Acrylamide and Glycidamide
Acrylamide (AA) has been identified as neurotoxic, mutagenic, and a probable genotoxic to animals and humans (Clin Chem, 2016). It is classified as a probable carcinogen to humans by the International Agency for Research on Cancer (IARC) and as a potential occupational carcinogen by the Occupational Safety and Health Administration (OSHA). People are exposed to acrylamide through certain occupational activities that involve production and use of acrylamide, such as in waste water treatment, ore processing, paper and textile industries and manufacturing of dyes and adhesives. AA exposure can occur from tobacco smoke and dry-heated food. The actual exposure of the general population to acrylamide and possible changes in this exposure over time are not known.
Glycidamide (GA), the primary metabolite of AA, has a higher reactivity towards nucleophilic reagents than AA. Results from animal studies suggest that genetic damage in somatic and germ cells is dependent upon the metabolism of AA to GA by Cytochrome P450 2E1 (CYP2E) (Clin Chem, 2016). To obtain comprehensive information about AA exposure and to assess potential health effects related to this exposure, it is necessary to measure both AA and GA exposures. Information on exposure to these chemicals in the general population is needed to assess potential health effects associated with this exposure and to monitor changes in exposure over time.
Examined participants aged 6 years and older from a one-third subsample were eligible.
This procedure describes a method to measure hemoglobin adducts of AA and its primary metabolite glycidamide in human whole blood or erythrocytes. Specifically, the reaction products with the N-terminal valine of the hemoglobin protein chains (N-[2-carbamoyl ethyl] valine and N-[2-hydroxycarbamoyl-ethyl] valine for AA and GA, respectively) are measured.
This method is based on the modified Edman reaction, which uses the effect of N-alkylated amino acids being able to form Edman products in neutral or alkaline conditions without changing the pH to acidic conditions required in conventional Edman reaction procedures. It was first described for N-terminal hemoglobin adducts of ethylene oxide, propylene oxide and styrene oxide and later optimized to increase yield of Edman products of these adducts. This optimized method was then successfully applied to adducts produced by other chemicals, such as AA and GA. This optimized method was further refined and modified in-house to increase sensitivity and enable automation.
The procedure described here consists of 4 parts:
1. Preparation of the specimen for measurement of hemoglobin adducts of AA and GA;
2. Measurement of total hemoglobin in the sample solution used for hemoglobin adduct measurements;
3. Modified Edman reaction in the sample solution and isolation of Edman products; and
4. Analysis of Edman products by high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) and results processing.
Because results are reported in pmol adduct per gram of hemoglobin, the amount of hemoglobin used for the modified Edman reaction needs to be known. Therefore, this procedure includes a measurement procedure for total hemoglobin. It is a commercial assay kit based on a well-established procedure commonly used in clinical chemistry. Quantitation of the AA and GA hemoglobin adducts is performed using octapeptides with the same amino acid sequence as the N-terminal of the beta-chain of hemoglobin with AA and GA attached at the valine.
Refer to the Laboratory Method Files section for a detailed description of the laboratory method used.
There were no changes to the lab method, lab equipment, or lab site for these components in the NAHNES 2015-2016 cycle.
Acrylamide and Glycidamide Lab Procedure Manual (December 2019)
Washed-packed red blood cell specimens were processed, stored, and shipped to the Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, GA for analysis.
Detailed specimen collection and processing instructions 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 protocols (QA/QC) meet the 1988 Clinical Laboratory Improvement Amendments mandates. Detailed quality control and quality assurance 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 QC 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 the 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’ QA/QC 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 2015-2016 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.
Subsample Weights
AA and GA were measured in a one-third subsample of participants 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 2015-2016 Demographics File contains demographic data, health indicators, and other related information collected during household interviews as well as the sample design variables. The recommended procedure for variance estimation requires use of stratum and PSU variables (SDMVSTRA and SDMVPSU, respectively) in the demographic data file.
The Fasting Questionnaire File includes auxiliary information, such as fasting status, the time of venipuncture, and the conditions precluding venipuncture.
This 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 named ended “LC” (ex., LBDACRLC) 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 LBX (ex., LBXACR) provides the analytic result for that analyte. For analytes with analytic results below the lower limit of detection (ex., LBDACRLC =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 pmol/g Hb) for AA and GA is:
|
Variable Name |
Analyte Description |
LLOD |
|
LBXACR |
Acrylamide |
3.90 |
|
LBXGLY |
Glycidamide |
4.90 |