Characterization of a glucuronide metabolite of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (nnk) and its dose-dependent excretion in the urine of mice and rats

Following analysis by reversed-phase HPLC, a previously uncharacterized metabolite of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) was found in the urine of A/J mice treated with NNK. Treatment with β-glucuronidase converted the metabolite to a peak that co-eluted with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL). Treatment with sulfatase or β-glucuronidase plus saccharic acid 1,4-lactone did not change the retention time of the metabolite. These data suggested that the unknown metabolite was a glucuronic acid conjugate of NNAL. Upon isolation and purification of larger quantities of the metabolite from the urine of A/J mice, CD-1 mice and F344 rats, ¹H and ¹³C NMR and MS confirmed that the unknown metabolite was 4-(methylnitrosamino)-1-(3-pyridyl)-1-butyl β-D-glucopyranosiduronic acid (NNAL Glu). To determine the quantitative relationship between NNK dose and NNAL Glu production and to compare the importance of glucuronidation relative to other metabolic pathways, [5-³H]NNK was administered to F344 rats and A/J mice at doses of 500-0.005 μmol/kg. At 500 μNNAL Glu accounted for 22% of the total urinary excretion of NNK in A/J mice, and for 8% in F344 rats 48 h after dosing. The proportions of excreted glucuronide and NNAL decreased with diminishing doses of NMK, yielding undetectable levels of each metabolite in both mice and rats at a dose of 0.005 μmol/kg NNK. Since substantial amounts of metabolites formed via α-hydroxylatlon and N-oxidation pathways were observed at the lower doses of NNK, these data demonstrate that NNAL glucuronidation is a quantitatively unimportant metabolic pathway at low doses of NNK.