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Browsing by Subject "glukuronidaatio"

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  • Glukuronidaation paikkaisomerian tutkimus johdoksenmuodostuksella hyödyntäen nestekromatografia-massaspektrometriaa 
    Mattila, Susanna (2012)
    The aim of the stydy was to evaluate how different chemical derivatization methods are suitable for characterization of regional isomers of different glucuronide conjugates. Glucuronidation is one of the phase II metabolic reactions where more water soluble and often inactive substances are produced. Different functional groups may be subjected to glucuronidation. It is important to determine the exact position of glucuronidation, as the isomers may possess different toxicological or pharmacological properties. For example morphine-6-glucuronide is pharmacologically more active than morphine itself. The glucuronide conjugates are commonly detected by liquid chromatography tandem mass spectrometry (LC-MS/MS) and/or nuclear magnetic resonance (NMR). MS/MSspectra of native molecule and glucuronidated molecule are usually similar because of an initial loss of 176 Da, i.e. monodehydrated glucuronic acid. This fact often makes it impossible to determine the site of glucuronidation. Samples of NMR-analysis requires larger amounts of sample materials than MS-analysis. Many of those derivatization reagents tested in this study were not reacting as they were supposed to react according to literature. O-phthalaldehyde (OPA) and 9-fluorenylmethyl chloroformate (FMOC) were forming derivatives as expected and those reagents are very suitable for glucuronide conjugates studies. At the end of the studies the site of the glucuronidation of dopamine- and serotonineglucuronides were evaluated by derivatization with OPA and FMOC. Derivatization with OPA and FMOC successfully gave information about the region of the glucuronide acid in dopamine- and serotoninemolecules. The assumptions supposed to be correct according to NMR-studies presented in literature.
  • Katekoliestradiolien glukuronidaatio 
    Hirvisaari, Laura (2012)
    Estradiol is a female sex hormone which is metabolized to two different catechol estradiols. 2-hydroxyestradiol (2-OHE2) is normally the major catechol estradiol metabolite but breast cancer patients have increased amounts of genotoxic 4-hydroxyestradiol (4-OHE2) and it arises to predominant metabolite with these patients. These catechol estradiols can form reactive quinones that can bind to DNA and lead to mutations and finally cause cancer. Catechol-O-methyl transferase can add methyl groups and UDP-glucuronosyl transferase (UGT) glucuronic acid groups to catechol estradiols. These phase II enzymes play important role in the inactivation of catechol estradiols because only non-conjugated catechol estradiols can be oxidized to quinones. The aim of this study was to find out which human UGTs catalyze glucuronidation of 2-OHE2 or 4-OHE2, how many different glucuronides are formed and in which part of the substrate glucuronic acid is added. To answer these questions chromatography methods for 2-OHE2 and 4-OHE2 glucuronides were developed using HPLC. Eleven UGT-enzymes glucuronidate 2-OHE2. UGTs 1A1, 1A7 and 1A10 form two different glucuronides and UGTs 1A3, 1A8, 1A9, 2A1, 2A2, 2A3, 2B7 and 2B15 form only the second glucuronide. It was possible to detect three different glucuronides for 4-OHE2 but the amount of the first glucuronide was under quantification limit. UGT1A10 catalyzed the formation of the second glucuronide and UGTs 1A7, 1A8, 1A9, 2B7 and 2B15 catalyzed the formation of the last glucuronide. One aim of the study was to find out which part of the substrate is glucuronidated but this aim was not achieved because suitable standards were not available.

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