Enzymatic Deconjugation Research Articles

Bioassay based screening of steroid derivatives in animal feed and supplements

Receptor binding transcription activation bioassays are valuable tools for the screening of steroid hormones in animal feed and supplements. However, steroid derivatives often lack affinity for their cognate receptor and do not show any direct hormonal activity by themselves. These compounds are thus not detected by these kinds of bioassays and need a bioactivation step in order to become active, both in vivo and in vitro. In this study a comparison was made between different in vitro activation methods for hormone esters and hormone glycosides. Testosterone acetate and testosterone decanoate were chosen as model compounds for the hormone esters, representing the broad range of steroid esters of varying polarities, while genistin was used as a substitute model for the steroid-glycosides. Concerning bioactivation of the steroids esters, the efficiency for alkaline hydrolysis was 90–100% and much better as compared to enzymatic deconjugation by esterase. As a result 1 μg testosterone ester per gram of animal feed could easily be detected by a yeast androgen bioassay. When comparing different enzyme fractions for deglycosilation, genistin was shown to be deconjugated most efficiently by β-glucuronidase/aryl sulfatase from Helix pomatia, resulting in a significant increase of estrogenic activity as determined by a yeast estrogen bioassay. In conclusion, chemical and enzymatic deconjugation procedures for ester and glycoside conjugates respectively, resulted in a significant increase in hormonal activity as shown by the bioassay readouts and allowed effective screening of these derivatives in animal feed and feed supplements.

Determination of Five Phthalate Monoesters in Human Urine Using Gas Chromatography-Mass Spectrometry

We have developed a gas chromatography-mass spectrometry (GC-MS) method to determine five phthalate monoesters (monoethyl phthalate (MEP), mono-n-butyl phthalate (MBP), mono-(2-ethylhexyl) phthalate (MEHP), monoisononyl phthalate (MINP) and monobenzyl phthalate (MBz)) in human urine. Human urine samples were subjected to enzymatic deconjugation of the glucuronides followed by extraction with hexane. The extracted phthalate monoesters were methylated with diazomethane, purified on a Florisil column and then subjected to GC-MS analysis. The recoveries from urine spiked with five phthalate monoesters were 86.3%-119% with coefficients of variation of 0.6%-6.1%. We measured phthalate monoester levels in human urine by analyzing 36 samples from volunteers. MBP and MEP were detected in all samples, and their median concentrations were 60.0 and 10.7 ng/mL, respectively. MBzP and MEHP were found in 75% and 56% of samples, and their median concentrations were 10.9 and 5.75 ng/mL, respectively. MINPs were not detected in most samples (6% detectable). Women had significantly (p < 0.05) higher mean concentrations of MBP and MEP than men. The estimated daily exposure levels for the four parent phthalates excluding diisononyl phthalate ranged from 0.27 to 5.69 mug/kg/day (median).

Metabolites of Protoberberine Alkaloids in Human Urine following Oral Administration of Coptidis Rhizoma Decoction

Coptidis Rhizoma has been used as a traditional Chinese herbal medicine to treat typhoid, pharyngolaryngitis, diabetes mellitus, gastroenteritis and secretory diarrhea for more than a thousand years in China. However, there is little information on the IN VIVO chemical constituents of Coptidis Rhizoma following oral administration. In this paper, the alkaloid constituents in urine were studied in humans following oral administration of Coptidis Rhizoma decoction. Using macroporous adsorption resin chromatography, open ODS column chromatography, and preparative high-performance liquid chromatography, twelve protoberberine alkaloid constituents were isolated. Their structures were elucidated by chemical evidence, enzymatic deconjugation and analyses of mass, (1)H-NMR and NOESY spectra. The identified alkaloid constituents include berberine ( P1), groenlandicine 3-O- β-D-glucuronide (M1), dehydrocheilanthifoline 2-O-β-D-glucuronide (M2), thalifendine 10-O-β-D-glucuronide (M3), jatrorrhizine 3-O-β-D-glucuronide (M4), columbamine 2-O-β-D-glucuronide (M5), berberrubine 9-O-β-D-glucuronide (M6), jatrorrhizine 3-O-sulfate (M7), demethyleneberberine 2-O-sulfate (M8), dehydrocorydalmine 10-O-sulfate (M9), 3,10-demethylpalmatine 10-O-sulfate (M10) and 2,3,10-trihydroxyberberine 2-O-sulfate ( M11). No other parent protoberberine alkaloids from Coptidis Rhizoma except for a trace of berberine were found in the urine. These findings suggested that the protoberberine alkaloids, which were absorbed in vivo following oral administration of Coptidis Rhizoma decoction, were mainly conjugated with glucuronic acid or sulfuric acid to form phase II metabolites directly or after biotransformation to phase I metabolites, and finally excreted in urine.

Development of a high-throughput LC/APCI-MS method for the determination of thirteen phytoestrogens including gut microbial metabolites in human urine and serum

The investigation into the potential usefulness of phytoestrogens in the treatment of menopausal symptoms requires large-scale clinical trials that involve rapid, validated assays for the characterization and quantification of the phytoestrogenic precursors and their metabolites in biological matrices, as large interindividual differences in metabolism and bioavailability have been reported. Consequently, a new sensitive high-performance liquid chromatography-mass spectrometry method (HPLC–MS) for the quantitative determination of thirteen phytoestrogens including their most important gut microbial metabolites (genistein, daidzein, equol, dihydrodaidzein, O-desmethylangolensin, coumestrol, secoisolariciresinol, matairesinol, enterodiol, enterolactone, isoxanthohumol, xanthohumol and 8-prenylnaringenin) in human urine and serum within one single analytical run was developed. The method uses a simple sample preparation procedure consisting of enzymatic deconjugation followed by liquid–liquid extraction (LLE) or solid-phase extraction (SPE) for urine or serum, respectively. The phytoestrogens and their metabolites are detected with a single quadrupole mass spectrometer using atmospheric pressure chemical ionization (APCI), operating both in the positive and the negative mode. This bioanalytical method has been fully validated and proved to allow an accurate and precise quantification of the targeted phytoestrogens and their metabolites covering the lower parts-per-billion range for the measurement of relevant urine and serum levels following ingestion of phytoestrogen-rich dietary supplements.

Confirmatory method for the determination of nandrolone and trenbolone in urine samples using immunoaffinity cleanup and liquid chromatography–tandem mass spectrometry

A confirmatory method for the simultaneous determination of nandrolone (α and β) and trenbolone (α and β) in urine samples by liquid chromatography electrospray mass spectrometry (LC–MS-MS) was developed. After an enzymatic deconjugation, the urine was subjected to a one-step cleanup on a commercially available immunoaffinity chromatography cartridge. The analytes were detected by liquid chromatography–positive ion electrospray tandem mass spectrometry using deuterium labelled internal standards. The analytical procedure was applicable to bovine and swine urine samples. The procedure was validated as a quantitative confirmatory method according to the Commission Decision 2002/657/EC criteria. The results obtained showed that the method was suitable for statutory residues testing regarding the following performance characteristics: instrumental linearity, specificity, precision (repeatability and intra-laboratory reproducibility), recovery, decision limit (CCα), detection capability (CCβ) and ruggedness. The decision limits (CCα) obtained, were between 0.54 and 0.60 μg L −1; the recovery was above 64% for all the analytes. Repeatability was between 1.6% and 5.7% and within-laboratory reproducibility between 1.6% and 6.0% for all the steroids.

Confirmatory method for the determination of resorcylic acid lactones in urine sample using immunoaffinity cleanup and liquid chromatography–tandem mass spectrometry

The presence of zeranol (α-zearalanol) in urine samples due to natural contamination or illegal treatment is under debate within the European Union. The simultaneous determination of zeranol, its epimer taleranol (β-zearalanol), zearalanone and the structurally related mycotoxin zearalenone with the corresponding α- and β-zearalenol metabolites appears to be critical in deciding whether an illegal use has occurred. The aim of this study is to develop and validate a simple analytical procedure applicable to bovine and swine urine samples for the determination of all six resorcylic acid lactones. After an enzymatic deconjugation, the urine was subjected to a one-step cleanup on a commercially available immunoaffinity chromatography cartridge. The analytes were detected by liquid chromatography–negative-ion electrospray tandem mass spectrometry using deuterium-labelled internal standards. The method was validated as a quantitative confirmatory method according to European Commission Decision 2002/657/EC. The evaluated parameters were: linearity, specificity, precision (repeatability and intra-laboratory reproducibility), recovery, decision limit, detection capability and ruggedness. The decision limits (CCα) obtained, were between 0.56 and 0.68 μg L −1; recovery above 66% for all the analytes. Repeatability was between 1.4 and 5.3% and within-laboratory reproducibility between 1.9 and 16.1% for the six resorcylic acid lactones.

Analysis of hydroxylated polycyclic aromatic hydrocarbons in urine using comprehensive two-dimensional gas chromatography with a flame ionization detector

The determination of polycyclic aromatic hydrocarbon (PAH) metabolites in human urine is the method of choice for assessing exposure to carcinogenic compounds. The objective of this study was the development of a comprehensive two-dimensional gas chromatography (GC × GC) method using a flame ionisation detector (FID) to simultaneously determine 10 hydroxylated PAH. The method was based on enzymatic deconjugation, liquid–liquid extraction, and trimethylsilyl (TMS) derivatization of the analytes by microwave heating. Satisfactory separation was achieved. The coefficient of variance was 3.8–12.8%. LOD was 0.03–0.18 μg/L, and LOQ was 0.1–0.5 μg/L. The mean recovery was 76%. The method was applied to the analysis of urine from smokers and non-smokers.

Isolation and Identification of Urinary Metabolites of Berberine in Rats and Humans

The urinary metabolites of berberine, an isoquinoline alkaloid isolated from several Chinese herbal medicines, were investigated in rats and humans. Using macroporous adsorption resin chromatography, open octadecyl silane column chromatography and preparative high-performance liquid chromatography, we isolated seven metabolites (HM1-HM7) from human urine and five metabolites (RM1-RM5) from rat urine after oral administration. Their structures were elucidated by enzymatic deconjugation and analyses of mass spectrometry, (1)H NMR, and nuclear Overhauser effect spectroscopy spectra. Besides the three known metabolites demethyleneberberine-2-O-sulfate (HM1 and RM3), jatrorrhizine-3-O-sulfate (HM5), and thalifendine (RM5), six new metabolites were identified, namely, jatrorrhizine-3-O-beta-D-glucuronide (HM2), thalifendine-10-O-beta-D-glucuronide (HM3), berberrubine-9-O-beta-D-glucuronide (HM4 and RM2), 3,10-demethylpalmatine-10-O-sulfate (HM6 and RM4), columbamin-2-O-beta-D-glucuronide (HM7), and demethyleneberberine-2,3-di-O-beta-D-glucuronide (RM1). These findings suggest that berberine undergoes similar biotransformation in rats and humans. Possible metabolic pathways of berberine in rats and humans are proposed.

Development of a high-throughput LC-APCI-MS method for the determination of 13 phytoestrogens (including gut microbial metabolites) in human urine and serum

Over the last few years the literature on the possible health benefits of phytoestrogens has expanded exponentially. However, potential use of phytoestrogens e.g. for the management of menopausal symptoms warrants further study in trials with rigorous scientific designs [1]. In view of the large interindividual variability in microbial metabolism of phytoestrogens in the large intestine, more consistent results for the use of phytoestrogens in a clinical environment can be expected, when there is a control for the absorption, metabolism and bioavailability. Large-scale studies require rapid, validated assays for the characterisation and quantification of the phytoestrogenic precursors and their metabolites in biological matrices. We report here a rapid, sensitive and specific HPLC-UV-atmospheric pressure chemical ionisation-mass spectrometry method for the simultaneous determination of 13 phytoestrogens (genistein, daidzein, equol, dihydrodaidzein, O-desmethylangolensin, coumestrol, secoisolariciresinol, matairesinol, enterodiol, enterolactone, isoxanthohumol, xanthohumol and 8-prenylnaringenin) in human urine and serum. The assay consists of a simple sample preparation procedure, using enzymatic deconjugation followed by liquid-liquid extraction (LLE) (urine) or solid phase extraction (SPE) (serum) and reversed-phase HPLC (XBridgeTM, 3.5µm, 150mm x 3.0mm i.d.) coupled to a photodiode array detector (DAD) and Multimode Source Detector (MSD) operating in APCI mode, using both positive and negative mode ionization. This method is suitable for the estimation of urinary and serum levels of the major phytoestrogens and their microbial metabolites (ppb-ppm range).

GC–MS methods for metabolic profiling of microbial fermentation products of dietary polyphenols in human and in vitro intervention studies

Flavonoids, a subclass of polyphenols, are major constituents of many plant-based foods and beverages, including tea, wine and chocolate. Epidemiological studies have shown that a flavonoid-rich diet is associated with reduced risk of cardiovascular diseases. The majority of the flavonoids survive intact until they reach the colon where they are then extensively metabolized into smaller fragments. Here, we describe the development of GC–MS-based methods for the profiling of phenolic microbial fermentation products in urine, plasma, and fecal water. Furthermore, the methods are applicable for profiling products obtained from in vitro batch culture fermentation models. The methods incorporate enzymatic deconjugation, liquid–liquid extraction, derivatization, and subsequent analysis by GC–MS. At the level of individual compounds, the methods gave recoveries better than 80% with inter-day precision being better than 20%, depending on the matrix. Limits of detection were below 0.1 μg/ml for most phenolic acids. The newly developed methods were successfully applied to samples from human and in-vitro intervention trials, studying the metabolic impact of flavonoid intake. In conclusion, the methods presented are robust and generally applicable to diverse biological fluids. Its profiling character is useful to investigate on a large scale the gut microbiome-mediated bioavailability of flavonoids.

Characterization of alkylphenol metabolites in fish bile by enzymatic treatment and HPLC-fluorescence analysis

Alkylphenol (AP) metabolites were characterized in the bile of Atlantic cod (Gadus morhua L.) after exposure to nine individual compounds (10mg/kg fish), 2-methylphenol (2-MP), 4-methylphenol (4-MP), 3,5-dimethylphenol (3,5-DMP), 2,4,6-trimethylphenol (2,4,6-TMP), 4-tert-butylphenol (4-t-BP), 4-tert-butyl-2-methylphenol (4-t-B-2-MP), 4-n-pentylphenol (4-n-PP), 4-n-hexylphenol (4-n-HexP) and 4-n-heptylphenol (4-n-HepP), and a mixture (total dose; 13.5mg/kg fish) of the nine APs by inter-muscular injection. The degree of alkylation ranged from methyl (C1) to heptyl (C7) and represents the types of APs present in produced water. Fish bile was collected on day 4 and 16 (exposure groups 2-MP, 3,5-DMP, 2,4,6-TMP and 4-t-B-2-MP) following exposure. Characterization of major metabolites was accomplished by enzymatic de-conjugation and analysis by high performance liquid chromatography connected to a fluorescence detector (HPLC-F) acquiring at ex/em 222/306nm. Two solid phase extraction (SPE) columns were evaluated for clean-up of samples prior to analysis. Independent of alkyl homologue, the glucuronide conjugated APs were the most abundant metabolites (73–100%), whereas sulfates, glucosides and unchanged compounds were excreted in amounts of 0–21%, 0–6.1% and 0–6.3%, respectively. The total concentration of measured metabolites in the bile, determined as their respective APs after de-conjugation, increased with increasing degree of alkylation (3.2±2.6μg/g bile for 2-MP and 571±81μg/g bile for 4-n-HepP) after exposure to an equal dose of AP. Comparison of metabolite concentrations in bile sampled 4 and 16 days after exposure, showed that the levels of 2-MP, 2,4,6-TMP and 4-t-B-2-MP were reduced by 55%, 30% and 45%, respectively whereas 3,5-DMP increased by 25% (not significant). This study suggests that analysis of de-conjugated metabolites in fish bile can be used to monitor AP exposure to fish, due to the relatively high and persistent level of these compounds. However, although HPLC-F is suitable for laboratory exposures, it might not be sufficient selective for field studies.

Urinary bisphenol-A concentration in infertile Japanese women and its association with endometriosis: A cross-sectional study

Bisphenol A (BPA), a raw material commonly used in the manufacture of resins such as polycarbonate and epoxy, is a possible xenoestrogen that is hypothesized to disrupt the human endocrine system. Humans are widely exposed to BPA. We investigated the urinary concentration of BPA in infertile Japanese women and its possible association with endometriosis. We recruited 166 women (aged 20-45) who had complained of infertility and visited a university hospital in Tokyo. The subjects were interviewed and their urine samples were obtained prior to a laparoscopic diagnosis of endometriosis between January 2000 and December 2001. Urinary total BPA concentration in 140 eligible urine samples was then measured using enzymatic deconjugation of glucuronide and sulfate and high-performance liquid chromatography isotope-dilution tandem mass spectrometry. Median (25th-75th percentile) unadjusted and creatinine-adjusted urinary BPA concentrations were 1.6 (0.69-2.8) μg/L and 0.80 (0.45-1.3) μg/g creatinine. No significant monotonic association of endometriosis with urinary BPA concentration was observed. Median urinary BPA concentration in women with stage 0-1 endometriosis (0.74 μg/g creatinine) did not significantly differ from that in those with stage II-IV endometriosis (0.93 μg/g creatinine) (p for difference=0.24). This study, based on a larger number of samples than those in previous studies in Japan and using the most reliable analytical method currently available, showed that urinary concentrations of BPA in women who consulted a physician for infertility were not higher than those in other populations. Moreover, no association between urinary BPA concentration and endometriosis was found in this cross-sectional study.

Quantitative Identification of Unknown Exposure Pathways of Phthalates Based on Measuring Their Metabolites in Human Urine

Humans are exposed to ubiquitous phthalates via multiple pathways. Exposures to phthalates have been estimated in some previous risk assessments in Japan based on point-of-contact measurement or scenario evaluation approaches. While the Japanese national government has regulated the use of di(2-ethylhexyl)phthalate (DEHP) and excluded several other phthalates from its regulation based on some of them, it is unclear whether such past exposure assessment studies fully assessed total human exposure to phthalates. In the present study, we measured their urinary metabolites, which show direct evidence of human exposure to phthalates. We recruited voluntary participants (N = 36) who agreed to donate urine samples, and measured the urinary concentrations of phthalate metabolites using enzymatic deconjugation, solid-phase extraction, and high-performance liquid-chromatography isotope-dilution tandem mass spectrometry. We then derived the daily intakes of their respective phthalates based on steady state assumption and finally compared them with the corresponding estimated daily intakes of each phthalate via diet and air derived from previous exposure or risk assessments in Japan. These comparisons showed that exposures to dimethyl phthalate, diethyl phthalate, and di-n-butyl phthalate via diet and air accounted for less than half of their respective total exposures. On the other hand, it appears that dietary intake was more predictive for the total exposure to n-butyl-benzyl phthalate and DEHP. The probabilities that the log normal distribution of each phthalate daily intake estimated from the present study exceeds the corresponding tolerable daily intake were estimated to be less than 10(-4).

Quercetin, but not its glycosidated conjugate rutin, inhibits azoxymethane-induced colorectal carcinogenesis in F344 rats.

The effect of the flavonoid quercetin and its conjugate rutin was investigated on (biomarkers of) colorectal cancer (CRC). Male F344 rats (n = 42/group) were fed 0, 0.1, 1, or 10 g quercetin/kg diet or 40 g rutin/kg diet. Two wk after initial administration of experimental diets, rats were given 2 weekly subcutaneous injections with 15 mg/kg body wt azoxymethane (AOM). At wk 38 post-AOM, quercetin dose dependently (P < 0.05) decreased the tumor incidence, multiplicity, and size, whereas tumor incidences were comparable in control (50%) and rutin (45%) groups. The number of aberrant crypt foci (ACF) in unsectioned colons at wk 8 did not correlate with the tumor incidence at wk 38. Moreover, at wk 8 post-AOM, the number and multiplicity of ACF with or without accumulation of beta-catenin were not affected by the 10 g quercetin/kg diet. In contrast, another class of CRC-biomarkers, beta-catenin accumulated crypts, contained less beta-catenin than in controls (P < 0.05). After enzymatic deconjugation, the plasma concentration of 3'-O-methyl-quercetin and quercetin at wk 8 was inversely correlated with the tumor incidence at wk 38 (r = -0.95, P </= 0.05). Rats supplemented with 40 g rutin/kg diet had only 30% of the (3'-O-methyl-) quercetin concentration of 10 g quercetin/kg diet-fed rats (P < 0.001). In conclusion, quercetin, but not rutin, at a high dose reduced colorectal carcinogenesis in AOM-treated rats, which was not reflected by changes in ACF-parameters. The lack of protection by rutin is probably due to its low bioavailability.

GC-MS method for detection of endogenous estrogens in urine samples

Breast cancer is the most common cause of death due to malignancy among women worldwide and the majority of the established risk factors are linked to estrogens as the case maybe the endogenous estrogen metabolism. The identification of specific alterations in endogenous estrogen metabolism as risk factor for breast cancer is a topic of extensive research. For simultaneus quantitation of all estrogens and estrogen metabolites of interest, an aforementioned gas-chromatography mass spectrometry method was optimised. After enzymatic deconjugation of estrogen conjugates using β-glucuronidase, solid-phase extraction on Sep-pak C18 columns and further sample purification by ion exchange chromatography on QAE-Sephadex cartridges in the acetat form, QAE-Sephadex cartridges in the borate form were used to separate estrogens into two fractions: one fraction containing estrogens without vicinal cis-hydroxyls and a second fraction containing estrogens with vicinal cis-hydroxyls (=catecholestrogens). After derivatisation estrogens were analysed by gas-chromatography mass spectroscopy in the selected ion monitoring mode.

Development and validation of a liquid chromatography–tandem mass spectrometry method for the separation of conjugated and unconjugated 17α- and 17β-boldenone in urine sample

Natural occurrence or illegal treatment of boldenone (BOLD) presence in cattle urine is under debate within the European Union. Separation of conjugated and unconjugated forms of 17α-boldenone (α-BOLD) and 17β-boldenone (β-BOLD) and presence of related molecules as androsta-1,4-diene-3,17-dione (ADD) appear critical points for the decision of an illegal use. The aim of this study is a new analytical approach of BOLD and ADD confirmation in cattle urine. The separation between conjugated and unconjugated forms of BOLD was obtained by a preliminary urine liquid–liquid extraction step with ethyl acetate. In this step the organic phase extracts only unconjugated BOLD and ADD, while BOLD in conjugated form remain in urine phase. Afterwards the urine phase, contains conjugated BOLD, was subjected to an enzymatic deconjugation. Solid-phase extraction (OASIS-HLB Waters) was used for the purification and concentration of analytes in organic and urine phases and liquid chromatography ion electrospray tandem mass spectrometry (LC–MS–MS) was applied for the confirmation of BOLD and ADD, using deuterium-labelled 17β-boldenone (BOLD-d3) as internal standard. The method was validated as a quantitative confirmatory method according to the Commission Decision 2002/657/CE. The results obtained demonstrate that the developed method show very high specificity, precision, trueness and ruggedness. Decision limits (CCα) smaller than 0.5ngmL−1 were obtained for each analyte.

Measurement of Urinary Monohydroxy Polycyclic Aromatic Hydrocarbons Using Automated Liquid−Liquid Extraction and Gas Chromatography/Isotope Dilution High-Resolution Mass Spectrometry

A method for the measurement of 24 hydroxylated polycyclic aromatic hydrocarbon metabolites (OH-PAHs) in urine has been developed. The method is based on enzymatic deconjugation, automated liquid-liquid extraction, and gas chromatography/isotope dilution high-resolution mass spectrometry after derivatization of the OH-PAHs to the trimethylsilylated derivatives. The metabolites included in the current method are formed from eight different parent compounds. The limits of detection were below 7 pg/mL when using a sample size of 2 mL of urine, except for 1- and 2-naphthols (18 and 12 pg/mL, respectively). The enzymatic deconjugation efficiency, verified by deconjugation of urine samples spiked with alpha-naphthyl beta-d-glucuronide sodium salt (1-NAP-GLU) and pyrene-1-sulfate potassium salt (1-PYR-SULF), was determined to be 97% for 1-NAP-GLU conjugate and 84% for 1-PYR-SULF. The overall coefficients of variance for six batches of quality control samples (n = 42), was 2.9-11%. Mean method recoveries of the 13C-labeled internal standards were 66-72%, except for 13C6-1-naphthol (46%). The throughput of this method has been determined to be 40 samples per day per analyst. This method is currently applied to epidemiological studies, such as the National Exposure and Nutrition Examination Survey (NHANES), to measure human exposure to PAHs.

PAH Metabolites in Bile Fluids of Dab (Limanda limanda) and Flounder (Platichthys flesus): Spatial Distribution and Seasonal Changes (7 pp)

Polycyclic aromatic hydrocarbons (PAH) are important environmental contaminants which may lead to increased levels of neoplastic aberrations or tumours in fish liver. Therefore, monitoring of PAH and their effects are part of several international environmental programmes. The aim of the present field study was to investigate the concentrations of the PAH metabolites in fish bile, to elucidate spatial, seasonal and species differences as well as to discuss different strategies of normalisation with regard to environmental monitoring. PAH metabolites were determined in the bile fluid of dab (Limanda limanda) and flounder (Platichthys flesus) caught in the North Sea and Baltic Sea between 1997 and 2004. After enzymatic deconjugation, two metabolites were determined by means of HPLC. The limit of detection and the limit of quantification were calculated. The accuracy of the method was tested with a standard reference material. Results were referred to bile volume as well as to biliverdin. The main metabolite, 1-hydroxypyrene, was determined in concentrations from < 0.7 to 838 ng/ml in bile of dab (Limanda limanda) and flounder (Platichthys flesus) caught between 1997 and 2004. The values for 1-hydroxyphenanthrene in fish bile were considerably lower (<0.4-87 ng/ml). Significant differences in the 1-hydroxypyrene levels were found between summer and winter surveys as well as between the sampling sites in the data set from 2004 (383 dabs and 62 flounders): Highest levels of PAH contamination were found in dab from the German Bight and in flounder from the Baltic Sea. Spatial differences in 1-hydroxypyrene concentrations between North Sea and Baltic Sea were discussed, as well as differences in relation to season, sex and species. Three parameters of normalisation (biliary protein, biliverdin and bile pigments) were discussed. Biliverdin was identified as a suitable parameter for the normalisation of PAH metabolites in field samples. Spatial differences in 1-hydroxypyrene concentrations of dab demonstrate the usefulness of PAH metabolites in fish bile as a monitoring parameter in marine regions. Significant differences in 1-hydroxypyrene concentrations were found between summer and winter sampling campaigns. This may be linked to an annual cycle of 1-hydroxyprene in dab. It is also possible that bile synthesis/release in dab differs between the seasons. There is no indication for a time trend from 1997 to 2004. It is recommended to relate PAH metabolites in fish bile to biliverdin concentrations. Although the concentrations are low in offshore regions and bile volumes are small, the method presented here allows one to measure PAH metabolites on an individual level which is a crucial prerequisite for meaningful monitoring studies.

Influence of Biotransformation of Luteolin, Luteolin 7-O-Glucoside, 3′,4′-Dihydroxyflavone and Apigenin by Cultured Rat Hepatocytes on Antioxidative Capacity and Inhibition of EGF Receptor Tyrosine Kinase Activity

Flavonoids are known as biologically active compounds. Although this has been shown by several in vivo studies, it is still elusive whether their metabolites exert similar activities. Herein we investigated the biotransformation of four different flavonoids, 3',4'-dihydroxyflavone, apigenin, luteolin and luteolin 7-O-glucoside, by cultured rat hepatocytes using a combination of enzymatic deconjugation, HPLC separation and high-resolution mass spectrometry. These flavonoids were chosen because they are active components of many plants, e. g., artichokes. All flavonoids showed rather complex metabolite patterns dominated by phase II metabolites, mainly sulfates, methyl sulfates and methyl glucuronides, but also of combined glucuronide and sulfate conjugates. Phase I metabolism by hydroxylation was rendered likely only for apigenin to form luteolin. When culture media containing the flavonoids and their metabolites were assayed for antioxidative capacity by the DPPH assay, only compounds with hydroxy groups in position 3' and 4' of the B ring were active. Thus, during metabolism of (inactive) apigenin a strong increase in the antioxidative effect was observed while that of the other three flavonoids decreased with time. Determination of EGF receptor tyrosine kinase activity likewise revealed strong inhibition in the presence of a catechol group at ring B. However, in this case the situation was much more complex resulting in a significant increase of the inhibitory activity of 3',4'-dihydroxyflavone and apigenin, but not of luteolin and luteolin 7-O-glucoside during 22 h of incubation. These results show that the biotransformation of flavonoids is very complex and may result not only in a loss but also in a gain of biological activity depending on the individual structural features.

Accumulation of catechins in bone and liver of mice fed green tea while under physical stress

Epidemiological evidence has linked increased habitual consumption of catechin rich tea to reduced risk of fractures and improved bone mineral density. The objective of this study was to determine the extent to which dietary tea catechins accumulate in bone and if induced osteopenia impacts catechin levels in bone. Male NIH Swiss mice were treated for 16 d in a 2x2 design: control diet (AIN 93G basal diet) or control diet supplemented with green tea water extract (30 g/kg), and with or without hindlimb suspension for 9 d. Following treatment, mice were euthanized and liver, muscle, femur, and tibia were harvested. Catechins were extracted by homogenization of tissue in organic solvent, followed by enzymatic deconjugation. HPLC-ECD analysis detected major tea catechins including epigallocatechin gallate (EGCG), epicatechin gallate (ECG), epigallocatechin (EGC), and epicatechin (EC) only in tissues of mice fed tea. Bone content (μmol/g, in the order of EGC, EC, EGCG, ECG, and total) was: 3.3±1.1, 1.8±0.6, 48.6±17.6, 8.2±3.5, and 61.9±21.9 for suspended mice; and 7.3±1.8, 2.2±0.9, 76.4±21.9, 8.6±3.2, and 94.4±26.3 in unsuspended mice. Liver content was: 1.8±1.1, 0, 0, 1.1±1.1, and 2.8±1.9 in suspended mice; and 4.0±2.5, 3.2±1.2, 28.7±11.8, 3.9±2.5, and 39.9±14.9 in unsuspended mice. Concentrations in bone were not significantly different between suspension treatments, while EC, EGCG, and total catechins in liver were significantly lower in suspended mice (p<0.05). These data confirm that dietary green tea catechins accumulate in bone tissue and that atrophy appears to impact tissue accumulation of these phytochemicals.