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. Acrylamide is formed during the cooking process, specifically when preparing french fries, potato chips and other fried products. Intake of acrylamide through the 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. Although acrylamide is known to cause adverse health effects, despite the existence of biomarkers to assess exposure to this chemical, limited data on the actual acrylamide exposure in the population exists. Filling this knowledge gap is especially important to properly assess the risks associated with the consumption of food containing high levels of acrylamide.

Examined participants aged 3 years and older were 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 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 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.

N-terminal hemoglobin adducts of acrylamide and glycidamide (October 2016)

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 Procedures Manual (LPM). Vials are stored under appropriate frozen (–70°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 LPM.

Mobile Examination Centers (MECs)
Laboratory team performance is monitored using several techniques. NCHS and contract consultants use a structured quality assurance evaluation during unscheduled 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 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’ 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.

Acrylamide and Glycidamide Data for 2005-2006
Measurements performed in this NHANES cycle were performed in isolated and washed red blood cells, which were prepared from EDTA-whole blood immediately after specimen collection. Measurements performed in the NHANES 2003-2004 cycle were conducted using frozen, stored whole blood. No information is available about the potential effect of these different specimen treatment and storage conditions on measurement results. These differences may need to be considered when comparing results from this NHANES cycle with those obtained from NHANES 2003-2004.

Refer to the 2005-2006 Laboratory Data Overview for general information on NHANES laboratory 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 2005-2006 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.