Table of Contents
1. Introduction
2. Nicotine and tobacco addiction
3. Acute effects of nicotine on the cardiovascular system
4. Metabolism of nicotine and cotinine
5. Quantification of nicotine and metabolites in urine
6. Biomarkers for tobacco use detection
7. Conclusion
Tobacco use is the leading cause of death globally. Nicotine, present in tobacco products such as cigarettes, cigar, smokeless tobacco & chew, is a substance that causes addiction in individuals leading to long-lasting use of tobacco in spite of determined efforts to quit. The main cause for this addiction is due to the presence of nicotine, which stimulates dopamine release and increases dopamine concentration, a mechanism that is thought to be the basis for addiction to drugs. Nicotine is the min alkaloid present in tobacco. The main acute effects of nicotine on the cardiovascular system are as follows: peripheric vasoconstriction, increased systemic arterial pressure, and increased cardiac frequency. In the nerve endings, it stimulates release of the neurotransmitters acetylcholine, dopamine, glutamate, serotonine, and gamma-aminobutyric acid (GABA) [Nicotine after entering the systemic absorption gets converted into various metabolites of which cotinine, 3-hyrodycotinine and 3-hydroxycotinine glucuronide are the major ones that can be detected quantitatively and qualitatively. Quantification of nicotine and its metabolites in urine while a patient is actively using a tobacco product is useful to express the concentrations that a patient achieves through self-administration of tobacco. Nicotine replacement dose can then be tailored to achieve the same concentrations early in treatment to assure adequate nicotine replacement so the patient may avoid the strong craving they may experience early in the withdrawal phase. This can be confirmed by measurement of urine nicotine and metabolite concentrations. Once the patient is stabilized on the dose necessary to achieve complete replacement and responding well to therapy, the replacement dose can be slowly tapered to achieve complete withdrawal. Apart from this nicotine presence is investigated among sports personnel as well.
Nicotine is rapidly metabolized in the liver to cotinine, exhibiting an elimination half-life of 2 hours. Cotinine exhibits an apparent elimination half-life of 15 hours. Patients using tobacco products excrete nicotine in urine in the concentration range of 1,000 to 5,000 ng/mL. Cotinine accumulates in urine in proportion to dose and hepatic metabolism (which is genetically determined); most tobacco users excrete cotinine in the range of 1,000 to 8,000 ng/mL. Urine concentrations of nicotine and metabolites in these ranges indicate the subject is using tobacco or is receiving high-dose nicotine patch therapy. Passive exposure to tobacco smoke can cause accumulation of nicotine metabolites in nontobacco users. Urine cotinine has been observed to accumulate up to 20 ng/mL from passive exposure.
Virtually all tobacco products contain nicotine in substantial concentration. Cotinine, a major metabolite of nicotine, can be easily detected in various body fluids like blood serum (plasma), urine and saliva. Cotinine is most commonly used as a marker to distinguish between tobacco users and non-smokers because of its greater sensitivity and specificity than other biochemical tests. Cotinine assessment in saliva is best preferred when multiple sample are available over a limited time as minimal protein binding and water solubility of cotinine in blood increases the concentration in saliva by upto 40 %. In blood all nicotine metabolites can be measured but as cotinine has longer half-life period compared to other nicotine metabolites it is used as the most preferred biomarker for measurements using blood samples. The most widely used biomarker in tobacco users is urine cotinine as it has higher sensitivity compared to blood cotinine. The size of the subject pool will influence the choice of body fluids for analysis.
Specific methods have been developed for the qualtitavive and quantitative detection of nicotine and it major metabolite namely cotinine, 3-hyrodycotinine and 3-hydroxycotinine glucuronide in plasma or serum of active and passive smokers. Half of the nicotine that enters the systemic system is excreted by urine in the form of these 3 metabolites. Cotinine which is one of the major metabolite of nicotine can be measured by gas chromatography (GC), liquid chromatography (LC), high performance liquid chromatography (HPLC), and colrimetic assay. As these method require expensive equipment, large amount of samples, reagents, skilled operators and in addition the procedure is time consuming making GC, LC and HPLC the least preferred method when bulk samples are to be screened.
In cases where the cotinine concentration has to be screened regularly, an alternative analytical immunological procedure known as enyme linked immunoabsorbent assay (ELISA), fluorescence immunoassay (FIA), and radioimmunoassay (RIA) can be adopted as the immunoassay method has several advantages. The ELISA method is a practically validated method for monitoring biological exposure to tobacco smoke.
Although measuring 3HC would be advantageous, the analysis of 3HC using GC–MS or HPLC is complicated, and the detection limit is too high; even solid phase extraction results in a detection limit of about 20 ng/mL. In contrast, the detection limit of the ELISA for IR-cotinine is 1.3 ng/mL, which is an important advantage, especially for monitoring passive smoking.
Gas Chromatography
A gas chromatography method for the detection and quantification of nicotine and its principal metabolites cotinine, trans-3-hydroxycotinine, nicotine–N′-oxide and cotinine–N-oxide in urine was developed. In order to measure the prevalence of nicotine exposure, the samples are collected and analyzed. Nicotine and/or metabolites were detected in urine sample, while concentration measurements indicated an exposure.
The determination of cotinine involves four extraction steps, the use of an internal standard, and an alkali flame ionization detector that is selective for phosphorous and nitrogen compounds. Numerous internal standard compounds are available in the market. Compounds with similar structure and physic-chemical behavior to the analytes serve as a better internal standards and results in more accurate quantification of the nicotine. Some commonly used internal standards are N-Ethylnornicotine, N-methylanabasine, quinolone and lidocaine. Manual injection of 2µl of the extract is sufficient for nicotine quantification with gas chromatographs maintained at different isothermal temperature while running the chromatograph. Lengthy extraction is required for the analysis of nicotine and cotinine to increase the volume of analyte by using several volumes of extracting solvent to remove contamination. With the aid of vaccum the liquid passes through the column and the analytes separate into the stationary phase. The nicotine and cotinine is finally eluted after a washing and drying step using water and 80% methanol. Nitrogen sensing detectors are used to measure trans-3-hydroxycotinine using N-ethylnornicotine for nicotine quantification and N-ethylnorcotinine for quantification of cotinine and trans-3-hydroxycotinine. The limits of nicotine and cotinine detection using this method is the range of 2 ng/ml to 10 ng/ml respectively.
High Performance Liquid Chromatography (HPLC)
Hplc offers no clear advantage over gas chromatography for the determination of nicotine and cotinine in biological samples as the sensitivity of HPLC is less compared to GC. The main advantage of HPLC is that it offers the analyst the option to screen nicotine and several of it metabolites simultaneously. After numerous pre-treatment to the urine sample, detection of nicotine and cotinine occurs within 1ng/ml to 3ng/ml. Isolation of the analyte from the matrices is accomplished by the liquid-liquid extraction and solid phase is used to remove the contamination. With some minor modification to mobile phase composition and the use of a single column, nicotine and its eight metabolites can be determined in both urine and plasma samples of smokers and non-smokers.
Immunological method of Analysis
The quantification of nicotine and cotinine using immunological technique involves use of either polyclonal or monoclonal antibodies raised from goat or rabbits which binds to the ligand in a different analytical formats. The ELISA kit contains numerous plates which are incubated with bovine serum albumin and coated with goat or rabbit anti-cotinine-4 bovine-globulin polyclonal antibody. To these plates 4-5 µl of urine is added along with 100 µl horseradish peroxidase labelled with cotinine. The range for ELISA is ng/ml to 500 ng/ml. The underlying principle in immunological method is the establishment of a dynamic equilibrium between antibody binding to labeled ligand and the competition for restricted antibody binding sites by unlabeled ligand present in the sample. The extent of competitive binding and binding inhibition is used to quantify the unlabeled ligand in the sample by comparing with a calibration curve.
This article is based on papers: Enzyme-linked immunosorbent assay of nicotine metabolites and Diagnostic Methods for Detection of Cotinine Level in Tobacco Users: A Review.