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.
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MRC Toxicology Unit, Medical Research Council Laboratories, Carshalton, Surrey, United Kingdom.
De Matteis
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D000143:Acids / Q000494:pharmacology; D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D007454:Iodides / Q000138:chemical synthesis; D017613:Iodine Compounds; D008297:Male; D008862:Microsomes, Liver / Q000378:metabolism; D011524:Protoporphyrins / Q000032:analysis* / Q000138:chemical synthesis / Q000378:metabolism; D051381:Rats; D013237:Stereoisomerism
D000143:Acids; D007454:Iodides; D017613:Iodine Compounds; D011524:Protoporphyrins; C010466:hydroiodic acid