Nicotine antibodies: comparison of ligand specificities of antibodies produced against two nicotine conjugates.

Conjugates between bovine serum albumin and (R,S)-2-aminonicotine were produced, and these conjugates were employed in rabbits and goats for the production of nicotine antibodies. In the assay of nicotine, an 125I-tyrosine methyl ester derivative of (R,S)-6-aminonicotine was employed as radioligand. The antibody-bound derivative was separated from the free derivative by charcoal adsorption (0.5% charcoal, 0.1% dextran T-70, 0.1% bovine serum albumin pH 7.3). Among the twenty five nicotine derivatives and metabolites examined, (R,S)-6-aminonicotine gave the highest cross-reaction. Cross-reaction with cotinine, a major mammalian metabolite of nicotine, was less than 0.1% for both the rabbit-derived and goat-derived antisera. Cross-reaction by other metabolites, such as (S)-nicotine-N'-oxide, (S)-nornicotine, and N-methylpyrrolidine was less than 1%. The antibodies produced were thus highly specific to nicotine. The radioimmunoassay for nicotine showed a maximum sensitivity of 10 ng/ml in 50-microliter plasma samples for both antisera. After the smoking of a single cigarette (1.2 mg nicotine content in mainstream) the peak of blood plasma level of nicotine in the subjects varied from 20--104 ng/ml, and high levels of nicotine were not necessarily found in heavy smokers.

Automated radioimmunoassay of nicotine.

We have developed an automated nonequilibrium procedure for the radioimmunoassay of nicotine. The use of a unique iodinated nicotine derivative in this procedure gave a sensitivity of 10 micrograms/l for nicotine with a between-run precision of 7.4% and within-run precision of 6.0%. Nicotine levels of 60 to 67 micrograms/ml were found in subjects 15 min after smoking one standard cigarette. The technique herein reported is a very rapid, and sensitive radioimmunoassay for nicotine and facilitates the determination of nicotine in smoking subjects during the actual process of smoking.

The interrelationship between the metabolism of (S)-continine-N-oxide and (S)-cotinine.

The metabolism of (S)-cotinine-N-oxide was studied in the rabbit and the dog. The pattern of Koenig-positive substances in the urine of the animals suggested the presence of cotinine, demethylcotinine, hydroxycotinine, and allohydroxycotinine, compounds already previously identified as metabolites of (S)-cotinine and (S)-nicotine in many mammalian species. In the dog, 34% of the administered oral dose of (S)-cotinine-N-oxide was recovered from the urine, and 21% was recovered from the urine of the rabbit. Confirmation of the presence of (S)-cotinine, (S)-demethulcotinine, hydroxycotinine, and allohydroxycotinine in the urine of the rabbits was obtained by isolation of the metabolites as themselves of as derivatives. The data, although establishing the possibilities of an intermediary role for (S)-cotinine-N-oxide in the metabolism of nicotine, do not clearly indicate whether the metabolites such as demethylcotinine arise via the route (S)-cotinine-N-oxide leads to (S)-cotinine leads to (S)-demethylcotinine or via the alternate route (S)-cotinine-N-oxide leads to (S)-demethylcotinine-N-oxide leads to (S)-demethylcotinine.

Nicotine-like actions of cis-metanicotine and trans-metanicotine.

The actions of the cis- and trans-isomers of metanicotine were observed on isolated rabbit aortic strips and ileal segments. The data are interpreted as showing a nicotine-like action on these preparations for both cis-metanicotine and trans-metanicotine. This hypothesis is supported in part by the demonstration that the action of the metanicotine isomers was affected by hexamethonium, cocaine, phenotolamine, reserpine and atropine in a manner similar to that previously seen in studies with nicotine. In dose-response studies on the aortic strip, trans-metanicotine was significantly less active than nicotine. cis-Metanicotine in turn was less active than trans-metanicotine and nicotine. Additionally, four pyridino compounds, 3-pyridylacet acid, N-(3 pyridlyacetyl) glycine, nicotinuric acid and trans-4-(3-pyridyl)-3-butenoic acid, were tested for both agonist and antagonist activity. No stimulatory activity was found with these compounds in either the aortic strip or ileal preparations. In aortic strip preparations, pretreatment with either 3-pyridylacetic acid or N-(3-pyridylacetyl) glycine provided a moderate to marked reduction in the contractile response to trans-metanicotine, whereas pretreatment with trans-4-(3-pyridyl)-3-butenoic acid caused a slight reduction.