Determination of nicotine N-1-glucuronide, a quaternary N-glucuronide conjugate, in human biological samples.

[Methyl-d3]-N-1-beta-D-glucopyranosyl-(+/-)-nicotinium inner salt ((+/-)-[methyl-d3]nicotine N-1-glucuronide) was synthesized from (+/-)-[methyl-d3]nicotine via reaction with methyl-2,3,4-tri-O-acetyl-1-bromodeoxy-alpha-D-glucopyranouronate, followed by deprotection with 1 M aqueous NaOH and purification by preparative TLC. Nicotine N-glucuronide was identified and determined directly in smokers' urine. A solid phase extraction method was used to partially isolate the material from urine. Subsequent determination was by thermospray-LC/MS using the synthetic d3-labeled nicotine N-glucuronide as internal standard. The identified urinary component had the same retention time as a synthetic standard and gave the same mass spectrum. The thermospray mass spectrum was characterized from the protonated molecular ion (m/z 339) and the protonated aglycone ion (m/z 163). Quantitative results from this direct method were compared with those from an indirect method, which calculated the nicotine glucuronide in the biological sample from the amount of nicotine released following treatment of the sample with the deconjugating enzyme, beta-glucuronidase. On average, the concentration of nicotine N-glucuronide determined by the direct method was 34% greater than that determined by the indirect method. Concentrations of nicotine N-glucuronide in urine ranged from 2.2 to 7.6 nmol/ml with a limit of detection of 1.3 nmol/ml.

Molecular recognition in nicotinic acetylcholine receptors: the importance of pi-cation interactions.

We explore the significance of pi-cation interactions in the binding of ligands to nicotinic acetylcholine receptors. Specifically, the Austin method of semiempirical molecular orbital theory is utilized to estimate the interaction of aromatic amino acid side chains with the cation-containing heterocyclic ring fragments of nicotinic ligands. Variational interaction energies (E(i)) of side chain-ligand fragment pairs are shown to be distance-dependent and follow a Morse-like potential function. The tryptophan side chain shows the most pronounced interaction with the cation fragments, followed by tyrosine and phenylalanine. For a given side chain, cationic fragments exhibit characteristically different E(i) profiles, with the azabicyclo[2.2.1]heptane fragment of the potent nicotinic ligand epibatidine eliciting the greatest interaction energy of the study set. Most significantly, the minimum energy values calculated for numerous fragments correlate with the binding affinity of the parent ligands- we show this to be the case for heteropentameric (alpha4beta2) and homopentameric (alpha7) nicotinic acetylcholine receptor subtypes. Furthermore, intermolecular distances corresponding to the Morse-like potential minimum also correlate with high-affinity binding. A number of parallel calculations were conducted at the Hartree-Fock 6-31G ab initio level of theory in an effort to substantiate these findings.

A further study of FTC yield and nicotine absorption in smokers.

The relationship between nicotine yield as determined by the FTC method and nicotine absorption was examined in 72 smokers in a more rigorous repetition of a previous study of 33 smokers. For this study, 113 smokers evenly distributed across four FTC "tar" yield ranges were recruited, only 72 demonstrated reasonable compliance with the study criteria with regard to sample collections and cigarette brand style consistency. Subjects recorded the number of cigarettes smoked daily and collected a 24-h urine sample and a saliva sample on 3 consecutive days. Nicotine absorption was determined by monitoring urinary excretion of nicotine and its metabolites. In addition, saliva samples were monitored for cotinine using radioimmunoassay (RIA). The correlation of the relationship for nicotine absorbed per cigarette was positive and significant (r = 0.31, P = 0.008) but weaker than in the previous study. Only smokers in the highest yield range showed any statistical difference from smokers in the lower ranges. Our results suggest that FTC nicotine yield is weakly related to nicotine absorption and that smoker-controlled factors exert a great influence on the amount of nicotine absorbed by smokers. Compensation is substantial but incomplete for the minority (by market share) of smokers at the low end of the yield scale. It is uncertain how well any alternative set of machine parameters would predict nicotine absorption for the majority of smokers, even if it were more predictive for the small number of smokers at the lower yield part of the range.

The heterocyclic substituted pyridine derivative (+/-)-2-(-3-pyridinyl)-1-azabicyclo[2.2.2]octane (RJR-2429): a selective ligand at nicotinic acetylcholine receptors.

The present report describes in vitro studies demonstrating that the heterocyclic substituted pyridine compound (+/-)-2-(3-pyridinyl)-1-azabicyclo[2.2.2]octane (RJR-2429) is extremely potent in activating human muscle nicotine ACh receptor (nAChR) (EC50 = 59 +/- 17 nM; Emax = 110 +/- 09% vs. nicotine). RJR-2429 is markedly less potent in activating nAChRs in the clonal cell line PC12, with EC50 = 1100 +/- 230 nM and Emax = 85 +/- 20% when compared with nicotine. The activation of a putative alpha 3 beta 4-containing nAChR in PC12 by RJR-2429 reveals a potency intermediate between nicotine and epibatidine (EC50 of 20,000 nM for nicotine and 30 nM for epibatidine). Dose-response curves for agonist-induced ileum contraction indicate that RJR-2429 is equipotent with nicotine, having an EC30 of approximately 2 microM. RJR-2429 binds with high affinity to alpha 4 beta 2 receptor subtype (Ki = 1.0 +/- 0.3 nM), and chronic exposure results in significant up-regulation of the high-affinity [3H]nicotine binding sites. In addition, RJR-2429 does not activate nAChRs present in rat thalamic preparations but is a potent inhibitor of this receptor subtype. It antagonizes nicotine-stimulated ion flux in thalamic synaptosomes with an IC50 of 154 +/- 37 nM. It also is a potent partial agonist at nAChRs mediating dopamine release from rat synaptosomal preparations (EC50 = 2 +/- 1 nM; Emax = 40%; epibatidine and nicotine have EC50 values of 0.4 and 100 nM, respectively). A model for the structure-activity profile of RJR-2429, nicotine and epibatidine was derived by molecular forcefield and quantum mechanics calculations and may provide important clues for the development of ligands selective for nAChR subtypes as probes in the life sciences or as potential therapeutic tools.

Correlation between hydrophobicity of short-chain aliphatic alcohols and their ability to alter plasma membrane integrity.

The quantitative relationship between chemical structure and biological activity has received considerable attention in the fields of pharmacology and drug development. More recently, quantitative structure-activity relationships (QSARs) have been used for predicting chemical toxicity. It has been proposed that alcohols may elicit their toxic effects through hydrophobic interactions with the cellular membrane. The objective of this study was to evaluate the role of hydrophobicity in the loss of membrane integrity following acute exposure to short-chain aliphatic alcohols in rat liver epithelial cells in vitro. The series of alcohols studied included methanol, ethanol, 1-propanol, 1-butanol, 1-pentanol, 1-hexanol, 1-heptanol, 1-octanol, 2-butanol, 2-methyl-1-propanol, and 2-methyl-2-propanol. The lactate dehydrogenase (LDH) assay was used to quantify membrane integrity. The logarithm of the octanol/water partition coefficient (log P) was used to quantify hydrophobicity. LDH50 values, representing alcohol concentrations yielding a 50% increase in LDH release relative to untreated controls (i.e., mild disruption of membrane integrity), and EC50 values, representing alcohol concentrations yielding 50% of the maximal release of LDH (i.e., moderate disruption of LDH release), were experimentally determined for each alcohol. The LDH50 and EC50 values were then used to derive the QSAR relationship. The aqueous alcohol concentrations yielding LDH50 or EC50 values ranged from 8.9 x 10(-4) m (LDH50 for octanol) to 3.5 m (EC50 for methanol), and the log P of the alcohols ranged from -0.77 (methanol) to 3.00 (octanol). From these data, we have derived two QSAR equations describing the role of hydrophobicity in the release of LDH from rat liver epithelial cells following a 1-hr alcohol exposure. The QSAR equation for LDH50 values, log (1/LDH50) = 0.896 log P + 0.117 (n = 11, SD = 0.131), was nearly identical to the QSAR equation for EC50 values, log (1/EC50) = 0.893 log P + 0.101 (n = 11, SD = 0.133], suggesting that similar structure-activity relationships exist at both mild and moderate levels of membrane disruption. Our data indicate that an increase in LDH release was positively and linearly correlated with the hydrophobicity (r = 0.993). These data may help predict the potential biological effects of other, as yet untested, aliphatic alcohols and aliphatic alcohol-like compounds (e.g., anesthetics) on the plasma membrane.

RJR-2403: a nicotinic agonist with CNS selectivity I. In vitro characterization.

Increasing evidence for an involvement of nicotinic cholinergic systems in neurodegenerative disorders has stimulated the search for compounds with selectivity for CNS nicotinic ACh receptors (nAChRs). To this end, we have evaluated a number of nicotinic agonists for their ability to 1) bind to and up-regulate high-affinity nAChRs, 2) release [3H]-dopamine or induce 86Rb+ efflux in synaptosomes, 3) activate nAChRs in PC12 cells, 4) activate muscle-type nAChRs in human TE671/RD cells and 5) induce contraction of guinea pig ileum. Our results indicate that (E)-N-methyl-4-(3-pyridinyl)-3-butene-1-amine (RJR-2403) binds with high affinity to rat brain cortex (Ki = 26 +/- 3 nM). Functional studies show that RJR-2403 is comparable to nicotine in activating rat thalamic synaptosomes (EC50 = 732 +/- 155 nM and Emax = 91 +/- 8% for RJR-2403; EC50 = 591 +/- 120 nM and Emax = 100 +/- 25% for nicotine) but is one-tenth as potent in inducing dopamine release (EC50 = 938 +/- 172 nM and Emax = 82 +/- 5% for RJR-2403; EC50 = 100 +/- 25 nM and Emax = 100 +/- 13% for nicotine). At concentrations up to 1 mM, RJR-2403 does not significantly activate nAChRs in PC12 cells, muscle type nAChRs or muscarinic receptors. Dose-response curves for agonist-induced ileum contraction indicate that RJR-2403 is less than one-tenth as potent as nicotine with greatly reduced efficacy. RJR-2403 does not antagonize nicotine-stimulated muscle or ganglionic nAChR function (IC50 > 1 mM). Chronic exposure of M10 cells to RJR-2403 (10 microM) results in an up-regulation of high-affinity nAChRs phenomenologically similar to that seen with nicotine. These results suggest that RJR-2403 interacts with higher potency at CNS nAChR sub-types than at muscle, ganglionic or enteric nAChRs and has higher selectivity for CNS vs. muscle or ganglionic nAChRs than does nicotine.

Comparison of measured and FTC-predicted nicotine uptake in smokers.

Cigarette smokers have a wide variety of "tar" and nicotine yields to choose from in the current market, ranging from 0.5 mg "tar" and less than 0.05 mg nicotine to 27 mg "tar" and 1.8 mg nicotine by the Federal Trade Commission (FTC) method. To understand better the relationship between FTC nicotine yields and actual nicotine uptake in smokers, we have studied nicotine uptake in 33 smokers of self-selected products representing four "tar" groupings: 1 mg "tar" (1MG), ultra-low "tar" (ULT), full-flavor low "tar" (FFLT), and full flavor (FF) cigarettes. These cigarette categories had mean FTC nicotine yields of 0.14, 0.49, 0.67, and 1.13 mg/cigarette, respectively. The subjects smoked their usual brand of cigarette ad libitum and provided a 24-h urine sample for total nicotine uptake analysis over a period during which the number of cigarettes smoked was recorded. Nicotine uptake was determined by monitoring urinary nicotine and its metabolites, including the glucuronide conjugates. Daily nicotine uptake was 9.1 +/- 7.3 mg (range 1-21 mg) for 1MG, 19.2 +/- 10.0 mg (range 4-42 mg) for ULT, 21.8 +/- 9.4 mg (range 13-38 mg) for FFLT, and 37.1 +/- 14.4 mg (range 21-60 mg) for FF smokers. On a per cigarette basis, yields were 0.23 +/- 0.11, 0.56 +/- 0.23, 0.60 +/- 0.18, and 1.19 +/- 0.43 mg nicotine, respectively. Although individual variability was fairly large (CVs of 0.39-0.80), means for the different groups showed that lower FTC yield smokers not only absorb less nicotine per 24-h period, but also per cigarette smoked. These data suggest that nicotine uptake is a function of individual smoking behavior within product design limits. We conclude from these data that, while FTC yield cannot precisely predict nicotine uptake for an individual smoker, it is useful in predicting and comparing actual nicotine uptake by smokers who select cigarettes with a particular FTC yield.

The genotoxic potential of nicotine and its major metabolites.

Nicotine is a naturally occurring alkaloid found primarily in members of the solanaceous plant family, which includes tobacco. Nicotine is rapidly absorbed by humans and then metabolized, primarily by cytochrome P450's. Studies on the genotoxic potential of these metabolites are limited. Nicotine and four of its major metabolites: cotinine, nicotine-N'-oxide, cotinine-N-oxide, and trans-3'-hydroxycotinine were evaluated for genotoxic potential in the Salmonella mutagenicity assay (strains TA98, TA100, TA1535, TA1537, and TA1538) at concentrations ranging from 0 to 1000 micrograms/plate and in the Chinese hamster ovary sister-chromatid exchange (SCE) assay at concentrations ranging from 0 to 1000 micrograms/ml. All assays were conducted with and without S9 metabolic activation. None of the five compounds increased the frequency of mutations or the frequency of SCEs. These results indicate that nicotine and its major metabolites are not genotoxic in the assays conducted.

Kinetic isotope effects on substrate association: reactions of phosphoenolpyruvate with phosphoenolpyruvate carboxylase and pyruvate kinase.

Kinetic isotope effects on association have been measured using the remote label methodology developed by O'Leary and Marlier (1979). The isotope effect on V/KA for the first substrate in an obligatorily ordered mechanism is an isotope effect on its second-order rate constant for association with the enzyme. With phosphoenolpyruvate carboxylase the 18(V/KPEP) when the bridging O is labeled decreases from 1.0056 +/- 0.0007 to 0.9943 +/- 0.0002 as the concentration of bicarbonate, the second substrate, increases from 2 to 200 mM. With pyruvate kinase the 18(V/KPEP) decreases from 1.0024 +/- 0.0014 to 0.9928 +/- 0.0027 as the concentration of ADP increases from 1.5 to 30 mM. These inverse kinetic isotope effects are best understood as arising from an isotope effect on the rate constant for forming the Michaelis complex of enzyme and substrate. The inverse value suggests that the bridging oxygen is in a vibrationally stiffer environment in the transition state for the association reaction.

Direct determination of cotinine-N-glucuronide in urine using thermospray liquid chromatography/mass spectrometry.

A thermospray liquid chromatographic/mass spectrometric method has been developed for direct determination of cotinine-N-glucuronide in the urine of smokers. Quantification was performed using methyl-d3-cotinine-N-glucuronide as internal standard and monitoring the protonated aglycons. Using a simple preparation, urine samples from four smokers were analyzed and the results compared favorably with those from a previously reported method that quantifies aglycon release following beta-glucuronidase treatment. Amounts of cotinine-N-glucuronide found in urine from smokers ranged from less than 0.7 to 21 nmol ml-1, indicating wide inter-individual variability in the metabolic production of this metabolite. Cotinine-N-glucuronide was found to be the second most abundant urinary nicotine metabolite. A similar method was developed for trans-3'-hydroxycotinine-N-glucuronide but this compound was not detected in smokers' urine.

Intragastric nitrosation of nicotine is not a significant contributor to nitrosamine exposure.

To determine the potential for intragastric nicotine nitrosation, we carried out a kinetic study of the reaction of nicotine with nitrous acid in aqueous solution. The reaction of nicotine with nitrous acid resulted in the formation of three products, NNA, NNN, and NNK. The three parallel reactions were first order of 10-6 L/mol/s. The optimum pH range for formation of NNA, NNN, and NNK was 2.4 to 3.1. Thiocyanate (100 mM) slightly increased the rate of formation of NNN and NNK but tripled the rate of formation of NNA at pH 3.5 at 37 degrees C. We have also studied the nitrosation of pseudooxynicotine, a bacterial and fungal metabolite of nicotine. This secondary amine nitrosated rapidly to produce NNK. Our proposed mechanism for the conversion of nicotine to NNK includes nine kinetically distinct steps and is in agreement with our experimental results. The rate limiting step involves the formation of nicotine-1',2'-iminium ion. This ion hydrolyzes to form pseudooxynicotine which undergoes rapid, irreversible nitrosation to NNK. Given the very slow rate of nicotine nitrosation, it is unlikely that nicotine itself contributes to exposure to nitroso compounds due to chemically mediated intragastric nitrosation.

EPR evidence for nitric oxide production from guanidino nitrogens of L-arginine in animal tissues in vivo.

Administration of Fe(2+)-citrate complex (50 mg/kg of FeSO4 or FeCl2 plus 250 mg/kg of sodium citrate) subcutaneously in the thigh or Escherichia coli lipopolysaccharide (LPS, 1 mg/kg) intraperitoneally, (i.p.) to mice induced NO formation in the livers in vivo at the rate of 0.2-0.3 micrograms/g wet tissue per 0.5 h. The NO synthesized was specifically trapped with Fe(2+)-diethyldithiocarbamate complex (FeDETC2), formed from endogenous iron and diethyldithiocarbamate (DETC) administered i.p. 0.5 h before decapitation of the animals. NO bound with this trap resulted in the formation of a paramagnetic mononitrosyl iron complex with DETC (NO-FeDETC2), characterized by an EPR signal at g perpendicular = 2.035, g parallel = 2.02 with triplet hyperfine structure (HFS) at g perpendicular. This allowed quantification of the amount of NO formed in the livers. An inhibitor of enzymatic NO synthesis from L-arginine, NG-nitro-L-arginine (NNLA, 50 mg/kg) attenuated the NO synthesis in vivo. L-Arginine (500 mg/kg) reversed this effect. Injection of L-[guanidineimino-15N2]arginine combined with Fe(2+)-citrate or LPS led to the formation of the EPR signal of NO-FeDETC2 characterized by a doublet HFS at g perpendicular, demonstrating that the NO originates from the guanidino nitrogens of L-arginine in vivo.

Characterization of the glucuronide conjugate of cotinine: a previously unidentified major metabolite of nicotine in smokers' urine.

Recent studies in our laboratories have confirmed that a major unidentified metabolite of nicotine in smokers' urine was susceptible to enzymatic degradation by beta-glucuronidase to afford (S)-(-)-cotinine. In order to establish the identity of this metabolite, the quaternary ammonium conjugate, viz., (S)-(-)-cotinine N-glucuronide, was synthesized. Reaction of methyl 2,3,4-tri-O-acetyl-1-bromo-1-deoxy-alpha-D-glucopyranuronate with (S)-(-)-cotinine at 60 degrees C for 3 days affords the fully protected conjugate as the bromide salt. Deprotection was accomplished in 1 M NaOH overnight at 25 degrees C. The deprotected inner salt was isolated by Dowex-50W cation-exchange chromatography. Electrospray mass spectra of the inner salt revealed the presence of ions with m/z 353 (M + H)+, 375 (M + Na)+, and 391 (M + K)+ as well as ions resulting from loss of water and cleavage of the glycosidic bond. Proton and carbon nuclear magnetic resonance spectra established that the position of glucuronidation was the pyridyl nitrogen. The magnitude of the coupling between H1" and H2" of the sugar ring (8.71 Hz) and nuclear Overhauser enhancements were consistent with the beta-isomer of the glucuronide conjugate. The synthetic (S)-(-)-cotinine N-glucuronide was susceptible to enzymatic hydrolysis by beta-glucuronidase to afford (S)-(-)-cotinine. Application of a cation-exchange high-performance liquid chromatographic method enabled the collection of a fraction containing (S)-(-)-cotinine N-glucuronide from a smoker's urine. The electrospray mass spectrum of this fraction contained ions consistent with the presence of (S)-(-)-cotinine N-glucuronide. The concentrated fraction was subjected to enzymatic hydrolysis by beta-glucuronidase to afford (S)-(-)-cotinine.(ABSTRACT TRUNCATED AT 250 WORDS)

Artifact formation during smoke trapping: an improved method for determination of N-nitrosamines in cigarette smoke.

Studies in our laboratory revealed artifactual formation of N-nitrosamines during trapping of mainstream and sidestream tobacco smoke by the method of Hoffmann and coworkers (2, 4). Both volatile and tobacco-specific N-nitrosamines were produced. This artifact formation took place on the Cambridge filter, which is part of the collection train used in the previously published procedure. When the filter was treated with ascorbic acid before smoke collection, artifact formation was inhibited. The improved method resulting from these studies was applied to a comparative analysis of N-nitrosamines in smoke from cigarettes that heat, but do not burn, tobacco (the test cigarette) and several reference cigarettes. Concentrations of volatile and tobacco-specific N-nitrosamines in both mainstream and sidestream smoke from the test cigarette were substantially lower than in the reference cigarettes.