Measurement Of Total Antioxidant Capacity Of Organisms And Its Association With GST Gene Polymorphism,Telomere Length And Related Clinical Indicators

Objectives:In order to evaluate the antioxidant capacity of organisms accurately and to explore the pathogenesis of oxidative stress related diseases,we established a new method for accurate and sensitive determination of antioxidant capacity of human urine based on I₂/KI redox couple,and evaluated redox status of clinical diabetic patients.From the perspective of precision and resolution,several methods to determine total antioxidant capacity of organisms were compared.Three methods were selected for combined detection of total antioxidant capacity of human serum,to achieve a comprehensive evaluation of redox state of human body.Determination of total antioxidant capacity of clinical chronic obstructive pulmonary disease?COPD?patients and the elderly were done by the combined detection method.Meanwhile,combined with GST gene polymorphism,telomere length and related clinical indicators,we explored the pathogenesis of COPD,and established a new index to determine the serum antioxidant capacity,i.e.unit albumin antioxidant capacity.Then in order to explore relationship between occurrence of clinical diseases and antioxidant capacity of organisms,the proportion of main antioxidants in serum and urine in total antioxidant capacity and the antioxidant mechanism of these substances were analyzed.Methods:1.A new method was established based on linear relationship between oxidation-reduction potential?ORP?and logarithm of concentration ratio of I₂/KI couple.Value of ORP of a known fixed concentration ratio of I₂/KI will be changed when I₂/KI solution reactes with urine.In order to measure the linearity of the method,both vitamin C and urine with equal difference concentration were applied to react with electrical couple solution of I₂/KI respectively.Referring to the document EP5 in National Committee for Clinical Laboratory Standards?NCCLS?,the precision of the new method was evaluated.The method of ORP was compared with the method of iodine titration to make methodology evaluation.Antioxidant capacity of urine in samples from 30 diabetes patients and 30 healthy subjects was measured to verify the clinical value of this method.2.Exogenous oxidants such as iodine,potassium permanganate and hydrogen peroxide were evaluated in the assay of total antioxidant capacity of serum by employing various detection methods.These methods include electrode potential,microtitration method,and agar diffusion.Based on the evaluation of repeatability,linearity,relative resolution,and detection conditions from seven methods——I₂/KI electric potential,Fe³⁺/Fe²⁺electric potential,H₂O₂ electric potential,iodine starch microtitration,KMnO₄ microtitration,iodine starch agar and KMnO₄ agar,we found that the combination of three assay methods were optimal to determine total antioxidant capacity of serum from diabetes patients and healthy participants.3.Using polymerase chain reaction?PCR?technology,GSTM1 and GSTT1 gene polymorphisms were detected in 33 COPD patients and 33 healthy people.Also,the relative length of telomere gene in them were measured by real time fluorescence quantitative PCR.Total antioxidant capacity?TAC?in serum was determined using I₂/KI potentiometry,KMnO₄ microtitration,and H₂O₂ potentiometric methods.Logistic regression analysis was carried out with biochemical screening indices,which was found to be closely related with the incidence of COPD,and then a further statistical analysis was made.Similarly,the total antioxidant capacity of the aged group and the young group was tested,and the clinical value of the new index was verified.4.By using the combined detection method of serum total antioxidant capacity,the antioxidant capacity of mixed serum and albumin standard with the same albumin concentration of mixed serum,the antioxidant capacity of mixed serum and total protein standard with the same total protein concentration of mixed serum,the antioxidant capacity of mixed urine and uric acid standard with the same uric acid concentration of mixed urine,the antioxidant capacity of mixed urine and urea standard with the same urea concentration of mixed urine,the antioxidant capacity of mixed urine and creatinine standard with the same creatinine concentration of mixed urine were determined.The proportion of albumin and total protein in serum total antioxidant capacity and the proportion of uric acid,urea and creatinine in total antioxidant capacity of urine were calculated based on the measured results.And the possible antioxidant mechanism of albumin was explored based on mass spectrometry.Results:1.There was a linear relationship between logarithm of concentration ratio of I₂/KI and ORP?R²=0.998?.On the condition of redox couple,vitamin C and ORP showed a linear relationship?R²=0.994?.Urine concentration and ORP showed a linear relationship?R²=0.986?.The precision results were in the acceptable range.The measurement results of ORP method were in agreement with the measurement results of iodine titration method.The two methods had a linear correlation?R²=0.9873?.The ORP values of the clinical diabetes group and the control group were found to be statistically significant?P<0.05?.2.Precision of the methods was acceptable and good linear relationships were observed between the measured values and the serum percent concentration.We conducted comparative analysis of assay results,i.e.,calculation of relative resolution,and combing the principle and applicable conditions of each method,we finally selected I₂/KI and H₂O₂ electrode potential,and KMnO₄ microtitration in combination as an optimized assay of total antioxidant capacity.Total antioxidant capacity of serum from30 diabetes patients was lower than that of 30 age-and sex-matched healthy participants and the difference was statistically significant?P<0.05?.3.GSTM1 and GSTT1 gene deletion rate in the COPD group was significantly higher than that in control group?P<0.05?.The differences in serum TAC between the COPD and control groups,GSTM1?+?and GSTM1?-?groups,and GSTT1?+?and GSTT1?-?groups were statistically significant?P<0.001?.Logistic regression analysis showed that the incidence of COPD was closely related to Z?M?and albumin,the regression parameters were statistically significant?P<0.05?.In addition,there was a significant difference in unit albumin antioxidant capacity either between the COPD and healthy groups,or between older and younger groups?P<0.05?,suggesting that this new index would have potential clinical value.Finally,logistic regression and comprehensive statistical analysis showed that it could correctly predict the occurrence of COPD according to three factors——GSTM1,GSTT1 gene expression and telomere length.4.The proportion of albumin?ALB?and total protein antioxidant activity in serum total antioxidant capacity was 63.5%and 82.5%respectively.The uric acid antioxidant activity in urine total antioxidant capacity was 50%,while urea and creatinine had no obvious antioxidant capacity.MALDI-TOF mass spectrometric analysis showed that the increased molecular weight of oxidized albumin was 879Da,which basically coincided with the theoretical analysis result?989Da?.Conclusions:1.The combined detection method of total antioxidant capacity in serum by using I₂/KI electric potential,H₂O₂ electric potential and KMnO₄ microtitration method was established and optimized.It could be used for the determination of serum antioxidant capacity in clinical diabetes and COPD patients and also has clinical application value.2.A new index,unit albumin antioxidant capacity,was established to evaluate the serum antioxidant capacity.It was found that this index will decrease in patients with related diseases,indicating that the index could be used to predict and evaluate the occurrence of related diseases,and have potential clinical application value.3.When the total antioxidant capacity of organism reduced to a certain extent pathologically,it is closely related to the deletion of GSTM1 and GSTT1 gene and the shortening of telomere length.Therefore,it can predict the antioxidant capacity and health level of the body on molecular level.4.Albumin in serum and uric acid in urine are two important antioxidants in these two body fluids,respectively.