| Budget Amount *help |
¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 1997: ¥1,600,000 (Direct Cost: ¥1,600,000)
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| Research Abstract |
Upon the phase of a drug discovery and development, construction of a model system reflecting the invivo metabolism for pre-clinical drug metabolism studies is desired. Moreover, a highly reliable method for the analysis of trace compounds in biological fluids is urgently needed. Therefore, we have been carried out on a comprehensive investigation by the technique of liquid chromatography-mass spectrometry (MS) and an immobilzed drug metabolizing enzyme.
1) A quantitative analytical method based on negative ion electrospray ionization (ESI) -MS for bile acid 3-glucuronides has been developed. Maximum sensitivity (200 fmol) could be achieved by recording the negative ion and mass spectra obtained were characterized by an intense-molecular ion [M-H]^- with a doubly charged ion [M-2H]^<2->, and the ratio of these negative ions were markedly influenced by the acidic component of salt added to a moble phase, acccording to a pKa value of an acidic moiety at C-24. Application of this technique
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demonstrate its usefullness, principally in the diagnosis of liver disease. The method has also applied to characterization of bile acid 24-glucuronides in incubation mixture with rat liver glucuronyltransferase.
2) For Obtaining sensitivity and specificity in ESI-MS for measuring the neutral and nonpolar substances that cannot be easily ionized in solution, reversed derivatization into ESI active substance by emloying immobilized recombinant sulfotransferase under physiological conditions has been studied The derivatization using dehydroepiandrosterone as a model compound was achieved quantitatively by incubation with the immobilized enzyme in the prsence of adenosine 3'-phosphate 5'-phosophosulfet at 37 ﾟC for 60 min. The spectrum of the sulfate exhibited a quasimolecular ion [M-H]^- as an inrence peak under the negative ion ESI-MS mode.
3) To creat a model system that are being used for the interpretation of prediction of drug metabolism and disposition, rat liver microsomal fractions containing cytoihrome P-450 enzymes have been noncovalently immobilized on an immobilized artificial membrane, and the enzyme activity was investigated by following the in vitro hydroxylation of estradiol. The method was found to be useful in phase I metabolic profiling and mechanistic studies of drugs. Less
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