Ethylene Oxide
Ethylene oxide (EO) is an important industrial chemical used in making consumer and non-consumer products and is used as a gaseous sterilant for medical devices. EO has been classified as a human carcinogen (Group 1) by the International Agency for Research on Cancer (IARC). EO has been detected in exogenous sources, such as tobacco smoke, automobile exhaust, and some food (Clin Chem, 2016). EO, is also formed endogenously in animals and humans as a result of Cytochrome P450 2E1 (CYP2E1) mediated metabolic oxidation of ethylene. It is also formed in vivo during normal physiological processes, such as methionine oxidation, lipid peroxidation, and via the metabolic activity of intestinal bacteria. Information on endogenous and exogenous EO exposure in the general population is very limited and 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 EO 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 EO adducts) 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 EO, propylene oxide, and styrene oxide and later optimized to increase yield of Edman products of these adducts. 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 EO;
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 EO hemoglobin adducts is performed using octapeptides with the same amino acid sequence as the N-terminal of the beta-chain of hemoglobin with EO 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 this component in the NHANES 2017-2018 cycle.
Ethylene Oxide Lab Procedure Manual (February 2023)
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 QC protocol for all the contract laboratories, which outlined the use of Westgard rules (Westgard et al, 1981) when testing 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 2017-2018 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. For example, in 2017-2018, approximately 80% of children aged 1-17 years who were examined in the MEC provided a blood specimen through phlebotomy, while 95% of examined adults aged 18 and older provided a blood specimen. 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
EO 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 2017-2018 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., LBDEOALC) 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., LBXEOA) provides the analytic result for that
analyte. For analytes with analytic results below the lower limit of detection
(ex., LBDEOALC =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 EO is:
| Variable Name | Analyte Description | LLOD |
|---|---|---|
| LBXEOA | Ethylene Oxide | 12.9 |
| Code or Value | Value Description | Count | Cumulative | Skip to Item |
|---|---|---|---|---|
| 9636.767387 to 1502431.3423 | Range of Values | 2452 | 2452 | |
| 0 | No Lab Result | 26 | 2478 | |
| . | Missing | 0 | 2478 |
| Code or Value | Value Description | Count | Cumulative | Skip to Item |
|---|---|---|---|---|
| 9.12 to 1460 | Range of Values | 2248 | 2248 | |
| . | Missing | 230 | 2478 |
| Code or Value | Value Description | Count | Cumulative | Skip to Item |
|---|---|---|---|---|
| 0 | detectable result | 2165 | 2165 | |
| 1 | below detectable limit | 83 | 2248 | |
| . | Missing | 230 | 2478 |