• Component Description
• Eligible Sample
• Description of Laboratory Methodology
• Laboratory Method Files
• Laboratory Quality Assurance and Monitoring
• Data Processing and Editing
• Analytic Notes
• References
• Codebook
  • SEQN - Respondent sequence number
  • WTSA2YR - Subsample A Weights
  • URX1NP - 1-Aminonaphthalene, urine (pg/mL)
  • URD1NPLC - 1-Aminonaphthalene, urine Comment Code
  • URX2NP - 2-Aminonaphthalene, urine (pg/mL)
  • URD2NPLC - 2-Aminonaphthalene, urine Comment Code
  • URX4BP - 4-Aminobiphenyl, urine (pg/mL)
  • URD4BPLC - 4-Aminobiphenyl, urine Comment Code
  • URXANS - o-Anisidine, urine (pg/mL)
  • URDANSLC - o-Anisidine, urine Comment Code
  • URXDMN - 2,6-Dimethylaniline, urine (pg/mL)
  • URDDMNLC - 2,6-Dimethylaniline, urine Comment Code
  • URXOTD - o-Toluidine, urine (pg/mL)
  • URDOTDLC - o-Toluidine, urine Comment Code

National Health and Nutrition Examination Survey

2013-2014 Data Documentation, Codebook, and Frequencies

Aromatic Amines - Urine (AA_H)

Data File: AA_H.xpt

First Published: February 2020

Last Revised: NA

Component Description

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).

Eligible Sample

Examined participants aged 6 years and older were eligible.

Description of Laboratory Methodology

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.

Laboratory Method Files

Aromatic Amines Laboratory Procedure Manual (February 2020)

Laboratory Quality Assurance and Monitoring

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).

Data Processing and Editing

The data were reviewed. Incomplete data or improbable values were sent to the performing laboratory for confirmation.

Analytic Notes

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:

References

• Bartsch, H., Caporaso, N., Coda, M., Kadlubar, F., Malaveille, C., Skipper, P. L., Talaska, G., Tannenbaum, S. R., Vineis, P., (1990) Carcinogen Hemoglobin Adducts, Urinary Mutagenicity, and Metabolic Phenotype in Active and Passive Cigarette Smokers. Journal of the National Cancer Institute, 82, 1826-1831.
  
• Bryant MS, Skipper PL, Tannenbaum SR, Maclure M. Hemoglobin Adducts of 4-Aminobiphenyl in Smokers and Nonsmokers. Cancer Research 1987; 47:602 8.
  
• Bryant MS, Skipper PL, Wishnok JS, Stillwell WG, Glogowski JA, Tannenbaum SR. Determination of Haemoglobin Adducts of Aromatic Amines by Gas Chromatography Mass Spectrometry. IARC Public Sci 1993; 109:281 92.
  
• Bryant, M. S., Vineis, P., Skipper, P. L., Tannenbaum, S. R., (1988) Hemoglobin adducts of aromatic amines: Associations with smoking status and type of tobacco. Proceedings of the National Academy of Sciences, 85, 9788-9791.
  
• Castelao, J. E., Yuan, J.-M., Skipper, P. L., Tannenbaum, S. R., Gago-Dominguez, M., Crowder, J. S., Ross, R. K., Yu, M. C., (2001) Gender- and Smoking-Related Bladder Cancer Risk. Journal of the National Cancer Institute, 93, 538-545.
  
• Caudill, S.P., Schleicher, R.L., Pirkle, J.L. Multi-rule quality control for the age-related eye disease study. Statist. Med. (2008) 27(20):4094-40106.
  
• Chiang, T. A., Pei-Fen, W., Ying, L. S., Wang, L. F., Ko, Y. C., (1999) Mutagenicity and aromatic amine content of fumes from heated cooking oils produced in Taiwan. Food and Chemical Toxicology, 37, 125–134.
  
• Dawson, R. F., (1952) Chemistry and Biochemistry of Green Tobacco. Industrial and Engineering Chemistry, 44, 266-270.
  
• DeBruin, L. S., Pawliszyn, J. B., Josephy, P. D., (1999) Detection of monocyclic aromatic amines, possible mammary carcinogens, in human milk. Chemical Research in Toxicology, 12, 78-82.
  
• Fischer, P. (1999) Chapter II, IIa Cigarette manufacture, tobacco blending. In Davis D.L. & Nielson M.T.  (Eds.), Tobacco Production, Chemistry and Technology (348). Oxford: Blackwell Science.
  
• Hammond SK, Coghlin J, Gann PH, Paul M. Taghizadeh K, Skipper PL, Tannenbaum SR. Relationship between Environmental Tobacco Smoke Exposure and Carcinogen Hemoglobin Adduct Levels in Nonsmokers. J Nab Cancer Inst 1993; 85:474 8.
  
• Heckman, R. A., Best, F. W., (1981) An investigation of the lipophilic bases of cigarette smoke condensate. Tobacco Science, 25, 33-39.
  
• IARC, (1993) 2,6-DIMETHYLANILINE (2,6-XYLIDINE). IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, 57, 323-335.
  
• IARC, (1999) ortho-ANISIDINE. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, 73, 49-58.
  
• IARC, (2000) ortho-TOLUIDINE. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, 77, 267-322.
  
• IARC, (2010) 2-NAPHTHYLANIME. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, 99, 369-405.
  
• IARC, (2010) 4-AMINOBIPHENYL. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, 99, 71-109.
  
• IARC, (2010) Some aromatic amines, organic dyes, and related exposures. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, 99, 1-678.
  
• Maclure M, Katz RB, Bryant MS, Skipper PL, Tannenbaum SR. Elevated Blood Levels of Carcinogens in Passive Smokers. Am J. Pub Health 1989; 79:1381-4.
  
• Neurath, G. B., Dünger, M., Pein, F. G., Ambrosius, D., Schreiber, O., (1977) Primary and secondary amines in the human environment. Food and Cosmetics Toxicology, 15, 275–282.
  
• Patrianakos, C., Hoffmann, D., (1979) Chemical Studies on Tobacco Smoke LXIV. On the Analysis of Aromatic Amines in Cigarette Smoke. Journal of Anlaytical Toxicology, 3, 150-154.
  
• Pereira, L., Mondal, P. K., Alves, M., (2015) Aromatic amines sources, environmental impact and remediation. In Pollutants in Buildings, Water and Living Organisms (pp. 297-346). Springer.
  
• Perera FP, Santella RM, Brenner D, Poirier MC, Munshi AA, Fischman HK, Ryzin JV. DNA Adducts, Protein Adducts, and Sister Chromatid Exchange in Cigarette Smokers and Nonsmokers.  J Natl Cancer Inst. 1987; 79:449-56.
  
• Riedel, K., Scherer, G., Engl, J., Hagedorn, H. W., Tricker, A. R., (2006) Determination of three carcinogenic aromatic amines in urines of smokers and nonsmokers. Journal of Analytical Toxicology, 30, 187-195.
  
• Schmeltz, I., Hoffmann, D., (1977) Nitrogen-Containing Compounds in Tobacco and Tobacco Smoke. Chemical Reviews, 77, 295-311.
  
• Skipper PL, Peng X, Soohoo CK, Tannenbaum SR. Protein Adducts as Biomarkers of Human Carcinogen Exposure.  Drug Metab Rev 1994; 26:111-24.
  
• Skipper PL, Tannenbaum SR. Molecular Dosimetry of Aromatic Amines in Human Populations.  Env. Health Perspec. 1994; 102:17-21.
  
• Stabbert, R., Schäfer, K. H., Biefel, C., Rustemeier, K., (2003) Analysis of aromatic amines in cigarette smoke. Rapid Communications in Mass Spectrometry, 17, 2125-2132.
  
• Talaska, G., Zoughool, M. A., (2003) Aromatic amine and biomarkers of human exposure. Journal of Environmental Science and Health. Part C, Environmental Carcinogenesis & Ecotoxicology Reviews, 21, 133-164.
  
• USHHS, (2012) Reporting Harmful and Potentially Harmful Constituents in Tobacco Products and Tobacco Smoke Under Section 904(a)(3) of the Federal Food, Drug, and Cosmetic Act. U.S. Department of Health and Human Services, Food and Drug Administration, Center for Tobacco Products (CTP), Silver Spring, MD.
  
• Vineis, P., (1994) Epidemiology of cancer from exposure to arylamines. Environmental Health Perspectives, 102, 7-10.
  
• Vitzthum, O. G., Werkhoff, P., Hubert, P., (1975) New volatile constituents of black tea aroma. Journal of Agricultural and Food Chemistry, 23, 999–1003.
  
• Westgard J.O., Barry P.L., Hunt M.R., Groth T. A multi-rule Shewhart chart for quality control in clinical chemistry. Clin Chem (1981) 27:493-501.
  
• Wishnok JS. Environmental Carcinogens: In Vivo Monitoring Using GC/MS. Anal Chem 1992; 64:1126-35.
  

Codebook and Frequencies