National Health and Nutrition Examination Survey

Acrylamide, a toxic and potentially cancer-causing chemical, is formed in high amounts in many types of food prepared/cooked at high temperatures. Because acrylamide is formed during the cooking process, specifically when producing French fries, potato chips and other fried products, intake of acrylamide through consumption of these foods can be high, thus exposing a large portion of the population to this chemical and putting them at risk of adverse health effects. Though acrylamide is known to cause adverse health effects and biomarkers exist to assess exposure to this chemical, no data on the actual acrylamide exposure in the population exist. Filling this knowledge gap is especially important to properly assess the risks associated with the consumption of food containing high levels of acrylamide.

Participants aged 3 year and older who do not meet any of the exclusion criteria are eligible.

This procedure describes a method to measure hemoglobin adducts of acrylamide 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-carbamoylethyl]valine and N-[2-hydroxycarbamoyl-ethyl]valine for acrylamide and glycidamide adducts, respectively) are measured.

This method is based on 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 acrylamide, glycidamide and acrylonitrile. 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 acrylamide and glycidamide;
2. Total hemoglobin measurement 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 HPLC/MS/MS and results processing

Because results are reported in pmol adduct per g 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 acrylamide and glycidamide hemoglobin adduct is performed using octapeptides with the same amino acid sequence as the N-terminal of the beta-chain of hemoglobin and with acrylamide and glycidamide attached at the valine (AA-VHLTPEEK, GA-VHLTPEEK) and the corresponding stable isotope labeled AA-Val(13C5 15N)-HLTPEEK and GA-Val(13C5 15N)-HLTPEEK as internal standards. Total hemoglobin measurement is performed using calibrators provided with the manufacture’s assay kit.

Packed red cells are processed, stored, and shipped to the Division of Environmental Health Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention for analysis.

Detailed specimen collection and processing instructions are discussed in the NHANES Laboratory/Medical Technologists 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 control and quality assurance protocols (QA/QC) meet the 1988 Clinical Laboratory Improvement Act mandates. Detailed quality control and quality assurance instructions are discussed in the NHANES Laboratory/Medical Technologists Procedures Manual (LPM). Read the LABDOC file for detailed QA/QC protocols.

The analysis of NHANES 2003–2004 laboratory data must be conducted with the key survey design and basic demographic variables.

The NHANES 2003–2004 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 Exam files may be linked to the laboratory data file using the unique survey participant identifier SEQN.

Demographic and Other Related Variables
The analysis of NHANES laboratory data must be conducted using the appropriate survey design and demographic variables. The NHANES 2003-2004 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. 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 other variable prefixed LBX (ex., LBXACR) provides the analytic result for that analyte.

The lower limit of detection (LLOD, in pmol/g) for acrylamide and glycidamide is:

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.