Chemistry of sulphur‐bound pyrrolic metabolites in the blood of rats given different types of pyrrolizidine alkaloid

Rats were injected with the pyrrolizidine alkaloids heliotrine, indicine, or anacrotine, and killed after 20 hr. Alkaloid metabolites conjugated to haemoglobin thiol groups were recovered in the form of pyrrolic monoethyl ethers, by treating blood samples with ethanolic silver nitrate under “buffered” conditions. Chemically prepared putative toxic metabolites of the alkaloids—dehydroheliotrine, dehydroindicine, and dehydroanacrotine—were also allowed to react in vitro with blood and with an immobilized thiol, thiol‐Sepharose, and subsequently the S‐conjugated pyrroles were again recovered as ethyl ethers. The recovered pyrrolic ethers were identified by comparing them with reference compounds prepared from ethanol and the dehydro‐alkaloids, and the structures of the S‐bound pyrroles were deduced. Blood from rats given the 9‐monoester alkaloids heliotrine or indicine contained pyrrolic residues, S‐bound at their 9‐position. Anacrotine‐treated rats yielded two diastereomeric 7‐ethers, showing that dehydrocrotanecine 7‐conjugates had been present in the blood. The products from alkaloid‐treated rats were identical with those from blood or thiol‐Sepharose treated with the corresponding dehydro‐alkaloids in vitro. This supported the view that proximal metabolites leading to S‐binding in vivo were the dehydro‐derivatives of the alkaloids. In each case the thiols were attacked by the most reactive centre of the dehydro‐alkaloid: the 9‐ester in dehydroheliotrine and dehydroindicine, and the 7‐ester in dehydroanacrotine. Accordingly, simple chemical reactions could account for the products formed in vivo. The identification of S‐conjugated pyrrolic metabolites on haemoglobin provided evidence of the type of alkaloid ingested. This would have diagnostic value for pyrrolizidine poisoning of livestock or humans. Thus, 9‐monoesters of heliotridine or retronecine would lead to 9‐conjugates; 7,9‐diesters (such as monocrotaline) would give 7‐conjugates; and anacrotine exposure would be distinguishable by giving two isomeric 7‐conjugates. The demonstration of S‐bound pyrroles on haemoglobin in rats exposed to indicine for less than 15 min illustrated the rapidity with which reactive metabolites are liberated after pyrrolizidine alkaloid ingestion.