Abstract
Earlier studies postulated that bioactivation of 4-ipomeanol by cytochrome P450 enzymes may occur through oxidation of its furan ring, following a mechanism similar to the bioactivation of other furan-containing compounds. This would lead to the formation of furan epoxides and α,β-unsaturated di-aldehyde-reactive metabolites that can conjugate with glutathione. These metabolites are thought to be responsible for the cytotoxic and anticancer properties of 4-ipomeanol. We hypothesized that if 4-ipomeanol is metabolized following this pathway, its glutathione conjugates would be isobaric (molecular ion mass = 492 Da) and would be excreted in bile. To investigate this hypothesis, we analyzed by liquid chromatography-tandem mass spectrometry the bile of rats administered d0/d6 4-ipomeanol (1:1 ratio) intravenously. Hexadeuterated 4-ipomeanol had all deuterium atoms incorporated on its aliphatic chain. Multiple reaction monitoring scans of bile for the mass transition: MH+/(MH - 129)+, which is characteristic of glutathione conjugates, detected four glutathione conjugates. The observation of the isotope cluster (M + 1)+ (d0)/(MH + 6)+ (d6) in a 1:1 molar ratio confirmed that these conjugates were derived from 4-ipomeanol. Retention of the six deuterium atoms in the glutathione conjugates detected, (MH + 6)+, indicates that the bioactivation of 4-ipomeanol took place on the furan ring moiety. Rat hepatic microsomal incubations provided additional evidence. From this study, the mass of the reactive metabolites of 4-ipomeanol can be inferred. The inferred mass (186 Da) matches the mass postulated. A pathway of 4-ipomeanol bioactivation is proposed here. This work represents one step forward to understanding the mechanism of bioactivation of 4-ipomeanol.
Footnotes
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doi:10.1124/dmd.104.000406.
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ABBREVIATIONS: P450, cytochrome P450; LC-MS/MS, liquid chromatography-tandem mass spectrometry; MRM, multiple reaction monitoring; HPLC, high-performance liquid chromatography; CAD, collisionally activated dissociation; L-754,394, N-[2-hydroxy-1-indanyl]-5-[2-[((1,1-dimethylethyl)amino) carbonyl]-4-[(furo[2,3-b]pyridin-5-yl)methyl]piperazin-1-yl]-4-hydroxy-2-(phenylmethyl)pentanamide.
- Received April 27, 2004.
- Accepted August 23, 2004.
- The American Society for Pharmacology and Experimental Therapeutics
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