Introduction Estrogen-induced cholestasis is a disease characterized by a failure of bile flow and bile production. It can develop in women after oral contraceptives use, hormone replacement therapy or during pregnancy. The estrogen metabolism is a complex process leading to formation of metabolites with different biological activities. It takes place primarily in the liver (Phase I and Phase II including hydroxylation, methylation, sulfation and glucuronidation). The enzymes from UDP-glucuronosyltransferases family , abbreviated UGT, are responsible for the glucuronidation of estrogens. Aims The objective of my work is to define estrogen metabolism and gene expression of UGT1A1, CYP1A2 and SULT1A1 and characterize cholestatic liver damage in the UGT1A1 deficient rat strain (Gunn rats) compared to rats with normal enzyme activity and try to define possible mechanisms responsible for the liver damage. Methods Adult female Gunn and corresponding heterozygous rats were treated with ethinylestradiol (EE, 5 mg/kg body weight SC) for 5 days, while control rats received propanediol (vehicle). Day six, the animals were sacrificed and plasma and liver tissue were collected for analysis. Markers of cholestasis and liver damage ALP, AST, ALT and bilirubin were determined using an automatic analyzer, total...

La cholestase intrahépatique de la grossesse (CIG) est la maladie du foie la plus répandue au cours de la grossesse. Elle est caractérisée par un prurit et est associée à une augmentation de la concentration des acides biliaires dans le sang, ce qui peut mener à un risque accru de conséquences périnatales indésirables, y compris un accouchement prématuré spontané et une augmentation des risques de mort de l’enfant à l’accouchement, entre autres. Le traitement médical de cette maladie repose actuellement sur l’acide ursodésoxycholique (UDCA) qui diminue le prurit et les anomalies biochimiques maternelles dans certains cas. Actuellement, le diagnostic de la CIG est posé suite à un test de quantification des acides biliaires sériques totaux par une méthode enzymatique. Nous émettons l'hypothèse que certains profils d’acides biliaires permettraient d’évaluer le risque de complications chez les femmes atteintes de CIG. En analysant les profilages, il pourrait être possible de déterminer la ou les espèces responsables de ces complications et ainsi déterminer des sous-groupes de patientes plus à risque de complications ou qui répondraient mieux au traitement. De plus, nous pensons que le traitement à l’UDCA, étant lui-même un acide biliaire, pourrait interférer lors de la quantification des acides biliaires totaux sériques, particulièrement dans les cas les plus problématiques de CIG où de fortes doses de ce composé sont administrées. Si c’était le cas, cela ferait en sorte que les valeurs de référence pourraient être modifiées en fonction du traitement administré. Le projet de recherche présenté vise au développement d’une méthode de quantification des acides biliaires sériques par la chromatographie liquide couplée à un spectromètre de masse en tandem (LC-MS/MS), qui permettrait un profilage des acides biliaires sériques chez les femmes enceintes atteintes de la CIG et qui permettrait également d’évaluer l’effet du traitement à l’UDCA sur ce profilage. Une méthode de quantification des acides biliaires par chromatographie liquide couplée à un spectromètre de masse en tandem a été développée et validée. Les surnageants obtenus par précipitation de protéines avec le méthanol ont été injectés sur le LC-MS/MS. La séparation est réalisée par chromatographie en phase inverse sur une colonne C18 de type interactions hydrophobes. Les transitions ioniques sur le spectromètre de masse ont été déterminées pour toutes les espèces d’acides biliaires au préalable et l’acide cholique deutéré, l’acide chénodésoxycholique deutéré ainsi que l’acide désoxycholique deutéré ont été utilisés comme standards internes. Quinze acides biliaires, y compris les acides biliaires conjugués et libres, ont été séparés et quantifiés par LC–MS/MS en utilisant l’ionisation par électro nébulisation (ESI) en mode ion négatif. La quantification a été réalisée en mode de surveillance de réactions multiples (MRM) avec des méthodes de courbes d'étalonnage externes. Les coefficients de corrélation des courbes standards pour tous les acides biliaires étaient supérieurs à 0,9966. La méthode développée a démontré une précision acceptable, avec une imprécision intra analyse inférieure à 3,2% pour toutes les espèces d’acide biliaire étudiées (pour des échantillons à 0,8 et 5 μg/mL) et une imprécision inter analyse inférieure à 15%. Une suppression d’ion moyenne de 8,2% a été observée, qui a été jugée acceptable. Une bonne corrélation a été obtenue entre la méthode LC-MS/MS et une méthode enzymatique (r=0,964). En conclusion, une méthode fonctionnelle, efficace et rapide a été développée pour quantifier les acides biliaires sériques individuels et différents profils d’acides biliaires représentant une large gamme de concentrations ont été comparés. La comparaison des profilages d’acides biliaires suggère que les acides biliaires principaux responsables de l’augmentation de la concentration des acides biliaires totaux dans le sang pour des échantillons à une concentration de plus de 10 μmol/L sont l’acide cholique glyco-conjugué (GCA), l’acide cholique tauro-conjugué (TCA) ainsi que l’acide ursodésoxycholique glyco- conjugué (GUDCA). Cette nouvelle méthode validée, et les données préliminaires sur les profils d’acides biliaires dans les échantillons cliniques, permettront de lancer des analyses cliniques prospectives pour évaluer l’effet du traitement par l’UDCA sur les concentrations totales d’acides biliaires sériques et sur les profils d’acides biliaires individuels chez les patientes atteintes de la CIG. / Intrahepatic cholestasis of pregnancy (ICP) is the most common liver disease during pregnancy. It is characterized by pruritus and is associated with an increased concentration of bile acids in blood, which may lead to an increased risk of perinatal consequences, including spontaneous preterm delivery and an increased risk of death at birth, among others. The medical treatment of this disease currently relies on ursodeoxycholic acid (UDCA) which reduces pruritus and maternal biochemical abnormalities in some cases. Currently, the diagnosis of ICP is made using an enzymatic assay to measure total serum bile acids. We hypothesize that profiling of the individual bile acids would make it possible to assess the risk of complications in women with ICP. By analyzing the bile acid profiles, it could be possible to determine which specie(s) is responsible for these complications and thus to distinguish subgroups of patients at higher risk of complications or who would respond better to treatment. In addition, we believe that UDCA treatment, being a bile acid itself, could interfere with the quantification of total serum bile acids, particularly in the most problematic cases of CIG where high doses of this compound are administered. If this was the case, it would mean that the reference values would need to be changed depending on the administered treatment. The research project aims to develop and validate a method for quantifying bile acids in serum by liquid chromatography coupled to a tandem mass spectrometer (LC-MS/MS), which would allow profiling of serum bile acids in affected women and which would also make it possible later to evaluate the effects of UDCA treatment on this profiling. A method for the quantification of bile acids by liquid chromatography coupled to tandem mass spectrometry has been developed and validated. The supernatants obtained by precipitation of proteins with methanol were injected onto the LC-MS/MS. The separation was carried out using reversed-phase chromatography on a C18 hydrophobic interactions type column. Ionic transitions on the mass spectrometer were determined for all bile acids species beforehand and deuterated cholic acid, deuterated chenodeoxycholic acid and deuterated deoxycholic acid were used as internal standards. Fifteen bile acids, including conjugated and free bile acids, were separated and quantified by LC–MS/MS using electrospray ionization (ESI) in negative ion mode. Quantification was performed in multiple reaction monitoring (MRM) mode with external calibration curve methods. Correlation coefficients for standard curves for all bile acids were greater than 0.9966. The method developed showed acceptable precision, with intra-assay imprecision of less than 3.2% for all the bile acid species studied (for samples at 0.8 and 5 μg/mL) and inter-assay imprecision under 15%. An average ion suppression of 8.2% was observed, which was judged acceptable. Finally, a good correlation was obtained between the LC-MS/MS method and an enzymatic method (r = 0.964). In conclusion, a functional, efficient and rapid method was developed to quantify the individual serum bile acids and different bile acids profiles representing a wide range of concentrations were compared. The comparison of the bile acid profiles suggests that the main bile acids responsible for the increase in total bile acids concentration in blood for samples at a concentration of more than 10 μmol/L are glycocholic acid (GCA), taurocholic acid (TCA), glycoursodeoxycholic acid (GUDCA). This new validated method, and the preliminary data on bile acid profiles in clinical samples, will allow us to initiate prospective clinical analyses to assess the effect of UDCA treatment on total bile acid concentrations and profiles in patients with intrahepatic cholestasis of pregnancy.

The aim of this thesis was to establish methods for selected PAPP-A (Pregnancy- Associated Plasma Protein A) gene polymorphisms analysis and to study genetic background of PAPP-A and biochemical background of PAPP-A and PlGF (Placental Growth Factor) in relation to risk pregnancy. Secondly, the aim was to establish method for two-dimensional (2D) electrophoresis of amniotic fluid. Methods for analysis of ten PAPP-A gene polymorphisms were established. These polymorphisms, PAPP-A and PlGF levels were studied in together 165 women in third trimester pregnancies complicated with threatening preterm labor (n=98), preeclampsia (n=35), IUGR (Intrauterine Growth Restriction) (n=34) and ICP (Intrahepatic Cholestasis of Pregnancy) (n=15). 114 healthy pregnant women served as controls. The method for 2D electrophoresis of amniotic fluid was established. Preeclamptic patients had significantly higher frequency of TT genotype of Cys327Cys (C/T) PAPP-A gene polymorphism compared to controls. Patients with ICP had increased serum levels of PAPP-A compared to controls, in patients with threatening preterm labor PAPP-A levels were rather decreased. PlGF levels did not differ from control group in patients with ICP and threatening preterm labor. Positive correlation was found between PAPP-A and PlGF in group of...

The aim of this thesis was to establish methods for selected PAPP-A (Pregnancy- Associated Plasma Protein A) gene polymorphisms analysis and to study genetic background of PAPP-A and biochemical background of PAPP-A and PlGF (Placental Growth Factor) in relation to risk pregnancy. Secondly, the aim was to establish method for two-dimensional (2D) electrophoresis of amniotic fluid. Methods for analysis of ten PAPP-A gene polymorphisms were established. These polymorphisms, PAPP-A and PlGF levels were studied in together 165 women in third trimester pregnancies complicated with threatening preterm labor (n=98), preeclampsia (n=35), IUGR (Intrauterine Growth Restriction) (n=34) and ICP (Intrahepatic Cholestasis of Pregnancy) (n=15). 114 healthy pregnant women served as controls. The method for 2D electrophoresis of amniotic fluid was established. Preeclamptic patients had significantly higher frequency of TT genotype of Cys327Cys (C/T) PAPP-A gene polymorphism compared to controls. Patients with ICP had increased serum levels of PAPP-A compared to controls, in patients with threatening preterm labor PAPP-A levels were rather decreased. PlGF levels did not differ from control group in patients with ICP and threatening preterm labor. Positive correlation was found between PAPP-A and PlGF in group of...