Materials and Methods

Chemicals.

S(−)-Nicotine ditartrate, UDP-glucuronic acid (UDPGA¹), Tris base, magnesium chloride, β-glucuronidase (type IX-A; 1,560,000 units/g, pH 6.8, from Escherichia coli), and alamethicin were purchased from Sigma (St. Louis, MO). [N-Methyl-¹⁴C]S(−)-nicotine (free base; specific activity, 55 mCi/mmol) and [N-methyl-¹⁴C]R(+)-nicotine (free base; specific activity, 55 mCi/mmol) were obtained from ARC (St. Louis, MO). S(−)-Nicotine N1-glucuronide was synthesized by modification of a previously reported procedure (Vashishtha et al., 2000). R(+)-Nicotine di-p-toluoyl tartrate and [glucuronyl-U-¹⁴C]UDPGA (specific activity, 252 mCi/mmol) were purchased from ICN Biomedical (Costa Mesa, CA). HPLC-grade methanol (EM Science, Gibbstown, ON, Canada) and reagent-grade sodium phosphate (BDH Chemicals, Toronto, ON, Canada) were also used. Scintillation cocktail Ultima Flow-M was obtained from Packard Instrument Co. (Meriden, CT). Double-distilled water (18 ± 0.05 ohm cm), deionized and purified by Milli-QTM Water System (Millipore Corporation, Bedford, MA), was used. HPLC mobile phase solvents were filtered through Millipore 0.45-μm filters before use.

Preparation of Liver Microsomes.

Human livers (Caucasian; two female and four males) were obtained from the International Institute for the Advancement of Medicine (Exton, PA). Microsomes were prepared from both individual and pooled livers (equal weight taken from each liver) by differential centrifugation using a literature procedure (Huskey et al., 1993). The microsomes were stored at −80°C until used. The protein content of the microsomal suspension was determined by the method of Lowry et al. (1951) using bovine serum albumin as a reference standard.

Biosynthesis of S(−)-NicotineN1-Glucuronide in Human Liver Microsomes.

The reaction mixture (500 μl) that consisted of MgCl₂ (10 mM), alamethicin (25 μg), UDPGA (3 mM), human liver microsomes (1 mg), Tris buffer (50 mM, pH 7.4), andS(−)-nicotine (1.25 mM) was incubated for 120 min at 37°C. The reaction was stopped by cooling on ice and adding acetonitrile (1.5 ml). The resultant mixture was centrifuged at 9000g for 15 min. The supernatant was evaporated under nitrogen. The residue was dissolved in 50% aqueous methanol and analyzed by electrospray ionization (ESI)-mass spectrometry.

ESI-mass spectra of the biosynthesized sample and synthesized reference standard (dissolved in 50% aqueous methanol) were acquired on a Thermoquest Finnigan TSQ7000 mass spectrometer operated in the positive ion mode. Desolvation of solvent droplets was aided by a heated capillary temperature of 250°C, and sheath and auxiliary gas pressures were set at 80 and 40 psi, respectively. Data acquisition and reduction were carried out using Xcalibur (version 1.2; Thermo Electron Corporation, Waltham, MA) loaded onto an NT workstation. The data were acquired over the mass range of m/z 150 to 400 at a scan time of 1 s. Samples dissolved in 50% aqueous methanol were infused into the mass spectrometer at a rate of 10 μl/min.

Glucuronidation Assays.

The incubation conditions of pooled microsomes forS(−)-nicotine initially were optimized with respect to pH, latency-disrupting agent concentration, and time of incubation and protein concentration required to give a linear rate of formation of the glucuronide. The effect of pH on the rate of glucuronidation was studied in the range of 5.5 to 9.5 (5.5, 6.5, 7.4, 8, 8.4, 9, and 9.5). Alamethicin was used as the latency-disrupting agent, and its concentration was varied over the range 0 to 50 μg/mg of protein (0, 2.5, 5, 10, 15, 20, 25, and 50 μg/mg of protein). The time of incubation and the protein concentration were varied from 15 to 120 min (15, 30, 45, 60, 90, and 120 min) and 125 to 625 μg/ml (125, 250, 375, 500, and 625 μg/ml), respectively.

The general procedure for kinetic determinations in pooled human liver microsomes under optimized conditions is now given. The final incubation mixture (100 μl) included MgCl₂ (5 mM), alamethicin (10 μg/mg of protein), Tris buffer (50 mM, pH 8.4), UDPGA (2 mM), and pooled human liver microsomes (500 μg). Variable concentrations of the substrate were added (4–0.02 mM, including 0.1 μCi of labeled substrate of S- or R-nicotine). The individual human liver microsomes, with respect to the determination of glucuronidation activities, were treated similarly except that only one substrate concentration (0.5 mMS-isomer; 0.3 mM R-isomer) was used. The mixture was incubated for 45 min, and protein was then precipitated by adding 100 μl of methanol followed by centrifugation at 9000g for 5 min. The supernatant (120 μl) was directly injected into the HPLC for radiometric analysis. In all experiments, incubations were carried out in triplicate, and in the case of the determination of kinetic constants, the experiment was repeated four to six times at each substrate concentration.

β-Glucuronidase Hydrolysis.

β-Glucuronidase treatment of incubated samples was studied. Incubated mixtures for kinetic determinations, as described above (i.e., 100 μl; optimized conditions, 0.02 mM S(−)-nicotine and 45 min of incubation), were centrifuged (9000g for 5 min) and then further incubated at 37°C for 30 h after the addition of anE. coli preparation (1500 U) as an enzyme source and adjustment to pH 7.4. The incubated mixtures were then treated by the usual work-up of addition of methanol and centrifugation before HPLC analysis. The control samples were treated in the same way, except that no β-glucuronidase was added.

HPLC Analysis.

HPLC analysis was carried out on a chromatographic system consisting of a Waters 600 multisolvent delivery system (Milford, MA) connected to a variable wavelength absorbance detector adjusted at 254 nm (Waters model 486) and a Packard 150TR flow scintillation analyzer. Samples were injected via an autosampler SCL-10A (Shimadzu Corp., Koyoto, Japan). Data acquisition and analysis were performed using Waters Millennium 32 (version 3.05.01) in which data were collected from both ultraviolet and radiometric detectors.

The separation and quantification of the glucuronide metabolite and the parent drug were achieved by gradient reversed phase chromatography on a Phenomenex (Torrance, CA) C₁₈ Luna analytical column (ODS 4.6 × 250 mm; 5-μm diameter particle). The analytical column was protected using Phenomenex Security Guard C₁₈ cartridges (4 × 3 mm). The gradient system used two solvents, A (5 mM sodium phosphate buffer, pH 4.5) and B (methanol). The gradient elution programmed run was as follows: A (100%)/B (0%) from 0 to 5 min, changed to A (90%)/B (10%) over 5 to 12 min, changed to A (100%)/B (0%) over 12 to 16 min, and maintained to the end of the 19-min run. The flow rate was maintained at 1.5 ml/min at all times. The retention times of nicotine and theN-glucuronide metabolite were 13.2 and 4.2 min, respectively.

Calculations.

V_max and K_mvalues were calculated according to Michaelis-Menten equations for one- and two-enzyme kinetics by nonlinear least-squares regression analysis (GraphPad Prism; GraphPad Software, San Diego, CA). TheV_max/K_m ratios were determined as a rough calculation of intrinsic clearance. Data are given as mean ± S.E.M.

Results and Discussion

A synthetic sample of the quaternary ammonium-linked glucuronide of S(−)-nicotine, where the site of the glucuronide moiety was previously proven by NMR analysis to be at the pyridine rather than the pyrrolidine nitrogen atom (Seaton et al., 1993), was used in the definitive identification of the metabolite. A polar metabolite isolated from the incubation of S(−)-nicotine with activated human liver microsomes was identified by comparing the positive ion ESI-mass spectra (M⁺ = 339) and HPLC retention times under various chromatographic conditions with that of the synthetic standard of S(−)-nicotineN1-glucuronide. The identity of the molecular ion peak was further confirmed by the daughter ion mass spectrum, which gave a peak at 163 mass units, indicative of the characteristic cleavage of the glycosidic bond (M-176)⁺, with transfer of a proton from the glucuronic acid moiety to the aglycone. Also, to ensure that the radioactivity peak used for the direct quantification of nicotine N1-glucuronide was due to the metabolite, two types of experiments were performed. First, the peak attributed to nicotineN1-glucuronide was at the same retention time irrespective of whether microsomal incubations were performed with the¹⁴C label on S(−)-nicotine or UDPGA. The second type of experiment to verify the identity of the metabolite HPLC peak involved typically incubated mixtures incubated further at pH 7.4 and 37°C with or without β-glucuronidase. NoN1-glucuronide was detected in the β-glucuronidase-treated samples. In contrast, after 30 h of incubation, the radioactivity counts due to the nicotine N1-glucuronide peak of the control incubation were 79% of the value at time 0. The reason(s) for the 21% degradation of nicotine N1-glucuronide in the control samples was not investigated; however, there is an indication in the literature that there is decomposition of cotinineN1-glucuronide to cotinine in urine under storage at temperatures such as 25 and 40°C (Hagan et al., 1997).

The radiometric chromatographic method used showed a complete resolution of the peaks of concern and was reproducible and sensitive for the range of substrate concentrations required for the kinetic study (Fig. 2). The stability of the assay samples was demonstrated in that no decrease inS(−)-nicotine N1-glucuronide values was detected when data for split deproteinized samples stored at 4^οC or room temperature for 48 h were compared with the data for otherwise identical samples obtained immediately after sample preparation. The incubation conditions for the formation of S(−)-nicotine N1-glucuronide in the pooled human liver microsomes (n = 6) were optimized.N-Glucuronidation catalysis of nicotine was low at or below pH 7.4, dramatically increased 6-fold over the pH 7.4 to 8.4 range, and only further increased 1.2-fold over the pH 8.4 to 9.5 range. The pH value of 8.4 that was used in all subsequent incubations has been previously used in UGT isoform studies of N-glucuronidation at a tertiary amine (Green et al., 1995, 1998). Alamethicin was investigated as a latency-disrupting agent because this pore-forming peptide has been successfully used with respect to the activation ofN-glucuronidation at an aromatic tertiary amine of other substrates (Vashishtha et al., 2001). In comparison with control values, there was an approximate 3-fold increase in the glucuronidation rate of nicotine at alamethicin concentrations of 5 to 50 μg/mg. An alamethicin concentration of 10 μg/mg was used in subsequent experiments. The incubation time and protein concentration were linear up to 60 min and 0.5 mg of protein, respectively.

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Figure 2

Radiochromatograms of extracts from incubations of S(−)-nicotine with human liver microsomes.

Incubations contained 0.02 mM S(−)-nicotine (0.1 μCi [N-methyl-¹⁴C]-S(−)-nicotine). Assays were conducted at 37°C for 45 min, as described underMaterials and Methods. a, incubation with UDPGA (2 mM); b, incubation without UDPGA.

For the pooled human liver microsomes examined, under linear reaction conditions, the N-glucuronidation of both S(−)- and R(+)-nicotine isomers conformed to singleK_m Michaelis-Menten kinetics (Fig.3). The apparent kinetic parameters obtained by nonlinear regression analysis of the data plotted between the rate of formation under the optimized conditions of the aromatic quaternary ammonium-linked glucuronide and substrate concentration are shown for both nicotine isomers in Table1. For the S-isomer, compared with the R-isomer, the K_m value was approximately half (0.11 versus 0.23 mM), whereas theV_max value was approximately double (132 and 70 pmol/min/mg of protein). Hence, there was a marked 4-fold difference in the apparent intrinsic clearance values (V_max/K_m) between the S- and the R-isomers (1.2 versus 0.31 μl/min/mg of protein). The only other report to the investigation of stereoselectivity in glucuronidation at a tertiary amine involved the aliphatic tertiary amine of the drug ketotifen (Mey et al., 1999). On the basis of the apparent intrinsic clearance values, a 60/40 ratio ofS(−)- to R(+)-ketotifenN-glucuronides was found to be formed in human liver. In the present case, such comparison of the enantiomers of nicotine gave a ratio of 80/20 in the formation of S(−)- toR(+)-nicotine N1-glucuronides. This difference indicates that N-glucuronidation is much more important in the hepatic metabolism of the S(−)-isomer of nicotine compared with the R(+)-isomer. A study was also performed to give preliminary indication of the interindividual variation in theN-glucuronidation of nicotine. The microsomes of the individuals (n = 6) of the pooled liver microsomes were examined at one substrate concentration under the optimum conditions for the pooled sample. The interindividual variations in the rate of glucuronidation of R(+)-nicotine (range, 6.93–64.87; mean, 22.91; pooled sample, 25.58 pmol/min/mg of protein) andS(−)-nicotine (range, 30.86–182.70; mean, 85.47; pooled sample, 93.24 pmol/min/mg of protein) were found to be 9.4- and 5.9-fold, respectively.

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Figure 3

Michaelis-Menten kinetics of glucuronidation of S(−)- and R(+)-nicotine by human liver microsomes.

There is a lack of data with which to compare the kinetic parameters obtained for S(−)-nicotine. No in vitro kinetic data has been reported for any of the glucuronide metabolites of nicotine. For only one drug, namely lamotrigine, has kinetic parameters forN-glucuronidation at an aromatic tertiary amine been reported for human liver microsomes (Furlan et al., 1999;n = 12, healthy livers; K_m, 5.5 mM; V_max, 960 pmol/min/mg of protein;V_max/K_m, 0.17 μl/min/mg of protein). Also, in vivo kinetic data for theN-glucuronidation of S(−)-nicotine are lacking. Certainly it is established that S(−)-nicotineN1-glucuronide is a moderately abundant metabolite because, when S(−)-nicotine is delivered via smoking or a patch, an approximate mean of 5% of the dose is eliminated in the urine as this metabolite (Byrd et al., 1992, 2000; Benowitz et al., 1994). Also, large interindividual variations were observed in these studies, both in terms of the percentage of the dose excreted as metabolite and the proportion of nicotine that is conjugated as the glucuronide. The lack of kinetic data for all glucuronide metabolites of nicotine hinders current investigation of in vitro-in vivo correlations. Moreover, there is a necessity to determine the UGT isoforms that catalyze these glucuronidation reactions. A major value of conducting in vitro studies, both with tissue preparations, including microsomes, and expressed UGT isoform preparations, is that this will enable delineation of the mechanistic basis of in vivo observations, including in cigarette smokers. For example, such methodologies are needed to investigate the recent observations in smokers of ethnic differences and polymorphic distributions in the N-glucuronidation of both nicotine and cotinine but not in the O-glucuronidation of trans-3′-hydroxycotinine (Benowitz et al., 1999). In summary, the methodologies used in the present study of nicotineN1-glucuronidation in human liver microsomes enabled identification of the resultant metabolite and delineation of stereoselective differences between nicotine enantiomers.