• Component Description
• Eligible Sample
• Description of Laboratory Methodology
• Laboratory Method Files
• Laboratory Quality Assurance and Monitoring
• Data Processing and Editing
• Analytic Notes
• References
• Codebook
  • SEQN - Respondent sequence number
  • LBXVIDMS - 25OHD2+25OHD3 (nmol/L)
  • LBDVIDLC - 25OHD2+25OHD3 comment code
  • LBXVD2MS - 25OHD2 (nmol/L)
  • LBDVD2LC - 25OHD2 comment code
  • LBXVD3MS - 25OHD3 (nmol/L)
  • LBDVD3LC - 25OHD3 comment code
  • LBXVE3MS - epi-25OHD3 (nmol/L)
  • LBDVE3LC - epi-25OHD3 comment code

National Health and Nutrition Examination Survey

2015-2016 Data Documentation, Codebook, and Frequencies

Vitamin D (VID_I)

Data File: VID_I.xpt

First Published: August 2021

Last Revised: NA

Component Description

The objectives of this component are to provide data for monitoring secular trends in measures of nutritional status in the U.S. population and to evaluate the effect of people's habits and behaviors, such as physical activity and the use of alcohol, tobacco, and dietary supplements on people's nutritional status.  Also, to evaluate the effect of changes in nutrition and public health policies, including welfare reform legislation, food fortification policy, and child nutrition programs on the nutritional status of the U.S. population.

These data will be used to estimate risks for deficiencies and toxicities of specific nutrients in the population and subgroups, to provide population reference data, and to estimate the contribution of diet, supplements, and other factors to serum levels of nutrients. Data will be used for research to further define nutrient requirements as well as optimal levels for disease prevention and health promotion.

Eligible Sample

Examined participants aged 1 years and older.

Description of Laboratory Methodology

The test principle for the CDC method utilizes high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) for the quantitative detection of 25-hydroxyvitamin D3 (25OHD3), 3-epi-25-hydroxyvitamin D3 (epi-25OHD3), and 25-hydroxyvitamin D2 (25OHD2) in human serum. The analytes are chromatographically separated generally on one of three pentaflurophenyl (PFP) columns (Thermo Scientific Hypersil GOLD PFP 2.1 x 100 mm, 1.9 µm particle size column, Phenomenex Kinetex PFP 2.1 x 100 mm, 1.7 µm, or Sigma-Aldrich Ascentis Express F5, 2.1 x 150 mm, 2.7 μm ). Mobile phase composition for optimized chromatography varies slightly for the three columns but is between 69% and 72% methanol in water. The composition of the solution added to the serum prior to extraction, the solution used for reconstitution, and the needle wash should match that used for the mobile phase.  

Serum samples are first treated by the addition of a 67% methanol solution containing three internal standards (IS) and additional solution of 69-72% methanol to allow enough volume to partition the organic and aqueous phases during extraction; this is followed by the addition of hexane.  Analytes are extracted from the aqueous phase into the hexane layer (liquid-liquid extraction), which is then dried under vacuum.  The extract is re-dissolved with 69-72% methanol.  An aliquot of the extract is injected onto the PFP column for the separation of 25OHD3, epi-25OHD3, 25OHD2, and the IS, 26,26,26,27,27,27-hexadeuterium-25-hydroxyvitamin D3, 6,19,19-trideuterium-3-epi-25-hydroxyvitamin D3, and 6,19,19-trideuterium-25-hydroxyvitamin D2. Detection is performed by using a triple quadrupole tandem mass spectrometer (Thermo TSQ Vantage system) using atmospheric pressure chemical ionization in the positive ion mode. Quantitation is accomplished by comparing the response ratio in the unknown with the response ratio of a known amount of analyte in a calibrator solution. Response ratios are based on the peak area of the analyte divided by the peak area of the internal standard. 

Refer to the Laboratory Method Files section for a detailed description of the methods used.

There were no changes to the lab method, lab equipment, or lab site for this component in the NHANES 2015-2016 cycle.

Laboratory Method Files

Vitamin D (August 2021)

Laboratory Quality Assurance and Monitoring

Serum 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 instructions on specimen collection and processing are discussed in the NHANES Laboratory Procedures Manual (LPM). Vials are stored under appropriate frozen (–30°C) conditions until they are shipped to the National Center for Environmental Health for testing.

The NHANES quality assurance and quality control (QA/QC) protocols meet the 1988 Clinical Laboratory Improvement Amendments mandates. Detailed QA/QC 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 are maintained.

Analytical Laboratories

NHANES uses several methods to monitor the quality of the analyses performed by the 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 periodically. The reports are reviewed for trends or shifts in the data. The laboratories are required to explain any areas of concern.

All QC procedures recommended by the manufacturers were followed. Reported results for all assays meet the Division of Laboratory Science’s 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

Four analytes are included in this dataset, total 25-Hydroxyvitamin D, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, and epi-25-hydroxyvitamin D3. Total 25-Hydroxyvitamin D is the sum of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3, but excludes epi-25-hydroxyvitamin D3.

Refer to the 2015-2016 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 2015-2016, approximately 79% of children aged 1-17 years who were examined in the MEC provided a blood specimen through phlebotomy, while 95% of examined adults age 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.

Demographic and Other Related Variables

The analysis of NHANES laboratory data must be conducted using the appropriate survey design and demographic variables. The NHANES 2015-2016 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. The SAS variable name ending in LC (ex., LBXVD2LC) 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., LBXVIDMS) provides the result for the analyte. For analytes with analytic results below the lower limit of detection (ex., LBXVIDLC=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 limits of detection (LLOD in nmol/L) for Vitamin D metabolites are:

Variable Name	Analyte Description	LLOD
LBXVD2MS	25-hydroxyvitamin D2	2.05 nmol/L
LBXVD3MS	25-hydroxyvitamin D3	2.23 nmol/L
LBXVE3MS	epi-25-hydroxyvitamin D3	1.64 nmol/L

Analysis of 25-Hydroxyvitamin D Data from NHANES III (1988-1994) and NHANES 2001-2006, with NHANES data collected since 2007

The LC-MS/MS-equivalent 25-Hydroxyvitamin D data are recommended for all analyses, but especially for analyzing secular trends for serum vitamin D involving NHANES III (1988-1994), NHANES 2001-2006 and NHANES data collected since 2007. Users are strongly encouraged to review the Analytical Note for 25-Hydroxyvitamin D Data Analysis (cdc.gov) prior to the analysis.

References

• Caudill, S.P., Schleicher, R.L., Pirkle, J.L. Multi-rule quality control for the age-related eye disease study. Statist. Med. (2008) 27(20):4094-40106.
• Westgard J.O., Barry P.L., Hunt M.R., Groth T. A multi-rule Shewhart chart for quality control in clinical chemistry. Clin Chem (1981) 27:493-501.

Codebook and Frequencies