Major Metabolite Of Nicotine Research Articles

Efficacy of Providing Nicotine in a Liquid Diet to Rats

To determine if rats would consume nicotine at psychoactive levels, a nutritionally balanced diet with 0, 20, 60, or 200 mg of nicotine tartrate per kg of diet was provided. Diet consumption and body weight differences were recorded for 14 days after which, following 16 hr of withdrawal, animals were given access to a two-bottle choice of the previously presented diet and a nicotine-free diet. Spontaneous horizontal motor activity was recorded 8, 16, and 24 hr after withdrawal. By Day 14, all animals showed a significant increase in diet consumption and significant weight gain compared to Day 1. Animals consumed an average of 2.1, 6.8, or 19.5 mg/kg/day of nicotine on the low, medium, and high-nicotine diets, respectively. However, animals receiving the high-nicotine diet consumed less diet and gained less weight than the control, low, and medium nicotine groups. During only the first 4 hr of the two-bottle choice (16–20 hr postwithdrawal), the high-nicotine group consumed significantly higher amounts of nicotine base than the other groups, but also consumed more of the control diet during the first 2 hr. In a replicate experiment, animals receiving the medium-nicotine diet showed an increased consumption of the nicotine diet and increased preference for nicotine following a 14-day exposure compared to the controlfed animals and compared to a baseline preference test. Also, this group showed differences in locomotor activity consistent with other studies using an injection regimen or subcutaneuos pumps to induce dependence. Finally, animals in all three groups exhibited high plasma nicotine and cotinine (a major nicotine metabolite) levels. Because animals in all groups tolerated the diet well, gained weight, selected the nicotine diet in a choice test, and showed withdrawal symptoms, we conclude that the liquid diet proved to be a satisfactory method of inducing nicotine dependence in rats.

Characterization of multiple products of cytochrome P450 2A6-catalyzed cotinine metabolism.

The primary metabolite of nicotine in smokers is cotinine. Cotinine is further metabolized to trans-3'-hydroxycotinine, the major urinary metabolite of nicotine in tobacco users. It was recently reported that cytochrome P450 2A6 catalyzes the conversion of cotinine to trans-3'-hydroxycotinine. In this work, we report that P450 2A6 metabolizes cotinine not only to trans-3'-hydroxycotinine but also to 5'-hydroxycotinine, norcotinine, and a fourth as yet unidentified metabolite. The products of baculovirus-expressed P450 2A6 [methyl-(3)H]cotinine metabolism were analyzed by radioflow HPLC. Three (3)H-labeled metabolites were detected and were present in approximately equal amounts. The identities of two of the metabolites were confirmed to be 5'-hydroxycotinine and trans-3'-hydroxycotinine by LC/MS/MS and LC/MS analysis and comparison to standards. The third product was not identified. A fourth product of P450 2A6-catalyzed cotinine metabolism was detected by LC/MS. It was identified by cochromatography with a standard and MS and MS/MS data to be norcotinine. An attempt was made to further characterize the unidentified (3)H-labeled metabolite by comparison to the cotinine metabolites generated by hamster liver microsomes. Hamster liver microsomes contain a P450, 2A8, which is closely related to P450 2A6, and have previously been shown to metabolize cotinine to three hydroxylated products, trans-3'-hydroxycotinine, 5'-hydroxycotinine, and N-(hydroxymethyl)norcotinine. We were unable to confirm that N-(hydroxymethyl)norcotinine was the unidentified cotinine metabolite generated by P450 2A6.

Prostaglandin E2 in Human Placenta: Its Vascular Effects and Activation of Prostaglandin E2 Formation by Nicotine and Cotinine

Tobacco smoking by pregnant women increases the frequency of spontaneous abortions and preterm births. Human labor is associated with enhanced intrauterine phospholipid metabolism and production of prostaglandin E₂ (PGE₂) which induces labor, initiates uterine contractions and maintains the homeostasis of placental blood flow. Therefore, we studied: (a) the influence of nicotine and cotinine on the effects of PGE₂ on placental vasculature in perfused human placental cotyledon, and (b) the activation of placental phospholipase A₂ (PLA₂) by nicotine and cotinine using 1-palmitoyl-2-[1-¹⁴C]arachidonyl-phosphatidylethanolamine (PE, 2.2 nmol) as substrate. These studies revealed that: (1) increasing concentrations of PGE₂ (10– 150 ng/ml) increased umbilical perfusion pressure by 170 ± 10% (n = 6) of control (100%). Cotinine (2 µg/ml) enhanced this effect at all concentrations of PGE₂. Nicotine (2 µg/ml) prevented the effect of PGE₂; (2) both cotinine (EC₅₀ 470–500 fmol/l) and nicotine (EC₅₀ 18–32 pmol/l) activated PLA₂ in human placental tissues. These observations indicated that cotinine was more potent than in nicotine activating PLA₂ and potentiating the vasoconstrictive effects of PGE₂ on fetal placental circulation. Nicotine activates nicotinic receptors and releases placental acetylcholine, a vasodilator of placental arteries. Acetylcholine stimulates muscarinic receptors of endothelial cells resulting in the release of endothelium-derived relaxing factor (EDRF), and possibly nitric oxide. Therefore, nicotine prevents or abolishes the vasoconstrictive effects of PGE₂ through the release of EDRF. Cotinine is inactive at nicotinic and muscarinic receptors. Therefore, accumulation of cotinine, the major metabolite of nicotine, in fetal circulation may contribute to production of PGE₂ and induction of preterm labor and spontaneous abortions.

The effect of cotinine or cigarette smoke co-administration on the formation of O6-methylguanine adducts in the lung and liver of A/J mice treated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)

4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a tobacco-specific nitrosamine, induces lung adenomas in A/J mice, following a single intraperitoneal (i.p.) injection. However, inhalation of tobacco smoke has not induced or promoted tumors in these mice. NNK-induced lung tumorigenesis is thought to involve O6-methylguanine (O6MeG) formation, leading to GC-->AT transitional mispairing and an activation of the K-ras proto-oncogene in the A/J mouse. NNK can be metabolized by several different cytochromes P450, resulting in a number of metabolites. Formation of the promutagenic DNA adduct O6MeG is believed to require metabolic activation of NNK by cytochrome P450-mediated alpha-hydroxylation of the methylene group adjacent to the N-nitroso nitrogen to yield the unstable intermediate, methanediazohydroxide. Nicotine, cotinine (the major metabolite of nicotine), and aqueous cigarette tar extract (ACTE) have all been shown to effectively inhibit metabolic activation of NNK to its mutagenic form, most likely due to competitive inhibition of the cytochrome P450 enzymes involved in alpha-hydroxylation of NNK. The objective of the current study was to monitor the effects of cotinine and cigarette smoke (CS) on the formation of O6MeG in target tissues of mice during the acute phase of NNK treatment. To test the effect of cotinine, mature female A/J mice received a single intraperitoneal injection of NNK (0, 2.5, 5, 7.5, or 10 mumole/mouse) with cotinine administered at a total dose of 50 mumole/mouse in 3 separate i.p. injections, administered 30 min before, immediately after, and 30 min after NNK treatment. To test the effect of whole smoke exposure on NNK-related O6MeG formation, mice were exposed to smoke generated from Kentucky 1R4F reference cigarettes at 0, 0.4, 0.6, or 0.8 mg wet total particulate matter/liter (WTPM/L) for 2 h, with a single i.p. injection of NNK (0, 3.75, or 7.5 mumole/mouse) midway through the exposure. Cigarette smoke alone failed to yield detectable levels of O6MeG. The number of O6MeG adducts following i.p. injection of NNK was significantly (p < 0.05) reduced in both lung and liver by cotinine and by cigarette smoke exposure. Our results demonstrate that NNK-induced O6MeG DNA adducts in A/J mice are significantly reduced when NNK is administered together with either cotinine, the major metabolite of nicotine, or the parental complex mixture, cigarette smoke.

A New Gas Chromatography–Mass Spectrometry Method for Simultaneous Determination of Total and Free trans-3′-Hydroxycotinine and Cotinine in the Urine of Subjects Receiving Transdermal Nicotine

trans-3'-Hydroxycotinine (THOC) has been recognized as the most abundant metabolite of nicotine. In an attempt to assess THOC and cotinine (COT) concentrations during nicotine transdermal therapy, we developed a new quantitative gas chromatography-mass spectrometry (GC-MS) method for simultaneous determination of total and free THOC and COT in human urine. The method utilizes the following: (a) hydrolysis of conjugated THOC and COT by beta-glucuronidase; (b) basic extraction of THOC and COT with mixed dichloromethane and n-butyl acetate; (c) derivatization of THOC with bis(trimethylflurosilyl)acetamide; and (d) separation and identification by GC-MS with selective ion monitoring. Lower limits of quantification for the assay were 50 and 20 microg/L for THOC and COT, respectively. The intra- and interassay CVs were 4.4% and 11% for THOC, and 3.9% and 10% for COT at 1000 microg/L. The results from six consecutive 24-h urine collections in 71 subjects administered daily transdermal nicotine doses of 11, 22, and 44 mg showed that, on average, free THOC was 76% of total THOC and free COT was 48% of total COT in all subjects. THOC is the major metabolite of nicotine and constitutes 20% of total nicotine intake at steady state, whereas urinary nicotine and COT excretion were 8% and 17%, respectively. The method is useful for simultaneous determination of free and total THOCand COT and can be used to assess the urinary excretion of these metabolites during transdermal nicotine therapy.

Inhibition of nicotinic responses by cotinine in bovine adrenal chromaffin cells.

We studied the effects of cotinine, the major metabolite of nicotine, on nicotine-induced increase in [3H]phorbol dibutyrate binding, activation of protein kinase C and [3H]noradrenaline release in primary cultured bovine adrenal chromaffin cells. Cotinine (1 mM, 15 min.) and nicotine (10 microM, 5 min.) increased the [3H]phorbol binding by 100% and 150%, respectively. Both a short-term (10 min.) and a long-term (24 hr) pretreatment with cotinine inhibited the effect of nicotine. A 24 hr pretreatment with cotinine (1 mM) also reduced the nicotine-induced increase in membrane-bound protein kinase C activity. Cotinine pretreatment (10 min.) dose-dependently inhibited the release of [3H]noradrenaline induced by nicotine and dimethylphenylpiperazinium. Cotinine pretreatment did not reduce the [3H]noradrenaline release induced by high extracellular potassium (56 mM) or veratrine (10 mg l-1). The results indicate that cotinine inhibits activation of protein kinase C and noradrenaline release induced by nicotinic agonists in primary cultures of bovine adrenal chromaffin cells. The results suggest that pre-existing cotinine could modify responses to acute exposure to nicotine in neural systems.

Ambulatory 24-h blood pressure monitoring in healthy, middle-aged smokeless tobacco users, smokers, and nontobacco users

Ambulatory 24-h blood pressure monitoring was conducted in 135 healthy, normotensive, middle-aged (35 to 60 years) men, with no antihypertensive medication, to study the influence of habitual smokeless tobacco use (n = 47) and smoking (n = 29) on diurnal blood pressure and heart rate. Comparisons were made with nonusers of tobacco (n = 59). Adjustments were made for differences in age, body mass index, waist-hip ratio, physical fitness, and alcohol intake. Daytime ambulatory heart rates were significantly ( P < .05) elevated in both smokeless tobacco users and smokers compared with nonusers (69 ± 14 and 74 ± 13 beats/min, respectively, versus 63 ± 12 beats/min). In subjects ≥ 45 years old, ambulatory daytime diastolic blood pressures were significantly elevated, on average by 5 mm Hg, in both smokeless tobacco users and smokers ( P < .001) compared with nonusers. Clinical measurements of heart rate and systolic blood pressure in smokers were significantly lower compared with the ambulatory mean values. Nighttime measurements showed only minor differences between the tobacco habit groups. The higher heart rates and blood pressures noted during the daytime in smokers and smokeless tobacco users were most likely due to the effects of nicotine. A strong positive relationship was found between cotinine (major nicotine metabolite) and blood pressure in smokeless tobacco users (systolic blood pressure, r = 0.48, P < .001; diastolic blood pressure, r = 0.41, P = .005), whereas an inverse relationship was found in smokers (systolic blood pressure, r = −0.12, P = .47; diastolic blood pressure, r = −0.03, P = .84), indicating additional and more complex influences on vascular tone in smokers than the influence of nicotine in smokeless tobacco users.

The Elimination Half-Life of Urinary Cotinine in Children of Tobacco-Smoking Mothers

Exposure to environmental tobacco smoke (ETS) is strongly associated with childhood morbidity. Cotinine, the major metabolite of nicotine, is a useful marker of tobacco smoke exposure. Cotinine levels in infants are higher than in older children or adults exposed to the same reported quantity of ETS. One hypothesis to explain this difference is that the urinary elimination half-life of cotinine is different between infants and older children. Urine was collected at admission, 12, 24 and 48 h, cotinine levels were subsequently measured and then standardized by correcting for creatinine excretion. Urinary elimination half-life of cotinine was calculated in 31 infants and 23 older children. The median half-life was 28.3 h (range 6.3–258.5 h) in infants, and 27.14 h (range 9.7–99.42 h) in older children. A Mann–Whitney U test showed no significant difference in the median half-life of cotinine between the two age groups (P=0.18). Multivariate linear regression analysis demonstrated no significant relationship between half-life of cotinine and corrected cotinine level (P=0.24). Our results support the hypothesis that higher cotinine levels in infants is due to greater exposure, rather than slower metabolism of cotinine.

Active and passive exposure status to tobacco smoke of department store employees measured by cotinine ELISA

Quantitation of urinary cotinine, a major metabolite of nicotine, by an enzyme-linked immunosorbent assay (ELISA), was performed in parallel with questionnaires containing items on smoking status, such as active and/or passive smokers, the number of cigarettes smoked, and the presence or absence of active smokers in the surroundings in a department store (517 employees). The cotinine values corrected by creatinine (cotinine-creatinine ratios, CCRs) approximately conformed to the extent of self-recognition of their exposure status to tobacco-smoke, and were low in the order of active smokers, passive smokers and non-smokers who felt they were not exposed to tobacco-smoke. Occupational differences of the CCRs were not found in the employees.In the active smokers, the CCRs were increasing according to the number of cigarettes per day they smoked, and the values were nearly proportional to nicotine contents of cigarette in the moderate smokers who smoked 11-20 cigarettes per day. The CCRs of males were higher than those of females in the active smokers, which also agreed well with the numbers of cigarettes they smoked per day. In the passive smokers, the CCRs were remarkably and significantly higher in subjects who felt they were exposed to tobacco-smoke both in their workplaces and homes.Urinary CCRs measured by ELISA are thus found to be a reliable and excellent objective indicator of both active and passive exposure-status to tobacco-smoke.

Development of a simple and rapid elisa of urinary cotinine for epidemiological application.

Highly stereospecific polyclonal antibodies (anti-CN) to cotinine (CN), a major metabolite of nicotine, were prepared from rabbit antisera to CN-linked keyhole limpet hemocyanin (KLH) by removing the antibodies to KLH and to its binding regions of CN. This was achieved by using immunoadsorbents consisting of insolubilized KLH onto CNBr-activated-Sepharose 4B. A new simple and rapid enzyme-linked immunosorbent assay (ELISA) of urinary CN was developed using the anti-CN. A brief outline of the method is as follows: CN-bovine thyroglobulin complex is coated onto wells of microtiter plates (1 ng/well), and then aliquots of urine samples or standard CN solutions were added followed by appropriate dilution of the anti-CN. The bound anti-CN antibodies are quantified spectrophotometrically with horseradish peroxidase-labelled anti-rabbit IgG and 2,2'-azino-di (3-ethylbenz- thiazoline)-6-sulfonic acid. Measurement of CN concentration in urine samples can be read off on a calibration curve drawn by using standard CN solutions. The standard curve ranged from 1 ng to 4 μ g /ml with an estimated lower limit of sensitivity of 7-8 ng/ml, resulting in within/between-assay CV (coefficient of variation) of lower than 10%. The method allowed one to assay more than 40 samples in duplicate by using just one plate, and is thus easily applicable to epidemiological investigations into exposure status to tobacco smoke.

Interovarian differences in levels of cotinine, a major metabolite of nicotine, in women undergoing IVF who are exposed to cigarette smoke.

Our purpose was to determine whether there is variation in levels of follicular fluid (FF) cotinine between the two ovaries of women undergoing IVF-ET who are exposed to cigarette smoke. In 61 women, there were two to four determinations of FF continine levels for each of two follicles, one from each ovary. For each woman a t test for significant difference between the means of both ovaries was done to test for interovarian variation. Thirty-seven nonsmokers, 8 passive smokers, and 16 active smokers differed greatly (P < 0.0001) in mean FF cotinine levels: 13.0, 91.1, and 420.3 ng/ml, respectively. Fourteen women had significant differences, at the P = 0.025 level or below, between their two ovaries. Five of them had differences significant at the 0.001 level. Even so, the correlation between the cotinine levels of the two ovaries was high. Cotinine uptake between the two ovaries of a woman may differ approximately one-fourth of the time. In spite of these differences, the overall correlation between ovaries is high. The clear distinction in levels of FF cotinine among active, passive, and nonsmokers demonstrates the reliability of FF cotinine testing. Detection of cotinine in a large proportion of nonsmokers shows how pervasive nicotine is in the environment.

Nicotine induces leukocyte rolling and adhesion in the cerebral microcirculation of the mouse

Nicotine and several related metabolites were examined for their ability to induce leukocyte rolling and adhesion in the cerebral microcirculation of the mouse. A cranial window was surgically prepared for the visualization of the pial microcirculation using an intra-vital microscopy imaging system. Using this technique rhodamine-labeled leukocytes could be visualized and video-recorded as they traveled within the microvessels, and the quantitation of their rolling and adhesion along the pial venule walls was assessed during an off-line video playback analysis. Nicotine was found to produce significant dose-related increases in leukocyte rolling and adhesion. Cotinine, a major nicotine metabolite, did not induce the same degree of leukocyte rolling and adhesion. Mecamylamine, a nicotine antagonist, was found to inhibit the nicotine-induced leukocyte rolling and adhesion. Anti-P-selectin antibody blocked nicotine-induced leukocyte rolling, while anti-CD18 antibody effectively inhibited leukocyte adhesion, but not rolling in similar experiments. Nicotine-induced leukocyte rolling and adhesion were also inhibited by superoxide dismutase and catalase. These data suggest that nicotine, the principle pharmacological agent in cigarette smoke and related tobacco products, acts via a ganglionic-type nicotinic receptor to enhance leukocyte rolling via P-selectin and reactive oxygen radical-dependent mechanisms in cerebral microcirculation of the mouse.

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.

Immunodetection of cotinine protein in granulosa-lutein cells of women exposed to cigarette smoke

To detect immunoreactivity to cotinine protein, a major metabolite of nicotine, in granulosa-lutein cells from patients exposed to cigarette smoke, as measured by levels of cotinine in follicular fluid (FF) samples. Controlled immunocytochemical study. Hospital IVF-ET program treating infertile patients. Twenty-eight women classified by self-reported smoking habits: active smokers (n = 17), passive smokers (n = 4), and nonsmokers (n = 7). Ovarian hyperstimulation. Grades of immunostaining intensity were assessed in granulosa-lutein cells. Patient scores of cell immunostaining were calculated and regressed on levels of FF cotinine. Cotinine levels in FF were higher in active smokers than in passive smokers or nonsmokers. Cotinine immunostaining was visualized in the nucleus and cytoplasm of granulosa-lutein cells. Mean grades and mean scores of immunostaining intensity were higher in active smokers than in passive smokers or nonsmokers. There was a strong positive correlation between scores of cell immunostaining and FF cotinine levels. The association between cotinine expression in granulosa-lutein cells and FF cotinine provides reliable evidence for a dose-related effect. This constituent of cigarette smoke appears to interact directly with and incorporate into these ovarian cells. Our approach seems useful for monitoring ovarian exposure to environmental toxins.

尿中ニコチン代謝物の定量によるタバコ煙暴露の評価と臨床応用

It is well known that tobacco smoke causes the health risks not only for smokers but for non-smoker as well. We thus evaluated the urinary cotinine, content, which is a major metabolite of nicotine, by high-performance liquid chromatography (HPLC). Such a determination is considered to provide a useful objective assessment of exposre to tobacco smoke. Using the Previously described method, non-smokers in a typical company were thus found to appareutly demonstrate cotinine concentrations due to passive smoking, Furthermore we also determined the urinary cotinine in an asthma patient who could not stop smoking. The results indicated that the measurement of the urinary cotinine level by HPLC could be effective as a strong motivating factor to stop smoking and also to form a system of nosmoking support groups. In the future, such continuous study will enable us to approximate the effect of passive-smoking on a disease based on the measurement of the urinary cotinine level.

Interference of Nicotine Metabolites in Cotinine Determination by RIA

Cotinine (COT), a major metabolite of nicotine (NIC), has been used as a biomarker in many studies of active and passive smoking (1)(2)(3)(4). Methods of analysis for COT in biological fluids include gas chromatography, gas chromatography–mass spectrometry, HPLC, HPLC–mass spectrometry, and immunoassays (5)(6)(7)(8)(9)(10). In particular, the RIA developed for COT and NIC by Langone and Van Vunakis (11) was the method reported by the International Agency for Research on Cancer (IARC) for determination of these analytes in several biological matrices (12)(13). In the description of the assay (11), no mention was made about cross-reactivity of anti-NIC and anti-COT antibodies with trans -3′-hydroxycotinine (THOC) and cotinine glucuronide (COT-G). Indeed, these two compounds were found to be the most abundant metabolites of NIC in urine of smokers (14). Other studies, however, have investigated the cross-reactivity of the anti-COT antiserum with THOC and other metabolites in an ELISA (15). The objective of this study was to investigate the cross-reactivity of the anti-COT and anti-NIC antisera with THOC and COT-G in the RIA (11)(12)(13). We also used urine samples from active and passive smokers to compare data from the RIA with data obtained by HPLC. The rabbit antiserum, (−)[3H]COT, and (−)[3H]NIC were …

Cotinine, a major metabolite of nicotine, is detectable in follicular fluids of passive smokers in in vitro fertilization therapy

To assess cotinine, a metabolite of nicotine, in follicular fluids (FF) of women who smoke either actively or passively or not all. Controlled clinical study. Infertile patients in a hospital IVF-ET program. One hundred eleven women classified by smoking habits: active smokers (n = 44), passive smokers (n = 17), or nonsmokers (n = 50). Ovarian hyperstimulation. Cotinine levels in FF. A strong correlation between number of cigarettes smoked and levels of FF cotinine was found. The levels of FF cotinine were: active smokers 710.4 +/- 128.2, passive smokers 76.3 +/- 56.5, and nonsmokers 4.2 +/- 2.0 ng/mL (mean +/- SEM). The level in active smokers was significantly greater than in other groups. The levels of FF cotinine in passive smokers differed significantly from nonsmokers. Eighty-four percent of nonsmokers actually were exposed to nicotine, with a mean value of 5.0 ng/mL. Cotinine was detectable in a dose-dependent manner in active and passive smokers. It was detected in all active smokers and in a majority of passive smokers and self-reported nonsmokers. A strong interindividual variation suggests differences in metabolism and smoking habits. Follicular fluid cotinine assessments are useful for infertility studies.

Serum Cotinine Levels, Smoking, and Periodontal Attachment Loss

Cigarette smoking and tobacco use have been the subjects of numerous studies for many years. Smoking has also been associated with periodontal disease. However, no relationship between a reliable biochemical marker and increased severity of the periodontal condition has yet been described. It was thus the aim of this study to apply the measurement of cotinine, the major metabolite of nicotine, as a quantitative method to assess levels of smoking, and to correlate serum levels of cotinine with severity of periodontal disease. The degree of association between smoking and periodontal attachment loss was investigated in a study including 79 patients 25 to 64 years old suffering from periodontitis. Patients were examined and the following parameters recorded: Gingival Assessment (GA), Probing Pocket Depth (PPD), Clinical Attachment Level (CAL), and Bone Crest Height (BCH). In addition, self-reported histories of tobacco use as well as blood samples for quantitative analysis of serum levels of cotinine were taken. The serum samples were analyzed for cotinine content by means of a competitive-inhibition ELISA technique. The differences in mean cotinine levels were statistically significant (p = 0.0001) between smokers and non-smokers, showing no overlap between the groups. Severity of periodontal attachment loss was positively correlated with serum levels of cotinine for both measures of periodontal disease (CAL p = 0.005; BCH p = 0.008). Results from the present study indicate that serum cotinine levels used as a biochemical marker of smoking status are correlated with severity of periodontal attachment loss.

Nicotine inhibits amyloid formation by the beta-peptide.

The 42-residues beta-(1-42) peptide is the major protein component of amyloid plaque cores in Alzheimer's disease. In aqueous solution at physiological pH, the synthetic beta-(1-42) peptide readily aggregates and precipitates as oligomeric beta-sheet structures, a process that occurs during amyloid formation in Alzheimer's disease. Using circular dichroism (CD) and ultraviolet spectroscopic techniques, we show that nicotine, a major component in cigarette smoke, inhibits amyloid formation by the beta-(1-42) peptide. The related compound cotinine, the major metabolite of nicotine in humans, also slows down amyloid formation, but to a lesser extent than nicotine. In contrast, control substances pyridine and N-methylpyrrolidine accelerate the aggregation process. Nuclear magnetic resonance (NMR) studies demonstrate that nicotine binds to the 1-28 peptide region when folded in an alpha-helical conformation. On the basis of chemical shift data, the binding primarily involves the N-CH3 and 5'CH2 pyrrolidine moieties of nicotine and the histidine residues of the peptide. The binding is in fast exchange, as shown by single averaged NMR peaks and the lack of nuclear Overhauser enhancement data between nicotine and the peptide in two-dimensional NOESY spectra. A mechanism is proposed, whereby nicotine retards amyloidosis by preventing an alpha-helix-->beta-sheet conformational transformation that is important in the pathogenesis of Alzheimer's disease.

Patterns of cigarette consumption and cotinine levels among African American women smokers.

Cigarette smoking intensifies a number of serious health problems, including lung cancer, hypertension, low birth weight, and infant mortality, that disproportionately affect black Americans. Cotinine, a major nicotine metabolite, is one indicator of smoke exposure. It has been reported that black women have higher mean cotinine levels than white women. This divergence may be attributed to biologic factors or to inaccuracy in reporting cigarette use. The purpose of this study was to characterize nicotine dependence and the relationship between self-reported cigarette use and cotinine levels among black women smokers. The sample consisted of 142 black women cigarette smokers recruited individually at urban health centers and worksites. A cotinine/cigarette ratio was determined for light, moderate, and heavy smokers. Underreporting of cigarette consumption, previously defined as cotinine value > 25 ng/ml/cigarette, ranged from 86% among light smokers to 70% among moderate smokers and 21% among heavy smokers. There were significant differences in cotinine/cigarette and nicotine dependence scores across levels of smoking. Average cotinine/cigarette values were higher in black women compared with previous reports for Mexican American women smokers. No comparable values are available for white women. Additional study is needed to begin to explain variations in levels of cotinine, as well as perceived nicotine dependence among black women.