High-performance liquid chromatography of N-alkylprotoporphyrins: an investigation of their formation and loss under chemical and enzymatic conditions in vitro.
A new technique for resolving N-alkylprotoporphyrins into each structural isomer is described. The technique has been used to investigate the rate of formation and loss of N-alkylporphyrins during reaction of the parent porphyrin with alkyl iodides and to establish the conditions required for optimal yields of the various isomers. Preferential loss of the isomers bearing the N-alkyl group on one of the vinyl-substituted pyrrole rings is observed on prolonged incubation and HI generated during the reaction has been shown to be responsible. A method for detection and partial resolution by HPLC of N-alkylprotoporphyrins produced by liver microsomes in vitro is also described. Microsomes from rats induced with 3-methylcholanthrene produce significantly more N-ethylprotoporphyrin from either 4-ethyl-3,5-diethoxycarbonyl-1,4-dihydro- 2,6-dimethyl-pyridine or ethylhydrazine than do microsomes from control animals, but the isomeric composition of the isolated N-alkylporphyrin differs from that reported in vivo. Evidence that authentic N-alkylporphyrins are lost during incubation with microsomes has been obtained, and here again, the isomers bearing the N-alkyl group on vinyl-substituted pyrrole rings are preferentially lost. Experiments with 14C-labeled N-methylprotoporphyrin show that approximately 40% of the lost porphyrin could be recovered bound covalently to the microsomal pellet.[1]References
- High-performance liquid chromatography of N-alkylprotoporphyrins: an investigation of their formation and loss under chemical and enzymatic conditions in vitro. Gibbs, A.H., De Matteis, F. Anal. Biochem. (1991) [Pubmed]
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