Introduction

Abstract Introduction Results & Discussion References

Extensive studies over a period of >40 years have elucidated the metabolic fate of nicotine (1-3), the principal alkaloid in tobacco products. This work represents the most detailed and exhaustive metabolic profile ever compiled for any known xenobiotic. However, almost all of the latter studies have focused primarily on the peripheral metabolism of nicotine, and relatively few investigations have addressed the important issue of whether nicotine biotransformation products are present in brain after peripheral nicotine administration. Nicotine metabolites in brain are of particular importance, because of their potential contribution to the neuropharmacological effects resulting from nicotine exposure.

Early work by Applegren et al. (4) and recently by Deutsch et al. (5) showed that only one major metabolite, cotinine, is found in the brain of mice and/or cats after an iv¹ injection of [¹⁴C-methyl]nicotine. However, autoradiographic TLC analysis of chloroform extracts of brain from [¹⁴C]nicotine-treated animals showed the presence of one major metabolite, cotinine, and four other unidentified minor metabolites (6). In other studies following peripheral [¹⁴C-methyl]nicotine administration, both cotinine and nicotine-N-1-oxide were detected in mouse brain by HPLRC (7). It is important to note the limitations of using methyl-labeled forms of radioactive nicotine as tracer molecules in these metabolic studies, due to the inability to detect normetabolites (i.e. N-demethylated biotransformation products) as a result of loss of radiolabel. In this respect, in our more recent studies in rats administered sc injections of (±)-[2-¹⁴C]nicotine (8), the nicotine metabolites (cotinine and nornicotine) were found by HPLRC analysis to be present in significant amounts in the brain. Taken together, these studies indicate that peripheral exposure to nicotine results not only in its uptake into the CNS, but also results in the presence of a variety of nicotine biotransformation products in brain. These metabolites may originate from nicotine biotransformation in brain and/or from uptake of peripheral nicotine metabolites into brain.

In addition to nicotine, it is well documented that several nicotine biotransformation products are pharmacologically active (9-13). In this respect, it is of importance to determine if such metabolites are present in brain after nicotine administration to ascertain their contribution to the overall neuropharmacological effects that result from nicotine exposure. We now report for the first time the detection of norcotinine as a nicotine CNS biotransformation product, along with the presence of nicotine, cotinine, and nornicotine in rat brain.

Methods

Compounds. (±)-[2-¹⁴C]Nicotine (specific activity: 56 mCi/mmol), S-()-[³H-N-methyl]nicotine (specific activity: 70 Ci/mmol), and S-()-[G-³H]cotinine (specific activity: 8 Ci/mmol) were obtained from Amersham Corporation (Arlington Heights, IL). The radiochemical purities of each of the radiolabeled compounds were >98%, as determined by HPLRC (14). SCINT-A XF scintillation cocktail was purchased from Packard Instrumental Co. (Meriden, CT). S()-Nicotine, analytical grade sodium acetate, and glacial acetic acid were purchased from Aldrich (Milwaukee, WI). Sodium phosphate (dibasic, anhydrous) was purchased from Sigma (St. Louis, MO). Triethylamine (HPLC grade) was purchased from J. T. Baker, Inc. (Phillipsburg, NJ). Methanol and acetonitrile (HPLC grade) were purchased from EM Scientific Co. (Gibbstown, NJ). S-()-Nornicotine and S-()-norcotinine were prepared as previously described (15, 16). S-()-Cotinine glucuronide and trans-3-hydroxycotinine were synthesized by the methods of Crooks et al. (17). All other metabolic standards were prepared as previously described (18).

In Vivo Metabolic Experiments. Male Sprague-Dawley rats (225-250 g) were obtained from Harlan Laboratories (Indianapolis, IN), and were housed two per cage with free access to food and water in the Division of Laboratory Animal Resources at the College of Pharmacy at the University of Kentucky. Experimental protocols involving animals were approved by the Institutional Animal Care and Use Committee at the University of Kentucky. Experiments were performed using groups of 4-7 rats. Rats were injected sc with 50 µCi of either (±)-[2-¹⁴C]nicotine, S-()-[³H-N-methyl]nicotine, or S-()-[³H-G]cotinine, in physiologically buffered saline (1.0 ml/kg). Rats treated with S-()-[³H-N-methyl]nicotine or S-()-[³H-G]cotinine were killed by decapitation 5, 30, 60, 240, 480, or 1,080 min later, and the trunk plasma and brain were quickly removed. Rats treated with (±)-[2-¹⁴C]nicotine were killed by decapitation at 240 min postinjection and similarly treated. The presence of radiolabeled nicotine and/or cotinine metabolites in brain was determined by homogenizing the brain in 3 volumes of ice-cold 1.15% w/v KCl using a polytron homogenizer. The homogenate was centrifuged at 3,000g for 15 min. The supernatant was treated with 2% w/v ZnSO₄ and the precipitated protein spun down at 30,000g for 60 min. Supernatants were analyzed directly for radiolabeled nicotine and/or cotinine and their respective metabolites by HPLRC using an Altex programmable HPLC system consisting of two Altex model 110A pumps, an Altex model 420 Solvent Programmer, an Altex model 153 analytical UV detector operating at 254 nm, and Linear dual channel recorder. Radioactivity in column effluents was determined by interfacing the Model HS Flow-one Radiomatic radioactive flow-through detector equipped with a Radiomatic ES Stream-splitter in series with the UV detector. Column effluent (50% split) was mixed in a 3:1 ratio by volume with Flow-Scint I scintillation cocktail before entry into the detector. Outputs from the UV and radioactivity detectors were recorded simultaneously.

Statistics. Student's t test was used to determine statistical differences between the mean amounts of cotinine appearing in brain from the groups of rats administered S-()-[³H-N-methyl]nicotine or (±)-[2-¹⁴C]nicotine.

	Results and Discussion

Abstract Introduction Results & Discussion References

HPLRC system 1 is able to quantitate nicotine and six nicotine metabolites in biological samples (8); the retention times for metabolic standards are S-()-nicotine, 50 min; S-()-cotinine, 25 min; nicotine-N-1-oxide, 36 min; (±)-trans-3-hydroxycotinine, 22 min; S-()-N-1-methylnicotinium iodide, 87 min; S-()-N-1-methylcotininium iodide, 53 min; and cotinine-N-1-glucuronide, 18 min. In the studies using S-()-[³H-N-methyl]nicotine as metabolic tracer, authentic standards of other possible metabolitessuch as nornicotine and norcotininewere not included in the analysis, because they would not be detected radiochromatographically when formed metabolically from S-()-[³H-N-methyl]nicotine, due to loss of the [³H] label. S-()-Nicotine and S-()-N-1-methylcotininium ion are not well resolved on analytical system 1 as previously described. Therefore, the [³H] label coeluting with these two standards was isolated and reanalyzed on a separate radiochromatographic system (system 2). This second system afforded a greatly improved resolution of S-()-nicotine (t_R = 18 min) and S-()-N-1-methylcotininium ion (t_R = 4 min). System 3 was used for the analysis of nicotine and nicotine metabolites (including normetabolites: nornicotine and norcotinine) in brain supernatant from animals that had been administered (±)-[2-¹⁴C]nicotine.

Figure 1 illustrates the time course of appearance of radiolabeled S-()-nicotine and at least two other radiolabeled bands in chromatograms of brain supernatants from representative rats that had been administered [³H-N-methyl]nicotine, after each of the six time points was examined. The total [³H] label in the band attributed to [³H]nicotine overlapped both the S-()-nicotine and the S-()-N-1-methylcotininium ion standards; this band was isolated and reanalyzed on analytical system 2 and was shown to consist only of S-()-[³H]nicotine (data not shown). S-()-N-1-Methylcotininium ion was not detected in brain at any of the time points analyzed (data not shown), which is consistent with the observation that this metabolite is not a peripheral biotransformation product of S-()-[³H-N-methyl]nicotine in Sprague-Dawley rats (19).

View larger version (12K): [in this window] [in a new window]   Fig. 1.   Time course of appearance of nicotine and nicotine metabolites in rat brain supernatant from representative animals after a single sc administration of 50 µCi of S-()-[3H-N-methyl]nicotine. Analyses were conducted by cation exchange HPLRC using chromatographic system 1 (see Methods for details). Additional data from this experiment is presented in table 1.

In terms of CNS distribution of the [³H] label, the relative amounts of S-()-nicotine and the two additional radiolabeled components (peak A and cotinine) (fig. 1 and table 1) changed markedly over the time course of the study. This is most likely due to a combination of S-()-nicotine efflux from the brain, and the rate of formation and CNS half-life of the other radiolabeled components. In the early part of the time course, S-()-nicotine is the most significant component in brain, whereas peak A appeared in brain at 5 min postinjection of S-()-[³H-N-methyl]nicotine and S-()-cotinine appeared at 30 min postinjection (table 1). However, at 4 hr postadministration, similar amounts of the three components are present in brain. At 18 hr postadministration, S-()-nicotine was not detected and peak A was still a major component. Thus, peak A has a remarkably long residence time in the CNS. Results suggest that peak A is not formed directly from S-()-cotinine, because it is detected in significant amounts at the 5-min time point, whereas, S-()-cotinine is not detected at 5 min. Thus, S-()-cotinine and peak A may be formed from S-()-nicotine by two separate pathways, consistent with the observed lack of metabolism of S-()-[³H-G]cotinine in brain (see herein).

S-()-Nicotine increased dramatically over the first 60-min post-sc administration, followed by a significant decrease (table 1). This decrease represents a relatively rapid efflux of nicotine out of the CNS, because only a small increase in cotinine was observed at 240 min. S-()-Nicotine could not be detected in brain at 8 hr postadministration. In plasma, S-()-nicotine also peaked at 60 min postadministration and declined to nondetectable levels at 8 hr (data not shown). Table 1 also illustrates a comparison of central pharmacokinetics of S-()-nicotine and S-()-cotinine after sc administration of S-()-[³H-N-methyl]nicotine. The rapid absorption of S-()-nicotine into the CNS from the sc site is clearly evident. These current observations are in good agreement with previous findings (20).

S-()-Cotinine has been detected previously in brain after peripheral S-()-nicotine administration in a number of animal species (4-8, 21). In the present study, S-()-cotinine could not be detected in brain at 5-min postnicotine administration; however, by the 60-min time point, a significant amount of S-()-cotinine was observed (fig. 1 and table 1). Furthermore, S-()-cotinine was a major component in brain at 4 hr and was still present in significant amounts 8 hr after S-()-nicotine administration. In agreement with the previous results, significant amounts of S-()-cotinine were present in plasma 5 min after S-()-nicotine administration. The amount of S-()-cotinine increased 5-fold, plateaued between 1 and 4 hr, and was still observable 18 hr after S-()-[³H-N-methyl]nicotine administration.

Several studies have clearly shown that S-()-cotinine is behaviorally active and that receptor sites other than cholinergic nicotinic receptors are likely mediators of these behavioral effects (11, 12, 22-26). Cotinine seems to activate the CNS serotonin system, such that increases in urinary 5-hydroxyindole acetic acid have been observed in smokers (22), and increased serotonin turnover in rat brain has been observed after peripheral cotinine administration (23). Thus, cotinine may modulate serotoninergic neurotransmission to produce its pharmacological effects. In operant behavioral studies, the rate-increasing effect of cotinine during fixed-interval responding was not attenuated by the centrally active cholinergic nicotinic receptor antagonist, mecamylamine (24). Also, it has been shown that S-()-cotinine possesses low affinity for nicotinic receptors, compared with S-()-nicotine (25, 26). Interestingly, cotinine administration (iv, in amounts that result in blood concentrations normally seen in moderately heavy smokers) to abstinent smokers was reported to reduce significantly self-reports of desire to smoke and irritability (12); however, comparisons with placebo were not made in this study, thus weakening the conclusions that cotinine produces a neuropharmacological effect. More recently, in a study by Keenan et al. (11), S-()-cotinine administration (iv) to abstinent cigarette smokers in doses sufficient to achieve cotinine serum levels commonly encountered during daily cigarette smoking, decreased subjective self-reported ratings of abstinence-induced restlessness, anxiety, tension, and insomnia when compared with the placebo group. Thus, cotinine seems to be behaviorally active under the conditions of the latter study, and was suggested to be partly responsible for mediating aspects of nicotine dependence and of tobacco withdrawal syndrome. Therefore, cotinine, the major metabolite of nicotine, may contribute to the neuropharmacological effects of smoking. Determination of the pharmacokinetics of cotinine accumulation in brain is relevant to understanding its neuropharmacological action and its role in the CNS effects of tobacco smoking.

Analysis of brain supernatant from animals treated with (±)-[2-¹⁴C]nicotine on chromatographic system 2 afforded the radiochromatographic profile shown in fig. 2. Because of the use of (±)-[2-¹⁴C]nicotine, normetabolic standards (i.e. demethylated metabolic standards) were included in the analysis, because the label will not be lost as a consequence of metabolic N-demethylation, as would occur in the [³H-N-methyl]nicotine experiments. In this respect, it should be noted that the [2-¹⁴C]-radiolabeled nicotine is only available as the racemate, and it is not known if the nicotine enantiomers differ in their CNS uptake and metabolism. In these (±)-[2-¹⁴C]nicotine metabolism studies, an appropriate time point for analysis of metabolites in brain was chosen based on the previous studies using S-()-[³H-N-methyl]nicotine, which showed that peak appearance of brain biotransformation products occurred at 4 hr after peripheral S-()-[³H-N-methyl]nicotine administration (fig. 1). In the (±)-[2-¹⁴C]nicotine experiment, the major radiolabeled band coeluted with superimposed metabolic standards of cotinine and norcotinine, and accounted for ~60% of total radiolabel compared with nicotine (24% of total radiolabel) (table 2). A radiolabeled band that coeluted with an authentic nornicotine standard was also detected, which accounted for ~15% of the total radiolabel. The supernatant was analyzed on chromatographic system 3, which allowed the efficient separation of nicotine, nornicotine, cotinine, and norcotinine (fig. 3). Results showed that, in addition to cotinine and nornicotine, norcotinine can also be detected as a minor metabolite of (±)-[2-¹⁴C]nicotine in brain supernatant 4 hr after (±)-[2-¹⁴C]nicotine administration and represents ~4% of total radiolabel in brain (table 2).

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Analysis of rat brain supernatant 4 hr after a single sc dose of 50 µCi of (±)-[2-14C]nicotine. Analyses were conducted by Partisil-10 ODS HPLRC using chromatographic system 2 (see Methods for details). NN, nornicotine; N, nicotine; NCOT, norcotinine; COT, cotinine.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Analysis of rat brain supernatant 4 hr after a single sc dose of 50 µCi of (±)-[2-14C]nicotine. Analyses were conducted by Partisil-10 C8 HPLRC using chromatographic system 3 (see Methods for details). NN, nornicotine; NCOT, norcotinine; N, nicotine; COT, cotinine.

If one compares the percentage of brain cotinine formed in the [³H]- and [¹⁴C]-metabolic experiments, a statistically significant (p < 0.01) decrease of ~20% in the proportion of cotinine in the S-()-[³H-N-methyl]nicotine experiment, relative to the (±)-[2-¹⁴C]-experiment, is observed (table 2). However, it should be noted that the specific activities of the [³H]- and [¹⁴C]-labeled nicotines used in the previous experiments are very different. Thus, the absolute mass of nicotine administered to rats in the [³H]-experiment is considerably smaller than that administered in the [¹⁴C]-experiment, and this may also affect the relative pharmacokinetics and metabolism of nicotine in brain. The early eluting peak A in the 4 hr [³H]-radiochromatogram (fig. 1) was not observed in the 4-hr [¹⁴C]-radiochromatogram, suggesting that substantial [³H]-dissociation during S-()-[³H-N-methyl]nicotine metabolism occurred in the [³H]-experiment. These differences in the metabolic profiles of the two radiolabeled nicotines may be accounted for as dissociated [³H] generated from oxidative metabolic cleavage of the N-methyl group. Therefore, peak A most likely results from a combination of metabolic cleavage of the N-methyl group and dissociated [³H] (i.e. it represents an oxidized 1-carbon unit, and indicates the extent of N-demethylation); thus, it is not observed in the [¹⁴C]-experiment. The long residence time of peak A in the CNS (fig. 1) suggests that the [³H] associated with this peak may be present as covalently bound label, perhaps to protein or other macromolecular structures, although such adducts would be expected to exhibit different partition characteristics than observed in the current study. However, this does not rule out the possibility of adducts with small molecular weight compounds, such as amino acids or peptides. In addition, formaldehyde is known to be rapidly detoxified to formate, which would be expected to exhibit similar partition characteristics as those observed for peak A. Thus, the use of these differently labeled nicotines in the present study provides important insights into the complex metabolic pathways for nicotine metabolism that would not have otherwise been obtained in a single radiolabeled nicotine study.

To determine if the CNS metabolite norcotinine is formed from N-demethylation of cotinine or 5-C-oxidation of nornicotine, S-()-[³H-G]cotinine was administered subcutaneously to rats, and the time course of appearance of cotinine and cotinine metabolites in brain was determined. If CNS metabolism of cotinine to norcotinine occurs, one would expect to detect both cotinine and its N-demethylated metabolite in brain supernatants over the time course studied. Brain supernatants were analyzed on HPLRC analytical system 3, which is capable of quantitating cotinine (t_R = 55 min), trans-3-hydroxycotinine (t_R = 26 min), and norcotinine (t_R = 33 min) (data not shown). The time course of appearance of S-()-[³H-G]cotinine in brain and plasma after sc injection is illustrated in fig. 4. S-()-Cotinine was detected in brain 5 min after peripheral bolus administration, and concentrations in both plasma and brain reached a maximum level at 30-60 min, then gradually declined. A significant amount of S-()-cotinine was still present in brain 18 hr after sc injection.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Time course of appearance of cotinine (COT) in plasma and brain supernatant in the rat after sc administration of S-()-[G-3H]cotinine. Samples were analyzed for cotinine content by Partisil-10 C8 HPLRC using chromatographic system 3 (see Methods for details).

If one compares CNS levels of nicotine and cotinine after their respective sc administrations, as would be expected from chemical considerations, nicotine uptake in brain is much more rapid than cotinine due to the relative polarities of the two molecules. Nicotine is a tertiary amine, and its unionized form is highly lipophilic, whereas cotinine contains a polar 2-oxy group, and is a more polar and less lipophilic molecule. Nevertheless, significant cotinine uptake into brain from the periphery is observed. In addition, peripheral cotinine formed from first-pass metabolism of nicotine may constitute part or all of the brain cotinine detected after sc administration of nicotine. Thus, cotinine could contribute to the neuropharmacological effects of smoking, because it has been demonstrated to be pharmacological active (10-12).

Results clearly show that S-()-cotinine is able to pass the blood-brain barrier and access the CNS; thus, cotinine formed as a result of peripheral biotransformation of nicotine may also be a source of CNS cotinine. However, it is not possible from these studies to ascertain whether in situ conversion of nicotine to cotinine via brain-metabolizing enzymes also occurs. In contrast to plasma, no cotinine metabolites were observed in brain supernatants at any of the time points examined over the time course (5-1,080 min), indicating that cotinine is not metabolized in the CNS. Results also indicate that norcotinine found in brain after sc administration of (±)-[2-¹⁴C]nicotine does not originate from N-demethylation of cotinine, and most likely results from 5-C-oxidation of initially formed nornicotine.

Nornicotine is a pharmacologically active metabolite of nicotine and a major alkaloid of tobacco (27, 28); it has been shown to inhibit high-affinity binding of S-()-[³H]nicotine to rat brain membranes with a K_i of 40-200 nM (29-31), supporting the contention that this nicotine analog acts at nicotinic receptor sites. In operant behavioral studies using food reinforcement in monkeys, dogs, and rats (10, 24, 32), S-()-nornicotine was found to be active and was suggested to contribute to the effect of nicotine. In our own work and in that of others, S-()-nornicotine evoked a concentration-dependent and calcium-dependent endogenous dopamine release from rat and mouse striatal slices or synaptosomes (9, 33). In more recent studies, we have found that S-()-nornicotine-evoked [³H]dopamine release from rat striatal slices was inhibited by mecamylamine and dihydro--erythroidine,² suggesting that S-()-nornicotine acts by stimulating nicotinic receptors to produce this effect. Far fewer studies have been performed to determine the pharmacological effects of norcotinine. Norcotinine does not seem to be pharmacologically active, because it neither evokes the release of [³H]dopamine from rat striatal slices nor inhibits [³H]dopamine uptake into rat striatal synaptosomes (unpublished results).

N-Demethylation is generally recognized as a metabolic pathway for nicotine, due to the isolation of both nornicotine and norcotinine in urine from a number of animal species after nicotine administration (15, 34-37). Norcotinine has also been reported as a urinary metabolite of nicotine in smokers (37-39). Other reports have shown that norcotinine occurs as a urinary metabolite of cotinine in dogs, mice, and rats, but could not be detected after administration of cotinine to humans (15, 33-37). Norcotinine is also formed in vitro from cotinine in hepatic, pulmonary, and renal tissues (40).

Mechanistically, formation of norcotinine could arise from 5-C-oxidation of nornicotine or from N-demethylation of cotinine. Both of these metabolites were present in brain after sc administration of (±)-[2-¹⁴C]nicotine in the present study. 5-C-Oxidation of nornicotine has been demonstrated in rat both in vivo and in vitro (41, 42), and norcotinine has been recognized as a major metabolite of nornicotine in the periphery (43). Formation of norcotinine via N-demethylation of cotinine should proceed via the N-hydroxymethyl intermediate, which is predicted to be relatively stable, due to the low pK_a of the pyrrolidone-N moiety. In this respect, Li and Gorrod (44) have recently isolated an in vitro metabolite of cotinine from hamster hepatic microsomes, which has been identified as N-hydroxymethylnorcotinine.

Figure 5 illustrates the origin of CNS nicotine metabolites in the rat after peripheral nicotine administration, based on our present studies. It is clear from previous studies that cotinine is metabolized to norcotinine in the periphery (15, 34-37). In the present study, norcotinine was not detected in the CNS after peripheral S-()-[G-³H]cotinine administration, even though S-()-[G-³H]cotinine readily crossed the blood-brain barrier. Taken together, the results indicate that the norcotinine observed in brain after peripheral nicotine administration must originate from the 5-C-oxidation of nornicotine in the brain. However, it is not possible in our present studies to ascertain the origin of central nornicotine after peripheral nicotine administration, because nicotine can be converted via N-demethylation to nornicotine in the periphery (18) and subsequently pass the blood-brain barrier; or alternatively, nornicotine in brain could originate from N-demethylation of nicotine locally in the brain. The availability of radiolabeled-nornicotine would be beneficial in this regard to elucidate the origin of brain nornicotine and norcotinine.

View larger version (13K): [in this window] [in a new window]   Fig. 5.   Current status of knowledge on the origin of CNS metabolites of nicotine after peripheral nicotine administration.

Metabolism of nicotine to nornicotine in the periphery seems to be a relatively minor pathway. In the study by Cundy and Crooks (18), (±)-[2-¹⁴C]nicotine was injected intraperitoneally into guinea pigs, and only small amounts of nornicotine (1.6%) were detected in the 24-hr urine void, and 77% of the administered radioactivity was traced. After single arterial doses of labeled nicotine to rats, it was found that nornicotine accounted for 8% of the total recovery of the administered radioactivity, when the total activity traced was 70% of the administered dose (38). These data, together with the results from our present studies, suggest that it is unlikely that the relatively high levels of nornicotine found in brain arise from peripherally formed nornicotine.

In conclusion, it seems that the half-life of nicotine is shorter in the periphery than in the brain, which probably reflects a greater metabolic activity in the periphery. Nicotine and four nicotine metabolites have been detected in rat brain 4 hr after sc injection of (±)-[2-¹⁴C]nicotine. Three of these metabolites have been identified as cotinine, nornicotine, and norcotinine. A fourth minor metabolite (peak B, fig. 3 and table 2) has not been identified, but is probably an N-demethylated metabolite, because it is not detected in the S-()-[³H-N-methyl]nicotine studies. In agreement with previous reports, cotinine was found to be a major metabolite of nicotine in brain. Nornicotine, a pharmacologically active nicotine metabolite, is also present in significant amounts in brain and is most likely formed from peripheral and/or central N-demethylation of nicotine. Norcotinine, a minor CNS metabolite of nicotine, is formed via 5-C-oxidation of nornicotine, because it cannot be detected in brain after sc administration of cotinine and is therefore unlikely to originate from cotinine N-demethylation. 3-Hydroxycotinine and its glucuronide conjugate were not detected as metabolites of either nicotine or cotinine in brain, even though these are major biotransformation products in the periphery (3). Thus, the present identification of pharmacologically active nicotine metabolites in brain after peripheral nicotine administration provides evidence in support of the contention that metabolites of nicotine contribute to the CNS effect of tobacco product usage, and affords new information pertinent to our understanding of the fundamental processes involved in nicotine's neurochemical and behavioral effects.