1502
Chem. Res. Toxicol. 2003, 16, 1502-1506
Communications N-Glucuronidation of trans-3′-Hydroxycotinine by Human Liver Microsomes Gwendolyn E. Kuehl and Sharon E. Murphy* Department of Biochemistry, Molecular Biology & Biophysics, University of Minnesota Cancer Center, Minneapolis, Minnesota 55455 Received August 15, 2003
trans-3′-Hydroxycotinine is the major nicotine metabolite excreted in the urine of smokers and other tobacco or nicotine users. On average, about 30% of the trans-3′-hydroxycotinine in urine is present as a glucuronide conjugate. The O-glucuronide of trans-3′-hydroxycotinine has been isolated from smokers urine and appears to be the major glucuronide conjugate of trans-3′-hydroxycotinine in urine. However, nicotine and cotinine are both glucuronidated at the nitrogen atom of the pyridine ring. We report here that human liver microsomes catalyze both the N-glucuronidation and the O-glucuronidation of trans-3′-hydroxycotinine. The N-glucuronide was purified by HPLC, and its structure was confirmed by NMR. Both N- and O-glucuronidation of trans-3′-hydroxycotinine were detected in 13 of 15 human liver microsome samples. The ratio of N-glucuronidation to O-glucuronidation was between 0.4 and 2.7. One sample only catalyzed N-glucuronidation, and one sample did not catalyze either reaction. The rates of N-glucuronidation varied more than 6-fold from 6 to 38.9 pmol/min/mg protein; rates of O-glucuronidation ranged from 2.8 to 23.4 pmol/min/mg protein. The rate of trans3′-hydroxycotinine N-glucuronidation by human liver microsomes correlated well with both the rate of nicotine and the rate of cotinine N-glucuronidation. trans-3′-Hydroxycotinine O-glucuronidation correlated with neither nicotine nor cotinine N-glucuronidation. These results suggest that the same enzyme(s) that catalyzes the N-glucuronidation of nicotine and cotinine may also catalyze the N-glucuronidation of trans-3′-hydroxycotinine in the human liver but that a separate enzyme catalyzes trans-3′-hydroxycotinine O-glucuronidation.
Introduction In smokers, nicotine is rapidly metabolized to cotinine, which in turn is metabolized to trans-3′-hydroxycotinine. Glucuronide conjugates of nicotine, cotinine, and trans3′-hydroxycotinine account for more than 40% of the nicotine dose in smokers and individuals on the nicotine patch (1, 2). Only the O-linked glucuronide of trans-3′hydroxycotinine, 1 (Scheme 1), has been identified in the urine of smokers and tobacco users (3). However, Nglucuronides of both nicotine and cotinine are present in smokers urine (4, 5) and are products of human liver microsomal metabolism (5-8). The N-glucuronide of trans-3′-hydroxycotinine, 2 (Scheme 1), has been synthesized (9), but it was not detected in smokers’ urine (10). UGTs1 catalyze the formation of glucuronide conjugates (11). Glucuronidation activity is highest in the liver, and as noted above, HLMs catalyze the N-glucuronidation of nicotine and cotinine. In this study, we characterized the specificity of trans-3′-hydroxycotinine glucuronidation by HLMs. The following questions were * To whom correspondence should be addressed. Tel: 612-624-7633. Fax: 612-626-5135. E-mail:
[email protected]. 1Abbreviations: HLM, human liver microsome; NNAL, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol; UDPGA, UDP-glucuronic acid; UGT, UDP-glucuronosyltransferase.
Scheme 1. N- and O-Glucuronides of trans-3′-Hydroxycotinine
addressed. Do HLMs catalyze both the N- and the O-glucuronidation of trans-3′-hydroxycotinine? If so, what is the ratio of N-glucuronidation to O-glucuronidation and does this vary among individual samples? In addition, the rates of trans-3′-hydroxycotinine glucuronidation were compared to the rates of nicotine and cotinine glucuronidation, which were previously determined for the same set of HLMs (8).
Materials and Methods Apparatus. The HPLC system used consisted of two Waters (Milford, MA) model 510 pumps, a Waters Lambda Max model 480 LC Spectrophotometer, a β-RAM radioflow detector (INUS,
10.1021/tx034173o CCC: $25.00 © 2003 American Chemical Society Published on Web 11/07/2003
Communications Inc., Tampa, FL), and a Waters 710 autoinjector. LC-ESI-MS/ MS was carried out on a TSQ 7000 instrument (Thermo Finnegan, San Jose, CA). 1H NMR spectra were acquired using a Varian 800 MHz spectrometer (NMR Facility, University of Minnesota). Chemicals and Enzymes. Reagents for glucuronidation assays including UDPGA, saccharolactone, and alamethicin were purchased from Sigma-Aldrich (St. Louis, MO). (3′R,5′S)3′-Hydroxycotinine (trans-3′-hydroxycotinine) and the N-glucuronide conjugate were purchased from Toronto Research Chemicals (Ontario, Canada). HLMs samples HLM 2, HLM 3, HLM 5, HLM 6, HLM 10, and HLM 12 were kindly provided by Dr. Rory Remmel (University of Minnesota). Microsomes for all other human liver samples were made from tissues provided by Dr. F. Peter Guengerich (Vanderbilt University) according to previously published protocols (12). Radiolabeled UDPGA [glucuronyl-14C(U)] (specific activity > 300 nCi/nmol) was purchased from American Radiolabeled Chemicals (St. Louis, MO). Glucuronidation by HLMs. Rates of trans-3′-hydroxycotinine glucuronidation were determined under the following conditions: 2 mg/mL protein, 0.1 mg/mL alamethicin, 5 mM trans-3′-hydroxycotinine, 2 mM 14C-UDPGA (5-15 nCi/nmol), 8.5 mM saccharolactone, and 10 mM MgCl2 in 100 µL of 50 mM potassium phosphate (pH 7.1). Reactions were incubated for 1 h at 37 °C in a shaking water bath before stopping with 1/10 volumes of 0.3 N ZnSO4 and 0.3 N Ba(OH)2. Samples were prepared for HPLC analysis as described previously (8). Filtered samples (50-100 µL) were injected on a 10 µm µBondpak C18 column (3.9 mm × 300 mm, Waters Corp.). Substrates and metabolites were eluted with 20 mM potassium phosphate, pH 7 (A), for 10 min, followed by a linear gradient to 25% methanol: 75% A in 50 min (HPLC system I). The rate of glucuronidation was linear with time and protein concentration. trans-3′Hydroxycotinine and its glucuronide conjugates were also quantified by separation on a 100 mm × 3 mm Hypersil Hypercarb 5 µm column (Phenomenex, Torrance, CA). Elution was with 100% water for 10 min, followed by a linear gradient to 50% methanol:water over 40 min (HPLC system II). The trans-3′-hydroxycotinine N-glucuronide standard eluted at 12 min. This Hypercarb column was used previously by Carmella and co-workers to characterize the N-glucuronide of NNAL (13). To confirm the presence of N-glucuronide or O-glucuronide conjugates, the metabolite peaks were collected from HPLC, the methanol was evaporated under a stream of nitrogen, and the fractions were treated with base or β-glucuronidase as described previously (8). The N-glucuronide is sensitive to cleavage by base, but the O-glucuronide is not (1, 13). Complete cleavage of the trans-3′-hydroxycotinine N-glucuronide by base was confirmed with the standard under the conditions used here. Collected fractions treated with either base or β-glucuronidase were reanalyzed by radioflow HPLC. LC-MS/MS Analysis. Glucuronides of trans-3′-hydroxycotinine were also analyzed by HPLC coupled with MS/MS detection in the positive ion mode. Reactions were carried out under conditions outlined above except with nonradioactive UDPGA. Metabolites were separated using HPLC system I, and glucuronides were analyzed by selected reaction monitoring for the neutral loss of m/z 176, glucuronic acid from m/z 369, the (M + 1)+ ion of the O-linked, and M+ of the N-linked 3′-hydroxycotinine glucuronide. A standard curve was generated for the trans3′-hydroxycotinine N-glucuronide standard. The MS was operated at a source voltage of 5 kV, a source current of 30 µA, and a capillary temperature of 250 °C. The collisional gas was maintained at 2 mTorr, and the collisional voltage was -20 V. Analysis of the N-Glucuronide by 1H NMR. Reactions in a total volume of 500 µL were carried out as above with nonradioactive UDPGA. The trans-3′-hydroxycotinine N-glucuronide was isolated using HPLC system II. The collected fraction was then evaporated to dryness under a stream of nitrogen and resuspended in D2O. 1H NMR spectrum of the glucuronide formed by HLMs was compared to the 1H NMR spectrum of the trans-3′-hydroxycotinine N-glucuronide standard.
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Figure 1. Radioflow HPLC analysis of the glucuronidation of trans-3′-hydroxycotinine by HLMs. HLM 129 (A) and HLM 3 (B) were incubated with 2 mM 14C-UDPGA (5 nCi/nmol) and 5 mM trans-3′-hydroxycotinine and analyzed using HPLC system I. The N- and O-glucuronide peaks are tentatively identified based on sensitivity to either β-glucuronidase or base.
Results and Discussion Studies of in vitro metabolism of trans-3′-hydroxycotinine glucuronidation have not previously been reported. In our initial experiment with two HLM samples (HLM 129 and HLM 3), we detected what appeared to be two glucuronide metabolites of trans-3′-hydroxycotinine. Samples were assayed with 14C-UDPGA, and glucuronide products were detected by radioflow HPLC. Two metabolite peaks that were dependent on the presence of both microsomal protein and trans-3′-hydroxycotininine were detected (Figure 1). The first glucuronide metabolite eluted at 10 min and the second at 28 min. The large radioactive peak eluting between 3 and 8 min is UDPGA. trans-3′-Hydroxycotinine N-glucuronide is a positively charged quaternary amine and would elute prior to the O-glucuronide conjugate of trans-3′-hydroxycotinine on reverse phase HPLC. Therefore, the 10 min peak was tentatively identified as the N-glucuronide and the 28 min peak as the O-glucuronide. At this time, standards for either the trans-3′-hydroxycotinine N-glucuronide or the O-glucuronide were not available. The identification of these metabolites as glucuronide conjugates was confirmed by their sensitivity to β-glucuronidase treatment. Both the 10 min and the 26 min peak were cleaved by β-glucuronide treatment. In addition, when the 28 min metabolite was collected from HPLC and treated with β-glucuronidase, 14C-glucuronic acid was quantitatively released. The peak at 10 min but not the peak at 28 min was sensitive to treatment with base. The N-glucuronide conjugates of nicotine, cotinine, and NNAL, which are structurally analogous to the trans-3′-hydroxycotinine N-glucuronide are all cleaved by base treatment (1, 13). These data are
1504 Chem. Res. Toxicol., Vol. 16, No. 12, 2003
Communications Table 1. Rates of trans-3′-Hydroxycotinine by HLMsa rateb (pmol/min/mg protein)
ratio of rates
sample
Nglucuronidation
Oglucuronidation
N-/Oglucuronidation
HLM 109 HLM 126 HLM 127 HLM 129 HLM 130 HLM 132 HLM 133 HLM 134 HLM 2 HLM 3 HLM 5 HLM 6 HLM 10 HLM 12
6.1 4.6 6.0 8.1 6.0 8.3 3.9 8.7 38.9 23.5 7.0 24.1 14.9 13.9
ND 6.2 12.9 20.1 2.8 4.5 5.7 6.3 23.4 11.5 6.5 22.0 16.9 5.2
0.7 0.5 0.4 2.1 1.8 0.7 1.4 1.7 2.0 1.1 1.1 0.9 2.7
a
Rates determined at 5 mM trans-3′-hydroxycotinine and 2 mM (5 nCi/nmol). b Values are the average of two determinations, which differed by less than 10%.
14C-UDPGA
Figure 2. LC-MS/MS analysis of trans-3′-hydroxycotinine glucuronidation. Glucuronidation reactions catalyzed by HLM 129 (A) and HLM 3 (B) were carried out with 2 mM UDPGA and 5 mM trans-3′-hydroxycotinine and analyzed using HPLC system I. Glucuronide conjugates were detected by ESI-MS/MS (m/z 369-176)+. The arrow indicates the retention time of the trans-3′-hydroxycotinine N-glucuronide standard.
consistent with the identification of the 10 min peak as the N-glucuronide conjugate and the 28 min peak as the O-glucuronide conjugate of trans-3′-hydroxycotinine. Identification of these metabolites as glucuronides of trans-3′-hydroxycotinine was confirmed by mass spectrometry. The 28 min peak was collected and analyzed by ESI-MS. The major ion detected was m/z 369, (M + 1)+ of trans-3′-hydroxycotinine glucuronide. MS/MS analysis of m/z 369 generated a single product ion, m/z 193, (M + 1)+ of trans-3′-hydroxycotinine. We were unsuccessful at purifying the 10 min metabolite free of UDPGA using C18 reverse phase HPLC. However, the formation of two glucuronide conjugates of trans-3′-hydroxycotinine by HLM was confirmed by the direct analysis of the HLM reaction by LC/MS/MS. The products of trans-3′-hydroxycotinine glucuronidation by HLM 129 and HLM 3 were analyzed by selected reaction monitoring of m/z 369 [M+ or (M + 1)+ of the Nand O-linked glucuronides of trans-3′-hydroxycotinine, respectively) to m/z 193 [(M + 1)+ of trans-3′-hydroxycotinine). Two metabolite peaks were detected with both samples (Figure 2). A standard of the trans-3′-hydroxycotinine N-glucuronide was now available, and the retention time of this standard was 4 min. One of the metabolites eluted at 4 min. This short retention time relative to the radioflow HPLC data (Figure 1) was due to the age of the C18 column. The amount of this metabolite produced by HLM 129 was 3-fold lower than that by HLM 3 (Figure 2A,B). This 3-fold difference in formation is consistent with the initial analysis of this sample by radioflow HPLC (Figure 1A,B). A standard curve for the N-glucuronide was generated, and the rates
of formation of this glucuronide by HLM 3 and HLM 129 were calculated to be 24 and 7 pmol/min/mg. These rates agree well with those determined by radioflow HPLC (Table 1). The second metabolite, most likely the Oglucuronide, eluted at 20 min. HLM 129 generated significantly more of this metabolite than did HLM 3 (Figure 2), consistent with the radioflow HPLC data (Figure 1). To separate the N-glucuronide completely from UDPGA, analysis was carried out on a Thermo Hypersil Hypercarb column (HPLC system II). This column has a graphite stationary phase and was used previously to characterize the N-glucuronide of NNAL (13). Two HLM samples were analyzed by radioflow HPLC using this column. Both samples formed a metabolite coeluting with the trans-3′-hydroxycotinine N-glucuronide standard (data not shown). The metabolite was collected from HPLC, and treatment with base quantitatively released glucuronic acid. The recovery and the complete cleavage of the glucuronide were confirmed by quantifying the trans3′-hydroxycotinine released from the N-glucuronide standard that was coinjected with the sample. Using HPLC system II, the N-glucuronide conjugate of trans-3′-hydroxycotinine formed by HLM was purified and an NMR spectrum was obtained. The spectra acquired for the HLM-generated metabolite were identical to the spectra of the N-glucuronide trans-3′-hydroxycotinine standard. The chemical shifts were assigned as follows: δ 9.0 (m, 2H, pyridinium H-2 and H-6), 8.4 (m, 1H, pyridinium H-4), 8.1 (m, 1H, pyridinium H-5), 5.7 (d, 1H, sugar H-1′), 5.1 (s, 1H, pyrrolidinium H-3), 4.6 (m, 1H, pyrrolidinium H-5), 4.0 (d, 1H, sugar H-5′), 3.7 (m, 2H, sugar H-3′ and H-4′), 3.6 (m, 1H, sugar H-2′), 2.7 (s, 3H, N-CH3), 2.45 (m, 1H, pyrrolidinium H-4a), and 2.4 (m, 1H, pyrrolidinium H-4b). All assignments were confirmed by COSY spectra. The sugar proton assignments differ from those reported by Crooks et al. (9) but agree with the assignments reported more recently for several nitrosamine glucuronides (14). Having identified the N-glucuronide conjugate as a product of HLM-catalyzed trans-3′-hydroxycotinine glucuronidation, we then determined the rate of glucuronidation by 15 HLM samples. Both O- and N-glucuronidation of trans-3′-hydroxycotinine were catalyzed by 13 of the 15 HLM samples tested (Table 1). The mean (( SD) rates of N- and O-glucuronidation by these 13 samples were 12.4 ( 10.0 and 11.1 ( 7.2 pmol/min/mg,
Communications
respectively. HLM 123 failed to produce any detectable rates of either N- or O-glucuronidation, and HLM 109 produced only the N-glucuronide. The 14 samples catalyzed N-glucuronidation at rates varying 10-fold, from 3.9 to 38.9 pmol/min/mg (Table 1). The rates of Oglucuronidation varied more than 8-fold from 2.8 to 23.4 pmol/min/mg. The ratio of the rate of N-glucuronidation to the rate of O-glucuronidation by the 13 samples that catalyzed both reactions ranged from 0.4 to 2.7. Therefore, in all samples, the N-glucuronidation of trans-3′hydroxycotinine accounts for a large percentage of the total glucuronidation, and in several samples, it is the predominate pathway. The rates of N- and O-glucuronidation of trans-3′hydroxycotinine did not correlate in these samples. These data suggest that separate enzymes probably catalyze these two glucuronidation reactions. The rates of neither N- nor O-glucuronidation of trans-3′-hydroxycotinine correlated with para-nitrophenol glucuronidation rates, which were determined previously on these samples (8). This small planar phenol is primarily glucuronidated by UGT1A6 and UGT1A9 and to a lesser extent by other UGTs (15). In human liver, UGT1A6 is likely the primary enzyme catalyzing the glucuronidation of para-nitrophenol (16). Therefore, it is unlikely that UGT1A6 is catalyzing the glucuronidation of trans-3′-hydroxycotinine. The rates of trans-3′-hydroxycotinine glucuronidation by these HLMs were compared with nicotine and cotinine glucuronidation rates that were determined previously with the same samples (8). The rates of trans-3′-hydroxycotinine N-glucuronidation were significantly correlated with nicotine (r ) 0.80, p ) 0.0004) and cotinine (r ) 0.76, p ) 0.0009) glucuronidation rates. The rates of trans-3′-hydroxycotinine O-glucuronidation correlated with neither cotinine nor nicotine glucuronidation. We recently reported that UGT1A4 is a good catalyst of the N-glucuronidation of nicotine and cotinine (8). The correlation data reported here suggest that UGT 1A4 may also catalyze trans-3′-hydroxycotinine N-glucuronidation. The N-glucuronide is a major glucuronide metabolite generated by HLMs; however, the N-glucuronide has not been detected in smokers’ urine (10). One study has looked at the metabolism of trans-3′-hydroxycotinine in vivo (17). In eight individuals, an average of 29% of the dose was excreted as a glucuronide conjugate. However, the urinary level of the conjugate was measured indirectly, as the difference in the level of trans-3′-hydroxycotinine before and after treatment with β-glucuronidase. Most of the data in the literature on the levels of trans3′-hydroxycotinine glucuronide excreted by smokers is also determined as a difference in free trans-3′-hydroxycotinine following β-glucuronidase treatment (2). Therefore, in these studies, it is unknown if any N-glucuronide is present. The assumption has been that the glucuronide is O-linked. The single study that looked for the presence of the N-glucuronide directly used thermospray LC/MS and did not detect this metabolite in the urine of four smokers (10). Cotinine N-glucuronide was detected in three of the four smokers using the same methodology. In preliminary experiments in our lab, we investigated the presence of the N-glucuronide in smokers’ urine by determining the difference in free trans-3′-hydroxycotinine before and after treatment with base. We detected no difference in 10 samples. Although these data are minimal, they suggest that the N-glucuronide may not
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be a urinary metabolite of nicotine in smokers. It is also possible that the N-glucuronide is unstable relative to the O-glucuronide and that while the trans-3′-hydroxycotinine N-glucuronide might be formed in the liver, it may be cleaved prior to excretion. Alternatively, the liver may not be the major site of trans-3′-hydroxycotinine glucuronidation in the body. Glucuronidation by other tissues may be the source of the trans-3′-hydroxycotinine O-glucuronide detected in smokers’ urine. We report here that trans-3′-hydroxycotinine N-glucuronide is a major product of trans-3′-hydroxycotinine glucuronidation by HLMs. However, to understand the role of this Nglucuronidation in nicotine metabolism in smokers requires further investigation.
References (1) Benowitz, N. L., Jacob, P., III, Fong, I., and Gupta, S. (1994) Nicotine metabolic profile in man: comparison of cigarette smoking and transdermal nicotine. J. Pharmacol. Exp. Ther. 268, 296-303. (2) Davis, R. A., and Curvall, M. (1999) Determination of nicotine and its metabolites in biological fluids: in vivo studies. In Analytical Determination of Nicotine and Related Compounds and Their Metabolites (Gorrod, J. W., and Jacob, P., III, Eds.), pp 583643, Elsevier Science, Amsterdam. (3) Gorrod, J. W., and Schepers, G. (1999) Biotransformation of nicotine in mammalian systems. In Analytical Determination of Nicotine and Related Compounds and Their Metabolites (Gorrod, J. W., and Jacob, P., III, Eds.), pp 45-67, Elsevier Science, Amsterdam. (4) Byrd, G. D., Chang, K. M., Greene, J. M., and deBethizy, J. D. (1992) Evidence for urinary excretion of glucuronide conjugates of nicotine, cotinine, and trans-3′-hydroxycotinine in smokers. Drug Metab. Dispos. 20, 192-197. (5) Caldwell, W. S., Greene, J. M., Byrd, G. D., Chang, K. M., Uhrig, M. S., deBethizy, J. D., Crooks, P. A., Bhatti, B. S., and Riggs, R. M. (1992) Characterization of the glucuronide conjugate of cotinine: a previously unidentified major metabolite of nicotine in smokers’ urine. Chem. Res. Toxicol. 5, 280-285. (6) Nakajima, M., Kwon, J. T., Tanaka, E., and Yokoi, T. (2002) HighPerformance Liquid Chromatographic Assay for N-Glucuronidation of Nicotine and Cotinine in Human Liver Microsomes. Anal. Biochem. 302, 131-135. (7) Ghosheh, O., and Hawes, E. M. (2002) N-glucuronidation of nicotine and cotinine in human: formation of cotinine glucuronide in liver microsomes and lack of catalysis by 10 examined UDPglucuronosyltransferases. Drug Metab. Dispos. 30, 991-996. (8) Kuehl, G., and Murphy, S. E. (2003) N-Glucuronidation of Nicotine and Cotinine by human liver microsomes and Heterologously-Expressed UDP-glucuronosyltransferases. Drug Metab. Dispos. 31, 1361-1368. (9) Crooks, P. A., Bhatti, B. S., Ravard, A., Riggs, R. M., and Caldwell, W. S. (1992) Synthesis of N-glucuronic acid conjugates of cotinine, cis-3-hydroxycotinine and trans-3-hydroxycotinine. Med. Sci. Res. 20, 881-883. (10) Byrd, G. D., Uhrig, M. S., deBethizy, J. D., Caldwell, W. S., Crooks, P. A., Ravard, A., and Riggs, R. M. (1994) Direct determination of cotinine-N-glucuronide in urine using thermospray liquid chromatography/mass spectrometry. Biol. Mass Spectrom. 23, 103-107. (11) Tukey, R. H., and Strassburg, C. P. (2000) Human UDPglucuronosyltransferases: Metabolism, expression, and disease. Annu. Rev. Pharmacol. Toxicol. 40, 581-616. (12) Fowler, B. A., Kleinow, K. M., Squibb, K. S., Lucier, G. W., and Hayes, W. A. (1994) Chapter 33sOrganelles as Tools in Toxicology. In Principles and Methods of Toxicology (Wallace Hayes, A., Ed.), pp 1267-1268, Raven Press, New York. (13) Carmella, S. G., Le, K. A., Upadhyaya, P., and Hecht, S. S. (2002) Analysis of N- and O-glucuronides of 4-(methylnitrosamino)-1(3-pyridyl)-1-butanol (NNAL) in human urine. Chem. Res. Toxicol. 15, 545-550. (14) Upadhyaya, P., McIntee, E. J., and Hecht, S. S. (2001) Preparation of pyridine-N-glucuronides of tobacco-specific nitrosamines. Chem. Res. Toxicol. 14, 555-561. (15) Ethell, B. T., Ekins, S., Wang, J., and Burchell, B. (2002) Quantitative structure activity relationships for the glucuronidation of simple phenols by expressed human UGT1A6 and UGT1A9. Drug Metab. Dispos. 30, 734-738.
1506 Chem. Res. Toxicol., Vol. 16, No. 12, 2003 (16) Ouzzine, M., Pillot, T., Fournel-Gigileux, S., Magadalou, J., Burchell, B., and Siest, G. (1994) Expression of the human liver UDP-glucuronosyltransferase UGT1*6 analyzed by specific antibodies raised against a hybrid protein produced in Escherichia coli. Arch. Biochem. Biophys. 310, 196-204.
Communications (17) Benowitz, N. L., Jacob, P., III (2001) trans-3′-Hydroxycotinine: Disposition kinetics, effects and plasma levels during cigarette smoking. Br. J. Clin. Pharmacol. 51, 53-59.
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