The Study Of Methodology And Applications Of Determination Of Total Bile Acid In Serum By Electrochemiluminescence

Objective:1. A method for determination of total bile acid in serum by electrochemiluminescence(ECL) was developed.2. A method for determination of total bile acid in serum by capillary electrophoresis combined with electrochemiluminescence(CE-ECL) was developed.3. To investigate a correlation of methods for determination of total bile acid in serum among ECL, CE-ECL and enzyme cycle method.4. To determine total bile acid in serum and in urine by ECL and by CE-ECL, and to research correlation between serum and urine.Methods:1. Serum total bile acid was analysed by ECL. Detection system was tri-electrode working system. A background electrolyte of 2.5 mmol/L tris(2,2'-bipyridyl)ruthenium(II) (TBR, Ru(bpy)32+), 833 U/L 3alpha- hydroxysteroid dehydrogenase (3α-HSD), 70 nmol/L nicotinamide adenine dinucleotide (NAD+) and 50 mmol/L phosphate buffer solution pH 8.0 was used under 25℃.The dilute sample was quantitated by external standard curve.2. Serum total bile acid was analysed by CE-ECL. The running buffer solution was 5 mmol/L phosphate buffer solution pH 7.0 containing 5 nmol/L NAD+. Detection system was tri-electrode working system. The background electrolyte of 75 mmol/L phosphate buffer solution pH 8.0 containing 5 mmol/L TBR, 167 U/L 3α-HSD, 70 nmol/L NAD+ was used. Electrophoresis was carried out at 25℃. The dilute sample was quantitated by external standard curve.3. Total bile acid was determined by enzyme cycle assay with automatic biochemistry analyzer.Results:1. ECL assay was linear in the range 1.0-100 fmol/L, correlation coefficient for linear regression was 0.997 5. The detection limit of bile acid in serum-based was 0.02 fmol/L. The within-day coefficients of variation ranged from 0.64-8.30% , the between-day coefficients of variation ranged from 2.50-9.08%. The average recoveries were 90.8-105.5%.2. CE-ECL assay was linear in the range 0.1-1 000 fmol/L, correlation coefficient for linear regression was 0.992 8. The detection limit of bile acid was 0.02 fmol/L. The within-day coefficients of variation ranged from 1.36-4.69%, the between-day coefficients of variation ranged from 2.93-5.64%. The average recoveries were 95.3-105.7%.3. A highly significant correlation were found between ECL method and enzyme cycle assay for total serum bile acid(r=0.708 6, p<0.000 0), and between CE-ECL method and enzyme cycle assay for total bile acid(r=0.721 6, p<0.000 0). The same positive trend were found between serum bile acid and urine bile acid (r=0.988 4 by ECL; r=0.996 4 by CE-ECL).Conclusion:1. A simple and sensitive ECL method was developed for the quantification of serum total bile acid.2. A simple and sensitive CE-ECL method was developed for the quantification of serum total bile acid.3. The results of total serum bile acid by ECL method and by CE-ECL method were found significant correlation with enzyme cycle method. Developed methods were novel, reliable and suitable for clinical practice.4. Urine bile acid concentration is a new real-time index for liver disease.