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Bile Samples (bile + sample)
Selected AbstractsCharacterization of the PCR inhibitory effect of bile to optimize real-time PCR detection of Helicobacter speciesFEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 2 2005Waleed Abu Al-Soud Abstract The inhibitory effect of human and porcine bile samples to detect Helicobacter DNA was studied by adding different concentrations of bile samples to PCR mixtures of six thermostable DNA polymerases containing cagA specific primers and Helicobacter pylori DNA. PCR products were amplified by using the Rotorgene system and SYBR Green I. Among the six DNA polymerases tested, rTth had the lowest sensitivity to bile inhibitors, whereas Taq and Tfl had the highest sensitivity. Bile proteins did not inhibit AmpliTaq DNA polymerase, whereas the fraction containing mainly bile acids and their salts inhibited the amplification capacity of AmpliTaq. Heating human bile at 98 °C and adding casein and formamide to the reaction mixture reduced the PCR inhibitory effect of bile. Therefore, a pre-PCR treatment based on dilution and heating of bile, adding casein and formamide to the reaction mixture of rTth DNA polymerase was found efficient to amplify DNA directly in bile. [source] Peptide antibiotic human beta-defensin-1 and ,2 contribute to antimicrobial defense of the intrahepatic biliary treeHEPATOLOGY, Issue 4 2004Kenichi Harada Human beta-defensins (hBDs) are important antimicrobial peptides that contribute to innate immunity at mucosal surfaces. This study was undertaken to investigate the expression of hBD-1 and hBD-2 in intrahepatic biliary epithelial cells in specimens of human liver, and 4 cultured cell lines (2 consisting of biliary epithelial cells and 2 cholangiocarcinoma cells). In addition, hBD-1 and hBD-2 were assayed in specimens of bile. hBD-1 was nonspecifically expressed immunohistochemically in intrahepatic biliary epithelium and hepatocytes in all patients studied, but expression of hBD-2 was restricted to large intrahepatic bile ducts in 8 of 10 patients with extrahepatic biliary obstruction (EBO), 7 of 11 with hepatolithiasis, 1 of 6 with primary biliary cirrhosis (PBC), 1 of 5 with primary sclerosing cholangitis (PSC), 0 of 6 with chronic hepatitis C (CH-C), and 0 of 11 with normal hepatic histology. hBD-2 expression was evident in bile ducts exhibiting active inflammation. Serum C reactive protein levels correlated with biliary epithelial expression of hBD-2. Real-time PCR revealed that in all of 28 specimens of fresh liver, including specimens from patients with hepatolithiasis, PBC, PSC, CH-C and normal hepatic histology, hBD-1 messenger RNA was consistently expressed, whereas hBD-2 messenger RNA was selectively expressed in biliary epithelium of patients with hepatolithiasis. Immunobloting analysis revealed hBD-2 protein in bile in 1 of 3 patients with PSC, 1 of 3 with PBC, and each of 6 with hepatolithiasis; in contrast, hBD-1 was detectable in all bile samples examined. Four cultured biliary epithelial cell lines consistently expressed hBD-1; in contrast these cell lines did not express hBD-2 spontaneously but were induced to express hBD-2 by treatment with Eschericia coli, lipopolysaccharide, interleukin-1, or tumor necrosis factor-,. In conclusion, these findings suggest that in the intrahepatic biliary tree, hBD-2 is expressed in response to local infection and/or active inflammation, whereas hBD-1 may constitute a preexisting component of the biliary antimicrobial defense system. Supplementary material for this article can be found on the Hepatology website (http:/interscience.wley.com/jpages/0270,9139/suppmat/index.html). (Hepatology 2004;40:925-932). [source] An algorithm for thorough background subtraction from high-resolution LC/MS data: application to the detection of troglitazone metabolites in rat plasma, bile, and urineJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 9 2008Haiying Zhang Abstract Interferences from biological matrices remain a major challenge to the in vivo detection of drug metabolites. For the last few decades, predicted metabolite masses and fragmentation patterns have been employed to aid in the detection of drug metabolites in liquid chromatography/mass spectrometry (LC/MS) data. Here we report the application of an accurate mass-based background-subtraction approach for comprehensive detection of metabolites formed in vivo using troglitazone as an example. A novel algorithm was applied to check all ions in the spectra of control scans within a specified time window around an analyte scan for potential background subtraction from that analyte spectrum. In this way, chromatographic fluctuations between control and analyte samples were dealt with, and background and matrix-related signals could be effectively subtracted from the data of the analyte sample. Using this algorithm with a ± 1.0 min control scan time window, a ± 10 ppm mass error tolerance, and respective predose samples as controls, troglitazone metabolites were reliably identified in rat plasma and bile samples. Identified metabolites included those reported in the literature as well as some that had not previously been reported, including a novel sulfate conjugate in bile. In combination with mass defect filtering, this algorithm also allowed for identification of troglitazone metabolites in rat urine samples. With a generic data acquisition method and a simple algorithm that requires no presumptions of metabolite masses or fragmentation patterns, this high-resolution LC/MS-based background-subtraction approach provides an efficient alternative for comprehensive metabolite identification in complex biological matrices. Copyright © 2008 John Wiley & Sons, Ltd. [source] Contradistinction between doxorubicin and epirubicin: in-vivo metabolism, pharmacokinetics and toxicodynamics after single- and multiple-dosing in ratsJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 7 2001Sandhya Ramanathan-Girish There is compelling in-vitro evidence that the evaluation of doxorubicin or epirubicin pharmacokinetics based solely on plasma concentration may not fully elucidate the differences between the two drugs. Both compounds bind to erythrocytes and their different binding to haemoglobin may influence their disposition in the body. The purpose of the present study was to compare the pharmacokinetics and metabolism of doxorubicin and epirubicin based on the plasma concentration, amount associated with blood cells and simultaneous monitoring of biliary and urinary elimination of unchanged drug and metabolites after single- and multiple-dose injections. The level of sarcoplasmic reticulum Ca2+ ATPase in the heart was also measured as a biomarker of cardiotoxicity. Male Sprague-Dawley rats were treated in a parallel design with doxorubicin or epirubicin on a multiple-dosing basis (4 mg kg,1 per week) or as a single dose injection (20 mg kg,1). Blood, urine and bile samples were collected periodically after each dose in the multiple-dosing regimen and the single dose injection, and at the end of each experiment the hearts were removed. The concentrations of each drug in plasma, blood cells, bile and urine samples were determined, and by simultaneous curve-fitting of plasma and bile data according to compartmental analysis, the pharmacokinetic parameters and constants were estimated. The concentration of drug associated with blood cells was analysed according to non-compartmental analysis. The bile and urine samples provided the in-vivo metabolic data. The level of Ca2+ ATPase in the heart, determined by Western blotting, was used as the toxicodynamic parameter to correlate with the kinetic data. Multiple-dosing regimens reduced the total plasma clearance and increased the area under the plasma concentration-time curve of both drugs. Also, the area under the curve of doxorubicin associated with blood cells increased with the weekly doses, and the related mean residence time (MRT) and apparent volume of distribution (Vdss) were steadily reduced. In contrast to doxorubicin, the MRT and Vdss of epirubicin increased significantly. Metabolic data indicated significant differences in the level of alcohol and aglycones metabolites. Doxorubicinol and doxorubicin aglycones were significantly greater than epirubicinol and epirubicin aglycone, whereas epirubicinol aglycone was greater than doxorubicinol aglycone. The area under the blood cells concentration-time curve correlated linearly with the changes in Ca2+ ATPase net intensity. The results of this study demonstrate the importance of the kinetics of epirubicin and doxorubicin associated with blood cells. Linear correlation between the reduction of net intensity of the biomarker with the area under the curve of doxorubicin associated with blood cells confirms that the differences between the two compounds are related to their interaction with blood cells. This observation together with the observed differences in metabolism may underline a significant role for blood cells in distribution and metabolism of doxorubicin and epirubicin. [source] Absence of glycochenodeoxycholic acid (GCDCA) in human bile is an indication of cholestasis: A 1H MRS studyNMR IN BIOMEDICINE, Issue 5 2009Omkar B. Ijare Abstract The utility of 1H MR spectroscopy in detecting chronic cholestasis has been investigated. The amide proton region of the 1H MR spectrum of human bile plays a major role in differentiating cholestatic (Ch) patterns from the normal ones. Bile obtained from normal bile ducts contains both taurine and glycine conjugates of bile acids , cholic acid (CA), chenodeoxycholic acid (CDCA), and deoxycholic acid (DCA). Absence of a glycine-conjugated bile acid glycochenodeoxycholic acid (GCDCA) has been observed in bile samples obtained from primary sclerosing cholangitis (PSC) patients. A total of 32 patients with various hepatobiliary diseases were included in the study. Twenty-one patients had PSC and 11 had normal cholangiograms. One PSC patient was excluded from the study because of a bad spectrum. Seventeen out of the 20 PSC patients showed an absence of GCDCA in their 1H MR spectrum of bile. Six of the 11 reference patients with normal cholangiogram also showed spectra similar to those of PSC, indicating the possibility of cholestasis. DQF-COSY and TOCSY experiments performed on bile samples from PSC patients also revealed absence of phosphatidylcholine (PC) in some of the bile samples, suggesting possible damage to the cholangiocytes by the toxic bile. These observations suggest that analysis of human bile by 1H MRS could be of value in the diagnosis of chronic Ch liver disorders. Copyright © 2008 John Wiley & Sons, Ltd. [source] Metabolite identification of a new antitumor agent icotinib in rats using liquid chromatography/tandem mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 14 2008Zhongmin Guan Icotinib, 4-[(3-ethynylphenyl)amino]-6,7-benzo-12-crown-4-quinazoline, is a new antitumor agent. The metabolic pathway of icotinib in rats was studied using liquid chromatography/tandem mass spectrometry (LC/MSn) analysis. Full scan and selected ion monitoring modes were used to profile the possible metabolites of icotinib in rat urine, feces and bile samples. Four phase I metabolites (M1,M4) and two phase II metabolites (M5, M6) were detected and characterized. Multiple-stage mass spectrometry and nuclear magnetic resonance (NMR) spectrometry were employed to elucidate structures of metabolites. Icotinib was metabolized to open the crown ether ring to form the main phase I metabolites. During metabolism, a reactive metabolite was formed. Using semicarbazide as a trapping agent, an intermediate arising from opening of the crown ether ring was detected as an aldehyde product by LC/MS/MS. These data indicated that ring opening of the crown ether was triggered by hydroxylation at the 8,-position of the ring to form a hemiacetal intermediate, which was further oxidized or reduced. Finally, the metabolic pathway of icotinib in rats was proposed. Copyright © 2008 John Wiley & Sons, Ltd. [source] The use of turbulent flow chromatography and the isocratic focusing effect to achieve on-line cleanup and concentration of neat biological samples for low-level metabolite analysisRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 5 2005J. L. Herman The use of turbulent flow chromatography in conjunction with column switching isocratic focusing was used to perform on-line sample cleanup and concentration of neat rat plasma for the identification of low-level metabolites. The concentration was achieved by focusing multiple injections, which were cleaned by a turbulent flow column, onto an analytical column prior to elution into the mass spectrometer. In addition, the first application of turbulent flow chromatography for on-line sample cleanup of neat bile samples is reported. The on-line cleanup and concentration method extracts and concentrates a sample 20-fold in 1,h, and is completely automated. Copyright © 2005 John Wiley & Sons, Ltd. [source] High-performance liquid chromatography/tandem mass spectrometry for the quantitative analysis of a novel taxane derivative (BAY59-8862) in biological samples and characterisation of its metabolic profile in rat bile samplesRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 19 2001Cristina Sottani A sensitive, specific, accurate and reproducible high-performance liquid chromatography (HPLC) analytical method was developed and validated for the quantification of the novel oral taxane analogue BAY59-8862 in mouse plasma and tissue samples. A fully automated solid-phase extraction procedure was applied to the plasma after internal standard (IS) addition, with only 0.2,mL volume of the sample loaded on a CN-Sep-pak cartridge. In the case of the tissues a very simple acetonitrile extraction was used to recover BAY59-8862 and its internal standard from liver. The procedure for the quantification of BAY59-8862 and its IS (IDN5127) is based on high-performance liquid chromatography/ion spray-tandem mass spectrometry, operating in selected ion monitoring mode. The retention times of BAY and IS were 7.21 and 10.36,min, respectively. In both plasma and tissue specimens the assay was linear in the range 50,5000,ng/mL (ng/g). The overall precision and accuracy were assessed on three different days. The results for plasma were within 6.1% (precision) and between 99 and 112% (accuracy), and for the liver samples within 7.3% and between 104 and 118%, respectively. The LOD was 5,ng/mL and 20,ng/g in the plasma and liver, respectively. In addition, the biliary excretion of the compound in rats was studied. The study showed that an oxidative chemical reaction was the preferred metabolic pathway for biliary excretion, and two sets of mono- and dihydroxylated metabolites were detected by LC/ISP-MS/MS experiments. With this method, BAY59-8862 pharmacokinetic was determined in mice. The combined results demonstrate that the methodology can be considered a valid approach to conduct pharmacokinetic and metabolic studies during preclinical and clinical investigations. Copyright © 2001 John7 Wiley & Sons, Ltd. [source] Identification of [(GS)2AsSe], in rabbit bile by size-exclusion chromatography and simultaneous multielement-specific detection by inductively coupled plasma atomic emission spectroscopyAPPLIED ORGANOMETALLIC CHEMISTRY, Issue 2 2002Jürgen Gailer Abstract An arsenic,selenium metabolite that exhibited the same arsenic and selenium X-ray absorption near-edge spectra as the synthetic seleno-bis(S -glutathionyl) arsinium ion [(GS)2AsSe], was recently detected in rabbit bile within 25,min after intravenous injection of rabbits with sodium selenite and sodium arsenite. X-ray absorption spectroscopy did not (and cannot) conclusively identify the sulfur-donor in the in vivo sample. After similar treatment of rabbits, we analyzed the collected bile samples by size-exclusion chromatography (SEC) using inductively coupled plasma atomic emission spectroscopy (ICP-AES) to monitor arsenic, selenium and sulfur simultaneously. The bulk of arsenic and selenium eluted in a single peak, the intensity of which was greatly increased upon spiking of the bile samples with synthethic [(GS)2AsSe],. Hence, we identify [(GS)2AsSe], as the major metabolite in bile after exposure of rabbits to selenite and arsenite. The reported SEC,ICP-AES method is the first chromatographic procedure to identify this biochemically important metabolite in biological fluids and is thus a true alternative to X-ray absorption spectroscopy, which is not available to many chemists. Copyright © 2001 John Wiley & Sons, Ltd. [source] Hepatic proliferation in Gunn rats transplanted with hepatocytes: effect of retrorsine and tri-iodothyronineCELL PROLIFERATION, Issue 3 2005F. J. Cubero However, a major problem in most transplantation studies to date has been the limited growth of transplanted cells in the recipient organ. We performed a strategy for selective proliferation of transplanted cells by interfering with the proliferative capacity of resident hepatocytes, using the pyrrolizidine alkaloid retrorsine and then transplanting liver cells in conjunction with repeated administration of triiodothyronine, an inducer of hepatocyte proliferation in rats. In the present study, foetal and adult syngeneic hepatocyte transplantation into spleen was performed in retrorsine-treated hyperbilirubinemic Gunn rats. In parallel, repeated injections of triiodothyronine were given to recipients. Rats were sacrificed at 1, 7, 30 and 90 days after transplantation and blood and bile samples were taken to assess the functionality of transplanted cells. The proliferative activity of transplanted hepatocytes was evaluated using proliferating cell nuclear antigen labelling index. In summary, both adult and foetal hepatocyte transplantation were effective in correcting a metabolic abnormality in Gunn rats for as long as 3 months. The RS/T3 model, as a measure to increase graft function, could represent an important advance to future clinical application of hepatocyte transplantation. [source] Disposition of isosteviol in the rat isolated perfused liverCLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 5-6 2010Hongping Jin Summary 1. The aim of the present study was to investigate the mechanisms involved in the clearance of isosteviol using the rat isolated perfused liver. 2. Six livers from male Sprague-Dawley rats were perfused with 15.7 ,mol isosteviol in a recirculating system. Perfusate and bile samples were collected for 60 min and the liver was collected at the end of the perfusion. All samples collected were incubated with ,-glucuronidase. Isosteviol,glucuronide was determined as equivalent isosteviol. Isosteviol concentrations were determined using a previously developed liquid chromatography,tandem mass spectrometry method. The final isosteviol liver/perfusate (L/P), bile/liver (B/L) and isosteviol-glucuronide in bile/liver (BG/LG) ratios were determined. 3. Isosteviol has a high clearance (21.4 ± 4.8 mL/min) from the perfusate, with a short half-life (13 ± 4 min). ,-Glucuronidase incubation revealed that isosteviol is conjugated in the liver and excreted into the bile. There was no isosteviol-glucuronide detected in perfusate samples. The total recovery of the rat isolated perfused liver system is 74 ± 14% and glucuronidated isosteviol accounted for 23 ± 4% of the administered dose. 4. In conclusion, we are the first to characterize the metabolism of isosteviol using rat isolated liver perfusion. Our results strongly suggest that the liver is the main organ of isosteviol elimination and that isosteviol is glucuronidated in the liver before it is excreted into the bile. 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