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Title: Characterisation of human hepatic cytochrome P450 : comparison of metabolic markers and antibody recognition
Author: Webster, Duncan F.
ISNI:       0000 0001 3565 116X
Awarding Body: University of Aberdeen
Current Institution: University of Aberdeen
Date of Award: 1997
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A cDNA library was produced in lambdagt11 from an induced human liver. Twenty-one clones were isolated. All clones were identified by two of the four antibodies used in the screening process. The phage insert was recovered by PCR with two specific primers. The PCR product was ligated into M13 vectors and sequenced. The DNA was a CYP2A sequence with highest identity to CYP2A6 but contained a point deletion mutation. Coumarin metabolism was measured in microsomal preparations of fifteen livers and was inhibited by three of the antibodies used in screening. RP1 monoclonal gave partial inhibition (60%) at ratios of 100:1. Two other antibodies IIB1 polyclonal and RP3 (monoclonal) were highly inhibitory at low ratios. IIB1 gave a 90% inhibition at 20:1 ratio, RP3 was 95% inhibitory at 6:1 ratio. O-dealkylation of alkoxyresorufins was determined for sixteen livers. BROD activity was partially inhibited by IIB1 polyclonal (60% at a 20:1 ratio). This supports the possibility that BROD is not a marker for CYP2B activity in humans and that BROD may be primarily metabolised by CYP4A. The results were compared with fifty-seven separate sets consisting of metabolism and antibody immunoquantification data. There were strong correlations between BROD activity and several CYP3A marker activities and protein levels identified by a polyclonal antibody that recognises P4503A. The metabolism of indomethacin was measured for fifteen livers. One of the metabolites of indomethacin, desmethylindomethacin, was produced by all livers. The metabolism could not be inhibited with antibodies as they interfered with the reaction in a non-specific manner. Comparisons with other metabolism in the same livers showed that indomethacin is probably a CYP3A substrate. The indomethacin method was modified ibuprofen and data was collected for fourteen livers. Two metabolites, I and II, were seen for each liver in 1:2 ratio respectively. These both correlated highly with tolbutamide metabolism.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Biochemistry