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PubMedDate not supplied

Interaction of cotinine with rat hepatic microsomal P-450. Comparison with metyrapone and immunomodulation of cotinine and metyrapone binding by monoclonal anti-cotinine antibodies.

The ability of the major nicotine metabolite, cotinine, to interact with rat liver microsomal cytochrome P-450 and the immunomodulatory effects of anti-cotinine antibodies were studied. Cotinine induced type II spectral changes with both microsomes from phenobarbital (PB)-induced rats and purified P-450 with apparent Ks values of 97 and 750 microM, respectively. In contrast, the Ks value was 0.3 microM for metyrapone and 5 microM for nicotine with both the microsomes and purified enzyme. The apparent Ki value for cotinine inhibition of 7-pentoxyresorufin O-dealkylase activity with the microsomes (87 microM) was approximately 87- and 870-fold higher than for nicotine and metyrapone, respectively. Monoclonal antibodies produced against cotinine cross-reacted equally well with metyrapone. They specifically blocked enzyme binding of both drugs based on dose-dependent inhibition of spectral changes, and reversed the metyrapone-induced inhibition of microsomal O-dealkylase activity. In contrast, antibodies to nicotine did not cross-react with cotinine or metyrapone and had no effect on their activity, although they did block the action of nicotine. These results demonstrate that cotinine binding to P-450 from PB-induced rats and inhibition of functional activity in vitro are qualitatively like the effects of metyrapone and nicotine, and that monoclonal anti-cotinine antibodies are useful molecular probes of the interactions between cotinine and metyrapone with the enzyme.

Animals↗

PubMedDate not supplied

Increased cotinine elimination and cotinine-N-oxide formation by phenobarbital induction in rat and mouse.

The metabolic fate of cotinine, the major metabolite of nicotine, was studied in phenobarbital-induced and non-induced isolated perfused rat lung and liver and in isolated hepatocytes of rats and mice. The non-induced lung tissue showed low cotinine metabolizing capacity while the perfused liver was approximately four times more active. After phenobarbital pretreatment the metabolism of cotinine was increased eight-fold in the intact liver. A substantial increase in cotinine metabolism was also found in isolated hepatocytes from PB-induced rats and in cultured mouse hepatocytes grown in a medium supplemented with PB. This was paralleled by an increased formation of cotinine-N-oxide which could be inhibited by 100 microM metyrapone. In contrast, the pulmonary elimination of cotinine was not affected by PB. A dominant role of primary N-oxidation of nicotine compared to C-oxidation was apparent in non-induced rat liver. After PB treatment the rate of nicotine-N'-oxide formation dropped markedly while the cotinine related pathways were increased causing an inversion of the N- to C-oxidation ratio. In the lung, cotinine formation was the preferred metabolic pathway of nicotine already in non-induced organs. The pattern of nicotine metabolites was not altered by PB induction. In conscious PB-induced rats receiving nicotine orally or intravenously, 3'-hydroxycotinine was found as the main urinary metabolite of nicotine while only a small fraction was excreted as cotinine-N-oxide. This discrepancy between the profile of nicotine metabolites in perfused liver and lung and in the urine in vivo indicates that extrahepatic organs other than the lung may be important sites of cotinine metabolism.

Animals↗

PubMed1987

Constant-rate infusion of nicotine and cotinine. I. A physiological pharmacokinetic analysis of the cotinine disposition, and effects on clearance and distribution in the rat.

The tissue partition of cotinine was measured by a GC-MS method following a 6-day constant-rate input of nicotine and cotinine to male rats by means of an osmotic minipump. The tissue-to-blood partition coefficients of cotinine were calculated for adipose (0.08), brain (0.48), heart muscle (0.51), following the cotinine infusion. When nicotine was infused the tissue partitioning of cotinine increased by a factor of 2.3-4.9, depending on the tissue sampled. Another group of animals were killed at timed intervals from 10 min to 30 hr, after having received a single intravenous bolus dose of 0.5 mg cotinine, and the washout of cotinine was traced in blood and tissues. A physiological model was used to simulate the disposition of cotinine. Generally, the model-predicted concentrations were consistent with those found experimentally. The fractional uptake of cotinine into various tissues was simulated. Blood, intestinal, and skeletal muscle tissues embodied more than 70% of the total body load of the drug. Clearance (Cl), volume of distribution (Vd), and the biological half-life (t1/2) were calculated both from the infusion study and by fitting a monoexponential model to the iv blood data of the rat. Significant differences were found in the apparent clearance calculated from the single iv bolus dose compared to the constant rate infusion. The volume of distribution was, however, consistent from both studies. The impact of a change in clearance was also simulated.

Animals↗

PubMed1989

High performance liquid chromatographic determination of nicotine and cotinine in plasma and nicotine and cotinine, simultaneously, in urine.

Three analytical procedures were developed to determine nicotine in plasma, cotinine in plasma and, simultaneously, nicotine and cotinine in urine. After liquid or solid-phase extraction, the purified aqueous phase is injected into a high performance liquid chromatograph equipped with an ultra-violet detector using a CN Spheri-5 micron cartridge-column with an inner diameter of 4.6 mm and a length of 10 or 22 cm. The limit of quantitation for nicotine in plasma was around 8 to 15 ng/ml, that of cotinine in plasma around 50 ng/ml and that of nicotine and cotinine in urine around 170 ng/ml and 70 ng/ml, respectively. The limit of detection of nicotine in plasma was around 1 ng/ml and that of nicotine and cotinine in urine around 20 ng/ml and 10 ng/ml, respectively. The passive exposure to cigarette smoke by non-smokers and the "resting levels" of nicotine in plasma and urine of smokers were studied. The analytical methods were set up to study the pharmacokinetics and bioavailability of nicotine in healthy volunteers following single and repeated administrations of different doses of transdermal nicotine systems.

Amphetamine↗

PubMed1990

Cotinine analytical workshop report: consideration of analytical methods for determining cotinine in human body fluids as a measure of passive exposure to tobacco smoke.

A two-day technical workshop was convened November 10-11, 1986, to discuss analytical approaches for determining trace amounts of cotinine in human body fluids resulting from passive exposure to environmental tobacco smoke (ETS). The workshop, jointly sponsored by the U.S. Environmental Protection Agency and Centers for Disease Control, was attended by scientists with expertise in cotinine analytical methodology and/or conduct of human monitoring studies related to ETS. The workshop format included technical presentations, separate panel discussions on chromatography and immunoassay analytical approaches, and group discussions related to the quality assurance/quality control aspects of future monitoring programs. This report presents a consensus of opinion on general issues before the workshop panel participants and also a detailed comparison of several analytical approaches being used by the various represented laboratories. The salient features of the chromatography and immunoassay analytical methods are discussed separately.

Animals↗

PubMed1997

Cotinine effects on nicotine metabolism.

BACKGROUND: Nicotine clearance and half-life are known to be significantly reduced in smokers compared to nonsmokers. Cotinine is the major primary metabolite of nicotine, and it accumulates in the body with regular smoking. Nicotine and cotinine appear to be metabolized by the same liver enzyme. Therefore we hypothesized that cotinine inhibits nicotine metabolism, resulting in slower nicotine clearance in smokers compared with nonsmokers. METHODS: This was a crossover, randomized, double-blind, and placebo-controlled study. The subjects were 12 healthy nonsmoking volunteers. They received two intravenous infusions of deuterium-labeled nicotine-d2 and cotinine-d4 (0.5 micrograms/kg/min), once with oral cotinine treatment of 0.25 mg/kg twice a day and once with placebo. Nicotine and cotinine pharmacokinetic parameters were determined for each infusion. RESULTS: During oral cotinine treatment, average plasma levels of cotinine ware 900 ng/ml, comparable to levels observed in some very heavy smokers. Cotinine had no effect on the disposition kinetics of nicotine-d2. The half-life of cotinine after low-dose cotinine-d4 infusion was comparable to that after high-dose cotinine described in previous studies. The half-life of labeled cotinine derived from nicotine was significantly longer than the half-life of cotinine administered as cotinine. CONCLUSIONS: Cotinine is not responsible for the lower nicotine clearance observed in smokers. Our data suggest that the pharmacokinetics of low-dose cotinine in nonsmokers do not differ from those of high-dose in smokers, and therefore cotinine levels can be used quantitatively in environmental tobacco exposure. The longer half-life of cotinine derived from nicotine suggests that slow release of nicotine from tissues is responsible for the apparent long half-life of cotinine in nonsmokers exposed to environmental tobacco smoke.

Adult↗

PubMed1994

Metabolism of nicotine to cotinine studied by a dual stable isotope method.

OBJECTIVES: (1) To determine the disposition kinetics of nicotine and cotinine, including the fractional conversion of nicotine to cotinine, (2) to compare the disposition kinetics of deuterium-labeled and unlabeled cotinine, and (3) to develop a pharmacokinetically based method for estimating daily intake of nicotine from cigarette smoking. STUDY DESIGN: Twenty cigarette smokers received a combined infusion of deuterium-labeled nicotine (d2) and cotinine (d4). Six nonsmokers received a combined infusion of unlabeled cotinine, cotinine-d2 and cotinine-d4. Daily intake of nicotine was estimated with use of the plasma cotinine concentration during ad libitum smoking, clearance of labeled cotinine, and fractional conversion of nicotine to cotinine. RESULTS: The kinetics of labeled versus unlabeled cotinine and of cotinine in smokers versus nonsmokers were similar. On average, 72% of nicotine was converted to cotinine, with a range from 55% to 92%. Subjects with lower clearances of nicotine had lower fractional conversion of nicotine to cotinine, indicating that this is the most rapid of the proximate metabolic pathways for nicotine. The equation for estimating daily intake of nicotine from smoking was: Dnic (mg/24 hr) = K x (Plasma Cot) (ng/ml), where K averaged 0.08, with a range from 0.047 to 0.102. Individual variability in the clearance of cotinine (coefficient of variation, 27.5%) accounts for more of the variability in K than does variability in the fractional conversion of nicotine to cotinine (coefficient of variation, 12.3%). CONCLUSIONS: Our study provides quantitative data on individual variability in the extent of C-oxidation of nicotine to cotinine and a quantitative perspective on the use of plasma cotinine as an indicator of daily intake of nicotine from tobacco.

Adult↗

PubMedDate not supplied

Saliva cotinine and recent smoking--evidence for a nonlinear relationship.

Cotinine concentration in various body fluids is considered to be among the most useful markers of nicotine exposure currently available. Despite the prevailing consensus concerning cotinine's usefulness, cotinine's large intrasubject variability has led some to question the value of a single-point measurement. Several individual differences (for example, age, race, sex, and so forth) may affect cotinine excretion, and a peculiar nonlinearity between the number of cigarettes smoked and cotinine concentration has been reported previously in the literature. The purpose of this investigation was to examine the nature of the association between cotinine and reported number of cigarettes smoked after adjustment for the relationship between cotinine and age, a key individual difference known to affect drug absorption, distribution, metabolism, excretion, and tissue sensitivity. The authors examined the relationship between saliva cotinine and daily cigarette consumption in 116 smokers (mean age = 37.4 years; average number of cigarettes smoked daily = 20.1) who logged each cigarette into a hand-held computer as part of a study on the accuracy of recall. The Pearson correlation between saliva cotinine and the logged number of cigarettes smoked in the previous 17 hours (the time window corresponding to the half-life of cotinine) accounted for significantly more of the variance in cotinine than did the average logged number of cigarettes smoked daily during 5 days. Age was also significantly associated with cotinine levels. Further examination of the relationship between cotinine and amount smoked in the previous 17 hours revealed evidence for a significant nonlinear component. Inclusion of both age and a cubic nonlinear component of daily cigarette consumption resulted in further significant improvement in the amount of variance accounted for in cotinine levels. These results suggest that adjustments forage and the inclusion of a nonlinear component for cigarette consumption will result in more precise use of cotinine as a validation tool for existing differences in smoking levels.

Adult↗

PubMed1990

Simulation and evaluation of nicotine intake during passive smoking: cotinine measurements in body fluids of nonsmokers given intravenous infusions of nicotine.

The technique of monitoring cotinine concentrations in body fluids as a means of measuring nicotine intake during passive smoking has been evaluated in two studies, both of which used intravenous infusion to stimulate nicotine intake. In the first study, nicotine and cotinine were given separately, for 1 hour in four different intravenous doses (3.2, 15.4, 30.9, and 61.7 nmol/min) to each nonsmoker. In the second study, nicotine and cotinine were infused for 4 hours; each subject received five different doses of nicotine (1.5, 3.1, 6.2, 10.8, and 15.4 nmol/min) and one of cotinine (10.8 nmol/min). The concentration of cotinine was constant in both plasma and saliva from 1 to 4 hours after the nicotine infusion; the plateau levels of cotinine were found to be linearly and directly related to the nicotine intake. The ratio of salivary to plasma cotinine was 1:1.27. A linear relationship was also found between nicotine and cotinine infusion rates and the AUC values for cotinine. The fraction metabolized to cotinine was found to be about 0.5. The results from these studies show that: (1) there is a linear relationship between the plateau concentration of cotinine and the amount of nicotine infused over a period of 1 up to 4 hours; (2) salivary cotinine provides the same information on nicotine intake as does plasma cotinine; and (3) single measurements of either plasma or salivary cotinine concentrations at 1 to 4 hours after the exposure could be used to predict the nicotine intake during 1 to 4 hours of environmental tobacco smoke exposure.

Adult↗

PubMed1996

Cotinine concentrations in plasma of smoking pregnant women and their infants.

In the Netherlands 30% of all women of reproductive age are habitual smokers. One third of these women continue to smoke during pregnancy. Tobacco smoke consists of more than 3600 different compounds. One of its chief pharmacologically active ingredients is nicotine of which 60% is metabolized to cotinine. Cotinine is the best available biochemical marker of nicotine consumption because it is specific for tobacco smoke exposure and it has a relatively long mean t1/2 of 15 hours. In the present study nicotine and cotinine concentrations were measured in 25 smoking and 25 non-smoking healthy pregnant women. In all 25 non-smoking pregnant women nicotine and cotinine levels were < 10 mg/l. Light smokers (< 10 cigarettes/day) were found to have nicotine blood concentrations < 10 mg/l and cotinine levels varying between 40 and 99 mg/l. Heavy smokers (> or = 10 cigarettes/day) had nicotine concentrations < 10 mg/l, but high cotinine levels varying from 115 to 199 mg/l. Cotinine was also determined in 25 neonates of non-smoking mothers and in 34 neonates of smoking mothers. In 9 of these 34 newborns the relationship between maternal and neonatal cotinine concentrations was investigated. Cotinine levels in neonates born to non-smokers and to women who smoked less than 10 cigarettes/day were below the detection limit of 10 mg/l. Cotinine values in neonates whose mothers smoked > or = 10 cigarettes/day were significantly higher than in those whose mothers smoked < 10 cigarettes/day, but significantly lower than in their mothers. The results of this study confirm that cotinine is more useful than nicotine in discriminating non-smokers, light and heavy smokers. Cotinine concentrations were significantly lower in the neonates than in their mothers, but there was a strong positive linear relationship between maternal and neonatal cotinine concentrations.

Cotinine↗

PubMed1993

Relation of passive smoking as assessed by salivary cotinine concentration and questionnaire to spirometric indices in children.

BACKGROUND: Previous studies of the effects of passive exposure to smoke on spirometric indices in children have largely relied on questionnaire measures of exposure. This may have resulted in underestimation of the true effect of passive smoking. Biochemical measures offer the opportunity to estimate recent exposure directly. METHODS: The relation between spirometric indices and passive exposure to tobacco smoke was examined in a large population sample of 5-7 year old children from 10 towns in England and Wales. The effects of passive exposure to smoke on lung function were assessed by means of both salivary cotinine concentration and questionnaire measurements of exposure. Analyses of the relation between spirometric values and cotinine concentrations were based on 2511 children and of the relation between spirometric values and questionnaire measures on 2000 children. RESULTS: Cotinine concentration was negatively associated with all spirometric indices after adjustment for confounding variables, which included age, sex, body size, and social class. The strongest association was with mid expiratory flow rate (FEF50), the fall between the bottom and top fifths of the cotinine distribution being 6%, equivalent to a reduction of 14.3 (95% confidence limits (CL) 8.6, 20.0) ml/s per ng/ml cotinine. Salivary cotinine concentrations were strongly related to exposure to cigarette smoke at home but 88% of children who were from non-smoking households and not looked after by a smoker had detectable cotinine concentrations, 5% being in the top two fifths of the cotinine distribution. A composite questionnaire score based on the number of regular sources of exposure was as strongly related to mid and end expiratory flow rates as the single cotinine measure. The fall in FEF50 per smoker to whom the child was exposed was 51.0 (26.5, 75.5) ml/s. The relationships between the questionnaire score and forced vital capacity (FVC) or forced expiratory volume in one second (FEV1) were not statistically significant. CONCLUSIONS: These effects of passive smoking on respiratory function are consistent with the results of previous studies and, although small in absolute magnitude, may be important if the effects of exposure are cumulative. In children aged 5-7 years the use of a single salivary cotinine concentration as a marker of passive exposure to smoke resulted in clear relationships between exposure and FVC and FEV1, whereas the associations were much weaker and not significant when based on the questionnaire score. The associations between exposure and mid or end expiratory flow rates were of similar magnitude for cotinine concentration and the questionnaire score. The use of salivary cotinine concentration in longitudinal studies may help to determine the extent to which these effects are cumulative or reversible.

Biomarkers↗

PubMed1997

Safety of cotinine in humans: physiologic, subjective, and cognitive effects.

Preliminary data suggest that cotinine, the major metabolite of nicotine, may be behaviorally active. Studies involving the administration of cotinine at doses that produce high blood concentrations (in excess of those produced by cigarette smoking) may be of interest. This inpatient, 10-day human study examined the safety and the effects from several high doses of oral cotinine fumarate (40, 80, or 160 mg) or placebo in abstinent cigarette smokers. All subjects smoked cigarettes ad lib during the first 2 days of the study, then were required to be abstinent beginning on the third day. All subjects were given placebo on this day to wash out nicotine before the administration of cotinine. Subjects were subsequently randomly assigned in a double-blind manner to cotinine or placebo for the next 3 days to determine the safety profile of cotinine. All subjects were given placebo on the final 3 days to examine cotinine withdrawal symptoms. The results showed no significant physiologic, subjective, or performance effects across the various doses of cotinine and placebo. Furthermore, no cotinine withdrawal effects were observed. This study demonstrates that short-term administration of cotinine to humans at levels as high as 10 times that attained from cigarette smoking is safe with no observable acute or withdrawal effects from cotinine in this setting.

Adult↗

PubMed1995

Distribution and retention of nicotine and its metabolite, cotinine, in the rat as a function of time.

Nicotine is oxidized to its major metabolite, cotinine, which has a long biological half-life (19-24 h). The plasma concentration of cotinine has been used as an index of tobacco smoke exposure. Cotinine possibly increases the turnover rate of platelet-activating factor (PAF) because it is a potent activator of PAF hydrolase, and it may play a significant role in tobacco-induced arterial thrombosis. Therefore, we studied the distribution and retention of nicotine as it was metabolized to cotinine in the rat. Nicotine (1 mg/kg, 5 microCi/kg) was administered into the femoral vein of male Sprague-Dawley rats under nembutal anesthesia. At different times (5-60 min) after nicotine administration, nicotine and its metabolite, cotinine, were determined by HPLC in plasma, liver, kidney, heart and brain. Within 5-10 min after administration, nicotine concentrations reached peak values in plasma (2,160 pmol/ml) and the organs analyzed. The plasma level of nicotine decreased by 50% within 20 min (half-time) after its intravenous administration. The half-time of nicotine in the brain was about 50 min. The half-times of nicotine for the other organs were about 20-25 min. The major metabolite, cotinine, accumulated in plasma, and by about 30 min the concentrations of nicotine and cotinine in plasma were about equal (890-1,000 pmol/ml). While cotinine accumulated in plasma, nicotine was eliminated by the kidney. While the nicotine concentrations decreased with time in all organs, cotinine concentrations remained constant. These observations indicate that nicotine is renally eliminated or metabolized to cotinine while cotinine exhibits a long retention time and accumulates in plasma.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Long-term systemic hemodynamic effects of cotinine in rats.

Cigarette smoking has been epidemiologically correlated with lower BP in humans, despite the acute BP effects of nicotine. Cotinine, a primary metabolite of nicotine, has been suggested as a mediator of the BP-lowering effect of smoking. To determine the cardiovascular effects of cotinine itself, arterial BP was monitored continuously in chronically instrumented Sprague-Dawley rats before, during, and after 14 days of intraarterial (i.a.) infusion of cotinine or placebo. Cardiovascular data were collected and analyzed by microcomputer for diurnal changes and for light- and dark-cycle averages. Heart rate (HR) was higher in both precotinine and placebo groups during the dark cycle than during the light cycle. HR was significantly lower (p < 0.025) during cotinine infusion in the cotinine group as compared with placebo. Five days after cotinine infusion, however, HR was not different from that of placebo controls. Arterial BP was not different between cotinine- and placebo-treated rats at any time period. The difference between HR but not arterial BP suggested that baroreceptor activity might differ after cotinine administration. Baroreceptor activity, assessed by analysis of HR changes evoked by phenylephrine, did not differ before and during cotinine administration, however. We conclude that cotinine did not decrease BP in rats under the present experimental conditions, but that cotinine was probably responsible for the observed bradycardia.

Animals↗

PubMed1996

Characterization of CYP2A6 involved in 3'-hydroxylation of cotinine in human liver microsomes.

Nicotine is primarily metabolized to cotinine, and cotinine is further metabolized to trans-3'-hydroxycotinine in human liver, which is a major metabolite of nicotine in humans. We studied the formation of trans-3'-hydroxycotinine from cotinine in human liver microsomes. trans-3'-Hydroxycotinine formation demonstrated single enzyme Michaelis-Menten kinetics (Km, 234.5 +/- 26.8 MicroM; Vmax, 37.2 +/- 2.4 pmol/min/mg protein). Significant correlation (r = .967, P < .001) between cotinine 3'-hydroxylase activities at low (50 microM) and high (1 microM) cotinine concentrations in 20 human liver microsomes suggested the contribution of a single enzyme to cotinine 3'-hydroxylation. The cotinine 3'-hydroxylase activity correlated significantly with immunoreactive cytochrome P450 (CYP)2A6 contents (r = .756, P < .01) and coumarin 7-hydroxylase activity (r = .887, P < .001). The cotinine 3'-hydroxylase activity was inhibited by coumarin, alpha-naphthoflavone, chlorzoxazone and anti-rat CYP2A1 antibodies. Microsomes of B-lymphoblastoid cells expressing human CYP2A6 exhibited cotinine 3'-hydroxylase activity. The Km value of the expressed CYP2A6 (264.7 microM) was almost identical to that of human liver microsomes. In conclusion, cotinine 3'-hydroxylation appears to be catalyzed solely by CYP2A6 in humans. Cotinine is a candidate for a new substrate for CYP2A6 in humans.

Aryl Hydrocarbon Hydroxylases↗

PubMed1995

Is serum cotinine a better measure of cigarette smoking than self-report?

OBJECTIVES: To address the question of whether serum cotinine is a better measure of cigarette smoking than self-reported behavior by examining the relation of biochemical, physical examination, and depression assessments to self-reported cigarette consumption and serum cotinine in a population-based sample. METHODS: Serum from 743 Mexican American participants in the Hispanic Health and Nutrition Examination Survey (HHANES) categorized by sex and number of cigarettes smoked per day (0, 1 to 9, 10 to 19, > or = 20) was analyzed for cotinine. HHANES results from hematocrit, hemoglobin, red blood cells (RBCs), white blood cells (WBCs), mean corpuscular volume (MCV), iron, transferrin, lead, erythrocyte protoporphyrin (EPP), vitamin E, vitamin A, cholesterol, body mass index (BMI), pulse rate, systolic and diastolic blood pressure (DBP), Center for Epidemiological Depression Scale (CES-D), and Diagnostic Interview Schedule (DIS) depression diagnosis were compared by category of cigarettes smoked per day and serum cotinine. RESULTS: Among women significant correlations were found between cigarettes per day and cotinine, respectively, and hematocrit (r = 0.148, r = 0.338), hemoglobin (r = 0.152, r = 0.342), WBCs (r = 0.160, r = 0.272), and BMI (r = -0.124, r = -0.164). Among men significant correlations were found between cigarettes per day and cotinine, respectively, and WBCs (r = 0.176, r = 0.296), MCV (r = 0.310, r = 0.264), lead (r = 0.105, r = 0.177), and BMI (r = -0.110, r = -0.192). Cotinine, but not cigarettes per day, was significantly correlated with hemoglobin (r = 0.179) and DBP (r = -0.146) in men and EPP (r = -0.135) and cholesterol (r = 0.105) in women. Mean CES-D score was correlated with cigarettes per day for both men (r = 0.106) and women (r = 0.158) but not with cotinine. CES-D caseness (score > or = 16) and a positive diagnosis of depression by DIS was not related to smoking behavior measures among men. Women smokers compared to nonsmokers had higher levels of depression. Multivariate regression models controlling for sex, age, and education indicated that serum cotinine was a significant predictor of hematocrit, hemoglobin, RBCs, WBCs, lead, and DBP; self-reported cigarettes was significant only for MCV. CONCLUSIONS: Serum cotinine may be a better method of quantifying risks from cigarette use in epidemiological studies.

Adult↗

PubMed1989

Idiotype-anti-idiotype hapten immunoassays: assay for cotinine.

Practical application of the idiotype-anti-idiotype reaction to hapten immunoassays has been demonstrated with cotinine as an example. The assay relies on the ability of cotinine, a major nicotine metabolite, to inhibit binding between a monoclonal anti-cotinine antibody (the idiotype) and a second monoclonal antibody (the anti-idiotype) specific for the antigen combining region on the idiotype. A solid phase enzyme-linked immunoadsorbent assay (ELISA) format was adopted in which fluid phase anti-cotinine and cotinine present either as a standard or in a test sample were incubated in microtiter plate wells coated with F(ab')2 fragments of the anti-idiotype. Horseradish peroxidase-labeled protein A and o-phenylenediamine were used to detect idiotype-anti-idiotype binding. Under optimal assay conditions, 0.9 ng cotinine inhibited immune binding by 50% and as little as 0.04 ng could be detected. In contrast, nearly 70 times more trans-3'-hydroxycotinine, a major urinary metabolite, and over 1000-fold more nicotine were required for 50% inhibition. Several other metabolites and structurally related compounds also were poor competitors. Assay reliability was good over a range of cotinine concentrations from 5 to 500 ng/ml saliva with intraassay coefficients of variation between 6 and 10% and interassay values between 6 and 13%. Also, there was a strong correlation (R2 = 0.994) between the cotinine levels found in saliva from 35 cigarette smokers with the idiotype-anti-idiotype assay and a cotinine-anti-cotinine ELISA. Because only monoclonal antibodies and antigen are required, the idiotype-anti-idiotype immunoassay offers a high degree of standardization without the need to prepare labeled hapten derivatives or macromolecular conjugates for solid phase assays.

Adult↗

PubMed1987

Comparison of monoclonal and polyclonal antibodies to cotinine in nonisotopic and isotopic immunoassays.

Monoclonal antibodies (McAb) were used to develop nonisotopic and radioimmunoassays (RIA) for quantitative determination of the major nicotine metabolite, cotinine, in physiological fluids. ELISAs and fluorescence immunoassays were carried out in microtiter plate wells coated with a conjugate of cotinine 4'-carboxylic acid bound covalently to poly-L-lysine. The detection systems were horseradish peroxidase (HRP)-labeled staphylococcal protein A, HRP-streptavidin-biotin, and biotinylated alkaline phosphatase-4-methylumbelliferyl phosphate. With the three McAb tested, I50 values ranged between 0.024-0.063 ng cotinine and as little as 0.005-0.015 ng gave 15% inhibition. These assays were 5-20 times more sensitive than similar assays using six rabbit antisera. With McAb the standard inhibition curves were steeper and complete inhibition of immune binding was achieved with approximately 1 ng cotinine. In contrast, 100-500 ng cotinine failed to give greater than 80-90% inhibition with rabbit antibodies either in the plate assays or in RIA using a 125I-labeled tyramine derivative of cotinine as the tracer. In this RIA, the sensitivity with McAb (mean I50 of 0.55 ng cotinine) was over three-fold greater than with rabbit antisera (mean I50 of 1.84 ng). The presence of antibodies directed to the amide linkage group common to the polylysine conjugate. 125I-tyramine derivative and the immunogen likely accounts for the inferior quality of assays using rabbit antisera. Consistent with this conclusion, superimposable inhibition curves were obtained in the RIA when monoclonal or rabbit antibodies were used with [3H]cotinine. Cotinine levels in saliva, serum and plasma from smokers and non-smokers determined with McAb-based assays showed a strong correlation with values obtained by RIA using rabbit antisera or by gas chromatography. Properly selected McAb offer distinct advantages over conventional antisera in nonisotopic immunoassays and RIAs for cotinine as a biochemical marker of active or passive smoking.

Antibodies↗