Sampling time for different matrices in stress assessment of farmed Atlantic salmon post-smolt.

The sustainability of commercial aquaculture production depends critically on prioritizing fish welfare management. Besides monitoring welfare parameters such as fish behaviour and water quality, fish stress level can also provide a reliable measure of the welfare status of farmed fish. Cortisol and 5 of its metabolites (5ß-THF, cortisone, 5ß-DHE, 5ß-THE, ß-cortolone) were previously identified by the authors as suitable stress biomarkers of farmed Atlantic salmon. Based on this knowledge, the present study aimed to investigate the time-related dynamics of these metabolites in plasma, skin mucus, bile and faeces over a 72 h- period. The objective was to determine the optimal sampling time for each matrix and to understand the clearance pathway of these metabolites following stress. An experiment was carried out using a total of 90 Atlantic salmon with an average weight of 438 (±132) g. The average sea temperature was 6.9 °C during the experimental period. A control group of 10 fish was first collected before the remaining 80 fish were submitted to a stress of netting and subsequent relocation into two separate cages. From each of these two stress groups, 10 fish were sampled at 1 h, 2 h, 4 h, 6 h and 12 h, 24 h, 48 h, 72 h after the stress event respectively. The concentrations of cortisol and its metabolites were measured at each of the sampling timepoint. The results demonstrated that plasma cortisol metabolites reached the highest concentration 4 h after stress and remained elevated despite the slight decrease for the remaining timepoints. The peak level was observed at 12 h post-stress in skin mucus and 24 h in bile and faeces. The findings suggest that these timepoints are the optimal for sampling Atlantic salmon post-smolt following stressful events in acute stress studies. Furthermore, the results reveal that analysing cortisol and its metabolites, both in free and conjugated forms, rather than free cortisol provides greater flexibility as their concentrations are less affected by sampling procedure. This study confirms the appropriateness of skin mucus and faeces as less-invasive sample matrices for fish stress evaluation and provides a basis for further developing low invasive tools for monitoring the welfare of farmed salmonid.

LC-MS/MS method for the determination of hormones: Validation, application and health risk assessment in various bovine matrices.

A new method, using liquid chromatography coupled to mass spectrometry (LC-MS/MS) for the detection of fourteen natural and synthetic hormones in muscles, was validated in other bovine matrices (liver, kidney, bile and hair) according to the Decision Commission 2002/657/EC. As result, this method demonstrates good linearity (R2 > 0.99) as well as accuracy with coefficients of variation for repeatability and reproducibility lower than 23%. Moreover, the values of decision limit (CCα) and detection capability (CCß) were determined indicating values ranging from 0.13 to 0.86 µg/kg and 0.25-1.72 µg/k for the majority of analytes. Recovery rate in the different matrices varied from 51.5 to 107%. Indeed, this method has been successfully applied to detect anabolic hormones in eighty-eight samples (muscle, liver, kidney, and bile) collected from different local slaughterhouses. Results showed that progesterone was found in 30 samples at concentrations ranging from 0.11 to 11.7 µg/kg, while testosterone was detected in 34 samples at concentrations ranging from 0.5 to 9.52 µg/kg. All bile samples contain epi-testosterone at concentration ranging from 0.89 to 280 µg/kg. These obtained data were used to calculate the estimated daily intake, hazard quotient and hazard index as exposure assessment.

Integration of biological effects, fish histopathology and contaminant measurements for the assessment of fish health: A pilot application in Irish marine waters.

This study investigates the use of a weight of evidence (WOE) approach to evaluate fish health status and biological effects (BEs) of contaminants for assessment of ecosystem health and discusses its potential application in support of the Marine Strategy Framework Directive (MSFD). External fish disease, liver histopathology and several BEs of contaminant exposure including 7-ethoxy resorufin O-de-ethylase (EROD), acetylcholinesterase (AChE), bile metabolites, vitellogenin (VTG) and alkali labile phosphates (ALP) were measured in two flatfish species from four locations in Ireland. Contaminant levels in fish were generally low with PCBs in fish liver below OSPAR environmental assessment criteria (EAC). There were consistencies with low PCB levels, EROD and PAH bile metabolite levels detected in fish. Dab from Cork, Dublin and Shannon had the highest relative prevalence of liver lesions associated with the carcinogenic pathway. An integrated biomarker response (IBR) showed promise to be useful for evaluation of environmental risk, although more contaminant parameters in liver are required for a full assessment with the present study.

LC-MS/MS and GC-MS methods in propofol detection: Evaluation of the two analytical procedures.

Propofol is a short-acting hypnotic agent that is commonly used to induce and maintain anesthesia. Propofol abuse and its involvement in suicide deaths have increased in recent years, especially among healthcare personnel. An example is the suicide of a 61-year-old nurse found with a propofol drip in his left arm. We describe the postmortem concentration of propofol in various tissues (femoral and cardiac blood, bile, urine, brain, and liver) and in the drip. The toxicological analyses were performed through two analytical methods, differing in derivatization reaction and in instrumentation: silylation for gas chromatograph-mass spectrometer (GC-MS), as routinely performed in our laboratory for this kind of analyses (lower limits of quantification-LLOQ-in urine and blood: 0.3 and 5ng/ml); for liquid chromatograph-tandem mass spectrometer (LC-MS/MS) an innovative azo-coupling derivatization (LLOQ: 0.0004 and 0.1ng/ml). This latter produces an azo-derivative (molecular composition: C18H22ON2; molecular weight: 282Da) highly ionizable in electro-spray ion source, both in negative and positive ionizations. These two methods were compared to evaluate the effectiveness of this new LC-MS/MS analysis. An acidic hydrolysis (HCl 6N, 100°C, and 1h) was performed for the biological samples (1ml or 1g) irrespective of the analytical method applied. The drip content was extracted adding phosphate buffer (pH 8) and a dichloromethane/ethylacetate 8:2 (v:v) mixture. Derivatization steps were: silylation with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA)+tetramethylammonium hydroxide (TMAH) for GC-MS; regarding LC-MS/MS, azo-coupling reaction with the aryl-diazonium salt (0-5°C, and 30min). The analyses were achieved in selected-ion monitoring for GC-MS (m/z, 235,250,73 propofol"; m/z, 252,267,27 propofol-d17) and in multiple reaction monitoring ([M-H](-): m/z 283→241,77, azo-propofol; m/z 299→251,77, azo-propofol-d17) for LC-MS/MS. Autopsy showed no significant findings. Propofol concentrations were (LC-MS/MS vs GC-MS, respectively): 15.1 vs 14.5mg/ml, drip content; 7.11 vs 6.07µg/ml, cardiac blood; 9.50 vs 7.19µg/ml, femoral blood; 0.64 vs 1.07µg/ml, bile; 0.042 vs 0.051µg/ml urine; 4.93 vs 5.89µg/g, brain; and 7.88 vs 6.80µg/g, liver. These values are comparable with the ones described in literature for death by acute propofol intoxication; the drip content is compatible with a diluted formulation of propofol available in Italy (20mg/ml injectable emulsion). The comparison shows an excellent fitting of the data (R(2): 0.9362). Toxicological results proved the cause of death as acute propofol intoxication. Furthermore, the new LC-MS/MS method showed an excellent effectiveness and reliability when compared to the routinely used GC-MS method.

Rapid Determination of Bile Acids in Bile from Various Mammals by Reversed-Phase Ultra-Fast Liquid Chromatography.

A valid and efficient reversed-phase ultra-fast liquid chromatography method was developed for the simultaneous determination of 13 bile acids in the bile of three mammal species, including rat, pig and human gallstone patients. Chromatographic separation was performed with a Shim-pack XR-ODS column, and the mobile phase consisted of acetonitrile and potassium phosphate buffer (pH 2.6) at a flow rate of 0.5 mL min(-1). The linear detection range of most bile acids ranged from 2 to 600 ng µL(-1) with a good correlation coefficient (>0.9995). The precision of each bile acid was <1.8% for intraday and <4.8% for interday. All bile acids were separated in 15 min with satisfactory resolution, and the total analysis time was 18 min, including equilibration. The method was successfully applied in rapid screening of bile samples from the three mammals. Significant metabolic frameworks of bile acids among various species were observed, whereas considerable quantitative variations in both inter- and intraspecies were also observed, especially for gallstone patients. Our results suggest that detecting the change of bile acid profiles could be applied for the diagnosis of gallstone disease.

In vitro assessment of marine Bacillus for use as livestock probiotics.

Six antimicrobial-producing seaweed-derived Bacillus strains were evaluated in vitro as animal probiotics, in comparison to two Bacillus from an EU-authorized animal probiotic product. Antimicrobial activity was demonstrated on solid media against porcine Salmonella and E. coli. The marine isolates were most active against the latter, had better activity than the commercial probiotics and Bacillus pumilus WIT 588 also reduced E. coli counts in broth. All of the marine Bacillus tolerated physiological concentrations of bile, with some as tolerant as one of the probiotics. Spore counts for all isolates remained almost constant during incubation in simulated gastric and ileum juices. All of the marine Bacillus grew anaerobically and the spores of all except one isolate germinated under anaerobic conditions. All were sensitive to a panel of antibiotics and none harbored Bacillus enterotoxin genes but all, except B. pumilus WIT 588, showed some degree of ß-hemolysis. However, trypan blue dye exclusion and xCELLigence assays demonstrated a lack of toxicity in comparison to two pathogens; in fact, the commercial probiotics appeared more cytotoxic than the majority of the marine Bacillus. Overall, some of the marine-derived Bacillus, in particular B. pumilus WIT 588, demonstrate potential for use as livestock probiotics.

Distribution of polycyclic aromatic hydrocarbon (PAH) residues in several tissues of edible fishes from the largest freshwater lake in China, Poyang Lake, and associated human health risk assessment.

The residual levels, tissue distribution and human health risk assessment of polycyclic aromatic hydrocarbons (PAHs) in edible fishes, bighead carp (Aristichthys nobilis) and silver carp (Hypophthalmichthys molitrix), from the largest freshwater lake in China, Poyang Lake, were studied. PAH concentrations ranged from 105 to 513ng g(-1)ww and from 53.9 to 401ng g(-1)ww in different tissues of bighead carp and silver carp, respectively. Low molecular weight (LMW) PAHs were the predominant compounds, suggesting the gill-water transfer might be the major exposure route for PAHs in the studied fish species. Tissue distribution indicated that the hepatobiliary system accumulated higher concentrations of PAHs than the extrahepatic tissues with bile being the most predominant tissue for both species. Composition analysis demonstrated that PAHs were from the combined petrogenic and pyrogenic origin, and the gasoline combustion might be the main source. A preliminary evaluation of human health risk using benzo[a]pyrene (BaP) potency equivalent concentration (PEC) as well as the incremental lifetime cancer risk (ILCR) indicated that PAHs in fish would induce potential carcinogenic effects.

Children's exposure to harmful elements in toys and low-cost jewelry: characterizing risks and developing a comprehensive approach.

Contamination problem in jewelry and toys and children's exposure possibility have been previously demonstrated. For this study, risk from oral exposure has been characterized for highly contaminated metallic toys and jewelry ((MJ), n=16) considering three scenarios. Total and bioaccessible concentrations of Cd, Cu, Ni, and Pb were high in selected MJ. First scenario (ingestion of parts or pieces) caused unacceptable risk for eight items for Cd, Ni, and/or Pb (hazard index (HI)>1, up to 75, 5.8, and 43, respectively). HI for ingestion of scraped-off material scenario was always <1. Finally, saliva mobilization scenario caused HI>1 in three samples (two for Cd, one for Ni). Risk characterization identified different potentially hazardous items compared to United States, Canadian, and European Union approaches. A comprehensive approach was also developed to deal with complexity and drawbacks caused by various toy/jewelry definitions, test methods, exposure scenarios, and elements considered in different regulatory approaches. It includes bioaccessible limits for eight priority elements (As, Cd, Cr, Cu, Hg, Ni, Pb, and Sb). Research is recommended on metals bioaccessibility determination in toys/jewelry, in vitro bioaccessibility test development, estimation of material ingestion rates and frequency, presence of hexavalent Cr and organic Sn, and assessment of prolonged exposure to MJ.

In vitro and in vivo assessment of the biocompatibility of an Mg-6Z(n) alloy in the bile.

There is a great clinical need for biodegradable bile duct stents. Biodegradable stents made of an Mg-6Zn alloy were investigated in both vivo animal experiment and in vitro cell experiments. During the in vivo experiments, blood biochemical tests were performed to determine serum magnesium, serum creatinine (CREA), blood urea nitro-gen (BUN), serum lipase (LPS), total bilirubin (TB) and glutamic-pyruvic transaminase (GPT) levels. Moreover, tissue samples of common bile duct (CBD), liver and kidney were taken for histological evaluation. In the in vitro experiments, primary mouse extrahepatic bile duct epithelial cells (MEBDECs) were isolated and cultured. Cytotoxicity testing was carried out using the MTT method. Flow cytometry analyses with propidium iodide staining were performed to evaluate the effect of Mg-6Zn alloy extracts on cell cycle. The in vivo experiments revealed no significant differences (P > 0.05) in serum magnesium, CREA, BUN, LPS, TB or GPT before and after the operation. Based on the HE results, hepatocytes, bile duct epithelial cells, renal glomerulus and renal tubule tissues did not present significant necrosis. In the in vitro experiments, the cell relative growth rate curve did not change significantly from 20 to 40 % extracts. In vitro experiments showed that 20-40 % Mg-6Zn extracts are bio-safe for MEBDECs. In vivo experiments showed that Mg-6Zn stents did not affect several important bio-chemical parameters or, harm the function or morphology of the CBD, kidney, pancreas and liver. Our data suggested that this Mg-6Zn alloy is a safe biocompatible material for CBD.

Determination of cnidilin and its two metabolites in rat bile and stool after oral administration by HPLC/electrospray ionization tandem mass spectrometry.

A simple, fast and sensitive method for the simultaneous determination of cnidilin and its two metabolites (M1 and M2) in rat bile and stool using HPLC coupled with electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) has been developed. The sample pretreatment was simple, because methanol was the only additive used for dilution of bile and ultrasound of stool. Pimpinellin was used as internal standard (IS). The separation was performed on a reverse phase C18 column with gradient elution consisting of 0.5‰ aqueous formic acid and methanol (containing 0.5‰ formic acid). The detection was in the multiple-reaction monitoring mode within 7 min. All the analytes were in accordance with the requirement of the validation of the method in vivo (linearity, precision, accuracy, limit of detection and limit of quantification). After oral administrating 24 mg/kg of the prototype drug cnidilin, M1 and M2 were determined in bile within 36 h, and in stool within 60 h. Cnidilin in bile was completely excreted in 24 h, and the main excretive amount of cnidilin was 80% in the first 6 h, but the drug recovery in bile within 24 h was <1.95%. In stool, the main excretive amount of cnidilin was 95.8% in the first 24 h, and the drug recovery within 48 h was lower than 1.48%.

Assessment of heavy metal and PAH exposure in largemouth bass (Micropterus salmoides) in the Reedy River watershed, South Carolina, USA: a multi-season assessment of metallothionein and bile fluorescence.

Biomarkers can be used as tools to help determine ecological exposure in watershed assessments. In the present study, metallothionein and fixed wavelength bile fluorescence for two-, four-, and five-ring hydrocarbons were used as biomarkers of exposure in largemouth bass (Micropterus salmoides) in the Reedy River watershed located in South Carolina, USA. Fish were sampled from three impoundments and a reference site over three seasons in the same year. Biomarker endpoints were compared to chemical concentrations at each site during each season. Results indicated that despite elevated concentrations of hydrocarbons being present in sediments, the hydrocarbons did not appear to be bioavailable based on bile fluorescence analysis. Bile fluorescence analysis also indicated that the hydrocarbons detected in this watershed were likely of petrogenic origin. Significantly elevated sediment concentrations of Cd, Cr, Cu, Pb, Ni, Ag, Zn, and metallothionein were found in one impoundment, Lake Conestee, compared with the reference site, indicating both the presence and bioavailability of these metals. Seasonal variability of bile fluorescence was limited; however, metallothionein showed elevated concentrations in the spring and summer compared with fall.

Bioavailability of pharmaceuticals in waters close to wastewater treatment plants: use of fish bile for exposure assessment.

Pharmaceuticals are ubiquitous in surface waters as a consequence of discharges from municipal wastewater treatment plants. However, few studies have assessed the bioavailability of pharmaceuticals to fish in natural waters. In the present study, passive samplers and rainbow trout were experimentally deployed next to three municipal wastewater treatment plants in Finland to evaluate the degree of animal exposure. Pharmaceuticals from several therapeutic classes (in total 15) were analyzed by liquid chromatography-tandem mass spectrometry in extracts of passive samplers and in bile and blood plasma of rainbow trout held at polluted sites for 10 d. Each approach indicated the highest exposure near wastewater treatment plant A and the lowest near that of plant C. Diclofenac, naproxen, and ibuprofen were found in rainbow trout, and their concentrations in bile were 10 to 400 times higher than in plasma. The phase I metabolite hydroxydiclofenac was also detected in bile. Hence, bile proved to be an excellent sample matrix for the exposure assessment of fish. Most of the monitored pharmaceuticals were found in passive samplers, implying that they may overestimate the actual exposure of fish in receiving waters. Two biomarkers, hepatic vitellogenin and cytochrome P4501A, did not reveal clear effects on fish, although a small induction of vitellogenin mRNA was observed in trout caged near wastewater treatment plants B and C.

Fat digestion and absorption in spice-pretreated rats.

BACKGROUND: A few common spices are known to stimulate secretion of bile with higher amount of bile acids which play a major role in digestion and absorption of dietary lipids. It would be appropriate to verify if these spices enable efficient digestion and absorption during high-fat intake. In this context, dietary ginger (0.05%), piperine (0.02%), capsaicin (0.015%), and curcumin (0.5%) were examined for their influence on bile secretion, digestive enzymes of pancreas and absorption of dietary fat in high-fat (30%) fed Wistar rats for 8 weeks. RESULTS: These spices enhanced the activity of pancreatic lipase, amylase, trypsin and chymotrypsin by 22-57%, 32-51%, 63-81% and 12-38%, respectively. Dietary intake of spices along with high-fat enhanced fat absorption. These dietary spices increased bile secretion with higher bile acid content. Stimulation of lipid mobilisation from adipose tissue was suggested by the decrease in perirenal adipose tissue weight by dietary capsaicin and piperine. This was also accompanied by prevention of the accumulation of triglyceride in liver and serum in high-fat fed rats. Activities of key lipogenic enzymes in liver were reduced which was accompanied by an increased activity of hormone-sensitive lipase. CONCLUSION: Thus, dietary ginger and other spice compounds enhance fat digestion and absorption in high-fat fed situation through enhanced secretion of bile salts and a stimulation of the activity pancreatic lipase. At the same time, the energy expenditure is facilitated by these spices to prevent the accumulation of absorbed fat.

Tracing of the Bile-chemotactic migration of juvenile Clonorchis sinensis in rabbits by PET-CT.

BACKGROUND: Adult Clonorchis sinensis live in the bile duct and cause clonorchiasis. It is known that the C. sinensis metacercariae excyst in the duodenum and migrate up to the bile duct through the common bile duct. However, no direct evidence is available on the in vivo migration of newly excysted C. sinensis juveniles (CsNEJs). Advanced imaging technologies now allow the in vivo migration and localization to be visualized. In the present study, we sought to determine how sensitively CsNEJs respond to bile and how fast they migrate to the intrahepatic bile duct using PET-CT. METHODOLOGY/PRINCIPAL FINDINGS: CsNEJs were radiolabeled with (18)F-fluorodeoxyglucose ((18)F-FDG). Rabbits with a gallbladder contraction response to cholecystokinin-8 (CCK-8) injection were pre-screened using cholescintigraphy. In these rabbits, gallbladders contracted by 50% in volume at an average of 11.5 min post-injection. The four rabbits examined were kept anesthetized and a catheter inserted into the mid duodenum. Gallbladder contraction was stimulated by injecting CCK-8 (20 ng/kg every minute) over the experiment. Anatomical images were acquired by CT initially and dynamic PET was then carried out for 90 min with a 3-min acquisition per frame. Twelve minutes after CCK-8 injection, about 3,000 (18)F-FDG-labeled CsNEJs were inoculated into the mid duodenum through the catheter. Photon signals were detected in the liver 7-9 min after CsNEJs inoculation, and these then increased in the whole liver with stronger intensity in the central area, presenting that the CsNEJs were arriving at the intrahepatic bile ducts. CONCLUSION: In the duodenum, CsNEJs immediately sense bile and migrate quickly with bile-chemotaxis to reach the intrahepatic bile ducts by way of the ampulla of Vater.

Plasma stability-dependent circulation of acyl glucuronide metabolites in humans: how circulating metabolite profiles of muraglitazar and peliglitazar can lead to misleading risk assessment.

Muraglitazar and peliglitazar, two structural analogs differing by a methyl group, are dual peroxisome proliferator-activated receptor-α/γ activators. Both compounds were extensively metabolized in humans through acyl glucuronidation to form 1-O-ß-acyl glucuronide (AG) metabolites as the major drug-related components in bile, representing at least 15 to 16% of the dose after oral administration. Peliglitazar AG was the major circulating metabolite, whereas muraglitazar AG was a very minor circulating metabolite in humans. Peliglitazar AG circulated at lower concentrations in animal species than in humans. Both compounds had a similar glucuronidation rate in UDP-glucuronic acid-fortified human liver microsomal incubations and a similar metabolism rate in human hepatocytes. Muraglitazar AG and peliglitazar AG were chemically synthesized and found to be similarly oxidized through hydroxylation and O-demethylation in NADPH-fortified human liver microsomal incubations. Peliglitazar AG had a greater stability than muraglitazar AG in incubations in buffer, rat, or human plasma (pH 7.4). Incubations of muraglitazar AG or peliglitazar AG in plasma produced more aglycon than acyl migration products compared with incubations in the buffer. These data suggested that the difference in plasma stability, not differences in intrinsic formation, direct excretion, or further oxidation of muraglitazar AG or peliglitazar AG, contributed to the observed difference in the circulation of these AG metabolites in humans. The study demonstrated the difficulty in doing risk assessment based on metabolite exposure in plasma because the more reactive muraglitazar AG would not have triggered a threshold of concern based on the recent U.S. Food and Drug Administration guidance on Metabolites in Safety Testing, whereas the more stable peliglitazar AG would have.

Evaluation of relative LC/MS response of metabolites to parent drug in LC/nanospray ionization mass spectrometry: potential implications in MIST assessment.

There is an increasing demand for quantitative data on metabolite exposure triggered by regulatory guidances. This contribution describes the accuracy of nanoelectrospray ionization mass spectrometry response of drug compounds and their metabolites from biological matrices compared with radiometric quantification. This is a comprehensive investigation of a set of real-life pharmaceutical compounds in relevant matrices such as urine, bile, feces and plasma. The data suggest that nanoelectrospray mass spectrometry can be used for semi-quantitation of metabolites in the absence of reference standards. Therefore, this approach is suitable to screen out non-relevant metabolites early in development as long as an adequate analytical error margin is applied thus balancing risks and resources.

Assessment of bile fluorescence patterns in a tropical fish, Nile tilapia (Oreochromis niloticus) exposed to naphthalene, phenanthrene, pyrene and chrysene using fixed wavelength fluorescence and synchronous fluorescence spectrometry.

Bile fluorescence patterns in Nile tilapia, a potential fish for biomonitoring tropical water pollution were assessed following exposure to selected polycyclic aromatic hydrocarbons (PAHs): naphthalene, phenanthrene, pyrene and chrysene. Non-normalized fixed wavelength fluorescence signals in the fish exposed to these PAHs reflected dose and/or time response relationships of their metabolism. Normalizing signals to biliverdin introduced deviations to these response patterns. The optimal wavelength pairs (excitation/emission) for synchronous fluorescence scanning measurements of bile metabolites of naphthalene, phenanthrene, pyrene and chrysene were identified as 284/326, 252/357, 340/382 and 273/382 respectively. This study supports the use of bile fluorescence in Nile tilapia by fixed wavelength fluorescence and synchronous fluorescence spectrometry with non-normalized data as a simple method for screening bioavailability of these PAHs.

1-Hydroxypyrene--a biochemical marker for PAH pollution assessment of aquatic ecosystem.

The aim of the present study was to assess aquatic contamination by polycyclic aromatic hydrocarbons (PAH), using the 1-hydroxypyrene (1-OHP) content in fish bile as a biochemical marker. A total of 71 chub (Leuciscus cephalus L.) were collected from seven locations on the Svitava and Svratka rivers in and around the industrial city of Brno, Czech Republic. The levels of 1-OHP were determined by reverse phase HPLC with fluorescence detection after deconjugation. Normalising the molar concentration of the biliary 1-OHP to the biliary protein content reduced sample variation. The content of 1-OHP was correlated with the PAH level in bottom sediment and semi-permeable membrane devices (SPMD), which was analyzed by a combination of HPLC/FLD and GC/MS methods. The highest mean values of 1-OHP were found in fish caught at the Svratka River at locations Modrice (169.2 ± 99.7 ng · mg(-1) protein) and Rajhradice (152.2 ± 79.7 ng · mg(-1) protein), which are located downstream from Brno. These values were significantly (P < 0.05) higher than those obtained from localities Knínicky (98.4 ± 66.1 ng · mg(-1) protein) and Bílovice nad Svitavou (64.1 ± 31.4 ng · mg(-1) protein). The lowest contents of PAH in sediment and SPMD were found at location Knínicky (1.5 mg · kg(-1) dry mass and 19.4 ng · L(-1), respectively). The highest contents of PAH in sediment and SPMD were found in Rajhradice (26.0 mg · kg(-1) dry mass) and Svitava before junction (65.4 ng · L(-1)), respectively. A Spearman correlation test was applied to determine the relationship between biliary 1-OHP and the sum of PAH in sediment and SPMD. A positive, but no statistically significant correlation was found. The main impact sources of elevated level of PAHs in sites located downstream from Brno are most probably intensive industrial and agricultural activities and domestic waste.

Bear bile: dilemma of traditional medicinal use and animal protection.

Bear bile has been used in Traditional Chinese Medicine (TCM) for thousands of years. Modern investigations showed that it has a wide range of pharmacological actions with little toxicological side effect and the pure compounds have been used for curing hepatic and biliary disorders for decades. However, extensive consumption of bear bile made bears endangered species. In the 1980's, bear farming was established in China to extract bear bile from living bears with "Free-dripping Fistula Technique". Bear farming is extremely inhumane and many bears died of illness such as chronic infections and liver cancer. Efforts are now given by non-governmental organizations, mass media and Chinese government to end bear farming ultimately. At the same time, systematic research has to be done to find an alternative for bear bile. In this review, we focused on the literature, laboratory and clinical results related to bear bile and its substitutes or alternative in English and Chinese databases. We examined the substitutes or alternative of bear bile from three aspects: pure compounds derived from bear bile, biles from other animals and herbs from TCM. We then discussed the strategy for stopping the trading of bear bile and issues of bear bile related to potential alternative candidates, existing problems in alternative research and work to be done in the future.

[Comparative assessment of bile and blood cholesterol, pigment, and protein metabolic parameters in patients with cholelithiasis in the presence and absence of opisthorchiasis].

AIM: to study the regularities of lithogenesis in patients with cholelithiasis (CL) and opisthorchiasis. SUBJECTS AND METHODS: Fifty-one patients with CL and opisthorchiasis were examined. Comparison groups comprised 32 with CL without opisthorchiasis and 14 patients with opisthorchiasis without CL. All the patients underwent a comprehensive examination involving laboratory and ultrasound studies. RESULTS: Opisthorchis invasion leads to increases in vesicular and hepatic bile cholesterol saturation and bile albumin levels due to intrahepatic ductal cholestasis, to a rise in bile unconjugated bilirubin concentration, which is caused by the activation of proliferative inflammation in the bile ducts and by the increased deposits of bilirubin complexes resulting from higher bile globulin levels. CONCLUSION: In opisthorchiasis, bile lithogenic factors mostly activate in the development of CH in patients with significant manifestations of cholestasis, angiocholitis with the most preserved concentration function of the gallbladder.