title: Radioimmunoassay of nicotine-delta 1'(5')-iminium ion, an intermediate formed during the metabolism of nicotine to cotinine.
abstract: Cotinine, one of the major metabolites of nicotine, is formed by two sequential enzyme reactions: [formula: see text] Chemical and immunological methods of analysis are available to quantify these two compounds (I and III). Study of the intermediate (III) is hampered because of its complex chemistry and lack of simple methods for its assay. Generally, (II) is trapped by addition of cyanide and analyzed as the 5'-cyanonicotine adduct. In order to develop an immunoassay for (II), rabbits were immunized with a 3'-succinylmethyl-5'-cyanonicotine-protein conjugate with the expectation that (II) would, after treatment with cyanide, be quantified as 5'-cyanonicotine. Unexpectedly, however, the antibodies recognized the cyano adduct and (II) to the same extent (limit of deletion = 1.2 pmol). Nicotine, cotinine, and several other metabolites do not significantly inhibit the antigen-antibody reaction. Inhibition studies with 5' substituted nicotine analogs indicate that the 5'-hydroxynicotine tautomer (IIb) is the species recognized by the antibodies. Inin vitro metabolic studies, (II) accumulates in the absence of aldehyde oxidase, but in its presence, is converted to cotinine. As judged by serological activity, dilute solutions (under 15 microM) of (II) are stable when allowed to stand for 6 days at pH 7 and 37 degrees C. However, at high concentrations of iminium ion (where NMR studies showed instability and formation of multiple products) serological activity is also lost. Iminium ions are generated during the metabolism of some tertiary amine xenobiotics. The use of alpha-cyanoamine haptens to elicit antibodies specific for iminium ions and/or their cyano adducts may permit development of immunoassays for these compounds.
authors.affiliation: Department of Biochemistry, Brandeis University, Waltham, MA 02254.
authors.forename: R S
authors.initials: RS
authors.lastname: Obach
authors.forename: H
authors.initials: H
authors.lastname: van Vunakis
mesh_terms: D000818:Animals; D000918:Antibody Specificity; D055598:Chemical Phenomena; D002621:Chemistry; D003367:Cotinine / Q000378:metabolism*; D005260:Female; D009682:Magnetic Resonance Spectroscopy; D009538:Nicotine / Q000031:analogs & derivatives* / Q000032:analysis* / Q000378:metabolism; D011760:Pyrrolidinones / Q000378:metabolism*; D011817:Rabbits; D011863:Radioimmunoassay; D013055:Spectrophotometry, Infrared; D013347:Subcellular Fractions / Q000378:metabolism
chemical_list: D011760:Pyrrolidinones; D009538:Nicotine; C034629:nicotine imine; D003367:Cotinine