Characterization of the glucuronide conjugate of cotinine: a previously unidentified major metabolite of nicotine in smokers' urine

Abstract

Recent studies in our laboratories have confirmed that a major unidentified metabolite of nicotine in smokers' urine was susceptible to enzymatic degradation by beta-glucuronidase to afford (S)-(-)-cotinine. In order to establish the identity of this metabolite, the quaternary ammonium conjugate, viz., (S)-(-)-cotinine N-glucuronide, was synthesized. Reaction of methyl 2,3,4-tri-O-acetyl-1-bromo-1-deoxy-alpha-D-glucopyranuronate with (S)-(-)-cotinine at 60 degrees C for 3 days affords the fully protected conjugate as the bromide salt. Deprotection was accomplished in 1 M NaOH overnight at 25 degrees C. The deprotected inner salt was isolated by Dowex-50W cation-exchange chromatography. Electrospray mass spectra of the inner salt revealed the presence of ions with m/z 353 (M + H)+, 375 (M + Na)+, and 391 (M + K)+ as well as ions resulting from loss of water and cleavage of the glycosidic bond. Proton and carbon nuclear magnetic resonance spectra established that the position of glucuronidation was the pyridyl nitrogen. The magnitude of the coupling between H1" and H2" of the sugar ring (8.71 Hz) and nuclear Overhauser enhancements were consistent with the beta-isomer of the glucuronide conjugate. The synthetic (S)-(-)-cotinine N-glucuronide was susceptible to enzymatic hydrolysis by beta-glucuronidase to afford (S)-(-)-cotinine. Application of a cation-exchange high-performance liquid chromatographic method enabled the collection of a fraction containing (S)-(-)-cotinine N-glucuronide from a smoker's urine. The electrospray mass spectrum of this fraction contained ions consistent with the presence of (S)-(-)-cotinine N-glucuronide. The concentrated fraction was subjected to enzymatic hydrolysis by beta-glucuronidase to afford (S)-(-)-cotinine.(ABSTRACT TRUNCATED AT 250 WORDS)

IDENTIFICATION OF METABOLITES IN URINE AND FECES FROM RATS DOSED WITH THE HETEROCYCLIC AMINE, 2-AMINO-3-METHYL-9H-PYRIDO[2,3-b]INDOLE (MeAαC)

“…Direct phase II conjugation of pyridine nitrogen with glucuronic acid has previously been reported. The major urinary metabolite of cotinine in smokers was identified as cotinine glucuronide (Caldwell et al, 1992), and the major metabolite in baboon urine after treatment with thiadiazinone was the quaternary N-glucuronide (McKillop et al, 1990). In both cases, analyses confirmed the site of glucuronic acid conjugation as the nitrogen in the pyridine ring, which results in a positive charge on the pyridine nitrogen.…”

Metabolism and Disposition Kinetics of Nicotine

“…5). Indeed, it appears that most of the reported urinary metabolites of nicotine are derived McKennis et al, 1963b;Neurath et al, 1987), 5Ј-hydroxycotinine (also called allohydroxycotinine) (Neurath, 1994), which exists in tautomeric equilibrium with the open chain derivative 4-oxo-4-(3-pyridyl)-N-methylbutanamide McKennis et al, 1962), cotinine N-oxide (Shulgin et al, 1987;Kyerematen et al, 1990b), cotinine methonium ion (McKennis et al, 1963a), cotinine glucuronide (Curvall et al, 1991;Caldwell et al, 1992), and norcotinine (also called demethylcotinine) (Bowman et al, 1959;Kyerematen et al, 1990b).…”

Glucuronidation of Nicotine and Cotinine by UGT2B10: Loss of Function by the UGT2B10 Codon 67 (Asp>Tyr) Polymorphism

“…Up to 31% of nicotine urinary metabolites are in the form of phase II glucuronidated compounds, with nicotine-glucuronide, cotinineglucuronide, and trans-3 ¶-hydroxycotinine glucuronide comprising the majority of these conjugates (1). Both cotinine and nicotine are glucuronidated on the nitrogen of the pyridine ring, and N-glucuronidation of both compounds is observed in human liver microsomes (HLM) and in the urine of smokers (2)(3)(4)(5). Whereas N-glucuronidation of 3 ¶-hydroxycotinine was observed in HLM, only its O-glucuronide was detected in the urine of smokers (6).…”