National Health and Nutrition Examination Survey

The specific aims of the component are: 1) to measure the prevalence and extent of tobacco use; 2) to estimate the extent of exposure to environmental tobacco smoke (ETS), and determine trends in exposure to ETS; and 3) to describe the relationship between tobacco use (as well as exposure to ETS) and chronic health conditions, including respiratory and cardiovascular diseases.

The tobacco component for NHANES will include questionnaire items on current and past use of cigarettes, pipes, cigars and smokeless tobacco. Exposure to ETS at home and at work and in-utero ETS exposure among children will also be obtained. ETS exposure will also be assessed for examinees 3 years of age and older, through the measurement of serum cotinine, a metabolite of nicotine. In addition, use of nicotine replacement products (e.g., gum and patch) will be collected, using questionnaires.

The tobacco-specific nitrosamine NNK (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone) is a significant component of tobacco and tobacco smoke. In the smoker’s body, NNK is rapidly reduced to its metabolite, NNAL (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanonol). A significant portion of NNAL may also exist in the glucuronide form NNAL-Gluc (NNAL-N-Gluc and NNAL-O-Gluc) in the urine. Both NNK and NNAL are potent lung carcinogens in rodents ( Smith C. J. and Hansch C. Food and Chemical Toxicology), and they are likely involved in the increased lung cancer risk in smokers ((Hecht S. S. and Hoffmann D., Carcinogenesis); (Hecht, Chem. Res.Toxicol); (Preston-(Martin, S, IARC Sci. Publ.) The NNAL metabolite has similar carcinogenicity to NNK (Hecht, S. S. Carcinogenesis,). NNAL (either free and/or total forms) may be used as a biomarker for exposure to NNK among active smokers, and also among nonsmokers following exposure to secondhand smoke (SHS).

Participants aged 3 years and older for cotinine and aged 6 years and older for total NNAL, who do not meet any of the exclusion criteria, are eligible.

Serum specimens and urine specimens are processed, stored, and shipped to the Division of Laboratory Sciences, National Center for Environmental Health, and Centers for Disease Control and Prevention for analysis.

Vials are stored under appropriate frozen (–20°C) conditions until they are shipped to National Center for Environmental Health for testing.

Cotinine is a major metabolite of nicotine that may be used as a marker for both active smoking, and as an index to Environmental Tobacco Smoke (ETS) exposure, or "passive smoking". Cotinine is generally preferred over nicotine for such assessments because of its substantially longer half-life. The half-life of cotinine in plasma has been estimated to be about 15–20 hrs; by contrast, the half-life of nicotine is only 0.5–3 hrs. Cotinine may be measured in serum, urine or saliva – the half-life of cotinine in all three fluids is essentially the same. Cotinine concentrations tend to be higher (3–8x) in urine than in serum; however, for studies requiring a quantitative assessment of exposure, plasma or serum is regarded as the fluid of choice. Therefore, serum was chosen for NHANES cotinine analyses.

Serum cotinine is measured by an isotope dilution-high performance liquid chromatography / atmospheric pressure chemical ionization tandem mass spectrometry (ID HPLC-APCI MS/MS). Briefly, the serum sample is spiked with methyl-D3 cotinine as an internal standard, and after an equilibration period, the sample is applied to a basified solid-phase extraction column. Cotinine is extracted off the column with methylene chloride, the organic extract is concentrated, and the residue is injected onto a short, C18 HPLC column. The eluant from these injections is monitored by APCI-MS/MS, and the m/z 80 daughter ion from the m/z 177 quasi-molecular ion is quantitated, along with additional ions for the internal standard, external standard, and for confirmation. Cotinine concentrations are derived from the ratio of native to labeled cotinine in the sample, by comparisons to a standard curve.

NNAL is measured by using liquid chromatography linked to tandem mass spectrometry (LC/MS/MS).  For “total” NNAL assays, the urine sample is fortified with an NNAL-¹³C₆ internal standard, and then hydrolyzed using β-glucuronidase in incubations for at least 24 hours.  The samples are then extracted and cleaned up on a specially-designed solid-phase molecularly-imprinted polymer (MIP) column, after which the analyte is eluted and analyzed by LC/MS/MS, monitoring the m/z 210->180 native, and m/z 216->186 internal standard transition ions.  NNAL concentrations are derived from the ratio of the integrated peaks of native to labeled ions by comparison to a standard calibration curve.  Free NNAL measurements are conducted in a similar manner, but with the omission of prior enzymatic hydrolysis.  Bound NNAL (i.e. NNAL-Gluc) may be estimated from the difference of (Total NNAL – Free NNAL).  This method has been described previously ( Xia Y, McGuffey JE, Anal Chem.)

There were no changes (from the previous 2 years of NHANES) to equipment, lab methods, or lab site. Detailed instructions on specimen collection and processing can be found in the NHANES Laboratory/Medical Technologists Procedures Manual (LPM).

Read the General Documentation on Laboratory Data file for detailed data processing and editing protocols. The analytical methods are described in the Description of Laboratory Methodology section above.

The NHANES quality assurance and quality control (QA/QC) protocols meet the 1988 Clinical Laboratory Improvement Act mandates. Detailed QA/QC instructions are discussed in the NHANES Laboratory/Medical Technologists Procedures Manual (LPM). Read the General Documentation on Laboratory Data file for detailed QA/QC protocols.

NHANES Survey Design:

The analysis of NHANES laboratory data must be conducted with the key survey design and basic demographic variables. The Household Questionnaire Data Files contain demographic data, health indicators, and other related information collected during household interviews. The Household Questionnaire Data Files also contain all survey design variables and sample weights required to analyze these data. The Phlebotomy Examination file includes auxiliary information on duration of fasting, the time of day of the venipuncture, and the conditions precluding venipuncture. The Household Questionnaire and Phlebotomy Examination files may be linked to the laboratory data file using the unique survey participant identifier SEQN.

In cases, where the result was below the limit of detection, the value for that variable is the detection limit divided by the square root of two.  The lower detection limit for cotinine was constant during this two-year cycle. Serum cotinine= 0.015 ng/mL.

The lower detection limit for NNAL was variable during this two-year cycle. For NNAL the variable named URD___LC indicates whether the result was below the limit of detection. There are two values: “0” and “1””. “0” means that the result was at or above the limit of detection. “1” indicates that the result was below the limit of detection.

Exam sample weights should be used for analyses. Please refer to the NHANES Analytic Guidelines and the on-line NHANES Tutorial  for further details on the use of sample weights and other analytic issues.