The direct barbituric acid assay for nicotine metabolites in urine: a simple colorimetric test for the routine assessment of smoking status and cigarette smoke intake

The qualitative direct barbituric acid (DBA) method of detecting urine nicotine metabolites was modified to make it quantitative. The performance of the quantitative DBA method was compared with the qualitative method and an established cotinine radio-immunoassay (RIA), using a panel of urines from 128 reported smokers and 383 reported non-smokers. The quantitative DBA method results were highly correlated with the cotinine RIA results, r = 0.85. The coefficients of variation for the two methods were 6% and 10%, respectively. Assuming that the reported smoking history was correct the qualitative DBA method gave a smoking detection rate of 91% and a false positive rate of 3%. At cut-off levels chosen to yield the same false positive rate the quantitative DBA method detected 93% of smokers, close to that of 98% detected with the cotinine RIA. The quantitative DBA method can be used to analyse over 170 samples per day compared to about 70 per day by RIA. It is therefore a fast and inexpensive alternative to cotinine assays for the assessment of smoking status and cigarette smoke intake.     

An automated colorimetric assay for urine nicotine metabolites: a suitable alternative to cotinine assays for the assessment of smoking status

We have modified the direct barbituric acid (DBA) test for urine nicotine metabolites so that it can be used on a continuous flow auto-analyser. The performance of the method was compared with a cotinine radio-immunoassay (RIA). The auto-analyser DBA method and cotinine RIA performed equally well in terms of assessing smoking status, yielding a false positive rate of 1.5% at a detection rate of 98% when compared to self-reported smoking information. The results obtained with the two methods were well correlated, r = 0.91, although the DBA method results were consistently higher than the corresponding cotinine RIA results; the method detects cotinine and other nicotine metabolites. The coefficient of variation for the DBA method was 3.4% compared with 10% for the RIA. The DBA method has a throughput of about 250 samples per day compared to about 70 per day by RIA and the reagents are readily obtained and inexpensive.     

Urinary biomarkers for secondhand smoke and heated tobacco products exposure

Concerns have recently grown about the health effects of secondhand smoke exposure and heated tobacco products. The analysis of tobacco smoke biomarkers is critical to assess the health effects of tobacco smoke exposure. For this purpose, the simultaneous determinations of exposure markers and health effect markers would provide a better evaluation of smoke exposure. In this study, nicotine metabolites (nicotine, cotinine, trans-3''-hydroxycotinine) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol in urine were analyzed as exposure markers. The DNA damage markers, 7-methylguanine and 8-hydroxy-2''-deoxyguanosine, were simultaneously measured as health effect markers. The results revealed significant levels of urinary nicotine metabolites and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol in the subjects exposed to secondhand smoke and heated tobacco products. In addition, the urinary levels of 7-methylguanine and 8-hydroxy-2''-deoxyguanosine tended to be high for secondhand smoke and heated tobacco products exposures, as compared to those of non-smokers. These biomarkers will be useful for evaluating tobacco smoke exposure.     

Disposition of nicotine and eight metabolites in smokers and nonsmokers: identification in smokers of two metabolites that are longer lived than cotinine

The disposition of a single intravenous dose of 14C-nicotine was investigated in six cigarette smokers and six nonsmokers. Plasma and urinary elimination of both nicotine and cotinine was faster in smokers than in nonsmokers. In the urine of both smokers and nonsmokers, we identified nicotine and eight metabolites, including two new metabolites: metabolite A (3-hydroxycotinine glucuronide) and metabolite G (demethylcotinine delta 2',3'-enamine). Metabolites A and G were of particular interest because, in smokers, they both persisted longer than cotinine. This property renders them more sensitive than cotinine as potential indicators of passive exposure to cigarette smoke.     

Relationship between smoking status and serum lipids in a hyperlipidemic population and analysis of possible confounding factors.

The aim of our study was to estimate the potential relationship between smoking behavior and other coronary heart disease risk factors in 250 hyperlipidemic patients. We present data obtained through self-reporting of the number of cigarettes smoked per day, measurements of three tobacco markers, and data on dietary habits and lipid variables. We measured cotinine (by HPLC) and thiocyanate and used a recent colorimetric assay for the indirect evaluation of the nicotine metabolites in a single urine specimen. Mean values of nicotine metabolites, expressed as cotinine equivalents, were 6.7, 39.9, and 79.4 mumol/L, respectively, for nonsmokers, light smokers (7.7 cigarettes per day), and heavy smokers (25.8 cigarettes per day). We found that light smokers have higher concentrations of cotinine and nicotine metabolites in proportion to the number of cigarettes smoked per day than do heavy smokers. Thus, the simple colorimetric assay can accurately evaluate smoking status. Hyperlipidemia and smoking are linked by an intricate network of multiple relations. The concentration of high-density lipoprotein (HDL) cholesterol is lower in heavy smokers, and the concentrations of triglycerides and cholesterol are higher. The 0.11 mmol/L difference in HDL cholesterol between light and heavy smokers is close to the results of previous papers; however, when gender, dietary habits (including alcohol intake), and data on body mass index are included in a multiple regression analysis, there is no longer an association between HDL cholesterol concentrations and smoking status. Therefore, these different dietary habits may be confounding factors that partly explain the pattern of lipid variables.     

Mentholated cigarette smoking inhibits nicotine metabolism

Smoking mentholated cigarettes has been suggested to convey a greater cancer risk compared with smoking nonmentholated cigarettes. Two of the possible mechanisms by which mentholated cigarette smoking could increase risk are by increasing systemic exposure to tobacco smoke toxins and by affecting the metabolism of nicotine or tobacco smoke carcinogens. To examine these possibilities, we performed a crossover study in 14 healthy smokers, one-half of whom were African-Americans and one-half whites. Subjects were randomly assigned to smoke mentholated or nonmentholated cigarettes for 1 week, then to cross over to the other type of cigarettes for another week. Subjects were confined to a Clinical Research Center for 3 days of each week, during which time blood levels of nicotine and carbon monoxide were measured throughout the day and an intravenous infusion of deuterium-labeled nicotine and cotinine was administered to determine the rate and pathways of nicotine metabolism. The systemic intake of nicotine and carbon monoxide was, on average, not affected by mentholation of cigarettes. Mentholated cigarette smoking did significantly inhibit the metabolism of nicotine (clearance: 1289 versus 1431 ml/min, two sided, p = 0.02). Inhibition of nicotine metabolism occurred both by slower oxidative metabolism to cotinine and by slower glucuronide conjugation. Our data do not support the hypothesis that mentholated cigarette smoking results in a greater absorption of tobacco smoke toxins. Our finding of impaired metabolism of nicotine while mentholated cigarette smoking suggests that mentholated cigarette smoking enhances systemic nicotine exposure.     

Cigarette smoking and hypertension influence nitric oxide release and plasma levels of adhesion molecules.

Progression of atherosclerosis is currently believed to involve interactions between leukocytes and vascular endothelium. Epidemiological risk factors for atherosclerosis such as hypertension and smoking are known to cause endothelial dysfunction, which is an early event in the atherosclerotic process; they also may be considered in the light of their effects on adhesion molecule expression and release. Little is known about the additive effect between these two risk factors on endothelial adhesion molecule expression and nitric oxide release. Soluble adhesion molecules and the nitric oxide were quantified in smoking hypertensive patients in comparison to those from patients with hypertension alone. Cotinine, a stable metabolite of nicotine, has been used to identify smokers. One hundred and three hypertensive patients were selected: 51 smokers (plasma cotinine levels >25 ng/ml) and 52 non-smokers. Plasma concentrations of soluble intercellular cell adhesion molecule-1 (sICAM-1), soluble endothelial leukocyte adhesion molecule-1 (sELAM-1) and soluble vascular cell adhesion molecule-1 (sVCAM-I) were quantified with ELISA methods. Plasma concentration of nitric oxide metabolites was measured by HPLC, whilst plasma concentration of cotinine was measured by RIA. Significant increases of sICAM-1 and sVCAM-1 were demonstrated in smokers (p<0.001 and p<0.05, respectively). In the same patients, a positive significant correlation between sVCAM-1 and plasma cotinine levels was observed (p<0.002). Nitric oxide metabolites were reduced significantly (p<0.04) in smokers. In conclusion, our data show that the two risk factors, smoking and hypertension, are additive risk factors in generating endothelial dysfunction and vascular damage, which plays a key role in atherogenesis.     

Nicotine metabolism and elimination kinetics in newborns

OBJECTIVE: To characterize the presence of and elimination kinetics of nicotine and its metabolites in newborns. METHODS: Blood samples from 13 newborns were collected during the first day of life and analyzed for nicotine and cotinine. Single daily urine samples were collected from nine newborns for up to 7 days and analyzed by gas chromatography-mass spectrometry for nicotine, cotinine, 3'-hydroxycotinine, and their conjugates. NONMEM was used to determine population half-life values. RESULTS: Blood and urine data gave similar results for nicotine and cotinine elimination kinetics. The elimination half-life for nicotine was 11.2 hours (95% confidence interval [CI], 8.0 to 18.9) based on blood data and 9.0 hours (95% CI, 7.0 to 12.4) based on urine data. The elimination half-life for cotinine was 16.3 hours (95% CI, 12.4 to 23.9) based on blood data and was 22.8 hours (95% CI, 19.5 to 25.8) based on urine data. The elimination half-lives for the other metabolites were 13 hours for conjugated nicotine; 19.8 hours for conjugated cotinine; 18.8 hours for 3'-hydroxycotinine; and 19.4 hours for conjugated 3'-hydroxycotinine. The half-life of nicotine is three to four times longer in newborns than in adults, whereas the half-life of cotinine is similar in newborns and adults. CONCLUSIONS: In adults, CYP2A6 is the predominant enzyme responsible for the metabolism of both nicotine and cotinine. The prolonged elimination of nicotine but not of cotinine in the newborn compared with that in the adult may be a result of different newborn CYP2A6 enzymatic substrate specificity, low CYP2A6 activity with another enzyme that is primarily responsible for cotinine metabolism, or differences in tissue distribution.     

Analysis of nicotine, 3-hydroxycotinine, cotinine, and caffeine in urine of passive smokers by HPLC-tandem mass spectrometry

BACKGROUND: A method is described for the simultaneous analysis of nicotine and two of its major metabolites, cotinine and 3-hydroxycotinine, as well as for caffeine from urine samples. The method was developed to assess exposure of restaurant and hotel workers to environmental tobacco smoke. METHODS: The method includes sample pretreatment and reversed-phase HPLC separation with tandem mass spectrometric identification and quantification using electrospray ionization on a quadrupole ion trap mass analyzer. Sample pretreatment followed standard protocols, including addition of base before liquid-liquid partitioning against dichloromethane on a solid matrix, evaporation of the organic solvent using gaseous nitrogen, and transferring to HPLC vials using HPLC buffer. HPLC separation was run on-line with the electrospray ionization-tandem mass spectrometric detection. RESULTS: The detection limits of the procedure were in the 1 microg/L range, except for nicotine (10 microg/L of urine). Still lower detection limits can be achieved with larger sample volumes. Recoveries of the sample treatment varied from 99% (cotinine) to 78% (3-hydroxycotinine). CONCLUSIONS: The method described is straightforward and not labor-intensive and, therefore, permits a high throughput of samples with excellent prospects for automation. The applicability of the method was demonstrated in a small-scale study on restaurant employees.     

Simultaneous and sensitive measurement of anabasine, nicotine, and nicotine metabolites in human urine by liquid chromatography-tandem mass spectrometry

BACKGROUND: Determination of nicotine metabolism/pharmacokinetics provides a useful tool for estimating uptake of nicotine and tobacco-related toxicants, for understanding the pharmacologic effects of nicotine and nicotine addiction, and for optimizing nicotine dependency treatment. METHODS: We developed a sensitive method for analysis of nicotine and five major nicotine metabolites, including cotinine, trans-3'-hydroxycotinine, nicotine-N'-oxide, cotinine-N-oxide, and nornicotine, in human urine by liquid chromatography coupled with a TSQ Quantum triple quadrupole tandem mass spectrometer (LC/MS/MS). Urine samples to which deuterium-labeled internal standards had been added were extracted with a simple solid-phase extraction procedure. Anabasine, a minor tobacco alkaloid, was also included. RESULTS: The quantification limits of the method were 0.1-0.2 microg/L, except for nicotine (1 microg/L). Cotinine-N-oxide, trans-3'-hydroxycotinine, nicotine, and anabasine in urine were almost completely recovered by the solid-phase extraction, whereas the mean extraction recoveries of nicotine-N'-oxide, cotinine, and nornicotine were 51.4%, 78.6%, and 78.8%, respectively. This procedure provided a linearity of three to four orders of magnitude for the target analytes: 0.2-400 microg/L for nicotine-N'-oxide, cotinine-N-oxide, and anabasine; 0.2-4000 microg/L for cotinine, nornicotine, and trans-3'-hydroxycotinine; and 1.0-4000 microg/L for nicotine. The overall interday method imprecision and recovery were 2.5-18% and 92-109%, respectively. CONCLUSIONS: This sensitive LC/MS/MS procedure can be used to determine nicotine metabolism profiles of smokers, people during nicotine replacement therapy, and passively exposed nonsmokers. This method avoids the need for a time-consuming and labor-intensive sample enrichment step and thus allows for high-throughput sample preparation and automation.     

Highly sensitive immuno-assays for the determination of cotinine in serum and saliva. Comparison between RIA and an avidin-biotin ELISA

Two immuno-assay methods (RIA and ELISA) have been developed for the accurate and sensitive measurement of cotinine in human body fluids (serum, saliva). RIA uses [3H]cotinine as antigen and charcoal/dextran for separating cotinine-bound antibodies from the free derivative. Another technique (ELISA) was developed to avoid the use of radio-labelled compounds and to determine cotinine in large populations, including passive or non-smokers who usually present very low concentrations. The two techniques were analytically validated. The detection limit was similar (0.1 micrograms/l) and the precision was better than 10% for both techniques. Non-smoker values ranged from 0.1 to 17 micrograms/l by ELISA and 0.1 to 27.5 micrograms/l by RIA, whereas smoker values ranged from 50 to 1000 micrograms/l (ELISA) and from 70 to 800 micrograms/l (RIA). The comparative analysis of cotinine in 96 human sera revealed a good correlation between the two methods (r = 0.97) and a reliable discrimination between the populations of non-smokers and smokers. As usual, the ELISA is more rapid (4 h 30 min) than the RIA (longer than 48 h). ELISA is proposed for use in the epidemiological investigation of the human tobacco risk.     

A sensitive enzyme immunoassay for measuring cotinine in passive smokers

BackgroundBoth active smoking and passive exposure to tobacco smoke are major risk factors for cardiovascular, pulmonary, and oncological diseases. The serum level of cotinine, a major proximate metabolite of nicotine, reflects active or passive exposure to tobacco smoke. However, currently available enzyme-linked immunosorbent assays (ELISAs) for cotinine have limited sensitivity, and a high-throughput quantification of the severity of passive exposure to tobacco smoke has not been possible thus far.MethodsWe generated a phage display of combinatorial antibody library, from which we selected a recombinant antibody against cotinine, developed a sensitive ELISA using this antibody, and evaluated the method in a clinical setting and an animal model.ResultsThe limits of detection and the lower limit of quantification were 31 pg/mL and 1 ng/mL cotinine, respectively. The intra- and inter-assay precisions based on three quality control samples were 3.8–13.5% and 14.0–15.0%, respectively. No significant interference from nicotine, trans-3'-hydroxy cotinine, tobacco alkaloids, or other serum components was found. When we applied our ELISA to serum samples from 36 volunteers, the serum cotinine levels were clustered into two groups, which exactly corresponded to their smoking behavior and this ELISA yielded reproducible and accurate results, which were comparable to those of LC/MS in a split assay. In animal studies, we were able to distinguish between rats injected with a nicotine dose equivalent to that of passive exposure to tobacco and rats without exposure.ConclusionThe competitive ELISA described here is useful for the detection and quantification of the severity of risk of passive smoking.     

Accelerated metabolism of nicotine and cotinine in pregnant smokers

Cigarette smoking is the foremost modifiable risk factor for adverse pregnancy outcomes. Nicotine is a suspected fetal neuroteratogen. There is concern that nicotine may achieve toxic levels during pregnancy if nicotine replacement therapies are prescribed at doses used in the nonpregnant state. Ten healthy, volunteer, pregnant smokers received infusions of deuterium-labeled nicotine and cotinine during pregnancy and again postpartum. From blood and urine measurements, the following were determined: clearance (renal and nonrenal) of nicotine and cotinine, clearance of nicotine via the cotinine pathway (an indicator of CYP2A6 activity), and daily intake of nicotine from smoking. The clearance of nicotine and cotinine was significantly higher (60 and 140%, respectively), and the half-life of cotinine was much shorter (8.8 versus 16.6 h, P < 0.01) during pregnancy. Although plasma levels of cotinine were lower during pregnancy (119 versus 202 ng/ml, P < 0.05), daily intake of nicotine from smoking was similar during pregnancy and postpartum. For a given level of intake, the pharmacologic and toxicologic effects of nicotine during pregnancy are anticipated to be less than expected from nicotine metabolism data in nonpregnant women. Our data indicate that no downward dose adjustment needs to be made for nicotine replacement therapy during pregnancy. Conversely, higher than usual doses of nicotine may be necessary to optimize efficacy. Lower cotinine levels observed during pregnancy do not necessarily reflect less smoke exposure, and cut-off levels used to classify nonsmokers, passive smokers, and active smokers need to be established for pregnancy.     

Effect of nicotine replacement and quitting smoking on circulating adhesion molecule profiles (sICAM-1, sCD44v5, sCD44v6)

BACKGROUND: Soluble ICAM-1 (sICAM-1; sCD54), sCD44v5 and sCD44v6 are circulating adhesion molecules, with immunomodulatory potential, that have been frequently attributed diagnostic, prognostic and aetiological significance in a number of inflammatory and malignant diseases. We have previously shown that systemic concentrations of these molecules are increased significantly in tobacco smokers, but reduce to within normal levels at 12 months following successful quitting. MATERIALS AND METHODS: We have been able to extend these observations by measuring levels before and 4, 8, 22 and 52 weeks after smoking cessation in subjects receiving high-dose nicotine replacement therapy (25 mg of nicotine; n = 34) or placebo patches (n = 34) for 26 weeks. Smoking cessation was confirmed by regular measurement of expired-air CO levels and by plasma cotinine analysis. RESULTS: Plasma sICAM-1, sCD44v5 and sCD44v6 concentrations all declined rapidly within 4 weeks of smoking cessation (P < 0.001 for all declines). Additionally, no differences were observed between those using nicotine replacement and those who were not for sICAM-1, sCD44v5, or sCD44v6. CONCLUSIONS: The recovery in smoking-associated adhesion molecule profiles represents an almost immediate beneficial effect of smoking cessation. Nicotine replacement therapy is an effective aid to quitting and does not affect these recoveries. The elevated levels of these important risk factors in smokers (sICAM-1, sCD44v5 and sCD44v6) are linked to noxious element(s) in tobacco smoke other than nicotine or nicotine metabolites.     

Liquid-chromatographic determination of nicotine and cotinine in urine from passive smokers: comparison with gas chromatography with a nitrogen-specific detector

We describe a simple, sensitive, and specific high-performance liquid-chromatographic method with ultraviolet detection (256 nm) for the simultaneous analysis of nicotine and cotinine in urine of passive smokers. The analytes are extracted and purified from the complex and impure matrix in two stages; first, by liquid-liquid extraction and followed by solid-phase extraction (C2 column). We used a "DB" C8 5-microns-particle column (25 x 0.46 cm) and a mobile phase of phosphate-citrate buffer and acetonitrile (91:9 by vol) containing 5 mL of triethylamine and 600 mg of heptanesulfonate per liter, adjusted to pH 4.4, to separate the compounds. Two internal standards (2-phenylimidazole and N-ethylnorcotinine) were used. The detection limit of the HPLC assay was less than 1 microgram/L for both analytes. The average interassay CV for nicotine was 7.6%, for cotinine 6.5%, in the concentration range 0-60 micrograms/L. The mean analytical recovery of nicotine with respect to the internal standard N-ethylnorcotinine was 102% and that for cotinine was 99%; the mean absolute recoveries of the compounds were as follows: nicotine 85%, cotinine 87%, and N-ethylnorcotinine 87%. To compare the HPLC assay results of 20 samples with the more-sensitive in-laboratory gas-chromatographic method with a nitrogen-phosphorus detector, we used both N-ethylnornicotine and N-ethylnorcotinine as internal standards. The mean correlation coefficient for nicotine values between the two methods was 0.934; for cotinine, 0.987.     

The influence of smoking on plasma homocysteine and cysteine levels in passive and active smokers.

Total plasma homocysteine (tHcy) and cysteine (tCys) levels are associated with cardiovascular diseases. One of the determinants that influence their levels is cigarette smoking. The aim of this study was to determine the relationship between plasma levels of both amino acids and urinary cotinine concentration as a reliable biomarker of tobacco smoke exposure. One hundred and seventeen volunteers (61 women and 56 men) aged 19-60 years (mean 40.3 +/- 11.0) were included in the study. The study subjects were qualified into non-smokers, passive smokers and active smokers based upon the urinary cotinine concentration. In each particular group, plasma tHcy and tCys levels were measured and evaluated in the whole population and separately in women and men. Statistically insignificant differences in plasma tHcy and tCys levels in the whole group of passive smokers in comparison with non-smokers were observed (11.47 vs. 10.94 micromol/l, p=0.414, and 253.0 vs. 266.9 micromol/l, p=0.163, respectively). However, statistically significant differences in plasma tHcy levels (13.29 vs. 10.94 micromol/l, p=0.011) and in plasma tCys levels (218.2 vs. 266.9 micromol/l, p<0.001) were found in the whole group of active smokers compared with non-smokers. The Pearson's coefficient (r) for the correlation between plasma tHcy level and urinary cotinine concentration was r=0.630 (p<0.001) in the whole group of active smokers and r=0.480 (p=0.003) in the whole group of passive smokers. The correlation between plasma tCys level and urinary cotinine concentration in both study groups was insignificant. Similar results were obtained when calculated separately for men and women. The results suggest that cigarette smoking is a strong determinant of plasma tHcy level, but it is not a determinant of plasma tCys level.     

Cigarette smoking and nicotine addiction.

Smokers smoke in large part because of the addictive effects of nicotine. Nicotine affects mood and performance and has been clearly implicated as the source of addiction to tobacco. People smoke to deliver desired doses of nicotine to their bodies, with certain rates of delivery and intervals between doses; these behaviors tend to be consistent for a person from day to day. Rational treatment of the pharmacologic aspects of tobacco addiction includes nicotine substitution therapy. New formulations of nicotine substitutes will provide more options to the physician for tailoring treatment to the needs of individual smokers. Other pharmacotherapies, particularly antidepressant drugs, hold promise for certain smokers.     

Assessment of the automated colorimetric and the high-performance liquid chromatographic methods for nicotine intake by urine samples of smokers' smoking low- and medium-yield cigarettes

We have compared the high-performance liquid chromatographic method with the direct barbituric acid test in an assessment of nicotine exposure. The effect of the endogenous colour in urine on the direct barbituric acid method was also studied. The applicability of methods was evaluated with urine samples from 15 smokers who smoked 5, 10 and 20 low- and medium-nicotine cigarettes per day. Assessments of nicotine intake with the methods were well correlated, although the high-performance liquid chromatographic method detects only cotinine and the direct barbituric method most of the nicotine metabolites. Before endogenous colour subtraction in the DBA method the correlation coefficient was 0.558 and after that 0.784. Both methods indicated similar changes in nicotine exposure with the change of brand and also a dose dependent relationship to the number of cigarettes smoked. The coefficients of variation for both of the methods were 3.4%. The endogenous colour determination lessened the capacity of direct barbituric acid method to one half being about 150 samples per day. With the high-performance liquid chromatographic method the capacity was about 50 samples per working day.     

Influence of tobacco abstinence on the disposition kinetics and effects of nicotine

Habitual tobacco smoking accelerates the metabolism of many drugs. With tobacco abstinence, it was expected that nicotine metabolism would be slower than when smoking. To test this hypothesis, the disposition kinetics of intravenous nicotine were studied in 20 healthy smokers while smoking, after abstaining from smoking for 1 week, and (in six subjects) when smoking again. Cardiovascular responses to nicotine were also measured. Unexpectedly, total and nonrenal clearance of nicotine increased by 36% and 39%, respectively, during abstinence. The increase in clearance after brief abstinence suggests that nicotine or its metabolites or another component of cigarette smoke inhibits nicotine metabolism in smokers. Cardiovascular responses to nicotine were greater after 1 week compared with overnight abstinence, consistent with loss of tolerance.     

Tobacco smoke chemicals attenuate brain-to-blood potassium transport mediated by the Na,K,2Cl-cotransporter during hypoxia-reoxygenation

Smoking tobacco, including cigarettes, has been associated with an increased incidence and relative risk for cerebral infarction in both men and women. Recently, we have shown that nicotine and cotinine attenuate abluminal (brain facing) K(+) uptake mediated by the Na,K,2Cl-cotransporter (NKCC) in bovine brain microvessel endothelial cells (BBMECs) after hypoxic/aglycemic exposure (stroke conditions). The purpose of the current study was to explore the effects of nicotine and tobacco smoke chemicals on K(+) movement through the blood-brain barrier during both hypoxia/aglycemia and reoxygenation. BBMECs were exposed to nicotine/cotinine, nicotine-containing cigarette smoke extract (N-CSE), or nicotine-free cigarette smoke extract (NF-CSE) in quantities designed to mimic plasma concentrations of smokers. Stroke conditions were mimicked in vitro in BBMECs through 6 h of hypoxia/aglycemia with or without 12 h of reoxygenation, after which NKCC-mediated K(+) uptake and paracellular integrity were measured with (86)Rb and [(14)C]sucrose, respectively. In addition, K(+) concentrations in brain extracellular fluid were estimated in (86)Rb-injected rats that were administered nicotine, N-CSE, or NF-CSE and on whom global ischemia/reperfusion by in vivo four-vessel occlusion was performed. Both in vitro and in vivo paradigms showed nicotine, the major alkaloid present in tobacco smoke, to be the determining factor of an inhibited response of abluminal NKCC in BBMECs during and after stroke conditions. This was measured as a decrease in abluminal brain endothelial cell NKCC activity and as an increase in brain extracellular K(+) concentration measured as the brain extracellular fluid (86)Rb/plasma ratio after in vivo four-vessel occlusion with reperfusion.