Title page for ETD etd-08272007-163623

Type of Document Dissertation
Author Liu, Xin
URN etd-08272007-163623
Title In vitro and in vivo studies on the biotransformation of β-nicotyrine, a minor tobacco alkaloid
Degree PhD
Department Chemistry
Advisory Committee
Advisor Name Title
Castagnoli, Neal Jr. Committee Chair
Kingston, David G. I. Committee Member
Merola, Joseph S. Committee Member
Riffle, Judy S. Committee Member
White, Robert H. Committee Member
  • metabolic bioactivation pathway - nicotine
Date of Defense 1995-06-05
Availability restricted

𝝱-Nicotyrine is a minor tobacco alkaloid found in both tobacco plants and tobacco smoke. Preliminary studies have shown that 𝝱-nicotyrine is pneumotoxic and that the toxicity is mediated via its metabolites formed in reactions catalyzed by cytochrome P-450. The in vitro metabolic fate of 𝝱-nicotyrine has been examined in rat liver microsomal preparations and rabbit lung and liver microsomal preparations utilizing HPLC UV-diode array analysis. Four metabolites have been identified from the incubation mixtures of 𝝱- nicotyrine with rabbit lung and liver microsomal preparations. The primary in vitro metabolites are two unstable pyrrolinone species, 1-methyl-5-(3-pyridyl)-4- pyrrolin-2-one and 1-methyl-5-(3-pyridyl)-3-pyrrolin-2-one, which exist in equilibrium. The pyrrolinones undergo autoxidation to form a secondary metabolite, 5-hydroxy-1-methyl-S-(3-pyridyl)-3-pyrrolin-2-one, and hydrolysis to form 5'-hydroxycotinine. The autoxidation is likely to involve a -free radical process. Evidence to support this proposal has been obtained by studies on 2- acetoxy-1-methyl-5-(3-pyridyl)pyrrole, a latent form of the pyrrolinones. Free radicals generated following hydrolysis of the acetoxypyrrole derivative have been trapped and detected by ESR analysis by spin trapping techniques. In contrast, only the starting substrate (𝝱-nicotyrine was found in the incubation mixture of 𝝱-nicotyrine with rat liver microsomes.

The in vivo metabolic fate of 𝝱-nicotyrine has been examined in rabbits and mice by HPLC UV-diode array and GC-EIMS analysis of urine extracts. Three metabolites have been identified. Besides S'-hydroxycotinine and 5-hydroxy-1-methyl-5-(3-pyridyl)-3-pyrrolin-2-one, observed in the in vitro studies, 3'-hydroxycotinine is also found and determined to be the principal urinary metabolite of 𝝱-nicotyrine in both species. The stereochemistry of this 3'- hydroxycotinine is cis. This is in contrast to the trans stereochemistry of the urinary metabolite of (S)-cotinine and (S)-nicotine. The mechanism of formation of cis-3'-hydroxycotinine from 𝝱-nicotyrine is proposed to involve a free radical process leading to the formation of the precursor 3-hydroxy-1-methyl-5-(3- pyridyl)-4-pyrrolin-2-one. This intermediate then undergoes a carbon-carbon double bond reduction to yield the final product. This proposal is supported by in vivo studies on 5-hydroxy-1-methyl-5-(3-pyridyl)-3-pyrrolin-2-one which undergoes reduction to 5'-hydroxycotinine. Furthermore,2-acetoxy-1-methyl-5- (3-pyridyl)pyrrole goes to cis-3'-hydroxycotinine as the major urinary metabolite.

The results of these studies point to a potentially novel metabolic bioactivation pathway of (S)-nicotine that could be relevant to some of the toxic effects observed in chronic tobacco users.

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