Studies on Novel Diagnostic and Predictive Biomarkers of Intrahepatic Cholestasis of Pregnancy Through Metabolomics and Proteomics.

Background: Intrahepatic cholestasis of pregnancy (ICP) usually occurs in the third trimester and is associated with increased risks in fetal complications. Currently, the exact mechanism of this disease is unknown. The purpose of this study was to develop potential biomarkers for the diagnosis and prediction of ICP. Methods: We enrolled 40 pregnant women diagnosed with ICP and 40 healthy pregnant controls. The number of placental samples and serum samples between the two groups was 10 and 40 respectively. Ultra-performance liquid chromatography tandem high-resolution mass spectrometry was used to analyze placental metabolomics. Then, we verified the differentially expressed proteins and metabolites, both placental and blood serum, in the first, second, and third trimesters. Results: Metabolomic analysis of placental tissue revealed that fatty acid metabolism and primary bile acid biosynthesis were enriched. In the integrated proteomic and metabolomic analysis of placental tissue, peroxisomal acyl-CoA oxidase 1 (ACOX1), L-palmitoylcarnitine, and glycocholic acid were found to be three potential biomarkers. In a follow-up analysis, expression levels of both placental and serum ACOX1, L-palmitoylcarnitine, and glycocholic acid in both placenta and serum were found to be significantly higher in third-trimester ICP patients; the areas under the ROC curves were 0.823, 0.896, and 0.985, respectively. Expression levels of serum ACOX1, L-palmitoylcarnitine, and glycocholic acid were also significantly higher in first- and second-trimester ICP patients; the areas under the ROC curves were 0.726, 0.657, and 0.686 in the first trimester and 0.718, 0.727, and 0.670 in the second trimester, respectively. Together, levels of the three aforementioned biomarkers increased the value for diagnosing and predicting ICP (AUC: 0.993 for the third, 0.891 for the second, and 0.932 for the first trimesters). Conclusions: L-palmitoylcarnitine, ACOX1, and glycocholic acid levels taken together may serve as a new biomarker set for the diagnosis and prediction of ICP.

Nitroalkene fatty acids modulate bile acid metabolism and lung function in obese asthma.

Bile acid profiles are altered in obese individuals with asthma. Thus, we sought to better understand how obesity-related systemic changes contribute to lung pathophysiology. We also test the therapeutic potential of nitro-oleic acid (NO2-OA), a regulator of metabolic and inflammatory signaling pathways, to mitigate allergen and obesity-induced lung function decline in a murine model of asthma. Bile acids were measured in the plasma of healthy subjects and individuals with asthma and serum and lung tissue of mice with and without allergic airway disease (AAD). Lung function, indices of inflammation and hepatic bile acid enzyme expression were measured in obese mice with house dust mite-induced AAD treated with vehicle or NO2-OA. Serum levels of glycocholic acid and glycoursodeoxycholic acid clinically correlate with body mass index and airway hyperreactivity whereas murine levels of ß-muricholic acid and tauro-ß-muricholic acid were significantly increased and positively correlated with impaired lung function in obese mice with AAD. NO2-OA reduced murine bile acid levels by modulating hepatic expression of bile acid synthesis enzymes, with a concomitant reduction in small airway resistance and tissue elastance. Bile acids correlate to body mass index and lung function decline and the signaling actions of nitroalkenes can limit AAD by modulating bile acid metabolism, revealing a potential pharmacologic approach to improving the current standard of care.

Clinical significance of small molecule metabolites in the blood of patients with different types of liver injury.

To understand the characteristic of changes of serum metabolites between healthy people and patients with hepatitis B virus (HBV) infection at different stages of disease, and to provide reference metabolomics information for clinical diagnosis of liver disease patients. 255 patients with different stages of HBV infection were selected. 3 mL blood was collected from each patient in the morning to detect differences in serum lysophosphatidylcholine, acetyl-L-carnitine, oleic acid amide, and glycocholic acid concentrations by UFLC-IT-TOF/MS. The diagnostic values of four metabolic substances were evaluated by receiver operating characteristic (ROC) curve. The results showed that the optimal cut-off value of oleic acid amide concentration of the liver cirrhosis and HCC groups was 23.6 mg/L, with a diagnostic sensitivity of 88.9% and specificity of 70.6%. The diagnostic efficacies of the three substances were similar in the hepatitis and HCC groups, with an optimal cut-off value of 2.04 mg/L, and a diagnostic sensitivity and specificity of 100% and 47.2%, respectively. The optimal cut-off value of lecithin of the HBV-carrier and HCC groups was 132.85 mg/L, with a diagnostic sensitivity and specificity of 88.9% and 66.7%, respectively. The optimal cut-off value of oleic acid amide of the healthy and HCC groups was 129.03 mg/L, with a diagnostic sensitivity and specificity of 88.4% and 83.3%, respectively. Lysophosphatidylcholine, acetyl-L-carnitine, and oleic acid amide were potential metabolic markers of HCC. Among them, lysophosphatidylcholine was low in the blood of HCC patients, and its diagnostic efficacy was better than that of acetyl-L-carnitine and oleic acid amide, providing reference metabolomics information in clinical diagnosis and future research.

UPLC-Q-TOF/MS-based plasma metabolome to identify biomarkers and time of injury in traumatic brain injured rats.

BACKGROUND: To identify the potent metabolic biomarkers and time of injury of traumatic brain injured (TBI). METHODS: A total of 70 Sprague-Dawley rats were used to establish the TBI model in this study. The serum was collected at 3 h, 6 h, 12 h, 24 h, 3 days and 7 days after surgery. Ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry was performed to analyze metabolic changes in the serum of the TBI rats from different groups. The differences between the metabolic profiles of the rats in seven groups were analyzed using partial least squares discriminant analysis. RESULTS: Metabolic profiling revealed significant differences between the sham-operated and other groups. A total of 49 potential TBI metabolite biomarkers were identified between the sham-operated group and the model groups at different time points. Among them, six metabolites (methionine sulfone, kynurenine, 3-hydroxyanthranilic acid, 3-Indolepropionic acid, citric acid and glycocholic acid) were identified as biomarkers of TBI to estimate the injury time. CONCLUSION: Using metabolomic analysis, we identified new TBI serum biomarkers for accurate detection and determination of the timing of TBI injury.

Usefulness of collagen type IV in the detection of significant liver fibrosis in nonalcoholic fatty liver disease.

INTRODUCTION/AIMS: Liver fibrosis assessment is a key issue in the evaluation of nonalcoholic fatty liver disease (NAFLD) patients. In the present study, we aimed to validate a noninvasive marker panel to assess significant and advanced fibrosis in these patients. METHOD: 126 biopsy-proven NAFLD patients were included. NAFLD diagnosis was based on histological criteria. Fibrosis stages were determined according to NASH-Clinical Research Network criteria. Clinical and laboratorial data were collected during the interval of three months before or after liver biopsy. Histological fibrosis stages were classified as significant fibrosis (F2-F4) and advanced fibrosis (F3-F4). Five serum biomarkers [hyaluronic acid (HA), collagen type IV (cIV), procollagen type III (PC III), laminin (LN) and cholylglycine (CG)] were assessed by chemiluminescence immunoassays. RESULTS: Most patients were female (61.61%), mean age: 55.7 ±â€¯9.13 years old and mean BMI was 32.1 ±â€¯5.9 kg/m2. Prevalence of diabetes, dyslipidemia, arterial hypertension, and metabolic syndrome was 68.75%, 82.29%, 63.54% and 81.05%, respectively. Patients with cIV above 30 ng/mL had a 5.57-times (IC: 1.86-16.69) the chance of having significant fibrosis and 7.61-times (IC: 2.27-25.54) the chance of having advanced fibrosis versus patients with values below 30 ng/mL. HA, PC III, LN and CG did not detect the presence of significant and advanced fibrosis. The AUROC of clV for detection of significant (0.718) and advanced fibrosis (0.791) was better than that of other serum biomarkers. CONCLUSION: Type 4 collagen could predict the presence of significant and advanced fibrosis in NAFLD patients and it would be a useful tool in routine clinical practice.

Reusable optofluidic point-of-care testing platform with lyophilized specific antibody for fluorescence detection of cholylglycine in serum.

A reusable optofluidic point-of-care testing platform (OPOCT) was successfully constructed through integrating evanescent wave fluorescence technology into an all-fiber-based optofluidic system. The compact design of the OPOCT allows it to be portable and suitable for on-site sensitive determination of biomarkers in serum without complicated and costly procedures. The sensitivity of 90.9 pM for antibody determination is observed thanks to the high transmission efficiency of excitation light and fluorescence in the OPOCT. The affinity constant between cholylglycine (CG) and anti-CG antibody was quantified using this platform based on the proposed theory. Using the lyophilized fluorescence-labeled specific antibody and reusable fiber optic biosensor, the OPOCT is applied to the one-step sensitive determination of CG in serum, which eliminates the dearth associated with liquid reagent handling, disposable biosensors, and user intervention. A limit of detection of 0.025 µg/mL for CG is obtained, which is far more than adequate for meeting diagnostic requirements. The matrix effect of serum samples on the evanescent wave-based optofluidic biosensor can be effectively reduced by simple dilution of serum samples. The performance of the OPOCT also compared favorably with that of a commercial turbidimetric inhibition immunoassay through analyzing multiple serum samples. This platform is ready to expand to measure any other biomarker by using its specific antibody. The simplicity, sensitivity, cost-effectiveness, and robustness of the OPOCT enable the early diagnosis of disease and making a timely clinical decision. Graphical abstract .

Dynamic Metabolomics Study of the Bile Acid Pathway During Perioperative Primary Hepatic Carcinoma Following Liver Transplantation.

BACKGROUND There are many situations of abnormal metabolism influencing liver graft function. This study aims to provide data for the development of liver function recovery after liver transplantation by dynamically analyzing metabolites of bile acids pathway in serum. MATERIAL AND METHODS A comprehensive metabolomics profiling of serum of 9 liver transplantation patients before transplantation, on the 1st, 3rd, and 7th days after liver transplantation, and healthy individuals were performed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS). Multivariate data and dynamic analysis were used to search for biomarkers between the metabolomics profiles present in perioperative liver transplantation and normal controls. RESULTS Thirty-three differential endogenous metabolites were screened by the threshold of variable importance in the projection (VIP) from an orthogonal partial least square discriminant analysis (OPLS-DA) greater than 1.0, q-value <0.05, and fold change (FC) ≤0.8 or ≥1.2 between the preoperative group and the normal controls in negative mode. The metabolite intensities of taurocholic acid, taurochenodeoxycholic acid, chenodeoxycholic acid glycine conjugate, and glycocholic acid pre-transplantation were significantly higher than those of normal controls. The average metabolite intensities of taurocholic acid and taurochenodesoxycholic acid on the first day after liver transplantation were lower than those observed pre-transplantation. The average metabolite intensities on day 3 after liver transplantation showed a sudden increase and then decreased after 7 postoperative days. The average metabolite intensities of glycocholic acid and chenodeoxycholic acid glycine conjugate showed an increasing trend on the 1st, 3rd, and 7th days after liver transplantation. CONCLUSIONS Use of taurocholic acid and taurochenodeoxycholic acid-related bile secretion, liver regeneration, and de novo bile acid synthesis may help clinical evaluation and provide data for the development of liver function recovery after liver transplantation.

Prospective Investigation of Serum Metabolites, Coffee Drinking, Liver Cancer Incidence, and Liver Disease Mortality.

BACKGROUND: Coffee has been consistently associated with lower risk of liver cancer and chronic liver disease, suggesting that coffee affects mechanisms underlying disease development. METHODS: We measured serum metabolites using untargeted metabolomics in 1:1 matched nested case-control studies of liver cancer (n = 221 cases) and fatal liver disease (n = 242 cases) in the Alpha-Tocopherol, Beta-Carotene Cancer Prevention cohort (n = 29 133). Associations between baseline coffee drinking and metabolites were identified using linear regression; conditional logistic regression models were used to identify associations with subsequent outcomes. RESULTS: Overall, 21 metabolites were associated with coffee drinking and also each subsequent endpoint; nine metabolites and trigonelline, a known coffee biomarker, were identified. Tyrosine and two bile acids, glycochenodeoxycholic acid (GCDCA) and glycocholic acid (GCA), were inversely associated with coffee but positively associated with both outcomes; odds ratios (ORs) comparing the 90th to 10th percentile (modeled on a continuous basis) ranged from 3.93 (95% confidence interval [CI] = 2.00 to 7.74) for tyrosine to 4.95 (95% CI = 2.64 to 9.29) for GCA and from 4.00 (95% CI = 2.42 to 6.62) for GCA to 6.77 (95% CI = 3.62 to 12.65) for GCDCA for liver cancer and fatal liver disease, respectively. The remaining six metabolites and trigonelline were positively associated with coffee drinking but inversely associated with both outcomes; odds ratio ranged from 0.16 to 0.37. Associations persisted following diet adjustment and for outcomes occurring greater than 10 years after blood collection. CONCLUSIONS: A broad range of compounds were associated with coffee drinking, incident liver cancer, and liver disease death over 27 years of follow-up. These associations provide novel insight into chronic liver disease and liver cancer etiology and support a possible hepatoprotective effect of coffee.

Serum Metabolomics and Incidence of Atrial Fibrillation (from the Atherosclerosis Risk in Communities Study).

We have previously identified associations of 2 circulating secondary bile acids (glycocholenate and glycolithocolate sulfate) with atrial fibrillation (AF) risk in 1,919 blacks in the Atherosclerosis Risk in Communities cohort. We aimed to replicate these findings in an independent sample of 2,003 white and black Atherosclerosis Risk in Communities participants, and performed a new metabolomic analysis in the combined sample of 3,922 participants, followed between 1987 and 2013. Metabolomic profiling was done in baseline serum samples using gas and liquid chromatography mass spectrometry. AF was ascertained from electrocardiograms, hospitalizations, and death certificates. We used multivariable Cox regression to estimate hazard ratios (HR) and 95% confidence intervals (95%CI) of AF by 1 standard deviation difference of metabolite levels. Over a mean follow-up of 20 years, 608 participants developed AF. Glycocholenate sulfate was associated with AF in the replication and combined samples (HR 1.10, 95% CI 1.00, 1.21 and HR 1.13, 95% CI 1.04, 1.22, respectively). Glycolithocolate sulfate was not related to AF risk in the replication sample (HR 1.02, 95% CI 0.92, 1.13). An analysis of 245 metabolites in the combined cohort identified 3 additional metabolites associated with AF after multiple-comparison correction: pseudouridine (HR 1.18, 95% CI 1.10, 1.28), uridine (HR 0.86, 95% CI 0.79, 0.93) and acisoga (HR 1.17, 95% CI 1.09, 1.26). In conclusion, we replicated a prospective association among a previously identified secondary bile acid, glycocholenate sulfate, and AF incidence, and identified new metabolites involved in nucleoside and polyamine metabolism as markers of AF risk.

Effect of Rifampicin on the Plasma Concentrations of Bile Acid-O-Sulfates in Monkeys and Human Liver-Transplanted Chimeric Mice With or Without Bile Flow Diversion.

The present study examined the significance of enterohepatic circulation and the effect of rifampicin [an inhibitor of organic anion-transporting polypeptide 1B (OATP1B)] on the plasma concentrations of bile acid-O-sulfates (glycochenodeoxycholate-O-sulfate, lithocholate-O-sulfate, glycolithocholate-O-sulfate, and taurolithocholate-O-sulfate) in monkeys and human liver-transplanted chimeric mice (PXB mouse). Rifampicin significantly increased the area under the curve of bile acid-O-sulfates in monkeys (13-69 times) and PXB mice (13-25 times) without bile flow diversion. Bile flow diversion reduced the concentration of plasma bile acid-O-sulfates under control conditions in monkeys and the concentration of plasma glycochenodeoxycholate-O-sulfate in PXB mice. It also diminished diurnal variation of plasma lithocholate-O-sulfate, glycolithocholate-O-sulfate, and taurolithocholate-O-sulfate in PXB mice under control conditions. Bile flow diversion did not affect the plasma concentration of bile acid-O-sulfates in monkeys and PXB mice treated with rifampicin. Plasma coproporphyrin I and III levels were constant in monkeys throughout the study, even with bile flow diversion. This study demonstrated that bile acid-O-sulfates are endogenous OATP1B biomarkers in monkeys and PXB mice. Enterohepatic circulation can affect the baseline levels of plasma bile acid-O-sulfates and modify the effect of OATP1B inhibition.

Quantification of glycocholic acid in human serum by stable isotope dilution ultra performance liquid chromatography electrospray ionization tandem mass spectrometry.

A rapid, accurate and sensitive stable isotope dilution ultra performance liquidchromatography electrospray ionization tandem mass spectrometry (ID-UPLC-ESI-MS/MS) method for the determination of glycocholic acid (GCA) in human serum was developed and validated. Serum samples were spiked with D5-glycocholic acid and then pretreated with protein precipitation. The analysis was performed on a Waters BEH C18 column (100 mm×2.1mm, 1.7µm), followed by ESI-MS/MS detection in negative ion mode under multiple reaction monitoring mode. The calibration curves covered a concentration range from 0.2 to 400ng/mL. The limit of detection and limit of quantification was 0.01ng/mL and 0.05ng/mL, respectively. The method showed satisfactory precision on intra-day (2.3-6.1%) and inter-day (2.4-4.6%) analyses and achieved good recovery at three spiked levels (103.7-114.3%). Moreover, this established method was successfully applied for quantification of GCA in serum samples from healthy volunteers, patients with hepatocellular carcinoma (HCC) and patients with other cancers. We demonstrated that the level of GCA in patients with HCC was significantly higher not only than that in healthy controls, but also than that in patients with other cancer, whereas no significant difference of GCA level was observed between healthy control group and other cancers group.

Dynamics of Bile Acid Profiles, GLP-1, and FGF19 After Laparoscopic Gastric Banding.

Context: An increase of bile acids (BAs), fibroblast growth factor 19 (FGF19), and glucagon-like peptide 1 (GLP-1) has been implicated in metabolic improvements after Roux-en-Y gastric bypass and vertical sleeve gastrectomy. However, data are still conflicting regarding their role after laparoscopic adjustable gastric banding (LAGB). Objective: To assess the fasting BA, FGF19, and GLP-1 concentrations in plasma before and after LAGB and to test for correlations with immunometabolic parameters. Furthermore, hepatic farnesoid X receptor (FXR) expression and regulation of FXR-dependent genes were analyzed. Design and Setting: Observational study at the University Hospital Innsbruck. Patients: Twenty obese patients. Interventions: Fasting plasma samples were taken before, 3, 6, and 12 months after LAGB. Liver biopsies were obtained at surgery and after 6 months postoperatively. Main Outcome Measures: BA profiles, GLP-1 and FGF19 levels, hepatic FXR expression and regulation of FXR target genes were determined. Results: Total, conjugated, and secondary BAs transiently increased 3 months after LAGB (P < 0.01). Only one BA, glycolithocholic acid sulfate, remained significantly elevated throughout the whole follow-up period (P < 0.05). GLP-1 had increased transiently 3 months after surgery (P < 0.01), whereas FGF19 levels increased continuously (P < 0.05). Insulin, homeostasis model assessment index, C-reactive protein, FGF19, and GLP-1 correlated positively with different BAs. No differences were seen in hepatic FXR expression and FXR-regulated genes. Conclusions: Our study results, not only identified LAGB-induced changes in BAs and BA-induced hormones, but also revealed associations between changes in BA profile with GLP-1 and FGF19.

Biomarkers of unstable angina pectoris and yangxin decoction intervention: An exploratory metabonomics study of blood plasma.

BACKGROUND: This study aimed to explore the related metabolic biomarkers and to observe the effects of Yangxin Decoction (YXD) on plasma metabolism of patients with unstable angina (UA). METHODS: In total, 10 patients with UA (intervention group) and 10 healthy participants (control group) were recruited for this study from January 2009 to December 2010. Plasma samples from both groups were analyzed using liquid chromatography mass spectrometry (LC-MS). Principle component analysis (PCA) and partial least squares (PLS) were used to explore the correlations between metabolic markers in patients with UA. RESULTS: The LC-MS results indicated that the serum levels of 5 potential metabolic markers, namely, ceramide, glycocholic acid, allocholic acid, lithocholic acid, and leukotriene (LT) B4, were significantly higher in the intervention group than those in the control group. CONCLUSION: The results of this study demonstrated potential metabolic markers that can be used to distinguish and diagnose patients with UA.

Serum Metabonomics of Mild Acute Pancreatitis.

BACKGROUND: Mild acute pancreatitis (MAP) is a common acute abdominal disease, and exhibits rising incidence in recent decades. As an important component of systemic biology, metabonomics is a new discipline developed following genomics and proteomics. In this study, the objective was to analyze the serum metabonomics of patients with MAP, aiming to screen metabolic markers with potential diagnostic values. METHODS: An analysis platform with ultra performance liquid chromatography-high-resolution mass spectrometry was used to screen the difference metabolites related to MAP diagnosis and disease course monitoring. RESULTS: A total of 432 endogenous metabolites were screened out from 122 serum samples, and 49 difference metabolites were verified, among which 12 difference metabolites were identified by nonparametric test. After material identification, eight metabolites exhibited reliable results, and their levels in MAP serum were higher than those in healthy serum. Four metabolites exhibited gradual downward trend with treatment process going on, and the differences were statistically significant (P < 0.05). CONCLUSION: Metabonomic analysis has revealed eight metabolites with potential diagnostic values toward MAP, among which four metabolites can be used to monitor the disease course.

Macromolecular Crowding Agents-Assisted Imprinted Polymers For Analysis Of Glycocholic Acid In Human Plasma And Urine.

Glycocholic acid (GCA) is a newly identified biomarker for hepatocellular carcinoma (HCC) patients. In this study, a method based on macromolecular crowding strategy has been applied for preparation of a molecularly imprinted polymer (MIP), which possesses high adsorption capacity for GCA. Polymethyl methacrylate was used as a macromolecular crowding agent, N-(3-aminopropyl)-methacrylamide hydrochloride as a functional monomer and ethylene dimethacrylate as a cross-linker. The morphology and binding characteristics of MIP were assessed by scanning electron microscopy and absorption experiments. The MIP was used as an adsorbent material to separate GCA, and the molecularly imprinted solid-phase extraction (MISPE) was carefully optimized. The MISPE combined with high-performance liquid chromatographic analysis was successfully used to determine the GCA in plasma and urine samples. When spiked levels ranged from 0.2 to 20 µmol L-1 , the recoveries were between 94.3 and 100.5%. As a proof of principle, this proposed method has been validated on a small subset of HCC patients (n = 10) and healthy volunteers (n = 10). The average GCA concentrations of HCC patients in plasma and urine were about 25 and 2.8 times than that of healthy volunteers. Copyright © 2016 John Wiley & Sons, Ltd.

Investigation of vitamin B6 inadequacy, induced by exposure to the anti-B6 factor 1-amino D-proline, on plasma lipophilic metabolites of rats: a metabolomics approach.

PURPOSE: Vitamin B6 status in the body is affected by several factors including dietary supply of the antivitamin B6 factor, 1-amino D-proline (1ADP), which is present in flaxseed. Owing to the prevalence of moderate B6 deficiency in the general population, a co-occurrence of 1ADP may lead to a further deterioration of B6 status. To this end, we applied a nontargeted metabolomics approach to identify potential plasma lipophilic biomarkers of deleterious effect of 1ADP on moderately vitamin B6-deficient rats using a high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry. METHODS: Twenty-four rats were fed with a semi-purified diet containing pyridoxine·HCl (PN·HCl) either 7 mg/kg diet (optimal B6) or 0.7 mg/kg diet (moderate B6). The rats were divided into four treatments (n = 6), and one treatment in each B6 diet group was also fed ad libitum with 10 mg/kg diet of synthetic 1ADP. After 5 weeks of study, plasma was collected from the rats and lipophilic metabolites were extracted using acetonitrile as a solvent for analysis. RESULTS: Ten potential plasma lipophilic biomarkers were identified out of >2500 detected entities, which showed significant differences between the treatments. Plasma glycocholic acid, glycoursodeoxycholic acid, murocholic acid, N-docosahexaenoyl GABA, N-arachidonoyl GABA, lumula, nandrolone and orthothymotinic acid concentrations were significantly elevated, while plasma cystamine and 3-methyleneoxindole concentrations were significantly reduced as a result of either low B6 status or 1ADP or their interaction. CONCLUSION: Changes in these metabolites revealed a potential defect in pathways linked with the biosynthesis and metabolism of bile acid components, N-acyl amino acids, analgesic androgens, anti-inflammatory and neuroprotective molecules. We also noted that the changes in these biomarkers can be alleviated by the application of adequate vitamin B6.

Exploration of novel predictive markers in rat plasma of the early stages of chronic renal failure.

To identify blood markers for early stages of chronic kidney disease (CKD), blood samples were collected from rats with adenine-induced CKD over 28 days. Plasma samples were subjected to metabolomic profiling by liquid chromatography-mass spectrometry, followed by multivariate analyses. In addition to already-identified uremic toxins, we found that plasma concentrations of N6-succinyl adenosine, lysophosphatidylethanolamine 20:4, and glycocholic acid were altered, and that these changes during early CKD were more sensitive markers than creatinine concentration, a universal indicator of renal dysfunction. Moreover, the increase in plasma indoxyl sulfate concentration occurred earlier than increases in phenyl sulfate and p-cresol sulfate. These novel metabolites may serve as biomarkers in identifying early stage CKD.

Evaluation of serum bile acid profiles as biomarkers of liver injury in rodents.

Bile acids (BAs) have been studied as potential biomarkers of drug-induced liver injury. However, the relationship between levels of individual BAs and specific forms of liver injury remains to be fully understood. Thus, we set out to evaluate cholic acid (CA), glycocholic acid (GCA), and taurocholic acid (TCA) as potential biomarkers of liver injury in rodent toxicity studies. We have developed a sensitive liquid chromatography-tandem mass spectrometry (LC/MS/MS) assay applicable to rat and mouse serum and evaluated levels of the individual BAs in comparison with the classical biomarkers of hepatotoxicity (alanine aminotransferase [ALT], aspartate aminotransferase [AST], glutamate dehydrogenase (GLDH), alkaline phosphatase, total bilirubin, gamma-glutamyl transferase, and total BAs) and histopathology findings in animals treated with model toxicants. The pattern of changes in the individual BAs varied with different forms of liver injury. Animals with histopathologic signs of hepatocellular necrosis showed increases in all 3 BAs tested, as well as increases in ALT, AST, GLDH, and total BAs. Animals with histopathologic signs of bile duct hyperplasia (BDH) displayed increases in only conjugated BAs (GCA and TCA), a pattern not observed with the other toxicants. Because BDH is detectable only via histopathology, our results indicate the potential diagnostic value of examining individual BAs levels in serum as biomarkers capable of differentiating specific forms of liver injury in rodent toxicity studies.

Application of ultra-performance liquid chromatography coupled with mass spectrometry to metabonomic study on spontaneously hypertensive rats and intervention effects of Ping Gan prescription.

This study describes the metabonomic characters of the spontaneously hypertensive rats (SHR) and intervention effects of Ping Gan prescription. Ultra performance LC coupled with Q-TOF MS (UPLC/MS-Q-TOF) was employed to develop a metabonomic method of the plasma of SHR. There was a significant difference in metabolic profiling observed between predose group of Wistar Kyoto rats and model group (SHR) by using the principal components analysis (PCA). Some significant changes in metabolites such as LysoPC(22:6), LysoPC(20:4), LysoPC(18:1), cholylglycine, PE(P-16:0e/0:0), sphingosine-1-phosphate, and 2-oxo-4-methylthio butanoic acid were identified. These biochemical changes were associated with the disturbance in sphingolipid metabolism and fat metabolism, which would be helpful to further understand the essence of hypertension and the therapeutic mechanism of Ping Gan prescription. This study suggests that the metabonomic method may be a valuable and feasible tool to explore the therapeutic effect and mechanism of traditional Chinese medicine (TCM).

[Clinical analysis of fetal death cases in intrahepatic cholestasis of pregnancy].

OBJECTIVE: To investigate the clinical features, critical laboratory parameters, and fetal monitoring methods in intrahepatic cholestasis of pregnancy (ICP). METHODS: A retrospective analysis of 21 cases of ICP suffered with fetal death in Women's hospital, School of Medicine, Zhejiang University from January 1999 to December 2010 were discussed. RESULTS: (1) The average age of ICP patients suffered with fetal death were (30.2 ± 4.6) years old. Among them, 4 cases were older than 35 years, six cases were multipara, one of them suffered stillbirth 2 year before. Twenty cases were singleton pregnancies and 1 case was twin pregnancy. (2) All 21 cases of fetal death occurred in the third trimester, 12 cases occurred before 37 weeks, 9 cases after 37 weeks.Nine cases were diagnosed by ultrasound in outpatient clinics, fetal heart beat disappeared in 9 patients after admission because of ICP, two disappeared after labor, one during anesthesia before emergent surgery. Perinatal mortality rate of ICP was 0.148% (21/14 184), and fetal death occurred from 29 to 41 weeks with an average gestational age of (33.8 ± 4.2) weeks. (3) Puritus occurred in all 21 cases while 11 of them had pruritus all over the body. Ten pregnant women felt the fetal movement decreased or disappeared before diagnosis of fetal death. The glycocholic acid levels increased in all of the 21 cases. Among them, glycocholic acid levels in 11 cases were (21.49 - 64.48) µmol/L, while in 10 cases were ≥ 64.48 µmol/L. Serum bile acid levels elevated in 16 cases which had been analyzed (the other 5 cases had not been checked), and the highest level reached 270 µmol/L. Serum alanine aminotransferase and aspartate aminotransferase were increased in 14 cases. Seven cases had their total bilirubin > 21 µmol/L, and 12 cases had their direct bilirubin levels significantly elevated. Among the 21 cases of ICP, 15 cases were in severe status, while the other 6 cases were mild. (4) Nine patients had no antepartum surveillance since fetal death were diagnosed before admission. The results of antepartum surveillance were as follows: 2 cases had nonreassuring nonstress test (NST), one had mild "V" type deceleration. Absence of diastolic flow in umbilical artery were found in 3 cases, and low fetal biophysical score was got in one case. (5) All 21 patients had vaginal delivery. Six of them delivered after natural contraction, and the remaining 14 cases delivered after oral intake of mifepristone and amniotic injection of ethacridine, or oxytocin induced labor within 48 hours, only one case delivered after additional dinoprostone suppositories. The appearance of fetus, placentas and membranes were normal, the lengths of umbilical cord were average. Four cases were found with cords binding the necks or the bodies. Eighteen cases had grade III amniotic fluid with meconium-stained, and 2 cases complicated by oligohydramnios. Ten cases had their fetuses and placentas examined by pathologist. Among them, one case had multiple malformations, no more obvious pathological abnormalities were found in other fetuses. Pathologic examination showed that fibrin deposited around chorion and deciduas basalis, large vessels accompanied by calcification, degeneration, hemorrhagic infarction, and increased focal syncytial nodules could be seen in all of the ten placentas. CONCLUSIONS: Fetal death in pregnant women with ICP often occurs after the contractions, Severe ICP may be a key factor that involved in the occurrence of fetal death. Up to now, there is no valid indicators in fetal monitoring, which can predict fetal death. Extensive assessment of the severity and careful antepartum surveillance should be achieved before timely termination of pregnancy.