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Technical Briefs |
Clin. Biochem. Dept., Istituto Superiore di Sanità, V.le Regina Elena 299, 00161 Rome, Italy
a author for
correspondence: fax ++ 39 6 4461961
Cotinine (COT), a major metabolite of nicotine (NIC), has been used as a biomarker in many studies of active and passive smoking (1)(2)(3)(4). Methods of analysis for COT in biological fluids include gas chromatography, gas chromatography–mass spectrometry, HPLC, HPLC–mass spectrometry, and immunoassays (5)(6)(7)(8)(9)(10). In particular, the RIA developed for COT and NIC by Langone and Van Vunakis (11) was the method reported by the International Agency for Research on Cancer (IARC) for determination of these analytes in several biological matrices (12)(13).
In the description of the assay (11), no mention was made about cross-reactivity of anti-NIC and anti-COT antibodies with trans-3'-hydroxycotinine (THOC) and cotinine glucuronide (COT-G). Indeed, these two compounds were found to be the most abundant metabolites of NIC in urine of smokers (14). Other studies, however, have investigated the cross-reactivity of the anti-COT antiserum with THOC and other metabolites in an ELISA (15).
The objective of this study was to investigate the cross-reactivity of the anti-COT and anti-NIC antisera with THOC and COT-G in the RIA (11)(12)(13). We also used urine samples from active and passive smokers to compare data from the RIA with data obtained by HPLC.
The rabbit antiserum, (-)[3H]COT, and (-)[3H]NIC were supplied by H. Van Vunakis (Brandeis University, Waltham, MA). The detectable range of measurement from the calibration curve was 0.5–50 µg/L for NIC and 0.2–20 µg/L for COT. The cross-reactivities of the anti-COT and anti-NIC antibodies with NIC metabolites different from THOC and COT-G were reported as <5%. In addition, urine samples of smokers were analyzed for COT and THOC with an HPLC method described by Zuccaro et al. (16). In brief, the method used reversed-phase chromatography with ultraviolet detection (254 nm) to measure NIC metabolites in urine samples that had undergone solid-phase extraction before chromatography. The limit of quantification with this method was 10 µg/L for NIC and 5 µg/L each for COT and THOC, and the assay showed good reproducibility, intra- and interday CVs being <4% for various concentrations of the analytes.
Calibrators for NIC, COT, THOC, and COT-G were prepared at several concentrations in Tris buffer (pH 7.4) and were used in the RIA to calculate inhibition curves by comparison with the calibration curves for COT and NIC. Duplicate or triplicate assays were performed on different days. The relative cross-reactivity was determined as calculated by Abraham (17). In particular, the percent of cross-reaction was calculated from the percent ratio between the concentration (µg/L) of COT (or NIC) and that of the other metabolites required to displace 50% of the (-)[3H]COT [or (-)[3H]NIC] bound to the antiserum.
Stock solutions of NIC, COT, and THOC were prepared in methanol, stored at <0 °C, and used with appropriate dilutions for chromatographic analyses. We also obtained urine samples from 13 smokers and 6 smoke-exposed nonsmokers, stored these at -20 °C, and used aliquots of these samples for both RIA and HPLC analyses. In particular, aliquots used for the RIA were diluted (from 1:50 to 1:400) so as to fall within the range of measurement reported for the assay.
In addition, we added known quantities of COT and THOC to blank urines obtained from nonsmokers who had not been exposed to environmental tobacco smoke. We analyzed these samples by RIA and HPLC to verify the results obtained for the smokers and the smoke-exposed nonsmokers.
The relative cross-reactivities of NIC, COT, THOC, and COT-G with the
anti-COT and anti-NIC antisera are reported in Table 1
. Results obtained for NIC and COT confirmed the data in the
literature (11). Further, we observed no cross-reactivity
of THOC and COT-G at increasing concentrations with anti-NIC antiserum,
nor of COT-G with anti-COT antiserum. However, the cross-reactivity of
THOC with anti-COT antiserum at 50% inhibition was 34%, high enough
to lead to an overestimation of COT in the presence of THOC—as
confirmed by the comparison between HPLC and RIA values of COT in blank
urine samples with added COT and THOC and in samples from smokers and
smoke-exposed nonsmokers (Table 2
). Moreover, the HPLC and RIA values of blank samples
supplemented with similar quantities of THOC and COT correlated highly:
r = 0.98 (P = 0.0003) for RIA vs HPLC
values for COT, and r = 0.99 (P =
0.0002) for RIA COT vs HPLC COT plus THOC. For samples from the smokers
and the exposed nonsmokers, RIA vs HPLC values for COT
(r = 0.8, P = 0.00002) and RIA COT vs
HPLC COT plus THOC (r = 0.8, P =
0.00001) still correlated, even though the RIA results for high values
of THOC differed from the true concentrations of COT and THOC (as
measured by HPLC) (Table 2
, subjects 7, 8, 10).
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In the recent years, COT has been used in epidemiological screenings as a biomarker to discriminate between active and passive smokers and to quantify the exposure to environmental tobacco smoke (2). Discrimination between active and passive smokers is reasonably easy, the mean concentrations of COT in the former group being as much as 10 times those in the latter. Much more difficult is differentiation between light smokers and highly exposed passive smokers, or between exposed passive smokers and nonexposed nonsmokers; in these subjects the COT values are low and may overlap, approaching cutoff values. For this situation, accuracy and precision of the measurements need to be improved, e.g., by adjusting urinary COT for the urinary creatinine concentration (18) or by using a more sensitive and specific method of detection such as isotope dilution liquid chromatography–tandem mass spectrometry (19) or a monoclonal antibody immunoassay tested for cross-reactivity with THOC.
Indeed, although cross-reactivity of THOC for COT antiserum might be useful from an epidemiological point of view (by indirectly increasing the sensitivity of the COT RIA), it represents an analytical bias, which should be indicated in reports of the characteristics of the assay. In any case, one must take into account the method of COT analysis when choosing the cutoff value to discriminate between exposed and nonexposed nonsmokers, to be able to correctly compare results obtained with methods based on different principles.
Finally, in the present study, we determined the cross-reactivity of THOC by assaying its racemic form, whereas the naturally occurring metabolite is (-)THOC; cross-reactivity of the natural metabolite with anti-COT antiserum, therefore, which is stereospecific (11), can be higher than values reported here.
References
4 years to
tobacco smoke—United States, 1988–1991. MMWR 1993;42:37–9..
The following articles in journals at HighWire Press have cited this article:
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  T. R. Gray, R. Magri, D. M. Shakleya, and M. A. Huestis Meconium Nicotine and Metabolites by Liquid Chromatography-Tandem Mass Spectrometry: Differentiation of Passive and Nonexposure and Correlation with Neonatal Outcome Measures Clin. Chem., December 1, 2008; 54(12): 2018 - 2027. [Abstract] [Full Text] [PDF]
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P. Jatlow, S. McKee, and S. S. O'Malley Correction of Urine Cotinine Concentrations for Creatinine Excretion: Is It Useful? Clin. Chem., November 1, 2003; 49(11): 1932 - 1934. [Full Text] [PDF]
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