John J. Langone

Stereospecific monoclonal antibodies to nicotine and cotinine and their use in enzyme-linked immunosorbent assays.

Stereospecific monoclonal antibodies (McAb) have been prepared against the tobacco alkaloid (S)-(-)-nicotine and its major metabolite (S)-(-)-cotinine. Nine anti-nicotine and 4 anti-cotinine hybridomas, selected by a screening procedure that utilized immunoprecipitation of the 3H-labeled natural isomers of nicotine or continine, were grown in the ascites fluid of pristane-primed syngeneic BALB/c mice. Antibodies in concentrations up to 7.5 mg/ml ascites and with binding affinities that generally exceeded 10(8) M-1 were obtained. Enzyme-linked immunosorbent assays (ELISAs) were developed in which nicotine or cotinine derivatives bound covalently to poly-L-lysine were coated onto wells of polyvinyl chloride microtiter plates. Coated wells were incubated sequentially with McAb in the presence or absence of inhibitor, rabbit anti-mouse immunoglobulin, then horseradish peroxidase-labeled protein A (HRP-SpA) before addition of substrate. The antibodies are highly specific and show minimal cross-reactivity with several nicotine metabolites and other structurally related compounds. In the respective assays, only 0.25 ng (S)-(-)-nicotine and 0.12 ng (S)-(-)-cotinine are required to give 50% inhibition of antibody binding, and as little as 0.05 ng nicotine and 0.02 ng cotinine give 15% inhibition. These assays are 5-10 times more sensitive than analogous ELISAs developed with rabbit antisera and HRP-SpA or conventional radioimmunoassays (RIAs) that utilize the rabbit antisera and 3H-labeled ligands. There was good correlation between the levels of nicotine (r = 0.967) and cotinine (r = 0.981) found in saliva samples from smokers and non-smokers assayed by McAb-based ELISAs and conventional RIAs.

NICOTINE ABSORPTION BY WORKERS HARVESTING GREEN TOBACCO

Green-tobacco sickness is an occupational illness of tobacco harvesters. Symptoms include nausea, vomiting, dizziness, and prostration. The disease is self-limited and of short duration, but recurs frequently in susceptible workers. The aetiology is not known, but nicotine has been suspected as a causative agent. Thirty-two workers on four North Carolina tobacco farms were studied during harvesting. None of these workers smoked or chewed tobacco. Urinary cotinine (the major metabolite of nicotine) levels were monitored over a 24-hour period to evaluate nicotine absorption. There was a tenfold rise in mean excretion of cotinine among workers who had greatest contact with the tobacco. Less cotinine was found in urine of workers who had less exposure. Levels of cotinine exceeded those found in novice smokers who smoked 3 cigarettes in succession. Absorption of nicotine from tobacco leaf is the likely cause of tobacco sickness.

Nicotine and its metabolites

Abstract A radioimmunoassay has been developed for the nicotine metabolite γ-(3-pyridyl)-γ-oxo- N -methylbutyramide. The sensitivity and specificity of the assay allow the determination of this compound at the picomole level in the presence of several structurally related molecules including nicotine and seven other nicotine metabolites. The assay has been used to characterize the enzyme system present in rabbit liver extract responsible for the conversion of cotinine to the oxoamide, and to measure oxoamide levels in urine, sera, and amniotic fluid of smokers. High-pressure liquid chromatography was used as an independent method to follow the enzymatic oxidation and in conjunction with the radioimmunoassay to analyze the urine samples.

Adriamycin and metabolites: Separation by high-pressure liquid chromatography and quantitation by radioimmunoassay☆

Abstract High-pressure liquid chromatography (HPLC) is used to separate adriamycin and the metabolites, adriamycinol, and adriamycin aglycone(s) found in the urine of patients treated with the drug. The concentrations of the isolated compounds are determined by radioimmunoassay (RIA). Their excretion rates decrease with time and their relative molar ratios vary in collections made at different times and from different individuals. This combined HPLC-RIA system offers an efficient and sensitive procedure for separation and estimation of these compounds that is not subject to interference from nonrelated endogenous materials which may affect results obtained with other methods.

Nicotine: Cotinine levels in blood during cessation of smoking * **

HE ROLE OF NICOTINE as a primary reinforcer and of nicotine withdrawal as a factor in maintaining cigarette smoking behavior has been recently reviewed by Jarvik. ~ In this paper, we present an exploration of the relationship between the amount of nicotine in cigarettes smoked per day, serum cotinine levels, and difficulty in modifying the smoking habit. Cotinine is the major nicotine metabolite. In contrast to the short half-life of nicotine itself, the half-life of cotinine in blood is far longer (30 hr versus 30 rain, respectively). Furthermore, cotinine is usually found in the blood at levels greater than that of nicotine, 2-s and cotinine levels remain fairly constant in individuals who smoke according to a consistent pattern. 2.4.r On this basis, we felt that serum levels of cotinine would be a better index of the degree of chronic nicotinism than measures of the parent alkaloid. Cotinine is approximately 50 times less toxic than nicotine in rats. s-lo In this study, participants in a smoking cessation program were asked to rate themselves in terms of the difficulty they experienced in giving up smoking. They also provided information on the number and type of cigarettes they had been smoking. We then examined the correlations between this information provided by the participants, their success in stopping cigarette use, and serum c0tinine levels taken at several points during the course of the participaIion in the cessation program. In addition, we attempted to ascertain activity of the sympathetic system by measuring plasma dopamine-beta-hydroxylase (DBH).

Radioimmunoassays for the Vinca alkaloids, vinblastine and vincristine.

Abstract Radioimmunoassays for the chemotherapeutic alkaloids, vinblastine (VLB) and vincristine (VCR) were developed that could determine less than 1 pmol of each compound in the homologous hapten-antibody reaction. The anti-VCR serum bound VCR 200 times more effectively than VLB, demonstrating a high specificity in spite of the structural similarity of the drugs. Several other chemotherapeutic drugs showed no significant cross-reactivity. The assays were used to measure plasma levels of the alkaloids in rabbits treated with the drugs and to determine immunoreactive drug equivalents in extracts of the periwinkle plant following fractionation by high-pressure liquid chromatography.

Nicotine and its metabolites: Radioimmunoassay for γ-(3-Pyridyl)-γ-oxo-N-methylbutyramide☆☆☆

Abstract A radioimmunoassay has been developed for the nicotine metabolite γ-(3-pyridyl)-γ-oxo- N -methylbutyramide. The sensitivity and specificity of the assay allow the determination of this compound at the picomole level in the presence of several structurally related molecules including nicotine and seven other nicotine metabolites. The assay has been used to characterize the enzyme system present in rabbit liver extract responsible for the conversion of cotinine to the oxoamide, and to measure oxoamide levels in urine, sera, and amniotic fluid of smokers. High-pressure liquid chromatography was used as an independent method to follow the enzymatic oxidation and in conjunction with the radioimmunoassay to analyze the urine samples.

Nicotine and its metabolites: radioimmunoassay for. gamma. -(3-pyridyl)-. gamma. -oxo-N-methylbutyramide. [/sup 125/I]

Abstract A radioimmunoassay has been developed for the nicotine metabolite γ-(3-pyridyl)-γ-oxo- N -methylbutyramide. The sensitivity and specificity of the assay allow the determination of this compound at the picomole level in the presence of several structurally related molecules including nicotine and seven other nicotine metabolites. The assay has been used to characterize the enzyme system present in rabbit liver extract responsible for the conversion of cotinine to the oxoamide, and to measure oxoamide levels in urine, sera, and amniotic fluid of smokers. High-pressure liquid chromatography was used as an independent method to follow the enzymatic oxidation and in conjunction with the radioimmunoassay to analyze the urine samples.