Serum bile acids as a sensitive biological marker for evaluating hepatic effects of organic solvents

Serum bile acids (SBAs) are suggested as a potentially sensitive and specific indicator of liver function which, accordingly, could provide an early indication of hepatobiliary dysfunction. This offers advantages over more traditional parameters of liver integrity/function. Recent studies have shown that occupational exposure to low levels of halogenated aliphatic or non-halogenated aromatic solvents is associated with significant increases in SBA levels. As this has often been evident in the absence of any effect on conventional parameters of hepatobiliary integrity/function, elevated SBA levels may well be regarded as a sensitive biological marker of exposure/effect of these compounds. In addition, it may be considered that they provide an early indicator of solvent-induced changes in hepatobiliary function. Extensive studies with experimental animals have also provided supporting evidence for these observations in solvent-exposed individuals. Investigations of the mechanisms at cellular and subcellular levels by which these increases occur have suggested that these effects are likely to be the result of selective, dose-related and reversible inhibition of bile acid uptake at the sinusoidal domain of the hepatocyte plasma membrane. Increased concentrations of SBA under low levels of exposure to different solvents have been demonstrated to be a short-lived and reversible effect which is not accompanied by any other evidence of liver damage. Therefore, it could be assumed that it is unlikely that there would be pathological sequelae to these effects, although the longer term ramifications of such effects have not been thoroughly investigated. Nevertheless, the available evidence indicates that investigation of SBA in solvent-exposed workers could provide useful indications of exposure and effect.     

Increased serum bile acids as a possible biomarker of hepatotoxicity in Brazilian workers exposed to solvents in car repainting shops.

The objective was to evaluate total serum bile acids (SBA) as a biological marker of hepatotoxicity in car painters exposed to organic solvents and to compare their performance with classic biochemical parameters of liver function. SBA were analysed in a selected group of workers (n=57) occupationally exposed to a mixture of organic solvents and in a control group (n=51). In addition, aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin (TB), gamma-glutamyltransferase (GGT) and alkaline phosphatase (ALP) were determined in the two groups. Urinary hippuric acid was measured in all samples. Statistical analysis of the data revealed a significant increase in the concentration of SBA, AST, ALP and TB in exposed workers compared with controls (Mann-Whitney, p相似文献   

Increased serum bile acids as a possible biomarker of hepatotoxicity in Brazilian workers exposed to solvents in car repainting shops

Abstract

The objective was to evaluate total serum bile acids (SBA) as a biological marker of hepatotoxicity in car painters exposed to organic solvents and to compare their performance with classic biochemical parameters of liver function. SBA were analysed in a selected group of workers (n=57) occupationally exposed to a mixture of organic solvents and in a control group (n=51). In addition, aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin (TB), gamma-glutamyltransferase (GGT) and alkaline phosphatase (ALP) were determined in the two groups. Urinary hippuric acid was measured in all samples. Statistical analysis of the data revealed a significant increase in the concentration of SBA, AST, ALP and TB in exposed workers compared with controls (Mann–Whitney, p≤0.05). However, SBA was the parameter most frequently altered in exposed workers and showed higher significance between the two groups (chi-square test) compared with the upper limit of the reference range (8?µmol?l^?1). In conclusion, SBA can be considered to be a sensitive parameter of hepatotoxicity induced by organic solvents than the traditional tests and it can be used as an biological marker of subclinical liver injury.     

Topological and functional characterization of the N-glycans of soybean (Glycine max) agglutinin.

Soybean agglutinin (SBA), is a noncovalently bound tetramer comprised of four identical subunits having a single N-glycan chain, Man9GlcNAc2, that is known to be essential for regeneration of the functional tetrameric structure from unfolded subunits. In this study, SBA was found to have strong affinity for concanavalin A, indicating that the N-glycans are extensively solvent-exposed. The susceptibilities of the N-glycans to alpha-mannosidase and endo-beta-N-acetylglucosaminidase revealed that their distal areas have nonreducing ends embedded among the subunits, whereas their proximal regions are solvent-exposed. Endo-beta-N-acetylglucosaminidase-digested SBA was unable to retain its conformation and gradually unfolded. Periodate-oxidized SBA, whose N-glycans closely correspond to the invariant pentasaccharide core, tended to dissociate into the subunits, but permitted to stay as folded monomers. This SBA species was capable of refolding from unfolded subunits but unable to form the functional tetramer. It seems probable that the proximal regions of the N-glycans function in the formation and stabilization of the subunit conformation, whereas the branches outside the invariant cores stabilize the tetrameric structure.     

Molecular aspects of bile formation and cholestasis

Recent insights into the cellular and molecular mechanisms that control the function and regulation of hepatobiliary transport have led to a greater understanding of the physiological significance of bile secretion. Individual carriers for bile acids and other organic anions in both liver and intestine have now been cloned from several species. In addition, complex networks of signals that regulate key enzymes and membrane transporters located in cells that participate in the metabolism or transport of biliary constituents are being unraveled. This knowledge has major implications for the pathogenesis of cholestatic liver diseases. Here, we review recent information on molecular aspects of hepatobiliary secretory function and its regulation in cholestasis. Potential implications of this knowledge for the design of new therapies of cholestatic disorders are also discussed.     

Hyperammonemia induces transport of taurine and creatine and suppresses claudin-12 gene expression in brain capillary endothelial cells in vitro

Ammonia is a key neurotoxin involved in the neurological complications of acute liver failure. The present study was undertaken to study the effects of exposure to pathophysiologically relevant concentrations of ammonium chloride on cultured brain capillary endothelial cells in order to identify mechanisms by which ammonia may alter blood-brain barrier function. Conditionally immortalized mouse brain capillary endothelial cells (TM-BBB) were used as an in vitro model of the blood-brain barrier. Gene expression of a series of blood-brain barrier transporters and tight junction proteins was assessed by quantitative real time PCR analysis. Exposure to ammonia (5mM for 72h) resulted in significant increases in mRNA levels of taurine transporter (TAUT; 2.0-fold increase) as well as creatine transporter (CRT; 1.9-fold increase) whereas claudin-12 mRNA expression was significantly reduced to 67.7% of control levels. Furthermore, [(3)H]taurine and [(14)C]creatine uptake were concomitantly increased following exposure to ammonia, suggesting that up-regulation of both TAUT and CRT under hyperammonemic conditions results in an increased function of these two transporters in TM-BBB cells. TAUT and CRT are respectively involved in osmoregulation and energy buffering in the brain, two systems that are thought to be affected in acute liver failure. Furthermore, claudin-12 down-regulation suggests that hyperammonemia may also affect tight junction integrity. Our results provide evidence that ammonia can alter brain capillary endothelial cell gene expression and transporter function. These findings may be relevant to pathological situations involving hyperammonemia, such as liver disease.     

Itch in Liver Disease: Facts and Speculations

Pruritus in hepatobiliary disease is commonly believed to be caused by retention of bile acids with their sequestration in the skin. HOwever, we have recently demonstrated that skin levels of bile acids in patients with cholestasis correlate poorly with pruritus. In this report, we present additional data concerning the relationship of pruritus to bile acid retention: (1) the urinary excretion of sulfated and nonsulfated bile acids was not significantly different in patients with cholestasis who itched compared to those who did not; (2) one patient with itch associated with a liver abscess had normal levels of bile acids in serum, skin, and urine; (3) patients with primary biliary cirrhosis who itched had lower serum bile acid levels than patients with mechanical biliary obstruction who did not itch.These studies support our premise that pruritus in hepatobiliary diseases is not directly related to bile acid retention. They suggest that the type of cholestatic disorder, and not simply the magnitude of the cholestasis, as estimated by the elevation of serum bile acids, is important. We propose that the agent responsible for pruritus is produced in response to cholestasis, possibly through activation of the alternate pathway of bile acid synthesis. Properties of the hypothetical pruritogen are discussed.     

Gas chromatography–mass spectrometry-based simultaneous quantitative analytical method for urinary oxysterols and bile acids in rats

A simultaneous quantitative assay method for urinary oxysterols and bile acids using GC–MS was developed to investigate the mechanism of liver toxicity induced by drugs or chemicals. Sample preparations were optimized by exploring various extraction solvents, derivatization reagents, and hydrolysis methods to achieve reliable and maximum sensitivity for these two different compound classes. As a result, satisfactory accuracy, precision, and sensitivity were obtained in the validation. The method was then applied to quantify urinary oxysterols and bile acids produced from liver toxicity induced by atorvastatin (250 mg/kg/day). From the results, increases in bile acid levels and decreases in the concentration ratio between cholic acid and chenodeoxycholic acid, which are the distinguishing phenomena observed in serum or bile for liver toxicity, were also observed in urine. Additionally, the mechanism of liver toxicity was investigated with the urinary concentration ratio of product to precursor in the metabolic pathway from cholesterol to bile acids. The results indicated that enzyme activities related to the production and degradation of bile acids, not oxysterols, were significantly changed from liver toxicity. Thus, it was concluded that urinary levels of oxysterols and bile acids could be useful tools for checking liver toxicity and investigating its mechanism.     

Cholic Acid Induces a Cftr Dependent Biliary Secretion and Liver Growth Response in Mice

The cause of Cystic fibrosis liver disease (CFLD), is unknown. It is well recognized that hepatic exposure to hydrophobic bile salts is associated with the development of liver disease. For this reason, we hypothesize that, CFTR dependent variations, in the hepatic handling of hydrophobic bile salts, are related to the development CFLD. To test our hypothesis we studied, in Cftr^-/- and control mice, bile production, bile composition and liver pathology, in normal feeding condition and during cholate exposure, either acute (intravenous) or chronic (three weeks via the diet). In Cftr^-/- and control mice the basal bile production was comparable. Intravenous taurocholate increased bile production to the same extent in Cftr^-/- and control mice. However, chronic cholate exposure increased the bile flow significantly less in Cftr^-/- mice than in controls, together with significantly higher biliary bile salt concentration in Cftr^-/- mice. Prolonged cholate exposure, however, did not induce CFLD like pathology in Cftr^-/- mice. Chronic cholate exposure did induce a significant increase in liver mass in controls that was absent in Cftr^-/- mice. Chronic cholate administration induces a cystic fibrosis-specific hepatobiliary phenotype, including changes in bile composition. These changes could not be associated with CFLD like pathological changes in CF mouse livers. However, chronic cholate administration induces liver growth in controls that is absent in Cftr^-/- mice. Our findings point to an impaired adaptive homeotrophic liver response to prolonged hydrophobic bile salt exposure in CF conditions.     

Trifluoroethanol-induced conformational change of tetrameric and monomeric soybean agglutinin: Role of structural organization and implication for protein folding and stability

2,2,2-Trifuoroethanol (TFE)-induced conformational structure change of a β-sheet legume lectin, soybean agglutinin (SBA) has been investigated employing its exclusive structural forms in quaternary (tetramer) and tertiary (monomer) states, by far- and near-UV CD, FTIR, fluorescence, low temperature phosphorescence and chemical modification. Far-UV CD results show that, for SBA tetramer, native atypical β-conformation transforms to a highly α-helical structure, with the helical content reaching 57% in 95% TFE. For SBA monomer, atypical β-sheet first converts to typical β-sheet at low TFE concentration (10%), which then leads to a nonnative α-helix at higher TFE concentration. From temperature-dependent studies (5–60 °C) of TFE perturbation, typical β-sheet structure appears to be less stable than atypical β-sheet and the induced helix entails reduced thermal stability. The heat induced transitions are reversible except for atypical to typical β-sheet conversion. FTIR results reveal a partial α-helix conversion at high protein concentration but with quantitative yield. However, aggregation is detected with FTIR at lower TFE concentration, which disappears in more TFE. Near-UV CD, fluorescence and phosphorescence studies imply the existence of an intermediate with native-like secondary and tertiary structure, which could be related to the dissociation of tetramer to monomer. This has been further supported by concentration dependent far-UV CD studies. Chemical modification with N-bromosuccinimide (NBS) shows that all six tryptophans per monomer are solvent-exposed in the induced α-helical conformation. These results may provide novel and important insights into the perturbed folding problem of SBA in particular, and β-sheet oligomeric proteins in general.     

Bile analysis as a tool for assessing integrity of biliary epithelial cells after cold ischemia--reperfusion of rat livers

Previous morphological studies failed to show appreciable injury of biliary epithelial cells (BEC) after cold ischemia of rat liver, although recent evidence indicated that BEC integrity and function were impaired in this model. We tested the hypothesis that analysis of bile for enzymes, such as lactate dehydrogenase (LDH), alanine transaminase (ALT), and aspartate transaminase (AST), can be used for assessing cold ischemic injury of BEC. Furthermore, we examined whether biliary gamma-glutamyltransferase (GGT) reflects warm ischemic injury of BEC and whether normothermic reperfusion aggravates the negative effect of cold ischemia on BEC integrity and function. Rat livers were reperfused after different periods of cold or warm ischemia using a blood-free perfusion model. Compared with controls, perfusate LDH, ALT, and AST levels and parameters of hepatocyte function, including hepatocyte tight junction permeability, were not significantly altered by 18-h cold ischemia. On the other hand, 9-h cold ischemia markedly increased biliary LDH, ALT, and AST levels. However, only LDH release into the bile was strongly dependent on the time of cold storage. Biliary GGT, LDH, and glucose levels decreased during the reperfusion period following 18-h cold ischemia. The results suggest that biliary LDH can be used for assessing injury of BEC in cold-preserved livers and that normothermic reperfusion does not aggravate preservation-induced injury of BEC after cold ischemic storage.     

Combining ASBT inhibitor and FGF15 treatments enhances therapeutic efficacy against cholangiopathy in female but not male Cyp2c70 KO mice

Therapeutic reduction of hydrophobic bile acids exposure is considered beneficial in cholestasis. The Cyp2c70 KO mice lack hydrophilic muricholic acids and have a human-like hydrophobic bile acid pool resulting in hepatobiliary injury. This study investigates if combining an apical sodium-dependent bile acid transporter inhibitor GSK2330672 (GSK) and fibroblast growth factor-15 (FGF15) overexpression, via simultaneous inhibition of bile acid synthesis and gut bile acid uptake, achieves enhanced therapeutic efficacy in alleviating hepatobiliary injury in Cyp2c70 KO mice. The effects of GSK, adeno-associated virus (AAV)-FGF15, and the combined treatment on bile acid metabolism and cholangiopathy were compared in Cyp2c70 KO mice. In female Cyp2c70 KO mice with more severe cholangiopathy than male Cyp2c70 KO mice, the combined treatment was more effective in reversing portal inflammation, ductular reaction, and fibrosis than AAV-FGF15, while GSK was largely ineffective. The combined treatment reduced bile acid pool by ～80% compared to ～50% reduction by GSK or AAV-FGF15, and enriched tauro-conjugated ursodeoxycholic acid in the bile. Interestingly, the male Cyp2c70 KO mice treated with AAV-FGF15 or GSK showed attenuated cholangiopathy and portal fibrosis but the combined treatment was ineffective despite reducing bile acid pool. Both male and female Cyp2c70 KO mice showed impaired gut barrier integrity. AAV-FGF15 and the combined treatment, but not GSK, reduced gut exposure to lithocholic acid and improved gut barrier function. In conclusion, the combined treatment improved therapeutic efficacy against cholangiopathy than either single treatment in the female but not male Cyp2c70 KO mice by reducing bile acid pool size and hydrophobicity.     

Unaltered hepatic oxidative phosphorylation and mitochondrial permeability transition in wistar rats treated with nimesulide: Relevance for nimesulide toxicity characterization

Nonsteroidal anti-inflammatory drugs have been associated with hepatotoxicity in susceptible patients. One such example is nimesulide, a preferential cyclooxygenase 2-inhibitor, widely used for the treatment of inflammation and pain. It was suggested that nimesulide could exert its hepatotoxicity by altering hepatic mitochondrial function, which was demonstrated in vitro. The objective of this study was to verify whether liver mitochondria isolated from rats treated with doses of nimesulide well above therapeutic levels possessed decreased calcium tolerance and oxidative phosphorylation, which indicates in vivo nimesulide mitochondrial toxicity. Male and female rats received nimesulide or its vehicle twice daily, for 5 days, and were killed on the seventh day for the isolation of liver mitochondria. Mitochondrial respiration, transmembrane electric potential, and calcium tolerance were characterized in all experimental groups. Nimesulide had no effect on liver mitochondrial function. Indexes of mitochondrial integrity, calcium loading capacity, and oxidative phosphorylation efficiency were unchanged between liver mitochondria from treated and control animals. In the animals tested, no evidence of degraded mitochondrial function due to nimesulide administration could be found. The results corroborate the notion that despite recognized in vitro mitochondrial toxicity, nimesulide does not cause detectable mitochondrial dysfunction in Wistar rats, even when administered in much higher concentrations than those known to have anti-inflammatory effects.     

Serum Lactic Dehydrogenase,Leucine Aminopeptidase and 5-Nucleotidase Activity: Observations in Patients with Carcinoma of the Pancreas and Hepatobiliary Disease

Serum lactic dehydrogenase, leucine aminopeptidase, 5-nucleotidase and alkaline phosphatase activities were investigated in a number of diseases involving the hepatobiliary system.Leucine aminopeptidase was found to be a sensitive indicator of biliary obstruction, serum 5-nucleotidase slightly less sensitive, and alkaline phosphatase appreciably less sensitive. Leucine aminopeptidase and 5-nucleotidase activities were often increased by malignant infiltration of the liver and primary hepatic disease even in the absence of jaundice.Serum lactic dehydrogenase was frequently increased in primary hepatic disease and malignant disorders but was not apparently affected by bile duct obstruction per se. Thirty-five of 45 patients with proved malignancy had increased lactic dehydrogenase levels.The highest leucine aminopeptidase levels were encountered in carcinoma of the head of the pancreas. The frequent increase in both serum lactic dehydrogenase and leucine aminopeptidase activities in patients with carcinoma of the head of the pancreas suggests that these combined estimations are useful laboratory procedures in the diagnosis of malignant extrahepatic obstruction.     

清胆颗粒利胆作用的实验研究

目的：通过研究清胆颗粒对正常大鼠及模型动物胆汁及病理组织的影响,确定其利胆功效,为临床应用提供依据.方法：采用胆管引流法测定清胆颗粒对正常大鼠胆汁量的影响;应用石胆酸（lithocholic acid,LCA）造成豚鼠胆囊炎模型,测定其对模型动物肝重、肝指数、胆囊容积及胆囊病理组织的影响.结果：清胆颗粒能显著增加正常大鼠胆汁流量,明显减轻模型动物肝湿重和肝指数,缩小胆囊容积,能明显降低胆汁中TB、DB及UCB/TB含量,对Ca^2＋增高有明显的抑制作用,对模型动物胆囊粘膜上皮增生有明显的抑制作用.结论：清胆颗粒具有显著的利胆作用.     

Determinants of biliary secretory pressure: the effects of two different bile acids

Biliary secretory pressure represents the force generated to deliver bile through the biliary system. Bile acid-induced toxicity may decrease canalicular bile formation and (or) induce back diffusion causing cholestasis. To determine if biliary secretory pressure is a sensitive indicator of bile toxicity, taurocholate was compared with a less cytotoxic bile acid, tauroursodeoxycholate. In fasted male Sprague-Dawley rats, the common bile duct was cannulated and the endogenous bile salt pool was removed by enteroclysis. Taurocholate (n = 35) or tauroursodeoxycholate (n = 35) in saline was infused for 1 h. Maximal biliary secretory pressure was then measured by attaching the biliary cannula to a column monometer and recording the maximum height to which bile rose. With taurocholate administration, bile flow and bile salt secretion linearly rose to a maximum infusion of 0.5 mumol/(min.g liver), above which hemolysis and death occurred. In contrast, tauroursodeoxycholate could be infused at higher rates with bile salt secretion plateauing at 1.25 mumol/(min.g liver] Both had similar choleretic potencies. Mean biliary secretory pressure at low (less than 0.15 mumol/(min.g liver] infusions was lower with taurocholate (22.5 cm bile) than tauroursodeoxycholate (25.2 cm). Further, increasing the taurocholate infusion decreased the biliary secretory pressure; yet for taurousodeoxycholate, pressure remained unchanged even at higher infusions. Thus, taurocholate but not tauroursodeoxycholate decreases biliary secretory pressure at high infusion rates, likely a reflection of its toxicity to the hepatobiliary epithelium.     

Complex physiological traits as biomarkers of the sub-lethal toxicological effects of pollutant exposure in fishes

Complex physiological traits, such as routine aerobic metabolic rate or exercise performance, are indicators of the functional integrity of fish that can reveal sub-lethal toxicological effects of aquatic pollutants. These traits have proved valuable in laboratory investigations of the sub-lethal effects of heavy metals, ammonia and various xenobiotics. It is not known, however, whether they can also function as biomarkers of the complex potential range of effects upon overall functional integrity caused by exposure to mixtures of chemicals in polluted natural environments. The current study used portable swimming respirometers to compare exercise performance and respiratory metabolism of fish exposed in cages for three weeks to either clean or polluted sites on three urban European river systems: the river Lambro, Milan, Italy; the rivers Blythe, Cole and Tame, Birmingham, UK; and the river Amstel, Amsterdam, The Netherlands. The UK and Italian rivers were variously polluted with high levels of both bioavailable heavy metals and organics, and the Amstel by mixtures of bioavailable organics at high concentrations. In both the UK and Italy, indigenous chub (Leuciscus cephalus) exposed to clean or polluted sites swam equally well in an initial performance test, but the chub from polluted sites could not repeat this performance after a brief recovery interval. These animals were unable to raise the metabolic rate and allocate oxygen towards exercise in the second trial, an effect confirmed in successive campaigns in Italy. Swimming performance was therefore a biomarker indicator of pollutant exposure in chub exposed at these sites. Exposure to polluted sites on the river Amstel did not affect the repeat swimming performance of cultured cloned carp (Cyprinus carpio), indicating either a species-specific tolerance or relative absence of heavy metals. However, measurements of oxygen uptake during swimming revealed increased rates of routine aerobic metabolism in both chub and carp at polluted sites in all of the rivers studied, indicating a sub-lethal metabolic loading effect. Therefore, the physiological traits of exercise performance and metabolic rate have potential as biomarkers of the overall sub-lethal toxic effects of exposure to complex mixtures of pollutants in rivers, and may also provide insight into why fish do not colonize some polluted environments.     

Identification of different transport systems for bile salts in sinusoidal and canalicular membranes of hepatocytes

The preservation of the functional polarity of hepatocytes in liver snips (1 x 2 x 4 mm) was demonstrated by fluorescent microscopic studies using the sodium salt of (N-[7-(4-nitrobenzo-2-oxa-1,3-diazol)]-3 beta-amino-7 alpha,12 alpha- dihydroxy-5 beta-cholan-24-oyl)-2-aminoethanesulfonic acid. This fluorescent bile salt derivative is not only taken up by hepatocytes of several cell layers at the surface of the snips but also secreted into bile canaliculi. The intact hepatobiliary transport of bile salts by hepatocytes of liver snips demonstrates that they are a useful system for the investigation of those transcellular transport processes which require the integrity of hepatic structure. Photoaffinity labelling of liver snips with the sodium salt of (7,7-azo-3 alpha,12 alpha-dihydroxy-5 beta-[3 beta-3H]cholan- 24-oyl)-2-aminoethanesulfonic acid revealed that the bile-salt-binding membrane polypeptides with apparent Mr values of 54,000 and 48,000 are exclusively located in the sinusoidal membrane, whereas a single bile-salt-binding polypeptide with an apparent Mr of 100,000 is located in the bile-canalicular membrane. Photoaffinity labelling of liver snips at 4 degrees C, when transcellular bile-salt transport is insignificant, resulted in the labelling of the two sinusoidal membrane polypeptides and practically no labelling of the polypeptide with an apparent Mr of 100,000. This latter polypeptide was also not labelled when Ca2 deprivation abolished bile secretion completely. These results indicate that the directed hepatobiliary transport of bile salts in hepatocytes is accomplished by transport systems which are different for sinusoidal uptake and canalicular secretion.     

A stable colorimetric assay to measure toxin elevation of inorganic phosphate in bile

A modified assay for the measurement of nanomole amounts of Pi in 50 microliters of bile is described. The assay is based on the formation of a complex between malachite green dye and phosphomolybdate under acidic conditions. Only three simple steps are required to produce a colored complex which remains stable for at least 3 h; these steps are precipitation with acid, addition of color reagent containing dye and surfactant, and incubation at 37 degrees C. Analysis of bile samples collected from a rat given the toxin 1,1-dichloroethylene demonstrated that the assay is well suited to routine assays of biliary Pi as an endogenous indicator of aberrant hepatobiliary function.