• 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

2011-2012 Data Documentation, Codebook, and Frequencies

Vitamin D (VID_G)

Data File: VID_G.xpt

First Published: October 2017

Last Revised: NA

Component Description

Vitamin D is functionally a hormone rather than a vitamin, and in conjunction with parathyroid hormone and calcitonin, it is one of the most important biological regulators of calcium metabolism. Vitamin D and its main metabolites may be categorized into two families of secosteroids (steroid B-ring open): cholecalciferol (vitamin D3) and ergocalciferol (vitamin D2). Both vitamins D3 and D2 are enzymatically hydroxylated in the liver to 25-hydroxy forms and then further metabolized in the kidney to the bioactive 1,25-dihydroxy forms.

Although 25-hydroxyvitamin D (25OHD) is not the bioactive form, it is the predominant circulating form of vitamin D, and thus, it is considered to be the most reliable index of vitamin D status (Olkowski, et al., 2003; Saenger, et al., 2006). Vitamin D3 is a naturally occurring form of vitamin D that is produced in the skin after 7-dehydrocholesterol is exposed to UV-B radiation. Commercially, vitamin D2 is produced by UV irradiation of plant-derived ergosterol. The two forms differ in the structures of their side chains, but they are metabolized identically. Good sources of vitamin D3 are fatty fish while mushrooms provide a good source of vitamin D2. Both forms are used for fortification of a limited selection of foods including milk, juice, margarines, cheese and nutrition bars.

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

Serum samples are first treated by the addition of an ethanolic solution containing three internal standards and a solution of 69-72% methanol followed by the addition of hexane. Analytes are extracted from the aqueous phase into the hexane layer (liquid-liquid extraction) which is 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 internal standards (IS), 26,27-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 MS-MS on a Thermo TSQ Vantage system using atmospheric pressure chemical ionization in the positive ion mode. Quantitation is accomplished by comparing the peak area of the analyte in the unknown with the peak area of a known amount of analyte in a calibrator solution. Calculations are corrected based on the peak area of the internal standard in the unknown compared with the peak area of the matched internal standard in the calibrator solution.

Refer to the Laboratory Method Files section for detailed laboratory procedure manual(s) of the methods used.

There were no changes to the lab method, lab equipment, or lab site for this component in the NHANES 2011-2012 cycle.

Laboratory Method Files

Vitamin D (25-Hydroxyvitamin D2, 25-Hydroxyvitamin D3, and 3-epi-25-Hydroxyvitamin D3) (October 2017)

Laboratory Quality Assurance and Monitoring

Serum specimens are 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. 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 Laboratory Procedures Manual.

Mobile Examination Centers (MECs)

Laboratory team performance is monitored using several techniques. NCHS and contract consultants use a structured quality assurance 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 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 quality control 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 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 quality control and quality assurance 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

In addition to total 25-Hydroxyvitamin D, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, and epi-25-hydroxyvitamin D3 are being released. Total 25-Hydroxyvitamin D is the sum of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3, but excludes epi-25-hydroxyvitamin D3.

Refer to the 2011-2012 Laboratory Data Overview for general information on NHANES laboratory 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 2011-2012 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. 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 other variable prefixed LBX (ex., LBXVIDMS) provides the result for the analyte.

The lower limits of detection (LLOD in nmol/L) for Vitamin D metabolites are:

SAS Label	SAS Variable	LLOD
25-hydroxyvitamin D2	LBXVD2MS	2.05 nmol/L
25-hydroxyvitamin D3	LBXVD3MS	2.23 nmol/L
epi-25-hydroxyvitamin D3	LBXVE3MS	1.64 nmol/L

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.

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.
  
• Olkowski AA, Aranda-Osorio G, and McKinnon J. Rapid HPLC method for measurement of vitamin D3 and 25(OH)D3 in blood plasma. Int J Vitam Nutr Res, (2003) 73(1):15-18.
• Saenger AK, Laha TJ, Bremner DE, and Sadrzadh SMH. Quantification of serum 25-hydroxyvitamin D2 and D3 using HPLC-tandem mass spectrometry and examination of reference intervals for diagnosis of vitamin D deficiency. Am J Clin Pathol, (2006)125:914-920.
• 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