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.
Aromatic amines are classified as carcinogens or possible carcinogens (Hammond SK, et al., 1993; Perera FP, et al., 1987; Skipper PL, et al., 1994; Wishnok JS, et al., 1992; Skipper PL, et al., 1994; and Maclure M, et al., 1989). The International Agency for Research on Cancer (IARC) has classified several AAs as carcinogenic to humans (group 1), or possibly carcinogenic to humans (group 2B). AAs such as o-toluidine, 2-aminonapthalene, and 4-aminobiphenyl, for example, are well-established human bladder carcinogens (IARC, 2010; Bartsch, H., et. al, 1990; Bryant, M.S., et. al, 1987). Aromatic amines are metabolized mainly in the liver, then travels to the bladder to be eventually excreted out of the body through urination. The amine functional groups may be metabolized in the liver to the acetylated and / or glucuronidated forms, or they may be oxidized to a hydroxylamine form, which undergoes further conversion via an acetylation reaction to form a N-acetoxy metabolite. The N-acetoxy metabolite is able to undergo non-enzymatic breakdown to yield the reactive nitrenium ion, nitrene or a free radical that can covalently bind to tissue macromolecules (proteins) and DNA to form adducts (IARC, 2000; Riedel, K., et. al, 2006; Talaska, G. Zoughool, M. A., 2003). Formation of the hydroxylamine metabolite is considered the primary pathway to AA carcinogenicity in the target organs (e.g., bladder) (Bryant MS, et al., 1987 and Bryant MS, et al., 1993). The acetylated and glucuronidated forms of AAs, which are excreted directly in urine, are the products of the body’s detoxification metabolism pathway. Consequently, total urinary concentrations of aromatic amines (free, acetylated and glucuronidated Aas) are effective surrogate biomarkers of AAs exposure or carcinogenic metabolites at the target tissues (bladder, liver, kidney, pancreas, spleen, thyroid, etc.).
Several AAs are on the FDA’s list of harmful and potentially harmful constituents (HPHCs) since the passage of The Family Smoking Prevention and Tobacco Control Act in 2009 and the creation of the Center of Tobacco Products (USHHS, 2012). Smoking tobacco and inhaling SHS may be major sources of exposure to several aromatic amines (AAs) (Bryant, et. al, 1988; Castelao, J. E., et. al, 2001; Vineis, P., 1994), which are suggested to be principal agents for the development of bladder cancer in humans (IARC, 2010). AAS are present in mainstream and side stream tobacco smoke, with the latter containing up to thirty times as much 4-aminobiphenyl (4-ABP) as mainstream smoke (Bryant MS, et al., 1987 and Bryant MS, et al., 1993). The total nitrogen content in tobacco leaves—as derived from nitrates, ammonia, amino acids, amides and alkaloids—ultimately contributes to the formation of AAs in tobacco smoke (Stabbert, R., et. al, 2003; Fischer, P., 1999; Dawson, R. F., 1952). Nitrate, which is introduced to the growing tobacco plant through the application of fertilizer, can be converted to ammonia, which, in turn, is converted to other nitrogenous organic compounds such as amino acids. Intermediate NH2 radicals, forming during the pyrolysis of ammonia during tobacco combustion, may react with aromatic CH groups (from compounds already present in the tobacco leaves) to form the AAs (Patrianakos, C., et. al, 1979). In addition to the pyrosynthetic mechanism, AAs may also be transferred directly from the tobacco leaves into the smoke via thermal degradation of alkaloids and amino acids (Schmeltz, I., Hoffman, D., 1977; Heckman, R. A., Best, F. W., 1981).
In addition to tobacco smoke, other exposure sources of AAs include several chemical industry sectors such as dyes and pigments (e.g., azo dyes, indigo dyes), pharmaceuticals, pesticides, herbicides, synthetic rubber and plastics (IARC, 2000; IARC, 1993; IARC – 4-Aminobiphenly, 2010; IARC – 2-Naphthylanime, 2010; IARC, 1999). In manufacturing these industrial chemicals, AAs are used as raw materials or intermediates, and therefore, they should not occur in the final products. As such, occupational exposure to AAs can occur by inhalation or skin contact during the production of chemicals that use AAs as raw materials or intermediates. AAs can also be found in environmental pollution such as diesel exhaust, combustion of wood chips and rubber, and substances in charcoal barbequed meats and fish (Debruin, L. S., et. al, 1999; Pereira, L., et. al, 2015). Natural occurrence of AA was reported, for example, in the aroma components of black tea, and certain vegetables (Vitzthum, O. G., et. al, 1975; Neurath, G. B., et. al, 1977). Other potential sources include emissions from cooking oils (e.g., vegetable, sunflower and refined lard oil) (Chiang, T. A., et. al, 1999).
Participants aged 18 years and older, who met the regular one-third subsample selection criteria, were included in this special subsample. Additionally, to oversample adult smokers, those participants aged 18 years and older, not in the regular one-third subsample, who smoked at least 100 cigarettes in their entire lifetime (SMQ020=1) and now smoke cigarettes every day (SMQ040=1), were also included in this special subsample.
Aromatic amines are quantified by an isotope-dilution gas chromatographic, tandem mass spectrometric method (ID GC-MS/MS). Urine samples are collected and stored at approximately -70±10°C. 13C and 2H internal standards are added, and the samples are hydrolyzed, cleaned up, and extracted on support liquid extraction (SLE) cartridges. The analytes are then derivatized to form pentafluoropropionamides, and analyzed by GC/MS/MS, using multiple reaction monitoring (MRM). The analyte concentrations are derived from the ratio of the integrated peaks of native to labeled ions by comparison to a standard curve.
Refer to the Laboratory Method Files section for a detailed description of the laboratory methods used.
This is a new component in the NHANES 2013-2014 cycle.
Aromatic Amines Laboratory Procedure Manual (February 2020)
Urine specimens are processed, stored, and shipped to the Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention for analysis.
Detailed instructions on specimen collection and processing are discussed in the NHANES Laboratory Procedures Manual (LPM). Vials are stored under appropriate frozen (–30°C) conditions until they are shipped to National Center for Environmental Health for testing.
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 LPM.
Mobile Examination Centers (MECs)
Laboratory team performance is monitored using several techniques. NCHS and contract consultants use a structured competency assessment evaluation during visits to evaluate both the quality of the laboratory work and the quality-control procedures. Each laboratory staff member is observed for equipment operation, specimen collection and preparation; testing procedures and constructive feedback are given to each staff member. Formal retraining sessions are conducted annually to ensure that required skill levels were maintained.
Analytical Laboratories
NHANES uses several methods to monitor the quality of the analyses performed by the contract laboratories. In the MEC, these methods include performing blind split samples collected on “dry run” sessions. In addition, contract laboratories randomly perform repeat testing on 2% of all specimens.
NCHS developed and distributed a quality control protocol for all CDC and contract laboratories, which outlined the use of Westgard rules (Westgard et al., 1981) when running NHANES specimens. Progress reports containing any problems encountered during shipping or receipt of specimens, summary statistics for each control pool, QC graphs, instrument calibration, reagents, and any special considerations are submitted to NCHS quarterly. The reports are reviewed for trends or shifts in the data. The laboratories are required to explain any identified areas of concern.
All QC procedures recommended by the manufacturers were followed. Reported results for all assays meet the Division of Laboratory Sciences quality control and quality assurance performance criteria for accuracy and precision, similar to the Westgard rules (Caudill, et al., 2008).
The data were reviewed. Incomplete data or improbable values were sent to the performing laboratory for confirmation.
Refer to the 2013-2014 Laboratory Data Overview for general information on NHANES laboratory data.
There are over 800 laboratory tests performed on NHANES participants. However, not all participants provided biospecimens or enough volume for all the tests to be performed. The specimen availability can also vary by age or other population characteristics. Analysts should evaluate the extent of missing data in the dataset related to the outcome of interest as well as any predictor variables used in the analyses to determine whether additional re-weighting for item non-response is necessary.
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.
Subsample Weights
Aromatic amines were measured in a one-third subsample of persons 6 years and older. Special sample weights are required to analyze these data properly. Specific sample weights for this subsample are included in this data file and should be used when analyzing these data.
Demographic and Other Related Variables
The analysis of NHANES laboratory data must be conducted using the appropriate survey design and demographic variables. The NHANES 2013-2014 Demographics File contains demographic data, health indicators, and other related information collected during household interviews as well as the sample weight variables. The recommended procedure for variance estimation requires use of stratum and PSU variables (SDMVSTRA and SDMVPSU, respectively) in the demographic data file.
The laboratory data file can be linked to the other NHANES data files using the unique survey participant identifier (i.e., SEQN).
Detection Limits
The detection limits were constant for all of the analytes in the data set. Two variables are provided for each of these analytes. The variable name ending in “LC” (ex., URD1NPLC) indicates whether the result was below the limit of detection: the value “0” means that the result was at or above the limit of detection, “1” indicates that the result was below the limit of detection. For analytes with analytic results below the lower limit of detection (ex. URD1NPLC =1), an imputed fill value was placed in the analyte results field. This value is the lower limit of detection divided by the square root of 2 (LLOD/sqrt[2]). The other variable prefixed URX (ex., URX1NP) provides the analytic result for the analyte.
The lower limit of detection (LLOD in pg/mL) for Aromatic Amines in urine is:
|
VARIABLE |
SAS LABEL |
LLOD |
|
URX1NP |
1-Aminonaphthalene, urine (pg/mL) |
1.29 |
|
URX2NP |
2-Aminonaphthalene, urine (pg/mL) |
2.79 |
|
URX4BP |
4-Aminobiphenyl, urine (pg/mL) |
1.75 |
|
URXANS |
o-Anisidine, urine (pg/mL) |
7.02 |
|
URXDMN |
2,6-Dimethylaniline, urine (pg/mL) |
15.67 |
|
URXOTD |
o-Toluidine, urine (pg/mL) |
111.22 |