Objective: To explore the clinical features, fetal outcomes and serum bile acids (BAs) metabolism in asymptomatic hypercholanemia of pregnancy (AHP), as well as the comparison with those in intrahepatic cholestasis of pregnancy (ICP) and normal pregnancies. Methods: A study containing 676 pregnant women was performed to investigate the clinical informations, routine biochemical features and obstetric outcomes of AHP by the comparison with ICP and normal pregnancies. Within the study subjects, 203 pregnant women received prospective determination for 55 serum individual BAs based on a validated UPLC-QTOF-MS/MS method. The differences in clinical features and serum BAs metabolism among the three groups were then investigated. Results: The risk of adverse fetal outcomes in AHP (28.3%) was significantly higher than that in normal pregnancies (8.9%, p < 0.001), but lower than that in ICP group (52.1%, p < 0.001). Multivariate statistics analysis indicated a distinctive serum BAs metabolic profiling among the three groups (PLS-DA, (RY)-Y-2 = 0.580, Q(2) = 0.537). Levels of serum BAs especially for deoxycholic acid species were found remarkably elevated in AHP as compared to those in ICP. Conclusions: AHP group had distinguished clinical features and serum BAs metabolism as compared to ICP group and normal pregnancies.

(Abstracted from Lancet Gastroenterol Hepatol 2021;6:547–558) Ursodeoxycholic acid (UA) is a common treatment for intrahepatic cholestasis of pregnancy (ICP), and its use is supported by the Society for Maternal-Fetal Medicine. However, evidence of the benefit of UA to fetal and neonatal outcomes in this patient population is unclear.

BACKGROUND:Asymptomatic hypercholanemia of pregnancy (AHP) is a controversial hypercholanemia, which is difficult to distinguish from intrahepatic cholestasis of pregnancy (ICP). Our aim is to elucidate the characteristics of urinary bile acid (BA) profiling of women with AHP and to find potential biomarkers for the diagnosis and differential diagnosis of AHP. METHODS:We developed a pseudo-targeted approach to perform metabolomics analysis of bile acids (BAs) using ultra-high performance liquid chromatography/hybrid quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS). Urinary BAs profiles were compared among AHP women (n = 20), ICP patients (n = 33) and normal controls (n = 35). RESULTS:The profiling of urinary BAs was significantly different among the AHP, ICP and control groups. Compared to the control group, the AHP group had higher levels of four possible sulfated BAs and trihydroxy BAs, including the species of muricholic acid (MCA), cholic acid (CA) and six possible BAs, whereas, 20 possible sulfated BAs, taurochenodeoxycholic acid (TCDCA), tetrahydrocannabinolic acid (THCA), and seven possible BAs were significantly lower in the AHP group than those in the ICP group. Based on the receiver operating characteristic (ROC) analysis, glycocholic acid (GCA) combined with T-ω-MCA were found to be the potential combination biomarker for the diagnosis (area under the curve was 0.960) of AHP, and mono-S, Gtri-S-2 combined with TLCA-S were found to be the potential combination biomarker for the differential diagnosis (area under the curve was 0.990) of AHP and ICP. CONCLUSIONS:The metabolisms of urinary Bas were altered in the AHP group compared with the ICP group and the control group. Urinary BA profiling analysis can serve as an effective tool for the diagnosis of AHP and the differential diagnosis of AHP and ICP.

Background: Intrahepatic cholestasis of pregnancy (ICP), as a pregnancy-specific liver disorder, obtains increasing recognition due to a series of adverse outcomes. ICP is generally characterized by pruritus and jaundice, and closely related to abnormalities in the metabolism and disposition of bile acids composition. Because of its elusive pathogenesis, ICP has become an intractable issue to be diagnosed and managed for obstetricians. Analysis of metabolic profile could reveal the state of small-molecule metabolites systematically and provide comprehensively metabolic information for diseases. We developed a pseudo-targeted approach to perform metabolomic analysis of bile acids in serum using an ultra-performance liquid chromatography-triple quadrupole time-of-flight tandem mass spectrometry (UPLC-Triple TOF-MS/MS) method. Methods: We investigated the metabolites of bile acids in 55 healthy pregnant women, 42 women with ICP and 11 women with ICP who persisted to accept ursodeoxycholic acid (UDCA) therapy. Results: The metabolic profiles of serum bile acids were significantly altered in ICP group compared with the control group. A screened potential combination biomarker, with a high diagnostic efficiency (area under the curve = 0.996, Youden index = 0.940), was superior to total bile acids for the diagnosis of ICP. Conclusions: The profiles of serum bile acids in women with ICP became more clear under the UDCA therapy, and were fully recovered after the delivery.

BACKGROUND:Intrahepatic cholestasis of pregnancy (ICP), a pregnancy-related liver disease, leads to complications for both mothers and fetuses. Metabolomic approach has been applied to maternal-fetal medicine. The global metabolomic alterations that are specific in ICP as yet have not been investigated. METHODS:Based on high performance liquid chromatography/hybrid quadrupole time-of-flight (HPLC/Q-TOF) mass spectrometry, the untargeted metabolomics was used to analyze the changes of urinary metabolites between ICP group and the control group. RESULTS:One hundred nine variables in positive model and 119 variables in negative model were significantly different (p<0.05) between the ICP group and the control group, with the VIP (variable importance in the project) score>1 by the orthogonal partial least squares discriminant analysis (OPLS-DA). 14 metabolites in positive model and 18 metabolites in negative model were selected and identified based on HMDB (human metabolome database). Most of these metabolites were involved in bile acids biosynthesis and metabolism, hormone metabolism and lipid metabolism. A metabolite panel (MG (22:5), LysoPE (22:5), l-homocysteine sulfonic acid, glycocholic acid and chenodeoxycholic acid 3-sulfate) was contrusted by the binary logistic regression analysis with high diagnostic accuracy for ICP. The area under the receiver operating characteristic curve was 0.988 with the sensitivity of 90.0% and specificity of 93.3%. CONCLUSIONS:Urinary metabolites allow for the discrimination of ICP from the controls by orthogonal partial least squares discriminant analysis. Therefore, these findings may provide deep insights for the etiopathogenesis of ICP. Moreover, the maternal urinary metabolite panel has the potential to be used as non-invasive biomarkers for the diagnosis of ICP.

目的:探讨基于母胎血清胆汁酸代谢轮廓的妊娠肝内胆汁淤积症(intrahepatic cholestasis of pregnancy,ICP)分度.方法:采用高效液相色谱-紫外检测法对61名正常孕妇,轻、重度ICP患者母、脐血的9种结合型胆汁酸成分进行测定,并将受试者分为建模集(n--40)与验证集(n=21),结合偏最小二乘判别分析对胆汁酸代谢轮廓进行分析、建模与验证.结果:母血胆汁酸代谢模型可区分正常孕妇组,轻度ICP组,与重度ICP组;脐血胆汁酸代谢模型可区分重度ICP组与非重度组.母、脐血分度模型的验证效率分别达90.5％与100.0％.牛磺熊去氧胆酸(P=0.004),牛磺胆酸(P=0.005)以及甘氨胆酸(P=0.032)在ICP孕妇中明显增高;甘氨石胆酸(P＜0.001)和牛磺脱氧胆酸(P=0.001)在ICP胎儿中明显增高.结论:ICP母、脐血各有其特征的胆汁酸代谢轮廓,均可用于对ICP分度.

Intrahepatic cholestasis of pregnancy (ICP), a pregnancy-related liver disease, leads to complications for both mother and fetus. Circulating microRNAs (miRNAs) have emerged as candidate biomarkers for many diseases. So far, the circulating miRNAs profiling of ICP has not been investigated. To assess the urinary miRNAs as non-invasive biomarkers for ICP, a differential miRNA profiling was initially analyzed by individual quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) assay in urinary samples from a screening set including 10 ICP and 10 healthy pregnancies. The selected candidate miRNAs were then validated by a validation set with 40 ICP and 50 healthy pregnancies using individual qRT-PCR assay. Compared with the expression in urine of healthy pregnant women, the expression levels of hsa-miR-151-3p and hsa-miR-300 were significantly down-regulated, whereas hsa-miR-671-3p and hsa-miR-369-5p were significantly up-regulated in urine from ICP patients (p < 0.05 and false discovery rate < 0.05). A binary logistic regression model was constructed using the four miRNAs. The area under the receiver operating characteristic curve was 0.913 (95% confidence interval = 0.847 to 0.980; sensitivity = 82.9%, specificity = 87.0%). Therefore, urinary microRNA profiling detection in ICP is feasible and maternal urinary miRNAs have the potential to be non-invasive biomarkers for the diagnosis of ICP.

Bile acids level in serum is a useful index for screening and diagnosis of hepatobiliary diseases. As bile acids concentration is closely related to the degree of hepatobiliary diseases, detecting it is a vital factor to understand the stage of the diseases. The prevalent determination for bile acids is the enzymatic cycling method which has low sensitivity while reagent-consuming. It is desirable to develop a new method with lower cost and higher sensitivity. An indirect electrochemical detection (IED) for bile acids in human serum was established using the screen printed carbon electrode (SPCE). Since bile acids do not show electrochemical signals, they were converted to 3-ketosteroids by 3-α-hydroxysteroid dehydrogenase (3α-HSD) in the presence of nicotinamide adenine dinucleotide (NAD+), which was reduced to NADH. NADH could then be oxidized on the surface of SPCE, generating a signal that was used to calculate the total bile acids (TBA) concentration. A good linear calibration for TBA was obtained at the concentration range from 5.00 μM to 400 μM in human serum. Both the precisions and recoveries were sufficient to be used in a clinical setting. The TBA concentrations in 35 human serum samples by our IED method didn't show significant difference with the result by enzymatic cycling method, using the paired t-test. Moreover, our IED method is reagent-saving, sensitive and cost-effective.

Capillary electrophoresis with electrochemiluminescence detection for the simultaneous analysis of cisatracurium besylate and its degradation products (laudanosine, quaternary monoacrylate) in pharmaceutical preparation was developed and fully validated. The significant parameters that influence capillary electrophoresis separation and electrochemiluminescence detection were optimized. The total analysis time of the analytes was 15 min. The linearities of the method were 0.1∼40.0 μg/mL for cisatracurium besylate and 0.04∼8.00 μg/mL for laudanosine, with correlation coefficients (r) of 0.999 and 0.998, respectively. The detection limits (S/N = 3) were 83.0 ng/mL for cisatracurium besylate and 32.0 ng/mL for laudanosine. The intraday relative standard deviations of the analytes were <3.0%, and the interday relative standard deviations were <8.0%. The developed method was cost-effective, sensitive, fast, and resource-saving, which was suitable for the ingredient analysis in pharmaceutical preparation.

A highly sensitive HPLC-MS/MS method for simultaneous determination of the quaternary ammonium pesticides chlormequat (CQ) and mepiquat (MQ) in pears and potatoes was developed and fully validated.

目的:探讨尿液硫酸化胆汁酸代谢轮廓分析在识别无症状高胆汁酸血症(asymptomatic hypercholanaemia of pregnancy, AHP)中的应用价值.方法:应用高效液相色谱-串联质谱法(HPLC-MS/MS)测定正常孕妇(13例),妊娠肝内胆汁淤积症(intrahepatic cholestasis of pregnancy,ICP)(17例)尿液硫酸化胆汁酸谱,采用不同扫描模式鉴别和分析尿液各种硫酸化胆汁酸,并用主成分分析法(principle component analysis,PCA)对尿液硫酸化胆汁酸代谢轮廓进行分析.结果:采用不同的扫描模式对尿液中15种可能的硫酸化胆汁酸进行鉴定并分析,经PCA对尿液硫酸化胆汁酸代谢轮廓分析,发现在ICP组中存在一类特殊的人群,其表现为无任何症状的胆汁酸水平升高而肝功能正常,但其尿液硫酸化胆汁酸代谢轮廓与正常孕妇类似.结论:尿液硫酸化胆汁酸代谢轮廓分析有助于AHP的识别.

建立了毛细管电泳-电化学发光/电化学检测(CE ECL/EC)法同时测定维生素B1 (VB1)和维生素B6(VB6)的新方法.在碱性条件下对VB1进行水解,可增强其电化学发光信号,并采用序贯均匀设计对毛细管电泳分离条件进行优化,测定VB1的电化学发光信号和VB6的电化学信号.VB1和VB6能在5 min内得到良好的分离.电化学发光强度与VB1浓度在5.0～5 000.0 μg/mL范围内具有良好的线性关系(r=0.997),检测限(S/N=3)为2.7μg/mL;电流强度与VB6浓度在10.0～1 000.0μg/mL范围内具有良好的线性关系(r=0.998),检测限(S/N=3)为6.8 μg/mL.VB1和VB6的加标回收率范围为96.0％～100.0％,相对标准偏差为1.4％～6.7％.用建立的方法对复合VB片中的VB1和VB6进行测定,结果较好.

Homocysteine (Hcy) and its metabolically related aminothiols cysteine (Cys), cysteinylglycine (CysGly), and glutathione (GSH) play important roles in the pathogenesis of uremia. Most of these aminothiols exist in plasma as disulphide and mixed disulphide forms. A new, simple, sensitive and reliable precolumn derivatization HPLC method, with fluorescence detection, for the determination of four kinds of aminothiols, including total, free and reduced fractions, in plasma simultaneously was established and validated. After a simple derivatization using N-(1-pyrenyl) maleimide (NPM), the assay was performed using a C-18 column with gradient elution. The method used for the total aminothiol determinations was linear in the range of 2.00–80.0, 10.0–1500, 1.00–120, and 3.00–240 μmol L−1 for GSH, Cys, Hcy, and CysGly, respectively. The method used for the reduced aminothiols was linear in the range of 0.10–8.00, 1.25–50.0, 1.25–50.0, and 0.01–4.00 μmol L−1 for GSH, Cys, Hcy, and CysGly, respectively. The method was successfully applied to the analysis of plasma from uremic patients (n = 29) and healthy subjects (n = 28). The results showed that the concentrations of the total, free and reduced forms of Hcy, Cys and CysGly were higher, while the concentrations of the three forms of GSH were lower (p < 0.05) in uremic patients than in the healthy controls. The concentrations of the three forms of Hcy and Cys were significantly decreased, but the concentrations of the three fractions of GSH and CysGly had no significant change, during hemodialysis. In conclusion, the developed method is simple, fast, accurate, and suitable for clinical measurements.

Objective:To investigate the effects of cold preservation reperfusion injury(CPRI)on the variation of bile acid composition in rats after liver transplantation.Methods:Reverse phase-high performance liquid chromatography with precolumn derivatization and fluorescence detection was used to determinate the bile acid composition in rat bile after the surgery by using the donor livers which were coldly preserved for 12 h,1 h and the control livers.Metabolite profiling analysis of bile acid was performed by the methods of principal component analysis and partial least squares-discriminant analysis.Results:It was obvious that CPRI for 12 h, 1 h groups and the control group can be separated with the metabolite profile of bile acids and bile duct injury was probably closely related with taurodeoxycholic acid.Hydrophobicity index of CPRI for 12 h group was increased significantly after liver transplantation and was closely correlated with cold preservation time of the donor livers.Conclusion:Metabolite profiling of bile acids in rat bile after liver transplantation is significantly influenced by CPRI.