No description available.

published_or_final_version / Paediatrics / Master / Master of Philosophy

Adsorption of bilirubin from aqueous buffer (pH = 7.8) by PVP, by cholestyramine and by amino acid containing pendants, which have been immobilized onto a polystyrene (Merrifield) resin or a water swellable polyamide resin using the solid phase peptide synthesis methods, have been studied. The adsorption of bilirubin by the pendants on the Merrifield resin was minimal while PVP and cholestyramine adsorbed some bilirubin. However, the best adsorbents were the immobilized amino acids on a water swellable polyamide resin. / A systematic study of the effect of the changes in the amino acid composition of the pendant, both in type and number, on the adsorption by the polyamide resins indicates that the change density, contributed by the R groups of the amino acids in the pendant, is the major factor in the adsorption process. However, some adsorption also occurs at the (alpha)-amino groups. Effects due to the conformation of the peptide chains are also indicated. Of the resins studied, those with peptide pendants containing arginine or lysine form the most efficient adsorbents for bilirubin in aqueous buffer solution. / Studies of the adsorption of bilirubin from bilirubin solutions containing bovine serum albumin as well as studies of desorption of bilirubin from the resins by bovine serum albumin indicates that some resins containing arginine in the pendants can successfully compete with albumin. Stoichiometric binding constants obtained for the polyamide resins by the method of Klotz are of the order of 1 x 10('3) M('-1) to 86 x 10('3) M('-1). These binding constants are lower than that of the reported values for the first binding site of bilirubin on albumin by a factor of 10('1) to 10('4) and lower by a factor of 10 to 10('3) than the values reported for the second binding site.

Doctor of Philosophy / Department of Chemistry / Ryan Rafferty / 6-Thiopurine (6TP), or 6-mercaptopurine, is an actively prescribed drug in the treatment of acute lymphocytic leukemia since 1952. Although 6TP has beneficial and promising therapeutic uses, severe toxicities are associated with its use such as jaundice and hepatotoxicity. These toxicities are due to the higher level of accumulation of bilirubin within the body. The bilirubin pathway involves the conjugation of water-insoluble bilirubin to two equivalents of UDPglucuronic acid (UDPGA) forming the water-soluble and excretable bilirubin diglucuronide species. The glucuronidation of bilirubin is catalyzed by the UDP-glucuronosyl transferase (UGT) enzyme, and the formation of substrate UDPGA is catalyzed by the UDP-glucose dehydrogenase enzyme (UDPGDH).The therapeutic activity of 6TP comes from two main routes: methylation of the thiol of 6TP and formation of a deoxy-6-thioguanosine triphosphate mimic that is incorporated into DNA resulting in apoptosis. In conjugation to its therapeutic metabolism, there are also detoxification pathways operating simultaneously that significantly reduce its therapeutic activity. In this pathway, oxidative metabolites of 6TP such as 8-hydroxyl-6-thiopurine (6TP-8OH), 6-thioxanthine (6TX) and 6-thiouric acid (6TU) can be formed by xanthine oxidase. It has been observed that the body retains 6TU well beyond 24-hour post 6TP treatment. Therefore, we proposed that the observed toxicity from 6TP administration comes from either 6TP or its oxidative excretion metabolites’ ability to inhibit one or both enzymes (UDPGDH, UGT) in the bilirubin excretion pathway. To investigate the toxicity resulting from the 6TP administration about these two enzymatic steps, inhibition analysis of these oxidative metabolites on UDPGDH was assessed using a robust UV-Vis method. The inhibition profile made with regards to varying UDP-glucose showed weak to no inhibition of 6TP towards UDPGDH with a K[subscript]i of 288 μΜ. However, 6TU, (the primary oxidative metabolite which is oxidized at C2 and C8) has increased inhibition towards UDPGDH with K[subscript]i of 7 μΜ. Inhibition was also observed with 6TX (oxidized at C2) and 8-OH-6TP (oxidized at C8) with K[subscript]i values 54 and 14 μΜ, respectively. To further confirm the results of the UV-Vis assessment, inhibition studies were carried out using an HPLC method that was developed and validated to separate all the analytes in the UDPGDH catalyzed reaction. Inhibition studies were performed via the HPLC method showed K[subscript]i values of 105 μΜ and 5 μΜ for 6TP and 6TU, respectively, towards UDPGDH. To assess the inhibition studies towards the UGT enzyme, an HPLC method was developed for the simultaneous determination of bilirubin and its mono/diglucuronides. The inhibition studies were carried to assess the formation of glucuronides and consumption of UDPGA in the presence of the inhibitors using the HPLC method developed. Neither 6TP nor 6TU were shown to inhibit UGT. Also, inhibition studies were carried out in vivo animal model, which further confirmed that 6TP and 6TU do inhibit UDPGDH, but no effect on UGT activity. With these results, we discovered that both 6TP and its oxidative metabolites inhibit UDPGDH. Furthermore, it was observed that C2 and C8 positions of 6TP are important for the toxicity towards UDPGDH. With the goal of developing a single multi-enzymatic assay, another HPLC method was developed to assess the UDPGDH and UGT catalyze reactions together. This method can be used as a standard method to assess interference of any molecule on bilirubin excretion. Given the findings in this study, efforts are being directed towards the synthesis of 8-substituted 6TP analogs that are proposed to retain its therapeutic efficacy but have limited to no toxicity.

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

No description available.

No description available.

Thesis (M. Phil.)--University of Hong Kong, 1988.

published_or_final_version / Medicine / Master / Master of Philosophy

The work described in this thesis falls into two areas of clinically significant binding the interaction of sodium nitroprusside with biological macromolecules, and the consequence of bilirubin to albumin binding in the assay for bilirubin. In Part 1; Chapter 1 is a general introduction reviewing the chemistry of sodium nitroprusside and its medical application. Chapter 2 is concerned with the evaluation of aquocobalamin as a cyanide trapping agent and the stability of sodium nitroprusside. Chapter describes 13C NMR and EHMO studies of the binding of cyanoferrates to cobalarains and cobaloximes. Chapter 4 reports on the pharmacological consequences of the interaction of nitroprusside with aquocobalamin. The second section of this chapter considers the interaction of nitroprusside with albumin and haemoglobins. In Part 2 of this thesis Chapter 5 reviews the methods used in bilirubin assay. Chapter 6 describes fluorescence and solid state studies investigating the binding of bilirubin to albumin and evaluates the ability of various "accelerators" to interfere with the binding of bilirubin to albumin in the diazo method of bilirubin assay. Chapter 7 considers the problems involved in using diazonium salts in the wet chemistry assays of bilirubin and describes an initial study evaluating the use of heterocyclic diazo compounds in the diazo dry base multi-layer film method of bilirubin assay.