National Health and Nutrition Examination Survey

Polybrominated diphenyl ethers (PBDEs) are included in a larger group of chemicals known as brominated flame retardants and they are added to products such as foam padding, textiles, or plastics to retard combustion. PBDEs are not chemically bound to the flame-retarded material, so they can enter the environment from volatilization, leaching, or degradation of PBDE-containing products. PBDEs are generally persistent in the environment and have been measured in aquatic sediments, house dust, and aquatic and terrestrial animals, especially in fish where PBDEs are known to bioconcentrate. Hence humans may be exposed though the diet, including breast feeding, and by contact with flame retarded products and contaminated house dust. The rate of elimination of PDBEs from the human body depends on the specific PDBE, with elimination half-lives that can vary from a couple weeks to several months.

Participants aged 12 years and older who met the subsample requirements.

The method described in this manual assesses human body burden of BFRs, specifically PBDEs in serum and/or plasma. This is done by measuring the concentration in serum/plasma through the use of solid-phase extraction (SPE) and subsequent sample clean-up. Final determination of target analytes is performed by isotope dilution gas chromatography high-resolution mass spectrometry GC/IDHRMS.

Concentrations of target analytes are reported on two different bases, i.e., (i) fresh weight basis (i.e., pg/g serum) and (ii) lipid weight basis (i.e., ng/g lipid). Lipid adjusted concentration values are preferable because (i) organohalogen compounds are lipophilic and hence distribute in the body mainly according to the tissues lipid content. Lipid adjusted concentrations correlates with the adipose tissue concentrations of the chemical. Normalization according to lipid content further reduces variability since differences in individuals serum lipid concentrations are cancelled out.

The samples are extracted using SPE, employing an automated SPE workstation (Rapid Trace^® , Caliper Life Sciences). Required sample pretreatment prior to extraction is performed on the Gilson 215 liquid handler, including automated addition of (i) internal standards, (ii) formic acid (denaturant) and (iii) water (diluent) with mixing in-between each addition by rotation. Formic acid is added to denature proteins in the sample enabling extraction of target compounds. During the extraction step the target analytes are transferred from a water medium to an organic solvent.

Sample cleanup, i.e., removal of co-extracted lipids, is obtained by elution (hexane; 8 mL) of the extract through a column containing from the top 0.1 g of silica and 1 g of silica/sulfuric acid (33% by weight). During this procedure serum lipids are degraded in the sulfuric acid layer while cholesterol is removed in the top layer consisting of activated silica gel. Without the activated silica gel layer cholesterol would eliminate water forming cholestene when coming in contact with the sulfuric acid. Cholestene would not be removed in the silica gel/sulfuric acid layer and interfere in the final HR-MS analyses, i.e., causing an ion suppression in the region of 2,2’,4,4’,5 pentabromodiphenyl ether (BDE-99) and 2,2’,4,4’,6-pentabromodiphenyl ether (BDE-100).

This sample cleanup is automated using the Rapid Trace^® (Caliper Life Sciences) also used for sample extraction. The samples are evaporated and transferred to GC vials. Evaporization is performed on the RapidVap® (LabConco), using vacuum, increased temperature and vortex action to aid the evaporization.

Serum concentrations are determined using gas chromatography isotope dilution high resolution mass spectrometry (GC/IDHRMS), which minimizes or eliminates many interferences associated with low-resolution measurement of organohalogen compounds. Splitless injection is used employing a short GC column (DB-5HT; 15 m length, 0.1 μm film thickness, 0.25 mm ID) enabling the determination of high molecular weight compounds such as decabromodiphenyl ether (BDE-209) having a molecular weight close to 1000 amu. Electron impact ionization (EI) is used. The two most abundant ions in the isotopic cluster (fragment or molecular ion) are monitored for the target analyte as well as for the 13C-labeled internal-surrogate standard. Quantification is made against a calibration curve covering the full concentration range of the target analytes.

Serum specimens 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 (–20°C) conditions until they are shipped to National Center for Environmental Health for testing.

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 person is observed for equipment operation, specimen collection and preparation; testing procedures and constructive feedback is given to each staff. Formal retraining sessions are conducted annually to ensure that required skill levels were maintained.

The NHANES QA/QC protocols meet the 1988 Clinical Laboratory Improvement Act mandates. Detailed QA/QC instructions are discussed in the NHANES LPM.

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 2.0% of all specimens.

NCHS developed and distributed a quality control protocol for all the contract laboratories which outlined the Westgard rules used 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 and Westat 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 Science’s quality control and quality assurance performance criteria for accuracy and precision (similar to specifications outlined by Westgard (1981).

Subsample weights

Measures of serum polybrominated diphenyl ethers (PBDE) were measured in a one third subsample of persons 12 years and over. 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.

Variance estimation

The analysis of NHANES 2003-2004 laboratory data must be conducted with the key survey design and basic demographic variables. The NHANES 2003-2004 Demographic Data File contains demographic and 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.

Links to NHANES Data Files

This laboratory data file can be linked to the other NHANES 2003-2004 data files using the unique survey participant identifier SEQN.

Detection Limits

The detection limits were variable for all of the analytes in the data set. Two variables are provided for each of these analytes. The variable named LBD___LC indicates whether the result was below the limit of detection. There are two values: “0” and “1”. “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 named LBX___ provides the analytic result for that analyte.

Please refer to the Analytic Guidelines for further details on the use of sample weights and other analytic issues.