Metabolic Profiling Analysis Of Urine Sulfated Bile Acids Applied In Diagnosis And Graduation Of Intrahepatic Cholestasis Of Pregnancy

BackgroundIntrahepatic cholestasis of pregnancy (ICP) is a cholestatic disorder inthe second or third trimester of pregnancy. The incidence of ICP is relevantwith ethnic groups and geographical variation. ICP has harm to fetal and isassociated with preterm delivery, meconium staining, fetal distress in labor.The etiology of ICP is not completely understood. ICP is correlative withfamilial clustering and can be reoccurred in subsequent pregnancies. Due tolack of unified criteria for diagnosis and clinical grading of ICP, thetreatment strategies for ICP patients is hard to choose. If we mistake severeICP for mild ICP, it will result in possibly perinatal death particularly“unexpected death”. In reverse, it will increase cesarean section toterminate pregnancy to avoid fetal death, and these will result in theunnecessary increase of cost and the waste of medical resource. Lab testsplay important role in diagnosis of ICP and serum bile acids is the mainbiomarkers at present, but the bile acids alone are not enough sensitive andspecific and can be influenced by many factors. Many authors studied thevalue of serum bile acids profiles to the diagnosis of ICP and providedsome important information to clinicians, but there is still lack of effectivebiomarkers to diagnose and discriminate patients with ICP. Sulfation is amajor metabolic pathway involved in the elimination and detoxification of bile acid, especially in hepatobiliary diseases. Sulfated bile acid has highwater solubility and is immediately excreted in the urine under cholestaticconditions, which indicated their efficient elimination in urine anddecreased damnification on liver. The formation of bile acids sulfatesincreases during ICP, so measurement of urine sulfated bile acids isexpected to be a useful test for diagnosis and clinical grading of ICP.High-performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS) has high sensitivity and specificity, and is an ideal methodfor detecting urine sulfated bile acids. It is possible for us to furtherunderstand the change of urine sulfated bile acids profiles in patients withICP.ObjectivesThe present study is to perform metabolic profiling analysis of urinesulfated bile acids in three groups of healthy pregnancy, mild ICP andsevere ICP by HPLC-MS/MS. The metabolic profiling of urine sulfatedbile acids and serum biomarkers in three groups are to be studied, in orderto investigate whether urine sulfated bile acids can be used as potentialbiomarkers for clinical diagnosis and grading of ICP, which helps cliniciansdetermine the management strategies for ICP.Method1. Establish the method of testing urine sulfated bile acids byHPLC-MS/MS3-sulfated lithocholic acid (3S-LCA),3-sulfated glycolithocholic acid(3S-GLCA) and3-sulfated taurolithocholic acid(3S-TLCA) were analyzedand the parameters of mass spectrometry ionization were optimized toobtain the maximum response for deprotonated ions of sulfated bile acids.Different mobile phase, gradient program were tested for optimalseparation of these sulfated bile acids. The method for urine sample preparation was also studied to establish optimal conditions for sampleprocessing. Based on these optimized parameters of mass spectrometry,chromatogram and sample preparation, the method of testing urine sulfatedbile acids were established. Detection limit, linearity, recoveries andprecision were performed to validate the method.2. Metabolic profiling analysis of urine sulfated bile acids inwomen with ICPAccording to present criteria of diagnosis and clinical grading of ICP,urine samples were collected from23patients with severe ICP,20caseswith mild ICP, and18cases of normal pregnant women. Urine sulfated bileacids in three groups of healthy pregnancy, mild ICP and severe ICP wereanalyzed by HPLC-MS/MS. metabolic profiling analysis of urine sulfatedbile acids were performed to search the potential biomarkers for diagnosisand clinical grading of ICP.Result1. A method for the determination of urine sulfated bile acids byHPLC-MS/MS was developed. Urine sample were extracted with OASISHLB SPE. The separations of sulfated bile acids was achieved on a Luna3u C18(150mm×2.00mm，3μm) column with mobile phase consist ofA (0.1%formic acid and2mmol/L ammonium acetate in methanol) and B(0.1%formic acid and2mmol/L ammonium acetate in acetonitrile) bygradient elution. The column was kept at40℃. The flow rate was0.2ml/min. Electrospray ionisation (ESI) was performed in the negative ionmode. The elutions were monitored in multiple reaction-monitoring mode.Total analysis time was18min for one sample including re-equilibrationtime of the column.2.The assay was linear in the range9.7-998.0ng/ml，9.0-1080.0ng/ml， 9.3-1123.3ng/ml for3S-TLCA,3S-LCA and3S-GLCA, with correlationcoefficients of standard curves for three sulfated bile acids better than0.999. The detection limits were5ng/ml for three sulfated bile acids. Theprecisions for three sulfated bile acids were CVs <4.0%for within-day andCVs <5.0%for between-day. The average recoveries for three sulfated bileacids were ranged from89.4-100.7%.3. CG, TBA, ALT and AST were raised significantly in the seraderived from both mild and severe ICP patients as compared with those inthe normal pregnant women. ALP were increased in three groups but nosignificant differences were found. DBIL and TBIL were increasedsignificantly in severe ICP group compared with normal pregnancy.4. Compared with normal pregnant women, Total urine3S-BA wasraised4.0-fold(p＜0.017)and9.0-fold (p＜0.017)in mild ICP and severeICP. Among them, total3S-Tauro BA and total3S-Tauro BA were3.2-fold and4.5-fold increase in mild ICP,11.6-fold and23.2-fold increase insevere ICP. The ratio of3S-Tauro BA/3S-Glyco BA was44.8%,62.1%and89.5%in normal pregnancy, mild ICP and severe ICP, respective.Sulfated unconjugated bile acids kept in low concentration in all groupsand3S-LCA was the lowest among sulfated bile acids. Sulfated Tauroconjugated bile acids and Glyco conjugated bile acids were the maincompositions and constituted90percent of sulfated bile acids in urine.Urine levels of3S-GCA,3S-TCA,3S-CA,3S-TLCA and3S-TDCA/3S-TCDCA/3S-TUDCA in the severe ICP patients were much higher thanthose in the normal pregnant women.5. Principal components analysis(PCA)showed that severe ICP groupcould be distinguished from normal pregnancy. Mild ICP could separatefrom severe ICP group and normal pregnancy. The correct rate for diagnosis of normal pregnancy and severe ICP reached88.9%and90.0%respectively by partial least-squares discriminant analysis (PLS-DA).ConclusionWe developed a method for the determination of urine sulfated bileacids by HPLC-MS/MS.15sulfated bile acids were identified by enhancedproduct ion (EPI) scan in a linear ion trap.Severe ICP patients could be distinguished from normal pregnantwomen by metabolic profiling analysis of urine sulfated bile acids, and twoasymptomatic hypercholanaemia of pregnancy (AHP) patients wereidenfified.Some valuable biomarkers for the diagnosis of severe ICP were foundin urine sulfated bile acids by partial least-squares discriminant analysis(PLS-DA).