| Objective:To establish potassium permanganate agar method and iodine starch agar method in good repeatability and linearity to determine the total antioxidants in urine and serum based on redox titration.The measurement of the urinary and serumal total antioxidants of ICU patients was conducted respectively to certificate whether the methods are suitable to be applied in the clinic.The urinary total antioxidants in coronary heart disease patients were determined by iodine starch agar method and the the urinary antioxidant mechanism was explored.The urinary and serumal total antioxidants of unselected patients with tumors were measured by potassium permanganate agar method and iodine starch agar method,the urinary and serumal antioxidant mechanism in tumor diseases were explored and the ROC curve in the diagnosis in tumor diseases was created.The relationship between urinary diffusion area and the indexes in routine urinalysis and serum biochemical test was analyzed,in the meanwhile,the relationshipbetween serumal diffusion area and the indexes mentioned above was analyzed too.The relationship between the urinary diffusion area as well as serumal diffusion area and the SIRT1 gene segments rs7895833 A>G polymorphic sites was discussed.Method:1.The potassium permanganate agar method based on redox titration in Mn7+/Mn2+ was established.The potassium permanganate solution in a certain concentration was mixed with agar solution to create a plate,urine or serum sample was added into the plate to react with the oxidant potassium permanganate in the agar.The area of the urinary or serumal diffusion area was calculated 24 hours later to evaluate the amount of total antioxidants in the sample.The precision and linearity of the method were evaluated under the guidance of document EP5-A2 of NCCLS.The method was applied to determine the amount of serumal total antioxidants of 30 ICU patients and 44 healthy people.2.The iodine starch agar method based on redox titration in I2/I-was established.The iodine starch solution in a certain concentration was mixed with agar solution to create a plate,urine or serum sample was added into the plate to react with the oxidant iodine in the agar.The area of the urinary or serumal diffusion area was calculated 24 hours later to evaluate the amount of total antioxidants in the sample.The precision and linearity of the method were evaluated under the guidance of document EP5-A2 of NCCLS.The method was applied to determine the amount of urinary total antioxidants of 30 ICU patients and 44 healthy people.3.The urinary antioxidants of 44 coronary heart disease patients were determined by the iodine starch agar method and the urinary antioxidant mechanism in coronary heart disease was explored.4.The urinary and serumal antioxidants of 44 unselected people with tumor diseases were determined by the two methods created before,the urinary and serumal antioxidant mechanism in tumor diseases were explored and the ROC curve in the diagnosis of tumor diseases was created.5.The relationship between urinary diffusion area and the indexes in routine urinalysis as well as serum biochemical test was analyzed with Spearman Bivariate Correlations and Linear Regression(backward method).In the meanwhile,the relationship between serumal diffusion area and the indexes mentioned above was analyzed too.6.The relationship between the urinary diffusion area/ serumal diffusion area and the SIRT1 gene segments rs7895833 A>G polymorphic sites was discussed with PCR-CTPP method.Results:1.In the precision experiment of the measurement of urinary total antioxidants in potassium permanganate agar method,the coefficient of variation(CV)of diffusion area for inner-run,between-run and between-day in high level was 3.8%,8.7% and 10.6%,respectively;the CV in low level was 2.6%,9.6% and 14.0%,respectively.In the linear experiment of the measurement of urinary total antioxidants in potassium permanganateagar method,the R2 in the equation of evaluating standard reducing agent Na S2O3 was0.994;the R2 in the equation of evaluating matrix substance was 0.902.In the precision experiment of the measurement of the serumal total antioxidants with potassium permanganate agar method,the CV of diffusion area for inner-run and between-run in high level was 2.9% and 12.3%,respectively;the CV in low level was 3.0% and 5.1%,respectively;the CV of the diffusion area in the two levels was not calculated because the variance between-day was 0.In the linear experiment,the R2 in the equation of evaluating standard reducing agent Na S2O3 was 0.994;the R2 in the equation of evaluating matrix substance was 0.987.The method was applied to determine the total antioxidants of serum in ICU patients and healthy people,the median diffusion area of ICU patients was 1.77 cm2,while it was 2.01 cm2 in healthy people,there was a difference between the two groups,where p= 0.002.2.In the precision experiment of the measurement of the urinary total antioxidants in iodine starch agar,the coefficient of variation(CV)of diffusion area for inner-run,between-run and between-day in high level was 5.1%,9.1% and 8.1%,respectively;the CV in low level were 2.7%,9.5% and 8.1%,respectively.In the linear experiment of the measurement in iodine starch agar method,the R2 in the equation of evaluating standard reducing agent Na S2O3 was 0.999;the R2 in the equation of evaluating matrix substance was 0.995.In the precision experiment measurement of the serumal total antioxidants in iodine starch agar method,the CV of diffusion area for inner-run,between-run an between-day in high level was 2.9%,5.4% and 7.1%,respectively;the CV in low level was 3.6%,5.9% and 14.9%,respectively.In the linear experiment,the R2 in theequation of evaluating standard reducing agent Na S2O3 was 0.999;the R2 in the equation of evaluating matrix substance was 0.990.The method was applied to determine the urinary total antioxidants of ICU patients and healthy people,the median diffusion area of ICU patients was 1.38cm2,while it was 1.13 cm2 in healthy people,there was a difference between the two groups,where p= 0.032.3.In the measurement of the urinary total antioxidants in coronary heart disease patients,the median diffusion area of coronary patients was 0.95 cm2,while the data was 1.13 cm2 in healthy people,there was a difference between the two groups,where p= 0.001.4.In the measurement of the urinary and serumal total antioxidants of unselected people with tumor diseases with the two methods,in the urinary diffusion area of potassium permanganate agar method,the median diffusion area of unselected people with tumor diseases was 1.83 cm2,while it was 1.54 cm2 in healthy people,there was a difference between the unselected people with tumor diseases and healthy people,where p= 0.038;in the urinary diffusion area of iodine starch agar method,the median diffusion area of unselected people with tumor diseases was 1.54 cm2,while it was 1.13cm2 in healthy people,there was a difference between the two groups,where p= 0.016.In the serumal diffusion area of potassium permanganate agar method,the mean diffusion area of unselected people with tumor diseases was 2.02±0.46 cm2,while it was1.97±0.17 cm2 in healthy people,but there was no difference between the two groups,where p= 0.158;in the serumal diffusion area of iodine starch agar method,the mean diffusion area of unselected people with tumor diseases was 3.20±0.40 cm2,while it was2.94±0.46 cm2 in healthy people,there was a difference between the two groups,where p= 0.005.The UMn,UI,SMn and SI were combined to create the ROC curve in the diagnosis of tumor diseases,the regression model was Y=0.850 UI +2.209 SI-7.691,the area under the curve(AUC)of the regression model was 0.787.5.The relationship between the urinary diffusion result and the indexes of the routine urinalysis/ serum biochemical test was analyzed with Spearman Bivariate Correlations,the urinary diffusion result was related to the indexes of urobilinogen,protein,glucose,p H value,specific gravity,turbidity and color in urine,where the p value was 0.048,<0.001,0.005,<0.001,<0.001,0.040 and <0.001,respectively;it was also related to the index glucose,urea,creatinine and albumin,globulin,cholesterol and triglyceride in serum,where the p value was 0.012,<0.001,0.017,0.001,0.016,0.043 and 0.038,respectively.While in the analysis of the relationship between the serumal diffusion result and the indexes of the routine urinalysis as well as serum biochemical test by Spearman Bivariate Correlations,the serumal diffusion result was related to the indexes of urobilinogen and color in urine,where the p value was 0.033 and 0.017,respectively;it was also related to the index glucose,uric acid,albumin,cholesterol and triglyceride in serum,where the p value was 0.011,0.004,<0.001,0.041 and 0.013,respectively.In the Linear Regression model related to the urinary diffusion,the indexes protein,p H value and color in urine and albumin and triglyceride in serum were selected into the model,where the p value was <0.001,<0.001,<0.001,0.056 and 0.080 respectively with Linear Regression(backward method)analysis.While in the Linear Regression model related to the serumal diffusion,the indexes urobilinogen,protein,p Hvalue,Leukocyte esterase and color in urine and urea,uric acid and albumin in serum were selected into the model,where the p value was 0.066,0.025,0.016,0.001,0.017,0.003,0.005 and <0.001 respectively with Linear Regression(backward)analysis.6.The genotype frequencies of the rs7895833 polymorphism were 3.1% for AA,43.1% for AG and 53.8% for GG.The frequency of the A allele was 24.6% and 75.4%for the G allele,which was in compliance with Hardy-Weinberg equilibrium(P =0.067),the serumal TAC was associated with AG type(p=0.009)and GG(p=0.010)type carriers.Conclusion:1.The potassium permanganate agar method and iodine starch agar method were established to determine the total antioxidants in urine and serum.The two methods can be applied to measure the total antioxidants in urine and serum in the clinic.2.The antioxidant mechanisms of the organism were different in specific diseases.In coronary heart disease,urinary antioxidants participate in the defense of redox stress.In tumor diseases,the urinary antioxidants were more sensitive to defense the redox stress compared with serumal antioxidants,the measurement of urinary antioxidants can be beneficial to the diagnosis of diseases.3.The antioxidant mechanisms of urine and serum could be independent.4.The genotype frequencies of the rs7895833 polymorphism were related to the serumal antioxidant capacity. |
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