Nicotine metabolite ratio as an index of cytochrome P450 2A6 metabolic activity

BACKGROUND: Nicotine and a variety of other drugs and toxins are metabolized by cytochrome P450 (CYP) 2A6. Our objective was to evaluate the use of oral nicotine with measurement of the trans-3'-hydroxycotinine (3HC)/cotinine (COT) metabolite ratio as a noninvasive probe of CYP2A6 activity. METHODS: Sixty-two healthy volunteers received an oral solution of deuterium-labeled nicotine (2 mg) and its metabolite cotinine (10 mg). Plasma nicotine and plasma and saliva cotinine and 3HC concentrations were measured over time. RESULTS: The 3HC/COT ratio derived from deuterium-labeled cotinine, measured in either plasma (2-8 hours after administration) or saliva (at 6 hours), was strongly correlated with the oral clearance of nicotine (r = 0.76-0.83, depending on the time of measurement). The 6-hour 3HC/COT ratio from nicotine derived from tobacco in 14 smokers was highly correlated with the ratio derived from deuterium-labeled nicotine (r = 0.88) and was also highly correlated with the oral clearance of nicotine (r = 0.90). Two subjects homozygous for inactive CYP2A6 alleles produced no 3HC, confirming the specificity of the metabolite ratio. The 3HC/COT ratio was also highly correlated with the clearance and half-life of cotinine, consistent with the fact that cotinine is also primarily metabolized by CYP2A6. CONCLUSIONS: The 3HC/COT ratio derived from nicotine either administered as a probe drug or from tobacco use, measured in either plasma or saliva, is highly correlated with the oral clearance of nicotine. The ratio appears to be a useful noninvasive marker of the rate of nicotine metabolism (which is important in studying nicotine addiction and smoking behavior), as well as a general marker of CYP2A6 activity (which is important in studying drug and toxin metabolism).     

Identification of novel CYP2A6*1B variants: the CYP2A6*1B allele is associated with faster in vivo nicotine metabolism

Cytochrome P450 2A6 (CYP2A6) is the human enzyme responsible for the majority of nicotine's metabolism. CYP2A6 genetic variants contribute to the interindividual and interethnic variation in nicotine metabolism. We examined the association between the CYP2A6*1B variant and nicotine's in vivo metabolism. Intravenous infusions of deuterium-labeled nicotine were administered to 292 volunteers, 163 of whom were White and did not have common CYP2A6 variants, other than CYP2A6*1B. We discovered three novel CYP2A6*1B variants in the 3'-flanking region of the gene that can confound genotyping assays. We found significant differences between CYP2A6*1A/*1A, CYP2A6*1A/*1B, and CYP2A6*1B/*1B groups in total nicotine clearance (17.2+/-5.2, 19.0+/-6.4, and 20.4+/-5.9, P<0.02), non-renal nicotine clearance (16.4+/-5.0, 18.5+/-6.2, and 19.8+/-5.7, P<0.01), and the plasma trans-3'-hydroxycotinine/cotinine ratio (0.26+/-0.1, 0.26+/-0.1, and 0.34+/-0.1, P<0.001). There were also differences in total nicotine (29.4+/-12.9, 25.8+/-0.12.9, and 22.4+/-12.4, P<0.01), cotinine (29.2+/-8.1, 32.2+/-9.1, and 33.0+/-6.6, P<0.01) and trans-3'-hydroxycotinine (32.4+/-9.1, 34.2+/-12.3, and 41.3+/-11.3, P<0.001) excreted in the urine. We report evidence that CYP2A6*1B genotype is associated with faster nicotine clearance in vivo, which will be important to future CYP2A6 genotype association studies.     

CYP2A6 genotype and the metabolism and disposition kinetics of nicotine

BACKGROUND AND OBJECTIVE: The liver enzyme cytochrome P450 (CYP) 2A6 is primarily responsible for the metabolism of nicotine. Variants in the CYP2A6 gene have been associated with altered nicotine metabolism and with effects on smoking behavior. Our objective was to determine the relationship between variant CYP2A6 genotypes and the disposition and metabolism of nicotine administered intravenously. METHODS: Intravenous infusions of deuterium-labeled nicotine and cotinine were administered to 278 healthy twin volunteers, most of whom were white. They were genotyped for CYP2A6*1, CYP2A6*2, CYP2A6*4, CYP2A6*7, CYP2A6*8, CYP2A6*9, CYP2A6*10, and CYP2A6*12. RESULTS: On the basis of the fractional clearance of nicotine to cotinine and on the plasma ratio of 3'-hydroxycotinine to cotinine, both shown to be indicators of CYP2A6 enzymatic activity, subjects were classified into 3 groups. Group 1 included wild-type variant CYP2A6*1/*1 (n=215) and was assumed to have 100% activity. Group 2 included *1/*9 (n=21) and *1/*12 (n=12), which averaged about 80% of normal activity. Group 3 included *1/*2 (n=10), *1/*4 (n=2), *9/*12 (n=3), *9/*4 (n=2), and *9/*9 (n=3), which averaged about 50% of normal activity. The mean total plasma clearance of nicotine (+/-SD) was 18.8+/-6.0, 15.5+/-4.9, and 11.7+/-5.1 mL.min-1.kg-1 in groups 1, 2, and 3, respectively, and group 1 had significantly faster clearance than group 2 (P<.05) and group 3 (P<.01). Overall, groups 2 and 3 also had lower total clearance of cotinine, had longer half-lives for nicotine and cotinine, and excreted in the urine a greater fraction of the nicotine dose as unchanged nicotine and nicotine glucuronide and excreted less as 3'-hydroxycotinine compared with group 1. CONCLUSIONS: We provide novel pharmacokinetic and metabolic data on nicotine after systemic dosing in relation to common CYP2A6 genotypes. Our data will enhance the interpretation of CYP2A6 genotypic data as used in association studies of smoking behavior and its health consequences.     

Simultaneous analysis of nicotine,nicotine metabolites,and tobacco alkaloids in serum or urine by tandem mass spectrometry,with clinically relevant metabolic profiles

BACKGROUND: Assessment of nicotine metabolism and disposition has become an integral part of nicotine dependency treatment programs. Serum nicotine concentrations or urine cotinine concentrations can be used to guide nicotine patch dose to achieve biological concentrations adequate to provide the patient with immediate relief from nicotine withdrawal symptoms, an important factor in nicotine withdrawal success. Absence of nicotine metabolites and anabasine can be used to document abstinence from tobacco products, an indicator of treatment success. METHODS: The procedure was designed to quantify nicotine, cotinine, trans-3'-hydroxycotinine, anabasine, and nornicotine in human serum or urine. The technique required simple extraction of the sample with quantification by HPLC-tandem mass spectrometry. RESULTS: The procedure for simultaneous analysis of nicotine, its metabolites, and tobacco alkaloids simultaneously quantified five different analytes. Test limit of quantification, linearity, imprecision, and accuracy were adequate for clinical evaluation of patients undergoing treatment for tobacco dependency. The test readily distinguished individuals who had no exposure to tobacco products from individuals who were either passively exposed or were abstinent past-tobacco users from those who were actively using a tobacco or nicotine product. CONCLUSIONS: Nicotine, cotinine, trans-3'-hydroxycotinine, nornicotine, and anabasine can be simultaneously and accurately quantified in either serum or urine by HPLC-tandem mass spectrometry with imprecision <10% at physiologic concentrations and limits of quantification ranging from 0.5 to 5 micro g/L. Knowledge of serum or urine concentrations of these analytes can be used to guide nicotine replacement therapy or to assess tobacco abstinence in nicotine dependency treatment. These measurements are now an integral part of the clinical treatment and management of patients who wish to overcome tobacco dependence.     

Inactivation of CYP2A6 and CYP2A13 during nicotine metabolism

Nicotine is the major addictive agent in tobacco. The primary catalyst of nicotine metabolism in humans is CYP2A6. However, the closely related enzyme CYP2A13 is a somewhat better catalyst. CYP2A13 is an extrahepatic enzyme that is an excellent catalyst of the metabolic activation of the tobacco-specific carcinogen 4-(methylnitrosamine)-1-(3-pyridyl)-1-butanone (NNK). Here we report that both CYP2A6 and CYP2A13 were inactivated during nicotine metabolism. Inactivation of both enzymes was dependent on NADPH and increased with time and concentration. Alternate substrates for CYP2A6 and CYP2A13 protected these enzymes from inactivation. Inactivation of CYP2A13 was irreversible upon extensive dialysis and seems to be mechanism-based. The K(I) of CYP2A13 inactivation by nicotine was 17 microM, the rate of inactivation, k(inact), was 0.1 min(-1), and the t(1/2) was 7 min. However, the loss in enzyme activity occurred after nicotine metabolism was complete, suggesting that a secondary or possible tertiary metabolite of nicotine may be responsible. [5-(3)H]Nicotine metabolism by CYP2A13 was monitored by radioflow high-pressure liquid chromatography during the course of enzyme inactivation; the major product was the Delta(1'(5'))iminium ion. However, cotinine was a significant metabolite even at short reaction times. The metabolism of the nicotine Delta(1'(5'))iminium ion to cotinine did not require the addition of aldehyde oxidase. CYP2A13 catalyzed this reaction as well as further metabolism of cotinine to 5'-hydroxycotinine, trans-3'-hydroxycotinine, and N-(hydroxymethyl)-norcotinine as enzyme inactivation occurred. Studies are on-going to identify the metabolite responsible for nicotine-mediated inactivation of CYP2A13.     

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.     

Nicotine metabolite ratio predicts efficacy of transdermal nicotine for smoking cessation

BACKGROUND: Nicotine is metabolized to cotinine, and cotinine is metabolized to 3'-hydroxycotinine (3-HC) by the liver enzyme cytochrome P450 (CYP) 2A6. More rapid metabolism of nicotine may result in lower nicotine blood levels from nicotine replacement products and poorer smoking cessation outcomes. This study evaluated the utility of the 3-HC/cotinine ratio as a predictor of the efficacy of nicotine replacement therapy as an aid for smoking cessation. METHODS: By use of an open-label design, 480 treatment-seeking smokers were randomly assigned to 8 weeks of transdermal nicotine or nicotine nasal spray use, plus behavioral group counseling. Assessments included demographics, smoking history, body mass index, and plasma nicotine, cotinine, and 3-HC concentrations, as well as CYP2A6 genotypes. Smoking cessation was biochemically verified at the end of treatment and at 6-month follow-up. RESULTS: The rate of nicotine metabolism, as indicated by pretreatment 3-HC/cotinine ratio derived from cigarette smoking, predicted the effectiveness of transdermal nicotine at both time points. The odds of abstinence were reduced by almost 30% with each increasing quartile of metabolite ratio (odds ratio, 0.72 [95% confidence interval, 0.57-0.90]; P=.005). Higher metabolite ratios also predicted lower nicotine concentrations (beta=-1.72, t(179)=-3.31, P<.001), as well as more severe cravings for cigarettes after 1 week of treatment (beta=0.32, t(190)=2.91, P=.004). The metabolite ratio did not predict cessation with use of nicotine nasal spray (odds ratio, 1.05 [95% confidence interval, 0.83-1.33]; P=.68). CONCLUSION: The nicotine metabolite ratio might be useful in screening smokers to determine likely success with a standard dose of transdermal nicotine.     

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.     

Cytochrome P450 2B6 activity as measured by bupropion hydroxylation: effect of induction by rifampin and ethnicity

AIM: The aim of this study was to investigate the activity of the drug-metabolizing enzyme cytochrome P450 (CYP) 2B6 before and after in vivo induction by rifampin (INN, rifampicin) in white subjects and Chinese subjects by use of the probe drug bupropion (INN, amfebutamone). METHODS: Healthy male white subjects (n = 9) and Chinese subjects (n = 9) (age range, 19-34 years) of known CYP2B6 genotype received orally administered bupropion (Zyban SR, 150 mg) alone and during daily treatment with rifampin (600 mg). Blood samples were taken for up to 72 hours after each bupropion dose, and plasma concentrations of bupropion and its active metabolites, hydroxybupropion, threohydrobupropion, and erythrohydrobupropion, were measured by HPLC. The subjects' CYP2B6 genotype was determined by use of a matrix-assisted laser desorption /ionization-time of flight (MALDI-TOF) mass spectrometry assay. RESULTS: Rifampin treatment increased the apparent clearance of bupropion in Chinese subjects and white subjects combined (n = 16) from 2.6 L x h(-1) x kg(-1) (95% confidence interval [CI], 2.3-3.0 L x h(-1) x kg(-1)) after bupropion alone to 7.9 L x h(-1) x kg(-1) (95% CI, 6.8-10.1 L x h(-1) x kg(-1)) during rifampin treatment. Rifampin treatment decreased the half-life of bupropion from 15.9 hours (95% CI, 13.5-20.4 hours) to 8.2 hours (95% CI, 6.7-12.4 hours). Rifampin treatment increased the hydroxybupropion maximum concentration from 395 ng/mL (95% CI, 341-497 ng/mL) to 548 ng/mL (95% CI, 490-638 ng/mL), decreased the area under the concentration-time curve extrapolated to infinity of hydroxybupropion from 14.7 microg x h/mL (95% CI, 12.7-18.4 microg x h/mL) to 8.4 microg x h/mL (95% CI, 7.4-10.2 microg x h/mL), and reduced the elimination half-life of hydroxybupropion from 21.9 hours (95% CI, 20.3-24.0 hours) to 10.7 hours (95% CI, 8.6-14.5 hours). There was no significant difference in the pharmacokinetics of bupropion or hydroxybupropion between white subjects and Chinese subjects before and after treatment with rifampin, once corrected for body weight. CONCLUSIONS: Rifampin significantly induces CYP2B6 activity in vivo, and the clinical consequences of potential interactions between rifampin and CYP2B6 substrates deserve further investigation. Rifampin appears to induce the elimination of hydroxybupropion. Differences in bupropion pharmacokinetics that were observed between white subjects and Chinese subjects can be attributed to differences in body weight, suggesting that, for a given subject weight, CYP2B6 activity is similar in white subjects and Chinese subjects.     

Effects of cigarette smoking and carbon monoxide on nicotine and cotinine metabolism

OBJECTIVES: To examine the effects of cigarette smoking on the disposition kinetics of nicotine and cotinine, to determine the effects of cigarette smoking on pathways of nicotine and cotinine metabolism, and to test the hypothesis that carbon monoxide inhibits the metabolism of nicotine. STUDY DESIGN: Twelve cigarette smokers were studied in three treatment conditions, each lasting 7 days, during which they smoked cigarettes, breathed carbon monoxide to achieve carboxyhemoglobin levels similar to cigarette smoking, or breathed air. In each treatment condition, subjects received a combined infusion of deuterium-labeled nicotine (d2) and cotinine (d4), with measurement of disposition kinetics and urine metabolite profile. RESULTS: Cigarette smoking significantly inhibited the metabolism of nicotine but had no effect on cotinine metabolism. Cigarette smoking markedly induced the O-glucuronidation of trans-3'-hydroxycotinine but had no effect on the N-glucuronidation of nicotine or cotinine. Carbon monoxide had no effect on nicotine or cotinine kinetics or metabolic profile. CONCLUSIONS: This study confirms previous observations that cigarette smoking inhibits nicotine metabolism but disproves the hypothesis that this effect is due to carbon monoxide. Induction of glucuronidation must be considered in understanding the effects of cigarette smoking on drug metabolism.     

Effect of grapefruit juice on cytochrome P450 2A6 and nicotine renal clearance

BACKGROUND AND OBJECTIVE: Grapefruit juice is an inhibitor of the cytochrome P450 (CYP) 3A4 enzyme and transporters such as P-glycoprotein and organic anion transporting polypeptides, leading to clinically important interactions. Our objective was to study the effect of grapefruit juice on the pharmacokinetics of nicotine, which is primarily metabolized by the CYP2A6 enzyme. METHODS: Ten volunteers were given a 2-mg oral dose of deuterium-labeled nicotine on 3 occasions together with 1 L of water, full-strength grapefruit juice, or half-strength grapefruit juice. Concentrations of nicotine and its metabolites were analyzed in plasma and urine for 8 hours. RESULTS: Grapefruit juice inhibited the formation of cotinine from nicotine (area under the plasma cotinine concentration-time curve from 0 to 8 hours of 6807 min.ng/mL, 7805 min.ng/mL, and 8007 min.ng/mL for full-strength grapefruit juice, half-strength grapefruit juice, and water, respectively; repeated-measures ANOVA, P=.009). The time to peak plasma concentration of cotinine was delayed (216 minutes, 159 minutes, and 147 minutes, respectively; ANOVA, P=.011), and the peak plasma concentration was lower with grapefruit juice compared with water (18 ng/mL, 21 ng/mL, and 22 ng/mL, respectively; ANOVA, P=.010). Oral clearance, peak plasma concentration, and time to peak plasma concentration of nicotine were not affected. Grapefruit juice increased the renal clearance of nicotine (231 mL/min, 219 mL/min, and 123 mL/min, respectively; ANOVA, P=.045) and cotinine (19 mL/min, 14 mL/min, and 16 mL/min, respectively; ANOVA, P=.002). CONCLUSIONS: Grapefruit juice inhibits the metabolism of nicotine to cotinine, a pathway mediated by CYP2A6, and increases the renal clearance of nicotine and cotinine. Nicotine oral clearance is not affected by grapefruit juice because the inhibition of hepatic metabolism is offset by the increase in the renal clearance of nicotine. However, other compounds metabolized by CYP2A6, as well as other drugs excreted via renal clearance mechanisms similar to those of nicotine, may be susceptible to significant pharmacokinetic grapefruit juice interactions.     

Female sex and oral contraceptive use accelerate nicotine metabolism

BACKGROUND: Several studies have reported that female smokers have a higher risk of lung cancer than male smokers. This could be related to sex differences in nicotine metabolism and related smoking behavior. This study tested the hypothesis that women metabolize nicotine more rapidly than men and that, among women, oral contraceptive users metabolize nicotine more rapidly than nonusers of oral contraceptives. METHODS: Two hundred seventy-eight healthy volunteers who were twins and 16 who were siblings of twins, recruited from the Northern California Twin Registry, received an infusion of deuterium-labeled nicotine and cotinine with frequent blood sampling. The plasma clearances of nicotine and cotinine, the clearance of nicotine to cotinine (an index of cytochrome P450 [CYP] 2A6 activity), and the ratio of trans-3'-hydroxycotinine to cotinine (another indicator of CYP2A6 activity) were measured. RESULTS: The clearances of nicotine and cotinine, the clearance of nicotine to cotinine, and the trans-3'-hydroxycotinine/cotinine ratio were significantly higher in women than in men (nicotine clearance, 15.6 +/- 4.3 mL.min(-1).kg(-1) in men versus 18.8 +/- 6.6 mL.min(-1).kg(-1) in women; P < .001); they were also higher among women taking oral contraceptives than in those who were not taking oral contraceptives (nicotine clearance, 22.5 +/- 6.6 mL.min(-1).kg(-1) in women taking oral contraceptives versus 17.6 +/- 6.1 mL.min(-1).kg(-1) in those who were not; P < .05). Women who were menopausal or postmenopausal were not different from men. Among oral contraceptive users, nicotine metabolism was accelerated among those taking combined and estrogen-only contraceptives but not progesterone-only contraceptives. CONCLUSIONS: Sex hormones influence nicotine metabolism. Nicotine and cotinine metabolism is faster in women than in men and is faster in women taking oral contraceptives compared with those who are not. Accelerated nicotine metabolism appears to be a result of estrogen. Sex-related differences in nicotine clearance could affect smoking behaviors, as well as response to nicotine medications, and could be a marker for altered metabolism of nicotine-derived carcinogens.     

Effects of passive smoking on theophylline clearance

Theophylline disposition was examined in seven passive smokers, defined as nonsmokers with long-term exposure to cigarette smoke, and seven age-matched nonsmokers with minimal smoke exposure. Subjects were given an intravenous infusion of aminophylline (6 mg/kg) and blood samples were drawn before and during the 48-hour postinfusion period. Clearance for passive smokers was 6.01 x 10(-2) L/hr.kg and for nonsmokers, clearance was 4.09 x 10(-2) L/hr.kg (p less than 0.025). Terminal elimination half-life for passive smokers was 6.93 hours versus 8.69 hours for nonsmokers (p less than 0.05). The mean residence time for passive smokers was 9.89 hours. For nonsmokers, the mean residence time was 13.11 hours (p less than 0.05). These measurements were statistically different, whereas there was no difference in volume of distribution between the groups, suggesting that passive smokers metabolize theophylline more rapidly than nonsmokers. Plasma and urine cotinine and nicotine concentrations were measured in all subjects. There was a significant difference between the subject groups in plasma (p less than 0.004) and urine (p less than 0.002) cotinine concentrations. Theophylline clearance correlated with both plasma (r = 0.73, p less than 0.01) and urine (r = 0.79, p less than 0.01) cotinine concentrations. Additional studies should be conducted to further define the pharmacokinetic characteristics of passive smokers and to assess the effects of passive smoking on drugs metabolized by the mixed function oxidase system.     

Acute Potassium Dichromate Poisoning: A Toxicokinetic Case Study

Case Report: A 48-year-old man drank 150 mL of an aqueous solution containing potassium dichromate 22.5 g in a suicidal attempt and was admitted 7 hours after the ingestion. Hemodialysis was promptly undertaken and chromium concentrations in serum, erythrocytes, and dialysate were determined during the treatment. Chromium elimination in urine was monitored during hemodialysis and the subsequent 400 hours. The total chromium eliminated via hemodialysis and urine was calculated as 36.7 mg or 0.16% of the ingested dose. Spontaneous urinary elimination proceeded according to an open one-compartment model. The elimination half-life was 71.37 hours ± 17.13 hours (95% CI). Chromium elimination from serum followed an open two-compartment model, with the half-lives of 3.16 hours ± 2.63 hours for phase 1 and 50 hours ± 27 hours (95% CI) for phase 2. Calcium-EDTA therapy had no influence on erythrocyte, serum, or urine chromium level. It contributed, however, to a significant increase in chromium elimination rate in the dialysate. Serum zinc was very low at admission and serum zinc, copper, and magnesium were controlled during the initial 30 hours.     

A novel polymorphism of human CYP2A6 gene CYP2A6*17 has an amino acid substitution (V365M) that decreases enzymatic activity in vitro and in vivo

Cytochrome P450 (CYP) 2A6 is a major CYP responsible for the metabolism of nicotine and coumarin in humans. We identified a novel allele, designated CYP2A6*17 , which contains A51G (exon 1), C209T (intron 1), G1779A (exon 3), C4489T (intron 6), G5065A (V365M, exon 7), G5163A (intron 7), C5717T (exon 8), and A5825G (intron 8). We developed a genotyping method by polymerase chain reaction-restriction fragment length polymorphism for the CYP2A6*17 allele, targeting the G5065A mutation. The allele frequency in black subjects (n = 96) was 9.4% (95% confidence interval [CI], 5.3%-13.5%). The allele was not found in white subjects (95% CI, 0%-0.9%; n = 163), Japanese subjects (95% CI, 0%-1.6%; n = 92), and Korean subjects (95% CI, 0%-0.7%; n = 209). To examine the effects of the amino acid change in the CYP2A6*17 allele on the enzymatic activity, we expressed a wild-type or variant (V365M) CYP2A6 together with NADPH-CYP reductase in Escherichia coli . For coumarin 7-hydroxylation, the apparent Michaelis-Menten constant value of variant CYP2A6 (1.06 +/- 0.11 micromol/L) was significantly (P < .005) higher than that of wild type (0.60 +/- 0.05 micromol/L). The maximum velocity values of the wild-type and variant CYP2A6 were 0.61 +/- 0.06 and 0.64 +/- 0.07 pmol . min -1 . pmol -1 CYP, respectively. For nicotine C -oxidation, the apparent Michaelis-Menten constant values of the wild-type or variant CYP2A6 were 31.6 +/- 2.9 micromol/L and 31.3 +/- 3.1 micromol/L, respectively. The maximum velocity value of variant CYP2A6 (0.72 +/- 0.21 pmol . min -1 . pmol -1 CYP) was significantly (P < .05) lower than that of the wild type (1.80 +/- 0.42 pmol . min -1 . pmol -1 CYP). Thus the intrinsic clearance values for coumarin 7-hydroxylation and nicotine C -oxidation by the variant were both significantly (P < .05) decreased to 40% to 60% compared with the wild type. Furthermore, cotinine/nicotine ratios after 1 piece of nicotine gum was chewed, used as an index of in vivo nicotine metabolism, were significantly (P < .05) decreased in heterozygotes of the CYP2A6*17 allele (5.4 +/- 2.7, n = 12) compared with homozygotes of the wild type (11.5 +/- 10.5, n = 37). A subject with CYP2A6*17 / CYP2A6*17 revealed the lowest cotinine/nicotine ratio (1.8). We found a novel allele in black subjects that affects the nicotine metabolism in vitro and in vivo.     

Determinants of the rate of nicotine metabolism and effects on smoking behavior

BACKGROUND: Studies on cytochrome P450 (CYP) 2A6 suggest that genotype affects the rate of nicotine metabolism and, consequently, cigarette consumption. However, known alleles of CYP2A6 associated with fast or slow metabolism are relatively uncommon, and there remains considerable variation in metabolic activity among those with presumed wild-type CYP2A6 alleles, suggesting that other genetic or environmental factors also influence the rate of nicotine metabolism. METHODS: We investigated determinants of the rate of nicotine metabolism and effects on smoking behavior in a United Kingdom cohort who participated in a placebo-controlled trial of smoking cessation via nicotine replacement therapy. Those who continued to smoke cigarettes at the 8-year follow-up formed our study group (N = 545). The ratio of the nicotine metabolite trans-3'-hydroxycotinine to cotinine in plasma was used as an index of CYP2A6 activity and thus as a marker of the rate of nicotine metabolism. RESULTS: The nicotine metabolite ratio was associated with sex (P < .0001), CYP2A6 genotype (*1B, *2, *4, *9, and *12) (P < .0001), CYP2B6 haplotype (*4-dominant) (P = .02), plasma nicotine concentration (P < .0001), and age (P = .02) but was not associated with dependence score (P > .20). The ratio also predicted the number of cigarettes smoked at will per day, although the association was weak (F(1, 492) = 4.05, P = .04). CONCLUSION: In this cohort the rate of nicotine metabolism is related to age, sex, CYP2A6 genotype, and CYP2B6 genotype and may affect the level of tobacco consumption.     

Relationship between interindividual differences in nicotine metabolism and CYP2A6 genetic polymorphism in humans

BACKGROUND: Nicotine is mainly metabolized to cotinine by cytochrome P450 (CYP) 2A6. Previously, we found that the CYP2A6 gene was deleted homozygously in one subject who was deficient in cotinine formation from nicotine. OBJECTIVE: Our objective was to clarify the relationship between interindividual differences in nicotine metabolism and CYP2A6 genetic polymorphism. METHODS: Nicotine was administered to 92 healthy Japanese subjects in the form of 1 piece of nicotine gum to investigate the potency of nicotine metabolism. The cotinine-nicotine ratio of the plasma concentration 2 hours after chewing was calculated as an index of nicotine metabolism. The genotypes of CYP2A6 gene, CYP2A6*1A, CYP2A6*1B, CYP2A6*2, CYP2A6*3, CYP2A6*4, and CYP2A6*5, were determined with polymerase chain reaction-restriction fragment length polymorphism. RESULTS: A large interindividual difference in nicotine metabolism was observed. Allele frequencies of CYP2A6*1A, CYP2A6*1B, and CYP2A6*4 were 42.4%, 37.5%, and 20.1%, respectively. The CYP2A6*2, CYP2A6*3, and CYP2A6*5 alleles were not found. Three subjects genotyped as CYP2A6*4/CYP2A6*4 were completely deficient in cotinine formation. The heterozygotes of the CYP2A6*4 allele tend to show lower capacities for cotinine formation. The subjects with CYP2A6*1A/CYP2A6*1B appeared to have higher capacities of cotinine formation than subjects with CYP2A6*1A/CYP2A6*1A, although the difference was not significant. The probit plot of the cotinine-nicotine ratio was not linear; this possibly indicated the existence of a novel mutation in the CYP2A6 gene genotyped as CYP2A6*1B/CYP2A6*4. CONCLUSIONS: The relationship between interindividual differences in nicotine metabolism and CYP2A6 genetic polymorphism in humans was proved.     

Improved methods for isolating cortisol metabolites from neonatal urine

Urine was collected from newborns, children, and adults who had received a tracer dose of [3H] cortisol. The free and different types of conjugated cortisol metabolites were separated by ion-exchange chromatography. A modified Amberlite XAD-2 procedure was used, which enabled quantitative extraction of the extremely polar cortisol metabolites from urine from newborns. Large differences between the ion-exchange chromatographic patterns for neonates and adults were observed. Urine from neonates was shown to contain quantitatively important amounts of an as-yet-unidentified conjugate that was absent from urine from adults. The effects of three different hydrolytic methods on the neonatal steroid conjugates were studied and it was found that the unknown conjugate in urine from the newborn could be hydrolyzed by solvolysis only.     

Pharmacokinetics of nicotine in kidney failure

BACKGROUND: Smoking is an important risk factor for cardiovascular and cerebrovascular complications in patients with chronic kidney failure. Very high plasma nicotine concentrations have been reported in patients with severe kidney failure, indicating that the disposition of nicotine in these patients may be different. The purpose of this study was to assess the pharmacokinetics of intravenously administered nicotine in healthy subjects and in patients with kidney failure. METHODS: Nine healthy subjects (glomerular filtration rate [GFR], 84 to 143 mL/min/1.73 m2), four patients with mild kidney failure (GFR, 63 to 73 mL/min/1.73 m2), five patients with moderate kidney failure (GFR, 18 to 36 mL/min/1.73 m2), and six patients with severe kidney failure (GFR, 1 to 10 mL/min/1.73 m2) were recruited. Three patients were treated with continuous ambulatory peritoneal dialysis. An intravenous infusion of nicotine (0.028 mg/kg) was given for 10 minutes. Nicotine and cotinine concentrations were measured in plasma, urine, and peritoneal dialysate from 0 to 24 hours after start of infusion RESULTS: There were significant correlations between GFR and total clearance, nonrenal and renal clearance of nicotine, area under the plasma concentration-time curve extrapolated to infinity, terminal elimination half-life, and mean residence time. Nonrenal clearance was 1303 mL/min and 661 mL/min in healthy subjects and patients with severe kidney failure, respectively. Only 1% to 2% of the nicotine dose was excreted unchanged in a 24-hour collection of peritoneal dialysate. The elimination of cotinine was also decreased in patients with kidney failure. CONCLUSION: Progressive kidney failure is associated with a gradual decrease of renal and nonrenal elimination of nicotine.     

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.