• 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
  • URXNAL - Urinary Total NNAL (ng/mL)
  • URDNALLC - Urinary Total NNAL Comment Code
  • URXNAB - N'-Nitrosanabasine, urine (ng/mL)
  • URDNABLC - N'-Nitrosanabasine (NAB) Comment Code
  • URXNAT - N'-Nitrosanatabine, urine (ng/mL)
  • URDNATLC - N'-Nitrosanatabine (NAT) Comment Code
  • URXNNN - N'-Nitrosonornicotine, urine (ng/mL)
  • URDNNNLC - N'-Nitrosonornicotine (NNN) Comment Code

National Health and Nutrition Examination Survey

2013-2014 Data Documentation, Codebook, and Frequencies

Tobacco-specific Nitrosamines (TSNAs) - Urine (TSNA_H)

Data File: TSNA_H.xpt

First Published: April 2019

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.

Tobacco-specific nitrosamines (TSNAs) are formed during tobacco smoking and tobacco curing. TSNAs are a leading class of carcinogens in tobacco products, thus becoming an important class of biomarkers for tobacco carcinogen uptake (Hecht and Hoffman, Carcinogenesis; International Agency for Research on Cancer, IARC; Shah and Karnes, Critical Reviews in Toxicology). There are seven major TSNAs identified from tobacco smoke, which includes 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), N’-nitrosonornicotine (NNN), N’-nitrosoanatabine (NAT) and N’-nitrosoanabasine (NAB). Due to their specificity to tobacco, the study of TSNA uptake will provide very useful insight into the mechanistic and epidemiologic role of these compounds in human cancer.

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 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.) NNAL 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 among nonsmokers following exposure to secondhand smoke (SHS).

Eligible Sample

Examined participants aged 6 years and older were eligible.

Description of Laboratory Methodology

Tobacco-specific nitrosamines in urine are measured by an isotope-dilution high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (ID HPLC-ESI MS/MS). For “total” TSNA assays, the urine sample is fortified with internal standards, and then hydrolyzed using β-glucuronidase in incubations for at least 24 hours. The samples are then extracted with liquid-liquid extraction followed by solid phase extraction, after which the analyte is eluted and analyzed by LC/MS/MS. The m/z 210/180, 210/93, and 216/186 are monitored for NNAL quantitation, confirmation, and ISTD, respectively. The m/z 178/148, 178/105, and 182/152 are monitored for NNN quantitation, confirmation, and ISTD, respectively. The m/z 190/160, 190/106, and 194/164 are monitored for NAT quantitation, confirmation, and ISTD, respectively. The m/z 192/162, 192/106, and 196/166 are monitored for NAB quantitation, confirmation, and ISTD, respectively. The concentrations are derived from their respective ratios of native to isotope-labeled in the sample by comparing to their standard curves. Free TSNA measurements are conducted in a similar manner, but with the omission of prior enzymatic hydrolysis.

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. NNAL has been removed from the COTNAL dataset from previous cycles and included in this new dataset.

Laboratory Method Files

TSNA Laboratory Procedure Manual (April 2019)

Laboratory Quality Assurance and Monitoring

Urine samples were processed, stored, and shipped to the Division of Laboratory Sciences, National Center for Environmental Health, and Centers for Disease Control and Prevention, Atlanta, GA for analysis.

Detailed instructions on specimen collection and processing are discussed in the NHANES Laboratory Procedures Manual (LPM). Vials were stored under appropriate frozen (–20°C) conditions until they were shipped to the 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.

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.

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., URDNALLC) 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. The other variable prefixed URX (ex., URXNAL) provides the analytic result for the analyte. For analytes with analytic results below the lower limit of detection (ex. URDNALLC =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 lower limit of detection (LLOD in ng/mL) for Tobacco-Specific Nitrosamines (TSNA) in urine:

References

• 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.
• Hecht, S. S. Biochemistry, biology and carcinogenicity of tobacco-specific N-nitrosamines. Chem. Res.Toxicol., 11: 559-603, 1998.
• Hecht, S. S. Human urinary carcinogen metabolites: biomarkers for investing tobacco and cancer. Carcinogenesis, 23: 907-922, 2002.
• International Agency for Research on Cancer. Smokeless tobacco and some tobacco-specific nitrosamines. In: IARC Monographs on the Evaluation of Carcinogenic Risks to Humans. Lyon, France: IARC 89:166-370, 2007.
• Preston-Martin, S. N-nitroso compounds as a cause of human cancer. IARC Sci. Publ., 84: 477-84, 1987.
• Shah K. A. and Karnes, H. T. A review of the analysis of tobacco-specific nitrosamines in biological matrices. Critical Reviews in Toxicology, 40 (4):305-327, 2010.
• Smith C. J. and Hansch C. The relative toxicology of compounds in mainstream cigarette smoke condensate. Food and Chemical Toxicology, 38:637-646, 2000.
• Wei B, Blount BC, Xia B, Wang L.,Assessing exposure to tobacco-specific carcinogen NNK using its urinary metabolite NNAL measured in US population: 2011-2012. J Expo Sci Environ Epidemiol. 26(3):249-56, 2016.
• 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.
• Xia B, Xia Y, Wong J, Nicodemus KJ, Xu M, Lee J, Guillot T and Li J. Quantitativeanalysis of five tobacco-specific N-nitrosamines in urine by liquid chromatography–atmospheric pressure ionization tandem mass spectrometry. Biomedical Chromatography, 28(3): 375–384, 2014.

Codebook and Frequencies