This thesis describes the development and application of solid phase microextraction (SPME) as a sample preparation technique for in vivo determination of pharmaceutical residues in fish tissue and wastewater. The occurrence, distribution and fate of pharmaceuticals in the environment are a subject of concern across the globe due to the impact they may have on human life and aquatic organisms. To address this challenge from an analytical perspective, a simplified and reliable analytical methodology is required to investigate and determine the concentration (bioconcentration factors) of trace pharmaceutical residue in fish tissue and environmental water samples (exposure). An improved SPME method, coupled with liquid chromatography with tandem mass spectrometry has been developed and applied to both controlled laboratory and field-caged fish exposed to wastewater effluent for quantitative determination of pharmaceutical residue in fish specific tissue. A new SPME configuration based on C18 thin film (blade) was developed and optimized to improve SPME sensitivity for in vivo determinations of trace pharmaceuticals in live fish. The C18 thin film extraction phase successfully quantified bioconcentrated fluoxetine, venlafaxine, sertraline, paroxetine, and carbamazapine in the dorsal-epaxial muscle of living fish at concentrations ranging from 1.7 to 259 ng/g. The reproducibility of the method in spiked fish muscle was 9-18% RSD with limits of detection and quantification ranging from 0.08 - 0.21 ng/g and 0.09 - 0.64 ng/g (respectively) for the analytes examined. Fish were sampled by in vivo SPME for 30 min to detect pharmaceutical uptake and bioconcentration, with experimental extracts analyzed using liquid chromatography coupled with tandem mass spectrometry. In addition, a simplified analytical methodology based on SPME was developed and optimized for determination and bioconcentration factor of different classes of pharmaceuticals residues in fish bile. The reproducibility of the method in spiked fish Rainbow Trout bile was 3-7% RSD with limits of detection (LOD) ranging from 0.3 – 1.4 ng/mL for the analytes examined. The field application of SPME sampling was further demonstrated in Fathead Minnow (Pimephales promelas), a small-bodied fish caged upstream and downstream of a local wastewater treatment plant where fluoxetine, atorvastatin, and sertraline were detected in fish bile at the downstream location. Also, a simple automated analytical method using high throughput robotic system was developed for the simultaneous extraction of pharmaceutical compounds detected in surface waters. The proposed method successfully determined concentrations of carbamazepine, fluoxetine, sertraline, and paroxetine in treated effluent at concentrations ranging from 240 - 3820 ng/L with a method detection limit of 2-13 ng/L, and a relative standard deviation of less than 16%. Application of the method was demonstrated using wastewater from pilot-scale municipal treatment plants and environmental water samples from wastewater-dominated reaches of the Grand River (Waterloo, ON). Finally, 4 and 8-d laboratory exposures were carried out with Rainbow Trout exposed to wastewater effluent collected from pilot scale at Burlington, ON. Additionally, wild fish, White Sucker (Catostomus commersonii) were collected and sampled from Waterloo and Kitchener downstreams containing local municipal effluent. Bioconcentration factors of the selected compounds were determined in both fish muscle and bile samples. The results show that anti-depressant drugs such fluoxetine, sertraline and paroxetine were uptake in the fish muscle and fish bile for both laboratory and field exposure. In summary, exposure of fish to micro-pollutants such as pharmaceuticals may be monitored through the analysis of bile, particularly at low concentration exposure of pharmaceuticals, where the sensitivity of analytical method may be challenged. SPME is a promising simple analytical tool which can potentially be used for monitoring of pharmaceuticals in fish tissue and wastewater.

名古屋大学博士学位論文 学位の種類 : 博士(医学)(課程) 学位授与年月日:平成7年3月27日 金子健一朗氏の博士論文として提出された

Thesis (Ph. D.)--University of Hong Kong, 2010. / Includes bibliographical references (leaves 142-175). Also available in print.

Diagnosis of acute pancreatitis (AP) is erroneous in up to one third of patients when based on clinical criteria and elevated serum amylase values. Furthermore, according to autopsy reports fatal pancreatitis remains clinically undiagnosed in 22 to 86 % of hospitalised patients. Consequently, search for better methods for the diagnosis of AP seems not only justified but urgent. The pancreas secretes an nonspecific lipase, the carboxylic ester hydrolase (CEH) with molecular properties different from other pancreatic secretory enzymes. These differences may imply that sites and rates of clearances from blood of pancreatic enzymes differ. Except for the pancreas this enzyme is secreted from the lactating mammary gland with milk. A sensitive and reproducible sandwich-ELISA for quantitative determination of CEH was developed. When establishing referent values it was noted that in individuals aged 20 to 65 years serum concentrations of CEH did not depend on age, gender, the time of the day or duration from food intake to blood sampling, or use of nicotine. The mammary gland did not contribute significantly to basal serum levels of CEH; enzyme levels in lactating women or women with mammary tumours were identical to those of the reference population. Seventy percent of patients with the diagnosis AP, based on elevated serum amylase levels and abdominal pain, had elevated CEH values. Among the patients with elevated amylase alone a probable cause of pancreatitis was lacking in the majority of patients. Contrastingly, a likely cause of AP could be identified in all patients presenting with abdominal pain and elevated CEH levels alone. These findings suggested that an elevated CEH level indicated AP more reliably than an elevated amylase level. In patients with AP diagnosed by contrast enhanced computed tomography (CECT) alone, or combined with histopathological diagnosis, serum CEH levels were elevated on admission in all but one patient, and in all within the next 24 h. Furthermore, in patients with severe pancreatitis CEH levels remained at a raised level from the second to at least the 10:th day following admission, whereas a significant decrease was noted in patients with mild pancreatitis. In contrast, serum amylase values were higher in patients with mild pancreatitis during the observation period than in those with severe pancreatitis. CEH levels were higher in patients with three or more Ranson signs than in those with less than three signs from the first day after admission. CEH levels were within referent range in 164 patients without known pancreatic disease admitted due to abdominal emergency conditions, or due to planned surgery for chronic extrapancreatic gastrointestinal diseases, and 16 patients having CECT without pathological findings in the pancreas. This suggests that AP can be excluded with very high degree of probability in presence of non-elevated CEH levels. A sandwich ELISA for determination of Guinea pig CEH and a model for graded pancreatitis in the same species were developed. CEH levels showed proportional to severity of inflammation, thus confirming previous clinical observations. CEH levels in bile were proportional to inflammation, while it was absent in urine. Amylase levels in urine were identical regardless of severity of inflammation, but low in bile. These results suggested differences in sites and rates of clearance between the two enzymes. Seemingly elevated CEH levels allowed identification of clinically significant pancreatitis following ERCP, which amylase levels did not. The presented studies have shown that quantitative determination in serum of CEH by the described method is a more reliable test for the diagnosis of AP than determination of amylase activity. The differences between CEH and amylase are, at least partly, due to differences in molecular properties determining rates and routes of clearances of the two enzymes from serum. / <p>Diss. (sammanfattning) Umeå : Umeå universitet, 1994, härtill 5 uppsatser.</p> / digitalisering@umu.se

Deoxycholic acid (DCA) is a secondary bile acid postulated to be involved in the etiology and the progression of colorectal cancer, but its specific mechanisms are not fully understood. DCA has been shown to induce apoptosis allowing selection for apoptosis-resistant cells, which highlights the importance of understanding the mechanisms of action of DCA. Previously, it has been demonstrated that DCA perturbs the plasma membrane, leading to the activation of receptor tyrosine kinases. Because the insulin-like growth factor-1 receptor (IGF-IR), a receptor tyrosine kinase, is demonstrated to play a significant role in protecting colorectal cancer cells from apoptosis, we hypothesized that DCA modulates IGF-IR functions in colorectal cancer cells. We demonstrated that DCA induced the dynamin-dependent endocytosis of IGF-IR through both clathrin-mediated and caveolin-1-dependent mechanisms. Endocytosis of IGF-IR sensitized cells to DCA-induced apoptosis, which demonstrated that IGF-IR played a role in protecting cells against DCA-induced apoptosis. Since DCA-induced endocytosis of IGF-IR was determined to be a caveolin-1 dependent process, caveolin-1 knockdown in HCT116 (HCT116-Cav1-AS) prevented the DCA-mediated endocytosis of IGF-IR. However, we observed an increased sensitivity of DCA-induced apoptosis in the Cav1-AS cells. This suggested that caveolin-1 knockdown altered the plasma membrane dynamics such that although IGF-IR was maintained at the plasma membrane, it facilitated a pro-apoptotic signal. We demonstrated that DCA induced the activation of the pro-apoptotic p38 signaling pathway in HCT116-Cav1-AS, but not in HCT116-Mock, via IGF-IR. Inhibition of both the IGF-IR and p38 independently in HCT116-Cav1-AS significantly decreased their sensitivity to DCA-induced apoptosis. These observations demonstrated that, in a caveolin-1 dependent manner, IGF-IR played a dynamic role in the DCA-mediated apoptosis. Finally, we provided preliminary evidence demonstrating that autophagy played a central role in protecting DCA-resistant cells from DCA-induced apoptosis.Since resistance to DCA also confers apoptosis-resistance, understanding the mechanisms that lead to or prevent DCA-induced cell death is significant, since they can lead to the development of novel therapeutic strategies to sensitize apoptosis-resistant colorectal cancer cells to undergo cell death.

The presence of inflammation in the progression of fatty liver disease induced by fasting was determined in mink. Tumour necrosis factor alpha (TNF-?), and monocyte chemoattractant protein 1 (MCP-1) liver mRNA levels were quantified by real-time PCR. Mink fasted for 5 and 7 days had significantly higher levels of TNF-? and MCP-1 liver mRNA, compared to mink fasted for 0, 1, and 3 days. Mink fasted for 7 days, but re-fed for 28 days had the lowest mRNA levels of both TNF-?, and MCP-1 demonstrating the liver’s ability to restore homeostasis post-fasting. TNF-? mRNA levels were correlated with MCP-1 liver mRNA and liver fat percent. To confirm the physical presence of inflammation, slides stained with haematoxylin and eosin were analyzed for bile ducts resulting in no significant differences. Results indicate that elevated MCP-1 and TNF-? expression are associated with fasting induced fatty liver in mink.

A better understanding of how dietary fibres impact the bioavailability of fat-soluble nutrients and nutraceuticals is required. The purpose of this research was to determine the influence of guar gum (GG) on the transfer processes impacting beta-carotene (BC) bioaccessibility (transfer to the aqueous phase) from an oil-in-water emulsion using an in vitro model simulating gastric and duodenal digestion. Canola oil emulsions (1.5 % soy protein isolate, 10 % canola oil and 0.1 % all trans BC, D4,3~160 nm) were prepared by microfluidization (40 MPa, 4 passes) and exposed, in the presence of 0.0, 1.0, 1.5, 2.0, or 4.0 % GG, to conditions representative of the stomach and duodenum in the fed state. Lipolysis, BC bioaccessibility, digestate apparent viscosities, droplet size, and bile acid (BA) binding were studied. With increasing concentration of GG, digestate viscosity was increased and lipolysis and bioaccessibility were decreased (P<0.05). Peak lipolysis was 56.2% vs. 21.6% for emulsions containing 0.0 % vs. 4.0 % GG, respectively. BC bioaccessibility was also lower in the presence of GG (i.e. 29.7 vs. 6.98 % for 0.0 vs. 4.0 % GG respectively). Thus, the presence of GG impacted digestive processes central to BC absorption. The impact of GG may be related to increased digestate viscosity entrapping mixed micelles or BAs and decreasing diffusion leading to decreased lipolysis and BC bioaccessibility. / NSERC, CFI

This work explores the usefulness of fish bile analysis in combination with biomarkers for identifying and evaluating new environmental contaminants in the aquatic environment. It illustrates how bile analysis can be used together with biomarkers to assess the causes of estrogenic effects, to identify chemicals in the aquatic environment that are taken up by fish, and to monitor environmental exposure. In a first application, fish exposed to sewage treatment plant effluent were studied. Elevated levels of vitellogenin in the exposed fish demonstrated that estrogenic effects occurred. Several estrogen disrupting substances were identified in the fish bile, and analysis of water samples confirmed that these substances were present in the effluent. The synthetic estrogen 17a-ethinylestradiol, which is known to be present in sewage treatment plant effluent, was shown for the first time to be taken up by fish. Considering the reported potencies of the detected substances, it was concluded that 17a-ethinylestradiol was the major contributor to the estrogenic effects. Chemical analysis of bile was used to identify rubber additives that were released from tires immersed in water. The bile of rainbow trout held in the water contained high levels of metabolites of PAHs and aromatic nitrogen compounds. Several biomarkers were also measured in the exposed fish, and EROD induction and oxidative stress were observed. Based on the bile analysis observations together with knowledge of toxicological mechanisms, it was postulated that the EROD induction was due to the PAHs, while aromatic nitrogen compounds caused the oxidative stress. Resin acids in fish bile proved to be a good indicator of exposure in a chronic long-term study of rainbow trout exposed to effluent from a total chlorine free (TCF) pulp mill. Elevated levels of GST (gluthatione-S-transferase) and GR (gluthatione reductase) activity, and the presence of DNA adducts after a two month recovery period, indicated that compounds in the pulp mill effluents have persistent effects. In addition to characterising the exposure of the fish to the effluent, the analysis of the resin acids in the bile provided evidence of accidents in the pulp mill that the existing process monitoring system had not detected. Resin acids in bile were also found to be a valuable indicator of exposure to pulp mill effluents for eelpout living in the Baltic Sea. A correlation between resin acid levels in bile and skewed sex ratios provided an important link in the chain of evidence that substances in the pulp mill effluents cause male bias of the eelpout embryos. A particularly good example of the potential of bile analysis was the identification of a previously unknown environmental contaminant. A large peak was observed in the bile extracts of fish that had been exposed to sewage treatment plant effluent. This peak was identified as triclosan, which demonstrated its presence in sewage treatment plant effluent. Other work went on to show that it is a common contaminant of the aquatic environment. The ability of fish to concentrate contaminant metabolites in bile to levels very much higher than in the environment, and the comparatively low levels of analytic interferences, make bile a particularly attractive matrix to search for new, unknown organic pollutants

This thesis describes the development and application of solid phase microextraction (SPME) as a sample preparation technique for in vivo determination of pharmaceutical residues in fish tissue and wastewater. The occurrence, distribution and fate of pharmaceuticals in the environment are a subject of concern across the globe due to the impact they may have on human life and aquatic organisms. To address this challenge from an analytical perspective, a simplified and reliable analytical methodology is required to investigate and determine the concentration (bioconcentration factors) of trace pharmaceutical residue in fish tissue and environmental water samples (exposure). An improved SPME method, coupled with liquid chromatography with tandem mass spectrometry has been developed and applied to both controlled laboratory and field-caged fish exposed to wastewater effluent for quantitative determination of pharmaceutical residue in fish specific tissue. A new SPME configuration based on C18 thin film (blade) was developed and optimized to improve SPME sensitivity for in vivo determinations of trace pharmaceuticals in live fish. The C18 thin film extraction phase successfully quantified bioconcentrated fluoxetine, venlafaxine, sertraline, paroxetine, and carbamazapine in the dorsal-epaxial muscle of living fish at concentrations ranging from 1.7 to 259 ng/g. The reproducibility of the method in spiked fish muscle was 9-18% RSD with limits of detection and quantification ranging from 0.08 - 0.21 ng/g and 0.09 - 0.64 ng/g (respectively) for the analytes examined. Fish were sampled by in vivo SPME for 30 min to detect pharmaceutical uptake and bioconcentration, with experimental extracts analyzed using liquid chromatography coupled with tandem mass spectrometry. In addition, a simplified analytical methodology based on SPME was developed and optimized for determination and bioconcentration factor of different classes of pharmaceuticals residues in fish bile. The reproducibility of the method in spiked fish Rainbow Trout bile was 3-7% RSD with limits of detection (LOD) ranging from 0.3 – 1.4 ng/mL for the analytes examined. The field application of SPME sampling was further demonstrated in Fathead Minnow (Pimephales promelas), a small-bodied fish caged upstream and downstream of a local wastewater treatment plant where fluoxetine, atorvastatin, and sertraline were detected in fish bile at the downstream location. Also, a simple automated analytical method using high throughput robotic system was developed for the simultaneous extraction of pharmaceutical compounds detected in surface waters. The proposed method successfully determined concentrations of carbamazepine, fluoxetine, sertraline, and paroxetine in treated effluent at concentrations ranging from 240 - 3820 ng/L with a method detection limit of 2-13 ng/L, and a relative standard deviation of less than 16%. Application of the method was demonstrated using wastewater from pilot-scale municipal treatment plants and environmental water samples from wastewater-dominated reaches of the Grand River (Waterloo, ON). Finally, 4 and 8-d laboratory exposures were carried out with Rainbow Trout exposed to wastewater effluent collected from pilot scale at Burlington, ON. Additionally, wild fish, White Sucker (Catostomus commersonii) were collected and sampled from Waterloo and Kitchener downstreams containing local municipal effluent. Bioconcentration factors of the selected compounds were determined in both fish muscle and bile samples. The results show that anti-depressant drugs such fluoxetine, sertraline and paroxetine were uptake in the fish muscle and fish bile for both laboratory and field exposure. In summary, exposure of fish to micro-pollutants such as pharmaceuticals may be monitored through the analysis of bile, particularly at low concentration exposure of pharmaceuticals, where the sensitivity of analytical method may be challenged. SPME is a promising simple analytical tool which can potentially be used for monitoring of pharmaceuticals in fish tissue and wastewater.

Type 1 diabetes (T1D) is a metabolic disorder characterized by destruction of the pancreatic beta-islet cells leading to complete loss of insulin production. Gliclazide is used in Type 2 diabetes (T2D) to stimulate insulin production but it also has beneficial extrapancreatic effects which make it potentially useful in T1D. In fact, some T2D patients continue to use gliclazide even after their diabetes progresses to T1D since it provides better glycemic control than insulin alone. About 30% of a gliclazide dose undergoes enterohepatic recirculation which may contribute to the observed high interindividual variability in its pharmacokinetics. This may limit its efficacy in T1D especially since diabetes can disturb the gut microbiota and give rise to changes in bile composition and enterohepatic recirculation. Improving the absorption of gliclazide through the use of bile acids and probiotics may reduce this variability and improve the efficacy of gliclazide in T1D. The aim of this thesis was to investigate the interaction between the semisynthetic bile acid, 12-monoketocholic acid (MKC) and gliclazide in terms of pharmacokinetics and hypoglycemic effects in a rat model of T1D with and without probiotic pretreatment. A parallel ex vivo (Ussing chamber) study was carried out to investigate the mechanism of the interaction. Sensitive LC-MS and HPLC methods (Chapter 2) were developed to determine the concentrations of gliclazide and MKC in Ringer's solution and rat serum. Diabetes was induced in male Wistar rats by intravenous (i.v.) alloxan (30 mg/kg). Rats with blood glucose concentration > 18 mmol/l and serum insulin concentration < 0.04 [mu]g/l, 2-3 days after alloxan injection were considered diabetic. A total of 280 male Wistar rats (Chapter 3) were randomly allocated into 28 groups (n=10) of which 14 were made diabetic. Then 7 groups of healthy and 7 groups of diabetic rats were gavaged with probiotics (10⁸ CFU/mg, 75 mg/kg) every 12 hours for three days after which single doses of gliclazide (20 mg/kg), MKC (4 mg/kg) or the combination were administered either by tail vein injection (i.v.) or by gavage. The other 14 groups (7 healthy and 7 diabetic) were gavaged with saline every 12 hours for three days and then treated in the same way. Blood samples were collected from the tail vein for 10 hours after the dose and analyzed for blood glucose, serum gliclazide & serum MKC concentrations. Serum concentration-time curves for gliclazide and MKC were used to determine pharmacokinetic parameters. In the parallel ex vivo study (Chapter 4), 88 rats were randomly divided into 22 groups (n=4 rats per group, 8 chambers per rat), of which 11 groups were made diabetic. Of the 22 groups, 8 groups (4 healthy and 4 diabetic) were pretreated with probiotics as described above to study their influence on gliclazide and MKC flux, 8 groups (4 healthy and 4 diabetic) were used to investigate the interaction between gliclazide and MKC during transport, and 6 groups (3 healthy and 3 diabetic) were used to study the influence of selective inhibitors of the drug transporters Mrp2, Mrp3 and Mdr1 on gliclazide flux. 10 cm piece of the ileum was removed from each rat, the underlying muscle layer and connective tissue removed and the epithelial sheets mounted into Ussing chambers. Gliclazide, MKC or a combination were added to either the mucosal or serosal side and samples collected from both sides for 3 h to determine mucosal-to-serosal absorptive flux (Jss[MtoS]) and serosal-to-mucosal secretory flux (Jss[StoM]) of gliclazide and MKC as appropriate. In diabetic rats, gliclazide alone had no effect on blood glucose levels (Ch3, exp2) whereas MKC reduced it from 23 � 3 to 18 � 3 mmol/l (Ch3, exp3) and the combination of gliclazide and MKC reduced it even further from 24 � 4 to 16 � 3 mmol/l (Ch3, exp4). In diabetic rats, probiotic treatment reduced blood glucose by 2-fold (Ch3, exp1) and enhanced the hypoglycemic effect of the combination of gliclazide and MKC (blood glucose decreased from 24 � 3 to 10 � 2 mmol/l). The bioavailability of gliclazide was higher in healthy rats (53.2 � 6.2%) than in diabetic rats (39.9 � 6.0%) (Ch3, exp2). In healthy rats, MKC enhanced gliclazide bioavailability (82.7 � 8.2%) but in diabetic rats MKC had no effect on gliclazide bioavailability (Ch3, exp4). In healthy rats, probiotic pretreatment significantly reduced gliclazide and MKC bioavailabilities (p<0.01) while in diabetic rats, probiotic pretreatment significantly increased the low bioavailability of gliclazide to a level similar to that in healthy rats (Ch3, exp2 & 3). MKC showed clear evidence of enterohepatic recycling and probiotics delayed and reduced its systemic absorption (Ch3, exp3). In ileal tissues from healthy rats, Ussing chamber studies showed gliclazide is most likely a substrate of Mrp2 and Mrp3 (Ch4, exp5) and MKC significantly reduced gliclazide Jss[MtoS] probably through Mrp3 inhibition (Ch4, exp1). In ileal tissue from diabetic rats, MKC had no effect on gliclazide Jss[MtoS] and Jss[StoM] (Ch4, exp2) and none of the inhibitors had any effect of gliclazide flux (Ch4, exp6). This suggests that these transporters are dysfunctional in this model of T1D. Probiotics and MKC have hypoglycemic effects that appear to be enhanced by gliclazide and all appear to interact at the level of ileal drug transporters. The combination of probiotic treatment, gliclazide and MKC exerted the greatest hypoglycemic effect in T1D rats. Accordingly, the application of this combination may have potential in improving the treatment of T1D.