National Health and Nutrition Examination Survey

Phytoestrogens are plant-derived polyphenolic compounds, such as isoflavones and lignans that are structurally similar to endogenous estrogens (Cornwell, et al. 2004). Phytoestrogens are capable of estrogen-receptor binding  (Cornwell et al., 2004) and may also influence other biologic pathways. The consumption of diets high in phytoestrogen-rich foods has been associated with lower rates of hormone-dependent cancers (Peeters et al., 2003; Adlercreutz et al., 2002; Davis et al., 1999), improved bone health (Ma et al., 2007; Coxam et al., 2003) and other healthy outcomes. Biomeasures of phytoestrogens are necessary to establish reference ranges for these compounds and to evaluate their potential effects on human health.

Participants aged 6 years and older who met the subsample requirements.

The test principle utilizes high performance liquid chromatography-atmospheric pressure photoionization-tandem mass spectrometry (HPLC-APPI-MS/MS) for the quantitative detection of genistein, daidzein, equol, O-desmethylangolensin, enterodiol, and enterolactone. Human urine samples are processed using enzymatic deconjugation of the glucuronidated phytoestrogens followed by size-exclusion filtration. Phytoestrogens are then separated from other urine components by reversed phase HPLC, detected by APPI-MS/MS, and quantified by isotope dilution. Assay precision is improved by incorporating carbon-13 labeled internal standards for each of the analytes, as well as a 4-methylumbelliferyl glucuronide and 4-methylumbelliferyl sulfate standards to monitor deconjugation efficiency. This selective method allows for rapid detection of phytoestrogens in human urine with limits of detection in the low parts per billion (ng/mL) range.

The assay method changed from 2003-2004 to 2005-2006. In 2003-2004 high performance liquid chromatography electrospry ionization mass spectrometric (LC-ESI-MS/MS) detection (Rybak, M. E. et al., 2008) was utilized and in 2005-2006 high performance liquid chromatography-atmospheric pressure photoionization-tandem mass spectrometry (LC-ESI-MS/MS) was utilized. Crossover studies comparing samples analyzed by LC-ESI-MS/MS and LC-APPI-MS/MS demonstrated high correlation coefficients (r>0.99) and regression slopes approximately equal to 1 and intercepts close to 0.

Urine specimens are processed, stored, and shipped to the Division of  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 are 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 Environmental Health Laboratory Sciences’ quality control and quality assurance performance criteria for accuracy and precision (similar to specifications outlined by Westgard (1981).

Subsample weights

Measures of urinary environmentals were measured in a one third subsample of persons 6 years and over. Special sample weights (WTSB2YR) 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  laboratory data must be conducted with the key survey design and basic demographic variables. The NHANES  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 data files using the unique survey participant identifier 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 URD___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 variables named URX___ provide the analytic result for that analyte.

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.