In recent investigations we have shown that (-)-nicotine and (-)-cotinine, a metabolite of (-)-nicotine, undergo pyridine-JV-methylation, which leads in the dog3 to the urinary excretion of the corresponding onium ions, nicotine isomethonium ion and (-)-cotinine methonium ion. In similar studies5 with 3-acetylpyridine the isolated quaternary ammonium urinary excretion product was N-methyl 3-(1 -hydroxyethyl)pyridinium ion. The results of the examination, although pointing to this quaternary ion as the predominant metabolic product in the methonium fraction of the urine, did not exclude the presence or intermediary participation of JV-methyl 3-acetylpyridin-ium ion, which may have been formed and metabolized further to give AT-methyl 3-(l-hydroxyethyl)pyridmium ion. Since the quantity of JV-methylpyridinium compound present in urine in these and other studies may be related, in part at least, to the relative stability or lability6 of the methyl group on the quaternary nitrogen atom, we were led to compare the ease with which such groups labelled with carbon-14 could enter into oxidative pathways, using respiratory carbon dioxide (labelled with carbon-14) of the rat as an indicator. Table 1. 24-H RECOVERY OF "C-AcTiviTY AFTER INTRAPERITONEAL ADMINISTRATION OF A7-METHYL-14C l/m PYRIDINIUM COMPOUNDS TO THE MALE RATCompound and dose (jM) No. of animals Percentage of 14C-activity recovered Respiratory CO2 Faeces Urine Total t3-Acetylpyridine methonium iodide (18) I (-)-Cotinine methonium iodide (24) Nicotinamide methonium iodide (20) Nicotinic acid methonium chloride (19-26) 3-Pyridylacetic acid methonium chloride (21) 4 3 3 32 3-80-8-41<0-01 0-13-0-57 0-03-0-11 0-16-0-18 0-40-9-36 0-78-1-21 0-94-1-530-72-1-07 0-22-0-56 74-1-84-4 87-6-96-2 94-6-97-7 i 95-5-98-5 94-0-97-6 1 95-7-98-8 92-0-97-0 92-8-98-0 62-9-67-9 63-6-68-3Faecal value determined for only two animals. t Range of total activity recovered per animal.For the examination a series of JV-methyl-14C 1/mpyridin-ium compounds were prepared by conventionally treating solutions of the corresponding pyridine compounds with methyl-14C iodide. The betaine trigonelline was used in the form of the hydrochloride, nicotinic acid methonium chloride. This and the betaine salt of 3-pyridylacetic acid, 3-pyridylacetic acid methonium chloride, were obtained via corresponding esters which were JV-alkyl-ated7'8 and afterwards hydrolysed. All compounds were injected intraperitoneally in neutral aqueous solution (1-1-5 ml.) into male albino Wistar strain rats (145-264 g). The animals were housed in glass metabolism cages, with water but no food, for collection of respiratory carbon dioxide in scintillator solution containing phenylethylamine9. Radioactive determinations on faeces (after combustion), urine (directly or following combustion), and respiratory carbon dioxide were corrected by an internal standard or by the channel-ratio10 procedure.The major mode of elimination of the radioactivity (Table 1) was by way of the urine; some radioactivity, in all cases, appeared in the faeces. The faecal values, however, must be considered high as the result of occasional contamination by urine. As a group, with the notable exception of 2V-methyl 3-acetylpyridinium ion, the JV-methyl-pyridinium compounds contributed very little radioactivity, less than 0-9 per cent of that administered, to the respiratory carbon dioxide. N-Methyl 3-acetylpyridinium ion, administered as the iodide (3-acetylpyridinium methiodide), led to the production of considerable quantities of radioactivity (3-8 per cent of the administered dose) in the form of respiratory carbon dioxide. The urine from the animals that received this acetyl quaternary ammonium ion contained material which co-chroraatographed on paper with the administered compound and, in addition, contained other radioactive components which require characterization. The presence of these components, an indication of active metabolism of the N-methyl 3-acetylpyridinium moiety, suggests again the possibility that the methonium compound, although never isolated in metabolic studies on 3-acetylpyridine, has an intermediary role in the metabolism of the ketone.The conversion of the methyl group of trigonelline to l4C-carbon dioxide was very limited in nature (approximately 0-1 per cent of the administered dose). The capacity of trigonelline to serve as a methyl donor has been subjected in the past to many investigations The work of du Vigneaud et al.1 suggests that if trigonelline had contributed appreciable quantities of one-carbon components to the labile methyl pool, this would have been reflected in greater quantities of radioactivity in the respiratory carbon dioxide. Handler and Dann11 noted that the feeding of trigonelline to rats was not effective in preventing fatty-liver formation. They noted, however, that the urine of the animals contained considerable quantities of acid-hydrolysable nicotinic acid derivatives. They concluded that rats have a capacity for demethylating trigonelline, and that the methyl group is not available for trans-methyl ation reactions. Moyer and du Vigneaud12, using growth of rats as a criterion, had noted previously that the methyl group was not available for significant methyl at ion of homocysteine. In contrast to the cited results with trigonelline, Najjar and Deal4, after finding that JV^methylnicotinamide exerted a lipotropic action in rats, suggested a possible demethylation of A^-methylnicotinamide, with the production of nicotinamide and a biologically active (labile) methyl group. The work of Keller, Wood and du Vigneaud13 failed to confirm this possibility. In our investigations 0-1-0-9 per cent of the radioactivity of iV1-methyl-14C-nicotinamide, administered in the form of nicotinamide methonium iodide, appeared as radioactive respiratory carbon dioxide. These results, although perhaps indicating the possibility of labile methyl group formation, are readily susceptible to other interpretations, including a degradation to methylamine and subsequent degradation14 to carbon dioxide. Only a more fully detailed investigation of the metabolites of the pyridinium compounds can provide the basis for a satisfactory interpretation.This work was supported by the American Tobacco Co., the Tobacco Industry Research Committee and the U.S. National Institutes of Health.