Bile UPLC-MS fingerprinting and bile acid fluxes during human liver transplantation

Bile flow restoration is a crucial step in the recovery process post transplantation of the liver. Here, metabolic trajectories based on changes in bile secretion - a known marker of functionality - have been utilised as an approach for discovering bile fluxes during transplantation. A total of ten liver transplants were monitored and from these 68 bile samples from both donors and recipients were collected and analysed using ultra-performance LC-MS in combination with multivariate statistical analysis. Based on the principal component scores constructed from the total bile fingerprint, differentiation of the bile acid concentrations before and after transplantation was detected. A trend was also observed, by constructing metabolic trajectories, whereby the post-transplant profiles approached the position of pre-transplant profiles within 30-60?min of the restoration of bile secretion function. The ten major conjugated bile acid salts were measured and a significant increase in concentrations of taurocholic acid and taurochenodeoxycholic acid were seen after transplantation. In addition, the ratios of secondary bile acids detected in gall bladder and hepatic bile were measured before and after transplantation. This study suggests that bile acid ratios in the donor liver at the pre-transplant and post-transplant stage may be important and that profiling of secreted bile after transplantation may aid clinical assessment and progress post-transplantation.     

Systematic characterization of the metabolites of echinacoside and acteoside from Cistanche tubulosa in rat plasma,bile, urine and feces based on UPLC‐ESI‐Q‐TOF‐MS

Echinacoside (ECH) and acteoside (ACT), as the most and major active components of Cistanche tubulosa, were reported to possess cardioactive, neuroprotective and hepatocyte protective effects, as well as antibacterial, antioxidative effects. Recently, more studies have focused on their pharmacological activities. However, their metabolic profiles in vivo have not been sufficiently investigated. This study proposes an approach for rapidly identifying the complicated and unpredictable metabolites of ECH and ACT in rat plasma, bile, urine and feces, and systematically and comprehensively revealing their major metabolic pathways, based on powerful ultra‐high performance liquid chromatography coupled with quadrupole time‐of‐flight tandem mass spectrometry. Plasma, bile, urine and feces were collected from rats after a single 200 mg/kg oral dose. A total of 49 metabolites were detected in rat biological samples. Through analyzing metabolites in bile samples, it was found that ECH and ACT were subjected to a marked hepatic first‐pass effect in liver. Copyright © 2016 John Wiley & Sons, Ltd.     

Hepatocyte organoids and cell transplantation: What the future holds

Historically, primary hepatocytes have been difficult to expand or maintain in vitro. In this review, we will focus on recent advances in establishing hepatocyte organoids and their potential applications in regenerative medicine. First, we provide a background on the renewal of hepatocytes in the homeostatic as well as the injured liver. Next, we describe strategies for establishing primary hepatocyte organoids derived from either adult or fetal liver based on insights from signaling pathways regulating hepatocyte renewal in vivo. The characteristics of these organoids will be described herein. Notably, hepatocyte organoids can adopt either a proliferative or a metabolic state, depending on the culture conditions. Furthermore, the metabolic gene expression profile can be modulated based on the principles that govern liver zonation. Finally, we discuss the suitability of cell replacement therapy to treat different types of liver diseases and the current state of cell transplantation of in vitro-expanded hepatocytes in mouse models. In addition, we provide insights into how the regenerative microenvironment in the injured host liver may facilitate donor hepatocyte repopulation. In summary, transplantation of in vitro-expanded hepatocytes holds great potential for large-scale clinical application to treat liver diseases.Subject terms: Adult stem cells, Self-renewal     

Serum metabonomics characterization of liver fibrosis induced by bile duct-ligated in rats and the intervention effects of herb compound 861

Liver fibrosis has been increasingly recognized as a cause for high morbidity and mortality of some diseases in humans. Herbal medicines have received great attention due to their low side effects and high safety. Herbal compound 861 (Cpd 861) has been effectively used for treating hepatic fibrosis for long time. Yet, its exact mechanism is still in fancy. Herein, we first established a liver fibrosis model by bile duct ligation (BDL), which led to the toxic accumulation of bile acids in animals, resulting in hepatic fibrosis. A serum metabonomics study on BDL-induced liver fibrosis rats after Cpd 861 treatment was performed using UHPLC-QTOF/MS. Multivariate analysis showed that Cpd 861 significantly reversed the metabolic perturbation induced by BDL to normal state, which is in agreement with the serum biochemical and histopathological findings. 15 metabolites were screened as potential biomarkers. Eight metabolic pathways were recognized as the most relevant pathways, involving dysfunction of amino acids metabolism and synthesis, fatty acid metabolism, phospholipids metabolism, and others. This is the first study to reveal the underlying mechanism of Cpd 861 based on metabonomics, which is complementary to biochemical analysis, and more importantly, a potentially powerful tool to interpret the mechanisms of extremely complex systems.     

Surface sampling capillary electrophoresis–mass spectrometry for a direct chemical characterization of tissue and blood samples

Capillary electrophoresis (CE) is a powerful separation tool for non-targeted analysis of chemically complex samples, such as blood, urine, and tissue. However, traditionally CE requires samples in solution for analysis, which limits information on analyte distribution and heterogeneity in tissue. The recent development of surface sampling CE–mass spectrometry (SS-CE–MS) brings these advantages of CE to solid samples and enables chemical mapping directly from the tissue surface without laborious sample preparation. Here, we describe developments of SS-CE–MS to increase reproducibility and stability for metabolite, lipid, and protein extraction from tissue sections and dried blood spots. Additionally, we report the first electrokinetic sequential sample injection for high throughput analysis. We foresee that the wide molecular coverage from a distinct tissue region in combination with higher throughput will provide novel information on biological function and dysfunction.     

Immobilizing sulfotransferase 1A1 on magnetic microparticles and their evaluation using capillary electrophoresis

Sulfotransferases are categorized as phase II metabolic enzymes. Human sulfotransferase 1A1 (SULT1A1) is involved in the sulfonation of xenobiotics with aid from the cofactor 3'‐phosphoadenosine‐5'‐phosphosulfate that acts as a sulfonate donor. In this study, we have attempted to immobilize SULT1A1 on magnetic microparticles (MMs). Different functionalized MMs were used to immobilize SULT1A1 and their enzyme activity was compared to the control (enzyme in solution). Paracetamol was used as model substrate. Separation of paracetamol and paracetamol sulfate by CE‐UV was optimized and validated. MMs with epoxy based immobilization of SULT1A1 showed better enzyme activity. Hence, they were tested for repeated usage to allow their implementation for the development of a CE immobilized micro enzyme reactor.     

Metabolic profiling for the identification of Huntington biomarkers by on‐line solid‐phase extraction capillary electrophoresis mass spectrometry combined with advanced data analysis tools

In this work, an untargeted metabolomic approach based on sensitive analysis by on‐line solid‐phase extraction capillary electrophoresis mass spectrometry (SPE‐CE‐MS) in combination with multivariate data analysis is proposed as an efficient method for the identification of biomarkers of Huntington's disease (HD) progression in plasma. For this purpose, plasma samples from wild‐type (wt) and HD (R6/1) mice of different ages (8, 12, and 30 weeks), were analyzed by C₁₈‐SPE‐CE‐MS in order to obtain the characteristic electrophoretic profiles of low molecular mass compounds. Then, multivariate curve resolution alternating least squares (MCR‐ALS) was applied to the multiple full scan MS datasets. This strategy permitted the resolution of a large number of metabolites being characterized by their electrophoretic peaks and their corresponding mass spectra. A total number of 29 compounds were relevant to discriminate between wt and HD plasma samples, as well as to follow‐up the HD progression. The intracellular signaling was found to be the most affected metabolic pathway in HD mice after 12 weeks of birth, when mice already showed motor coordination deficiencies and cognitive decline. This fact agreed with the atrophy and dysfunction of specific neurons, loss of several types of receptors, and changed expression of neurotransmitters.     

Electromigration of a heteroconjugated imidazole-acetate complex in ACN

ACN is an extremely poor hydrogen bond donor and therefore the anions dissolved in it are solvated mainly by other hydrogen bond donors (e.g. uncharged acids) possibly present in the solution. Under properly selected experimental conditions stabilization via hydrogen bonding can be used for separation in CE as has been demonstrated for uncharged acids by several authors. Electromigration based on heteroconjugation can be of importance, e.g. when aqueous separation medium cannot be used due to stability reasons. It also allows CE to be used as a tool for solution chemistry measurements, if the required physicochemical properties of the studied system are known or they can be predicted with sufficient accuracy by existing theories. In the present work we showed that also an uncharged base can stabilize an anion via hydrogen bonding in ACN. In the setup imidazole was chosen as a model base and acetate ion as complexing anion in equimolar acetic acid-acetate buffer. The resulted hydrogen-bonded imidazole-acetate complex (i.e. heteroconjugate) possesses a charge and can thus migrate in CE. It was shown that the studied complexation in ACN is sensitive to competition by other hydrogen bond donors such as water and methanol. On the other hand, acetone, which is a poor hydrogen bond donor, did not have much effect on the complexation. To take the effect of ionic strength on mobility into account, mobilities of the imidazole-acetate complex measured at various ionic strengths were corrected to zero ionic strength by the aid of conductivity equation. A fit of the 1:1 binding isotherm to the ionic strength corrected mobility versus acetate concentration data led to rather good correlation. However, x-reciprocal linear transformation of the binding isotherm showed nonlinearity, which could be partly explained by homoconjugation of acetic acid and acetate ion. Since the homoconjugation constant for acetic acid under present experimental conditions was not available, theoretical simulations were used to demonstrate the effects of homoconjugation. The possibility of multiple complexation of imidazole was discussed as well.     

Metal cations for the determination of fluorescent phosphoinositides by capillary electrophoresis

Phosphatidylinositol (PI) and its phosphorylated derivatives known as phosphoinositides (PIPs), are essential regulators of cell signaling and membrane trafficking, cytoskeletal dynamics, and nuclear functions. Disruption of PI metabolism is associated with disorders such as immune dysfunction, cardiovascular disease, and cancer; therefore, there is currently great interest in studying PIPs and their metabolic enzymes. Here, we describe a method for the separation of fluorescent PI and its seven fluorescent phosphorylated derivatives by CE‐LIF. The CE method utilizes a Tris buffer and sodium deoxycholate in the presence of 30% 1‐propanol and 5% of a dynamic coating reagent, EOTrol^TM low reverse (EOTrol LR). It is simple, fast, highly sensitive, and it offers LODs in the order of 1.5 amol. The effect of cations such as lithium, sodium, potassium, cesium, barium, manganese, zinc, magnesium, calcium, spermine, and gentamicin were evaluated. Calcium and magnesium provided the best selectivity and resolution for the separation of the analytes while magnesium offered the best data reproducibility. The developed CE method would be useful in the studies of enzymatic activity in the PI and PIPs metabolic pathways using CE‐based in vitro and CE cell‐based assays, and/or for drug screening.     

Clinical significance of serum bile acid radioimmunoassay in hepatobiliary diseases--with special reference to the CG/SLCG ratio (author's transl)

Both serum cholylglycine (CG) and sulfolithocholylglycine (SLCG) levels were radioimmunoassayed by PEG method in 209 samples (204 hepatobiliary diseases, 5 normal controls). 1) The results revealed that serum bile acid levels were excellent indicators for hepatic dysfunction in comparison with the conventional liver function tests. 2) Means of 19.4 +/- 9.3 microgram/dl for CG and 21.7 +/- 6.7 microgram/dl for SLCG were obtained in controls. Most hepatobiliary diseases demonstrated abnormally high bile acid levels, with extremely high CG values in conditions with bile stasis. 3) To differentiate various hepatobiliary diseases more clearly, the ratio of the primary and secondary bile acids (CG/SLCG ratio) was introduced (1.0 +/- 0.6 for controls). In cases of bile stasis, CG/SLCG ratios ranged from 7.8 +/- 4.8 for intrahepatic cholestasis to 34.8 +/- 27.6 for congenital biliary atresia, while other hepatic disorders demonstrated relatively low values. We conclude that the CG/SLCG ratio is a useful index for cholestasis. Diagnosis of the congenital biliary atresia could be possible.     

Molecular complexes of crown ethers. part 2: Complexes between crown ethers and π acceptors

The intermolecular charge transfer (CT) complexes of four crown ethers (CE), viz. B15C5, B18C6, DB18C6 and DB24C8 (as donors), and three acceptors, i.e. tetracyanoethylene (TCNE) 7,7,8,8-tetracyanoquinodimethane (TCNQ) and chloranil were studied in the UV-Visible region in 1,2-dichloroethane (DCE) at 298.2 K. Solid complexes were also prepared and the ratio of the donor to the acceptor was 1:1. Results indicate that the _max of the CT band decreases whileK _c (formation equilibrium constant) increases following the systematic change of the electron affinity of the acceptors. TheK _c values suggest that the phenyl group in CE plays a major role in the formation of a CT complex.     

The Induction and Analysis of Micronuclei and Cell Killing by Ultraviolet-B Radiation in Human Peripheral Blood Lymphocytes

The DNA-damaging potential of ultraviolet-B (UVB) radiation was investigated by analyzing the frequency and origin of micronuclei (MN) in cytokinesis-blocked, binucleated (BN) peripheral blood lymphocytes (PBL) and cloning efficiencies (CE) of PBL after exposure to different fluences of UVB. In total, PBL obtained from five normal donors were investigated. The PBL from all donors showed a dose-related, linear-quadratic increase in the frequency of MN per 1000 BN cells and in the frequency of micronucleated BN cells. In two experiments the origin of UVB-induced MN was studied by analyzing MN for the presence or absence of centromeres by applying the MN assay in combination with a centromeric probe and fluorescence in situ hybridization. This revealed, for the first time, that UVB-induced MN were centromere negative, indicating that UVB acted exclusively as a clastogenic agent in the tested dose range. The PBL from all donors showed a clear dose-dependent decrease in CE, after UVB exposure. The UVB-exposed PBL from all donors showed an inverse relationship between the induction of MN and the decrease in CE, but regression analysis revealed no correlation between the induction of MN and the decrease in cell survival. It is concluded that UVB has a clastogenic and cytotoxic effect on PBL.     

Simultaneous determination of serotonin and creatinine in urine by combining two ultrasound-assisted emulsification microextractions with on-column stacking in capillary electrophoresis

This article describes the development of a rapid, simple, and sensitive analytical approach for the simultaneous determination of serotonin (5‐hydroxytryptamine) and creatinine in urine samples by combining two ultrasound‐assisted emulsification microextractions (USAEMEs) in series with on‐column stacking in CE. This serial USAEME procedure comprises analytes extraction from the donor solution (urine with K₂CO₃ additive) to an organic solvent followed by a back‐extraction from the organic phase into a small volume of hydrochloric acid. After 15 min of sample pretreatment, the acidic acceptor solution was analyzed directly on CE in the mode of capillary zone electrophoresis. The adoption of HCl as the acceptor phase not only provided effective back‐extraction but also facilitated pH‐mediated on‐column stacking in CE analysis. About 360‐fold sensitivity enhancement was achieved for serotonin detection. The limits of detection were 7.9 nM for serotonin and 13.3 μM for creatinine, respectively. Satisfactory results were obtained with respect to precision and recovery. The proposed method has been demonstrated to be convenient and effective for the analysis of real urine samples. We believe that two USAEMEs in series will find wide applications in simplified sample pretreatment prior to CE analysis.     

Mass spectrometry for the detection of differentially expressed proteins: a comparison of surface-enhanced laser desorption/ionization and capillary electrophoresis/mass spectrometry

The discovery of biomarkers is currently attracting much interest as it harbors great potential for the diagnosis and monitoring of human diseases. Here we have used two advanced mass spectroscopy based technologies, surface enhanced laser desorption ionization (SELDI-MS) and capillary electrophoresis/mass spectrometry (CE/MS), to obtain proteomic patterns of urine samples from patients suffering from membranous glomerulonephritis (MGN) and healthy volunteers. The results indicate that CE/MS analysis is able to display a rich and complex pattern of polypeptides with high resolution and high mass accuracy. In order to analyze these patterns, the MosaiqueVisu software was developed for peak identification, deconvolution and the display of refined maps in a three-dimensional format. The polypeptide profiles obtained with SELDI-MS from the same samples are much sparser and show lower resolution and mass accuracy. The SELDI-MS profiles are further heavily dependent on analyte concentration. SELDI-MS analysis identified three differentially expressed polypeptides, which are potential biomarkers that can distinguish healthy donors from patients with MGN. In contrast, approximately 200 potential biomarkers could be identified by CE/MS. Thus, while SELDI-MS is easy to use and requires very little sample, CE/MS generates much richer data sets that enable an in-depth analysis.     

Potential of capillary electrophoresis, tandem mass spectrometry and coupled capillary electrophoresis-tandem mass spectrometry as diagnostic tools

Urine and blood samples from patients with known metabolic disorders have been analyzed by CE, MS-MS and CE-MS-MS. For the identification of defects in acylcarnitine metabolism, blood spots on filter paper were analyzed using an MS-MS "neonatal screening" approach. Direct CE-MS-MS analysis was used for the analysis of urine samples from patients with different metabolic disorders, including galactosemia, neuroblastoma, Zellweger syndrome, propionic acidemia and alcaptonuria. The sensitivity of the CE-MS-MS method was increased by use of multiple reaction monitoring.     

Identification of the absorbed components and their metabolites of Tianma‐Gouteng granule in rat plasma and bile using ultra‐high‐performance liquid chromatography combined with quadrupole time‐of‐flight mass spectrometry

Tianma‐Gouteng granule (TGG), a Chinese herbal formula preparation, is clinically used for the treatment of cardio‐cerebrovascular diseases such as hypertension, cerebral ischaemia, acute ischaemic stroke and Parkinson's disease. Although few reports have been published concerning the absorbed prototype components of TGG, the possible metabolic pathways of TGG in vivo remain largely unclear. In this study, a method using UPLC–Q/TOF MS was established for the detection and identification of the absorbed prototype components and related metabolites in rat plasma and bile after oral administration of TGG at high and normal clinical dosages. A total of 68 components were identified or tentatively identified in plasma and bile samples, including absorbed prototypes and their metabolites. The major absorbed components were gastrodin, isorhynchophylline, rhynchophylline, isocorynoxeine, corynoxeine, geissoschizine methyl ether baicalin, baicalein, wogonoside, wogonin, geniposidic acid, leonurine, 2,3,5,4′‐tetrahydroxystilbene‐2‐O‐β‐d ‐glucoside and emodin. The main metabolic pathways of these components involved phase I (isomerization, hydrolysis and reduction) and phase II (glucuronidation and sulfation) reaction, and the phase II biotransformation pathway was predominant. The present study provides rich information on the in vivo absorption and metabolism of TGG, and the results will be helpful for further studies on the pharmacokinetics and pharmacodynamics of TGG.     

Determination of bile acids in pig liver, pig kidney and bovine liver by gas chromatography-chemical ionization tandem mass spectrometry with total ion chromatograms and extraction ion chromatograms

An effective method has been developed for quantitative determination of six bile acids including lithocholic acid (LCA), deoxycholic acid (DCA), chenodeoxycholic acid (CDCA), hydodeoxycholic acid (HDCA), cholic acid (CA) and ursodeoxycholic acid (UDCA) in biological tissues including pig liver, pig kidney and bovine liver by gas chromatography-chemical ionization/tandem mass spectrometry (GC-CI/MS/MS). Camphor-10-sulphonic acid (CSA) was proposed as effective catalyst for bile acid derivatization. Reactions were accelerated ultrasonically. The effects of different catalysts and reaction times on derivatization efficiency were evaluated and optimized. Bile acids were determined as methyl ester-trimethylsilyl ether and methyl ester-acetate derivatives. The efficiency of trimethylsilylation and acetylation was evaluated. Trimethylsilylation was done with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) as the trimethylsilyl donating reagent in a ultrasonic bath for 20 min. Acetylation was done in pyridine with acetic anhydride at 40-45°C for 4 h. The former reaction was faster than the latter. Thus, trimethylsilylation was employed for the quantitative analysis. Negligible interferences from sterols in biological matrices were observed when the biological samples were treated with solid phase extraction before GC-CI/MS/MS. The linearity, reproducibility, detection limit and recovery were evaluated under the optimized conditions. Satisfactory results were obtained when bile acid derivatives of LCA, CDCA, HDCA, and UDCA were determined with total ion chromatograms (TIC) while DCA and CA were determined with extracted ion chromatograms (EIC), respectively. The detection limits (S/N=3) for six bile acids in biological tissues were ranging from 0.40 to 1.6 ng/mL and the recoveries indicated that the proposed method was feasible for the determination of trace bile acids in the biological samples studied. The experimental results for the animal tissues purchased from five different markets were compared. Interestingly, all of the six bile acids were present in pig liver while only the dihydroxy bile acids, DCA, CDCA and HDCA were found in pig kidney. In addition to DCA and CDCA, trihydroxy bile acid, CA, are the major bile acids in bovine liver.     

Assessing the suitability of capillary electrophoresis‐mass spectrometry for biomarker discovery in plasma‐based metabolomics

The actual utility of capillary electrophoresis‐mass spectrometry (CE‐MS) for biomarker discovery using metabolomics still needs to be assessed. Therefore, a simulated comparative metabolic profiling study for biomarker discovery by CE‐MS was performed, using pooled human plasma samples with spiked biomarkers. Two studies have been carried out in this work. Focus of study I was on comparing two sets of plasma samples, in which one set (class I) was spiked with five isotope‐labeled compounds, whereas another set (class II) was spiked with six different isotope‐labeled compounds. In study II, focus was also on comparing two sets of plasma samples, however, the isotope‐labeled compounds were spiked to both class I and class II samples but with concentrations which differ by a factor two between both classes (with one compound absent in each class). The aim was to determine whether CEMS‐based metabolomics could reveal the spiked biomarkers as the main classifiers, applying two different data analysis software tools (MetaboAnalyst and Matlab). Unsupervised analysis of the recorded metabolic profiles revealed a clear distinction between class I and class II plasma samples in both studies. This classification was mainly attributed to the spiked isotope‐labeled compounds, thereby emphasizing the utility of CE‐MS for biomarker discovery.     

CE–MS for anionic metabolic profiling: An overview of methodological developments

The efficient profiling of highly polar and charged metabolites in biological samples remains a huge analytical challenge in metabolomics. Over the last decade, new analytical techniques have been developed for the selective and sensitive analysis of polar ionogenic compounds in various matrices. Still, the analysis of such compounds, notably for acidic ionogenic metabolites, remains a challenging endeavor, even more when the available sample size becomes an issue for the total analytical workflow. In this paper, we give an overview of the possibilities of capillary electrophoresis‐mass spectrometry (CE–MS) for anionic metabolic profiling by focusing on main methodological developments. Attention is paid to the development of improved separation conditions and new interfacing designs in CE–MS for anionic metabolic profiling. A complete overview of all CE–MS‐based methods developed for this purpose is provided in table format (Table 1) which includes information on sample type, separation conditions, mass analyzer and limits of detection (LODs). Selected applications are discussed to show the utility of CE–MS for anionic metabolic profiling, especially for small‐volume biological samples. On the basis of the examination of the reported literature in this specific field, we conclude that there is still room for the design of a highly sensitive and reliable CE–MS method for anionic metabolic profiling. A rigorous validation and the availability of standard operating procedures would be highly favorable in order to make CE–MS an alternative, viable analytical technique for metabolomics.