Trapping and measurement of short-lived alkylating agents in a recirculating flow system.
A procedure has been developed for estimating the survival time of short-lived alkylating agents in a flow system at physiological temperature and pH. The system simulated the slow release into the bloodstream of a reactive compound formed in the liver. A solution of the reactive compound was injected slowly into a fast stream of aqueous fluid and immediately mixed. After a delay (up to 1 min) determined by a length of tube, during which hydrolysis took place, the surviving reactive compound was trapped on a column of immobilized thiol (thiol-Sepharose), and the fluid was recirculated via a reservoir. The system was used to study the hydrolysis of the pyrrolizidine alkaloid metabolites dehydromonocrotaline, dehydro-anacrotine and dehydroretrorsine. The S-bound pyrrolic moiety in the trap was measured colorimetrically and hydrolysis rates were estimated after a series of 1-h runs with different delay times. Hydrolysis of dehydroretrorsine was very rapid, whereas the hydrolysis of dehydromonocrotaline and dehydro-anacrotine fitted biphasic first-order reactions, with a faster first phase. By isolating and identifying the trapped products from dehydromoncrotaline it was shown that the two phases involved hydrolysis of the 7- and 9-ester groups, respectively. The results supported the view that a proportion of the reactive metabolites from the alkaloids monocrotaline and anacrotine would be able to survive long enough to be transported from the liver to the lungs of a rat. The flow system would be applicable to the study of other types of short-lived metabolites.
MRC Toxicology Unit, Medical Research Council Laboratories, Carshalton, Surrey, U.K.
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Mattocks
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Jukes
D000477:Alkylating Agents / Q000032:analysis*; D055598:Chemical Phenomena; D002621:Chemistry; D004867:Equipment Design; D006868:Hydrolysis; D007700:Kinetics; D016686:Monocrotaline / Q000031:analogs & derivatives*; D011763:Pyrrolizidine Alkaloids / Q000032:analysis; D012685:Sepharose / Q000031:analogs & derivatives; D013438:Sulfhydryl Compounds; D013997:Time Factors
D000477:Alkylating Agents; D011763:Pyrrolizidine Alkaloids; D013438:Sulfhydryl Compounds; C053599:dehydroanacrotine; C020272:dehydroretrorsine; C014114:monocrotaline pyrrole; D016686:Monocrotaline; D012685:Sepharose