Acute pancreatitis is an inflammatory process that initiates with the damage of the pancreatic acinar cells and can also involve other organs and peripancreatic tissues. Vascular alterations are proposed as factors that contribute significantly to the morbidity of patients with pancreatitis. However, few studies were directed to understand these alterations and the underlying mechanisms. In this way, the objective of this study was to investigate vascular changes caused by acute pancreatitis induced by common bile duct obstruction (CBDO) on the vascular reactivity of rat mesenteric artery, in order to generate scientific knowledge about the mechanisms involved in this condition and enable future approaches that lead to a better treatment of this disease in humans. For this purpose, adult male Wistar rats were used (220-300 g) and divided into two groups: (i) sham animals, which were submitted to surgery procedure without obstruction of the duct and (ii) CBDO animals which were submitted to surgery procedure and obstruction of the common bile duct. After 24 or 48 h of this surgery, animals were anesthetized and the superior mesenteric artery was removed. Serum samples were also obtained for amylase determination. Mesentery artery rings were obtained (1-2 mm) and used for in vitro reactivity experiments and measurement of nitric oxide (NO) and superoxide anion (O2 -). For the reactivity experiments, the rings were mounted in isolated organ chambers and cumulative concentration-response curves to acetylcholine or phenylephrine were obtained. For evaluating the production of NO and O2 -, rings were incubated with specific fluorescent dyes. Levels of serum amylase were augmented in animals with CBDO after 24 (p<0.05) or 48 h (p<0.001) of obstruction, when compared with sham group, confirming the induction of pancreatitis. Mesenteric rings from CBDO animals showed decreased relaxing response for acetylcholine or contractile response to phenylephrine after 24 or 48 h of induction of pancreatitis, when compared with sham group. Our data also showed an increase in NO (p<0,05) and O2 - (p<0,05) production in the rings collected after 48 h of the surgical procedure, but not after 24 h. These findings showed that acute pancreatitis induction by CBDO impaired relaxation and contractile response, possibly by mechanisms that are triggered by increased NO and O2 - production, probably associated with the inflammatory process. Additional studies are needed to identify the mechanisms underlying these vascular effects of acute pancreatitis. / A pancreatite aguda é um processo inflamatório que se desenvolve a partir de lesão dos ácinos pancreáticos e também pode envolver tecidos peripancreáticos e outros órgãos. Alterações na função vascular são propostas como fatores que contribuem sobremaneira para a morbidade dos pacientes com pancreatite, entretanto poucos estudos foram direcionados para entender estas alterações e os mecanismos subjacentes. Com base nessas premissas, o objetivo do presente estudo foi investigar as alterações vasculares provocadas pela pancreatite aguda induzida por obstrução do ducto biliopancreático (ODBP) sobre a reatividade de artéria mesentérica de ratos, com o intuito de gerar conhecimento científico sobre os mecanismos envolvidos nesta condição e permitir abordagens futuras que levem ao melhor tratamento desta doença em humanos. Para tanto, foram utilizados ratos machos adultos Wistar (220-300 g) que foram divididos em grupos falso operado (Sham), no qual somente houve a cirurgia, sem obstrução do ducto, ou ODBP, o qual foi submetido a cirurgia e obstrução do ducto biliopancreático. Em seguida, os animais dos diferentes grupos foram eutanasiados após 24 ou 48 h e a artéria mesentérica superior foi removida, bem como soro foi separado para dosagem de amilase. Desta artéria foram obtidos anéis com endotélio (1-2 mm) que foram utilizados para a realização dos experimentos de reatividade in vitro e mensuração de óxido nítrico (NO) e ânion superóxido (O2 -). Para os experimentos de reatividade, os anéis foram montados em sistema de banho de órgão isolado e foi testada sua resposta relaxante ou contrátil, mediante estimulação com acetilcolina (ACh) ou fenilefrina (Fen) respectivamente. Para avaliar a produção de NO e O2 -nos anéis dos animais dos diferentes grupos, foram utilizadas sondas fluorescentes específicas para estes radicais livres. Nos animais com ODBP foram encontradas concentrações séricas de amilase elevadas após 24 (p<0,05) ou 48 h (p<0,001), quando comparados ao grupo sham, confirmando a indução da pancreatite. Os anéis dos animais com ODBP demonstraram uma diminuição da resposta relaxante à acetilcolina ou contrátil à fenilefrina após 24 ou 48 h da indução da pancreatite, quando comparados aos animais do grupo sham. Também foi observado aumento na produção de NO (p<0,05) e de O2 - (p<0,05) nos anéis coletados após 48 h, mas não 24 h da indução da pancreatite. Estes achados mostraram que a indução da pancreatite aguda por ODBP prejudica tanto a resposta relaxante quanto a contrátil de anéis de artéria mesentérica, por mecanismos que possivelmente são desencadeados pelo aumento na produção de NO e O2 -, provavelmente associado com o processo inflamatório. Entretanto, experimentos adicionais são necessários para identificar os mecanismos pelos quais a pancreatite aguda causa estes efeitos.

Die Hämoxygenase-1 (HO-1) ist das geschwindigkeitsbestimmende Enzym des Hämabbaus und ist wichtiger Regulator inflammatorischer Prozesse. Der Verlauf einer experimentellen akuten Pankreatitis (AP) konnte im Tiermodell durch HO-1 Induktion abgemildert werden. Die Aktivierung und Proliferation pankreatischer Stellatum Zellen (PSC) wird durch eine experimentelle HO-1 Induktion inhibiert und kann so möglicherweise vor der Fibrosierung des Pankreasparenchyms bei chronischer Pankreatitis (CP) schützen. Die Transkription der HO-1 wird durch einen GT-Repeat beeinflusst, der im Promoter lokalisiert ist. Diese Arbeit untersuchte, ob Varianten des GT-Repeat oder weitere genetische Varianten der HO-1 mit verschiedenen Pankreatitisformen assoziiert sind. Der GT-Repeat und der SNP rs2071746 wurden mit fluoreszensmarkierten Primern bzw. mit Schmelzkurvenanalyse bei 285 Patienten mit AP, bei 208 Patienten mit alkoholischer CP (ACP), bei 207 mit idiopathischer/hereditärer CP (ICP/HCP), 147 Patienten mit Alkoholischer Leberzirrhose (ALZ) und bei 289 Kontrollen untersucht. Bei den ACP Patienten wurde die GT-Repeat Analyse auf insgesamt 446 Patienten erhöht. Zusätzlich wurden die kodierenden HO-1 Abschnitte mittels DNA-Sequenzierung bei 145 Patienten mit ACP, 138 Patienten mit ICP/HCP, 147 Patienten mit ALZ und bei 151 Kontrollen analysiert. Das Exon 3 wurde darüber hinaus bei zusätzlichen ICP/HP Patienten und Kontrollen untersucht. Die Längenverteilungen des GT-Repeat, die Allelverteilung des SNP rs2071746 und die Verteilung der bei der DNA-Sequenzierung gefundenen synonymen und nicht synonymen Varianten waren bei allen untersuchten Gruppen nicht signifikant unterschiedlich. Obwohl die funktionellen Daten einen Einfluss von HO-1 Varianten auf die Pathogenese der verschiedenen Pankreatitis-Formen nahelegen, konnte unsere umfangreiche genetische Analyse keine Assoziation nachweisen. Genetische Varianten der HO-1 haben keinen Einfluss auf die Entwicklung einer AP, ACP, ICP/HCP und ALZ.:Inhaltsverzeichnis Vorbemerkung ..................................................................................................... 3 Bibliographische Beschreibung.......................................................................... 4 Abkürzungen/Abbildungen ................................................................................ 6 1. Einleitung........................................................................................................9 1.1 Akute Pankreatitis ......................................................................................................................... 9 1.2 Chronische Pankreatitis ............................................................................................................... 11 1.3 Genetische Aspekte der Chronischen Pankreatitis ...................................................................... 12 1.3.1 Kationisches Trypsinogen (PRSS1) ...................................................................................... 12 1.3.2 Anionisches Trypsinogen (PRSS2) ....................................................................................... 14 1.3.3 Serinproteaseinhibitor, Kazal Typ1 (SPINK1)..................................................................... 14 1.3.4 Chymotrypsin C (CTRC) ...................................................................................................... 15 1.3.5 CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) ...................................... 15 1.4 Hämoxygenase-1 ......................................................................................................................... 16 1.4.1 Physiologische Bedeutung der Hämoxygenase-1 (HO-1) .................................................... 16 1.4.2 Genetische Varianten der Hämoxygenase-1 ........................................................................ 18 1.4.3 Hämoxygenase-1 und Pankreatitis....................................................................................... 20 1.5 Hypothese/Fragestellung ............................................................................................................. 21 2. Publikation ..................................................................................................... 22 3. Zusammenfassung der Arbeit ...................................................................... 23 4. Literaturverzeichnis...................................................................................... 28 5. Danksagung.................................................................................................... 35 6. Erklärung über die eigenständige Abfassung der Arbeit .......................... 36 7. Lebenslauf ...................................................................................................... 37

Sulfur amino acids have critical function as intracellular redox buffers and maintain homeostasis in the external milieu by combating oxidative stress. Synthesis of glutathione (GSH) is regulated at a substrate level by cysteine, which is synthesized by homocysteine via the transsulfuration pathway. Oxidative stress and diminished glutathione pools play a sustained role in the pathogenesis of acute pancreatitis. One of the aims of this study was to experimentally address the temporal relationship between plasma sulfur amino acid levels in patients suffering from acute pancreatitis. The data indicated low concentration of cysteine initially, at levels similar to those of healthy controls. Glutathione was found reduced whilst cysteinyl-glycine and ¿- glutamyl transpeptidase activity were increased in both mild and severe attacks. As the disease progressed, glutathione and cysteinyl-glycine were further increased in mild attacks and cysteine levels correlated with homocysteine and ¿-glutamyl transpeptidase activity. The progress of severe attacks was associated with glutathione depletion, reduced ¿-glutamyl transpeptidase activity and increased cysteinyl-glycine, that correlated with glutathione depletion. The corollary that ample supply of cysteine and cysteinly-glycine does not contribute towards glutathione synthesis in acute pancreatitis poses an important issue that merits resolution. Heightened oxidative stress and depletion of glutathione rationalized the progression of disease in severe attacks. An upsurge that reactive oxygen species can shift redox state of cells is determined by the ratio of the abundant redox couples reduced and oxidized glutathione (GSH: GSSG) in cell. The study reported a novel methodology for quantification of total oxidized glutathione (tGSSG) and total glutathione (tGSH) in whole blood using reverse phase high performance liquid chromatography. The novelty of the method is ascertained by the use of a mercaptan scavenger 1, methyl-2-vinyl-pyridinium trifluromethanesulfonate for the total oxidized glutathione determination. The results reported permit quantitation of tGSSG and tGSH and was applied to a control group. Finally, the study was also focussed in developing a liquid chromatography-mass spectrometric method to evaluate free and conjugated bile acids in patients suffering from various degrees of cholestatic-hepatobiliary disorders. The study reported low levels of ursodeoxycholic acid (UDCA) and slightly high levels of lithocholic acid (LCA). All the primary bile acids seem to be conjugated with glycine and taurine amino acid.

La pancréatite, qui désigne l’inflammation du pancréas, est une condition grave qui survient lorsque les enzymes pancréatiques, servant normalement à la digestion des aliments, digèrent le pancréas. Les causes les plus fréquentes sont la consommation excessive d’alcool et la migration de lithiases vésiculaires dans les voies biliaires. On reconnait également des formes moins fréquentes qui contribuent au fardeau de la maladie et la pancréatite induite par un médicament est l’une d’entre elles. En cours d’hospitalisation et lors du suivi, cette étiologie cause des difficultés aux médecins et aux pharmaciens qui doivent travailler de concert pour d’abord la diagnostiquer, mais également identifier le médicament déclencheur afin d’éviter la récurrence. Néanmoins, cette identification se voit complexifiée, dû à un manque de données probantes fiables et récentes concernant d’une part l’épidémiologie, mais aussi l’étiologie de cette condition. L’objectif principal de ce projet est d’étudier les cas de pancréatites aiguës médicamenteuses hospitalisés au Centre intégré universitaire de santé et services sociaux du Saguenay–Lac-Saint-Jean afin d'obtenir un portrait réel de cette étiologie en plus d’ajouter de nouvelles preuves à la littérature. La méthodologie repose sur la révision des dossiers médicaux des cas hospitalisés pour au moins une pancréatite médicamenteuse dans les six hôpitaux du Saguenay–Lac-Saint-Jean entre 2006 et 2014. Les données recueillies et leurs analyses ont permis de documenter les médicaments ayant causé une pancréatite aiguë médicamenteuse au Saguenay–Lac-Saint-Jean en plus de participer à l’avancement des connaissances actuelles par l’identification de médicaments non associés à la pancréatite aiguë jusqu’à maintenant. / Pancreatitis, which refers to the inflammation of the pancreas, is a serious medical condition in which the pancreatic enzymes, that normally digest food, start to digest the pancreas. The most common causes are excessive alcohol consumption and gallstone migration into the bile ducts. Less common causes also contribute to the burden of the disease and drugs are one of them. Diagnosis of drug-induced pancreatitis and identification of the triggering drug in order to avoid recurrence is a challenge to both physicians and pharmacists. Moreover, identification of the causative drug is complex due to a lack of reliable and recent evidence concerning the epidemiology and the etiology of this condition. The main objective of this project is to study drug-induced acute pancreatitis cases hospitalized at the Centre intégré universitaire de santé et services sociaux of the Saguenay–Lac-Saint-Jean in order to obtain an accurate representation of this etiology in addition to adding new evidences to the literature. The methodology relies on the review of medical records of hospitalized cases for at least one drug-induced acute pancreatitis in the six hospitals of the Saguenay–Lac-Saint-Jean between 2006 and 2014. The data collected and their analyzes document drugs involved in acute pancreatitis in the Saguenay–Lac-Saint-Jean region in addition to participating in the advancement of our current knowledge by describing drugs that had not been associated with acute pancreatitis until now.