Table of Contents

Component Description

Ethylene Oxide

Ethylene oxide (EO) is an industrial chemical used in the making of consumer and non-consumer products and is used as a gaseous sterilant for medical devices. EO has been detected in tobacco smoke, automobile exhaust, and some food (Clin Chem, 2016).  EO, which is a metabolite of ethylene, is 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 the metabolizing 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.

Eligible Sample

Examined participants aged 6 years and older from a one-third subsample were eligible.

Description of Laboratory Methodology

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.

This is a new component in the 2013-2014 survey cycle.

Laboratory Method Files

Acrylamide and Glycidamide Lab Procedure Manual (December 2019)

Laboratory Quality Assurance and Monitoring

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 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 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 QC 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).

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.

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 2013-2014 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

12.90

References

Codebook and Frequencies

SEQN - Respondent sequence number

Variable Name:
SEQN
SAS Label:
Respondent sequence number
English Text:
Respondent sequence number
Target:
Both males and females 6 YEARS - 150 YEARS

WTSA2YR - Subsample A weights

Variable Name:
WTSA2YR
SAS Label:
Subsample A weights
English Text:
Subsample A weights
Target:
Both males and females 6 YEARS - 150 YEARS
Code or Value Value Description Count Cumulative Skip to Item
16284.37488 to 530325.34726 Range of Values 2724 2724
0 No Lab Result 31 2755
. Missing 0 2755

LBXEOA - Ethylene Oxide (pmol/g Hb)

Variable Name:
LBXEOA
SAS Label:
Ethylene Oxide (pmol/g Hb)
English Text:
Ethylene Oxide (pmol/g Hb)
Target:
Both males and females 6 YEARS - 150 YEARS
Code or Value Value Description Count Cumulative Skip to Item
9.12 to 1720 Range of Values 2485 2485
. Missing 270 2755

LBDEOALC - Ethylene Oxide comment code

Variable Name:
LBDEOALC
SAS Label:
Ethylene Oxide comment code
English Text:
Ethylene Oxide comment code
Target:
Both males and females 6 YEARS - 150 YEARS
Code or Value Value Description Count Cumulative Skip to Item
0 detectable result 2398 2398
1 below detectable limit 87 2485
. Missing 270 2755