the antioxidative capacities in the older group measured with thismethod revealed significantly lower antioxidant capacity than in the younger group(p<0.05).5. Organic compounds could be measured by these four methods, and the statisticsindicated that the most important antioxidant section in serum may be attributed to theproteins.6. The data on the antioxidative capacities in the older group measured with thesefour methods all revealed significantly lower antioxidant capacity than in the youngergroup; But the natural antibody showed a higher level in the old group, and the value ofP is less than0.05; Also, compared with younger group, there was a significantdifferences in clinical laboratory paramaters between the older group and younger group(P<0.05), the TP, ALB,A/G is lower in the olds, but AST, LDH, Urea, Glucose andChol were higher in the old group.7. In the correction analysis, we found that there is no relation between theantioxidant ability in serum and the level of natural antibody; but there is a significantcorrelation between the ability of antioxidant and the level of LDH, TP, ALB, A/G. Thevalue of p is lower than0.05; HLA-DQB1*05sites have significant influences onantioxidant ability.Conclution:1. The antioxidant capacity in human serum can be determined accurately by thesefour methods.2. HLA-DQB1*05sites have significant influences on antioxidant ability.