Rates of excretion of cotinine, nicotine glucuronide, and 3-hydroxycotinine glucuronide in rat bile.

A new HPLC assay was adapted for radiometric detection of nicotine metabolites in rat bile. Two glucuronides were identified as the principal biliary metabolites of nicotine. In addition to nicotine glucuronide and 3-hydroxycotinine glucuronide, cotinine was also detected in bile after administration to rats of a single subcutaneous dose of (-)-S-nicotine (0.2 or 1.0 mg/kg) that contained a tracer dose of rac-[pyrrolidine-2'-14C]nicotine (20 microCi). Biliary metabolites accounted for only 3% of the [14C]nicotine dose, but phenobarbital pretreatment (100 mg/kg ip for 3 days) increased the amount of [14C]nicotine-derived radioactivity recovered in bile to 8% and also accelerated rates of biliary excretion of all three nicotine metabolites. Dose-dependency of nicotine metabolism occurred: less nicotine glucuronide was excreted at the low dose than at the high dose.

Nicotine metabolism in stumptailed macaques, Macaca arctoides.

Stumptailed macaques received a single i.v. dose of 14C-nicotine (5 microCi and 300 micrograms/kg) on several separate occasions to establish baselines. Then they were pretreated with phenobarbital (2.0 mg/kg/day im for 7 days) and 8 weeks later pretreated with cimetidine (8.5 mg/kg/day im for 4 days). Nicotine in the same dose as before was readministered after each pretreatment. The baseline pattern of nicotine metabolism observed in plasma and urine, highly reproducible within and between macaques, closely resembled that previously reported in humans using GC and HPLC analysis. In urine, nicotine and eight metabolites were identified, including high concentrations of metabolites A and G. Recently discovered in humans, metabolites A and G are of special interest for their long duration in the body. Phenobarbital pretreatment accelerated rates of production of all nicotine metabolites except nornicotine and nicotine-1'-N-oxide, whereas cimetidine retarded rates of production of five metabolites. Sex differences in nicotine metabolism resembled those reported in humans. Collectively, these results suggest that the stumptailed macaque may be a useful model for certain aspects of nicotine metabolism in humans.

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.

A new quantitative thermospray LC-MS method for nicotine and its metabolites in biological fluids.

A rapid thermospray liquid chromatography-mass spectrometry (TSP LC-MS) method is described for the simultaneous determination of nicotine and 17 of its metabolites. Chemical ionization of nicotine and its metabolites separated by reversed-phase HPLC is achieved by postcolumn addition of ammonium acetate buffer with the filament of the ion source turned off. Quantification is accomplished by selectively monitoring the unique protonated molecular ion of each metabolite. Trideuterated cotinine serves as an internal standard. Linear responses for cotinine, demethylcotinine, and trans-3'-hydroxycotinine were observed over a concentration range of 20-8000 ng/mL, and 80-8000 ng/ml for nicotine and nicotine-1'-N-oxide. Of the 17 metabolites examined, only nicotine, cotinine, demethylcotinine, and trans-3'-hydroxycotinine were detected in smokers' urine.

Metabolism of nicotine by hepatocytes.

The profile of nicotine metabolites produced by freshly isolated hepatocytes from rats, hamsters, guinea pigs, mice and humans was investigated after a 30-min exposure to nicotine ([2-14C]pyrrolidine). Large species differences occurred in the extent of nicotine metabolism; these ranged from 95% metabolism in guinea pig hepatocytes to only 30% metabolism in human and rat hepatocytes. The spectrum of metabolites formed also varied widely in different species. In hepatocytes from obese human subjects, nicotine was metabolized most extensively in smokers, least in nonsmokers, and to an intermediate degree in exsmokers, suggesting that cigarette smoking enhances the rate of nicotine metabolism. Pretreatment of all nonhuman species studied with phenobarbital and beta-naphthoflavone and with Aroclor in rats produced distinctive inductive patterns. Phenobarbital pretreatment of nonsmokers for 2 days prior to liver biopsy doubled the extent of nicotine conversion to cotinine by their hepatocytes. Rat and hamster hepatocytes exhibited sex and stereoselectivity differences in nicotine metabolism. Collectively, these studies indicate that hepatocytes offer some advantages over in vivo systems in investigating certain aspects of nicotine metabolism.

Sexual dimorphism of nicotine metabolism and distribution in the rat. Studies in vivo and in vitro.

Interpretation of sex differences in nicotine metabolism and disposition in rats required studies both in vivo and in vitro to provide both metabolic and pharmacokinetic data. In each of four rat strains studied in vitro, males metabolized nicotine faster than did females. In Sprague-Dawley rats, studies of nicotine kinetics after a single iv dose of [14C]nicotine revealed a larger nicotine volume of distribution in females than in males. A prolonged plasma nicotine half-life in females balanced the larger volume of distribution, so that no sex difference appeared in plasma clearance of nicotine. Nevertheless, sex differences in nicotine metabolism are indicated inasmuch as 1) females had lower plasma cotinine concentrations than did males; 2) urinary recoveries of nicotine were higher in female than in male rats; 3) total urinary output of nicotine metabolites was higher in male than female rats, consistent with the enhanced N- and C-oxidation of nicotine by male rats observed in vitro. In female rats the reduced rate of nicotine metabolism, as well as a larger volume of distribution of nicotine, explains in part the reported increased lethality of female compared with male rats.

Pharmacokinetics of nicotine and 12 metabolites in the rat. Application of a new radiometric high performance liquid chromatography assay.

A new radiometric assay for nicotine and 12 of its metabolites disclosed that plasma nicotine and cotinine t1/2 beta were independent of dose after single intraarterial nicotine doses of 0.1, 0.5, or 1.0 mg/kg. At high doses, nicotine AUC and clearance tended to exhibit a small degree of dose dependency. The longest lived metabolites, cotinine-N-oxide and a previously unidentified metabolite now revealed to be allohydroxydemethylcotinine, persisted for 96 hr after nicotine injection, whereas cotinine was detected for only 48 hr. Cotinine, formerly considered the longest lived nicotine metabolite, serves widely as the most sensitive indicator of prior exposure to small concentrations of nicotine. The present studies disclose new, longer lasting metabolites that may perform this function more sensitively, at least in the rat. At the 3 doses of nicotine administered, plasma nicotine half-life ranged from 0.9 to 1.1 hr; total body clearance of nicotine ranged from 2.9 to 3.9 liters.hr-1.kg-1; and apparent volume of distribution of nicotine from 4.7 to 5.7 liters.kg-1. Also at these 3 doses, mean half-lives of urinary excretion of cotinine, cotinine-N-oxide, and allohydroxydemethylcotinine ranged from 4.8 to 5.3 hr, from 7.9 to 8.2 hr, and from 9.9 to 11.0 hr, respectively.

Radiometric-high-performance liquid chromatographic assay for nicotine and twelve of its metabolites.

A sensitive, reproducible radiometric-high-performance liquid chromatographic assay has been developed to measure concentrations of nicotine and twelve of its metabolites in biological fluids. Following administration of nicotine ([2-14C]pyrrolidine) to rats, the assay was used in a pharmacokinetic investigation.

An integrated approach to the pharmacological evaluation of traditional materia medica.

A triple-tiered working model is projected for the pharmacological evaluation of crude drugs prescribed in traditional medical practices. Of the proposed component tests and procedures, the majority have had their usefulness already validated in natural product pharmacological research. First-level investigations (herbalist consultations/professional attestations, hippocratic screening and literature surveys) provide first-hand pharmacological information about traditional materia medica. Second-level investigation comprising bioassay-modelled tests (brine shrimp toxicity test, opiate receptor-binding studies, fertilized sea urchin egg test, platelet adenyl cyclase/phosphodiesterase assays, hexobarbital-induced sleeping time test, antimicrobial assays, isolated guinea pig ileum test and pharmacodynamic screening) are designed to extend the mode(s) of drug action suggested by the first-level procedures. Based on data from Level 1 and Level 2 findings, third-level experiments are specific tests tailored to confirm the pharmacodynamic/kinetic properties and clinical efficacy of traditional drugs. Level 2 and Level 3 tests may be combined in monitoring fractionations and subsequent isolation of unique crude drug constituents with potential application in conventional therapeutics.

Preliminary chemical characterization of pharmacologically active compounds in aqueous plant extracts.

A test system is described which, based on the use of ion exchangers, gel filtration on Sephadex, and extraction with organic solvents, gives information about the stability, molecular size, charge, and polarity of pharmacologically active compounds in aqueous plant extracts. Knowledge of these properties permits development of a suitable isolation method for the compounds. The method is illustrated by the identification of quaternary ammonium salts as responsible for the contracting activity on the isolated guinea pig ileum of an aqueous extract of roots of Maeura subcordata.

Time-dependent induction of hepatic drug metabolism in rats by cotinine.

Cotinine, administered twice a day for 2 days in a dose of 20 or 40 mg/kg i.p., induced rat hepatic drug metabolism between 22% to 62%. Induction decreased to 26-37% after 4 days of cotinine (40 mg/kg bid). No significant induction was observed after 8 days of this dose of cotinine. Explanations are offered for decreasing induction by cotinine with time.

Influence of different forms of tobacco intake on nicotine elimination in man.

In each of three separate experiments mean plasma nicotine t1/2 beta was slightly, but statistically significantly, shorter in habituated compared to naive cigarette smokers. Two of these experiments involved nicotine administration by smoking a cigarette containing a standardized amount of nicotine, whereas in the third experiment nicotine was injected intravenously as a single tracer dose of 14C-nicotine. In contrast to cigarette smokers, naive and habituated snuff dippers had similar mean plasma nicotine t1/2 beta. Habituated pipe smokers tended to have very slightly, but not statistically significantly, shorter plasma nicotine t1/2 beta S than naive pipe smokers. These distinctions are related to special features that characterize each form of tobacco intake.

Smoking-induced changes in nicotine disposition: application of a new HPLC assay for nicotine and its metabolites.

A sensitive, rapid high-pressure liquid chromatographic assay was developed to compare the disposition of an intravenous dose of 14C-nicotine in normal, carefully matched smokers and nonsmokers. The elimination half-lifes of nicotine and cotinine were shorter in smokers than in nonsmokers. Also consistent with an inductive effect of smoking was the increased nicotine elimination rate constant in smokers, but smoking induced more complex kinetic changes: nicotine volume of distribution was diminished in smokers, whereas nicotine clearance and area under the concentration-time curve were unchanged. The presence of nicotine and its principal metabolites in a morning specimen of urine obtained from nonsmokers before 14C-nicotine administration suggests ubiquitous, passive exposure to and absorption of chemicals present in cigarette smoke.