| Objective:1. To establish a method of solid phase extraction-reverse phase high performance liquid chromatography and ultraviolet detection (SPE-HPLC- UV) for determination of nine conjugated bile acids in serum .2. The developed method was applied to determination of conjugated bile acids in maternal serum and umbilical cord serum of patients from mild, severe intrahepatic cholestasis of pregnancy (ICP) and healthy pregnancies to get the profiles of bile acids, which can be used in diagnosis and grading of ICP. Then to investigate the changes in maternal-fetal bile acid balance.Methods :1. Bile acids were extracted from serum samples using Chromabond C18 ec cartridges.2. Bile acids were eluted using methanol/phosphate buffer (60μmol/L, pH3.3) (66:34, v/v)as mobile phase on a Waters Nova C18 column (3.9×150 mm,5μm). The column was kept at 25℃. The flow rate was 0.8 ml/min. The eluate was monitored by a ultraviolet detection at 197 nm. The analysis time was 20 min.3. Conjugated bile acids in maternal serum and umbilical cord serum of 16 patients with severe ICP, 11 patients with mild ICP, and 14 healthy pregnancies were determined by HPLC.Results:1.The nine types of conjugated bile acids were completely analyzed in twenty minutes by this method. The assay was linear in the range 5~500μmol/L, with the correlation coefficients for linear regression ranged from 0.9993 to 0.9998.The detection limits ranged from 1.25 to 3.13μmol/L. The within-day coefficients of variation were below 5.95%, and the between-day coefficients of variation were below 7.92% for all bile acids. The recoveries were 83.11%~101.34%.2.Maternal serum:The levels of TCA, TCDCA and TDCA in serum of patients with mild ICP were significantly higher (p<0.05) than those of healthy pregnancies. The level of TCA, GCA, GLCA, TCDCA and TDCA in serum in patients with severe ICP was significantly higher (p<0.05) than those of healthy pregnancies. The level of TCA, GCA, GLCA and TDCA in serum in patients with severe ICP was significantly higher (p<0.05) than those of mild ICP.Umbilical cord serum: The level of TCA, GCA and TDCA in serum in patients with severe ICP was significantly higher (p<0.05) than those of healthy pregnancies. The level of TCA, GCA and TDCA in serum in patients with severe ICP was significantly higher (p<0.05) than those of mild ICP. There were no significant differences of the bile acids between mild ICP and healthy pregnancies.Conclusion :1. The developed method was accurate, sensitive, rapid, specific and suitable for determining simultaneously of conjugated bile acids in maternal serum and umbilical cord serum and for the routine clinical practice and the research practice.2. The results of detection show that mild and severe ICP patients have specific bile acids profiles. It can be used to diagnose and grading of ICP.3. This research found the concentrations of total bile acid between maternal serum and umbilical cord serum have positive correlations in all of the three groups. The concentrations of total bile acid , GCA and TCA of normal pregnancies were higher in umbilical cord serum than in maternal serum; however, the concentrations of total bile acid and TCA of mild and severe ICP were significantly elevated in maternal serum compared with umbilical cord serum. The results show the changes in transplancental transport system of bile acid contribute the changes in maternal-fetal bile acid balance . |
