• 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
  • LBDFOTSI - Serum total folate (nmol/L)
  • LBDFOT - Serum total folate (ng/mL)
  • LBXSF1SI - 5-Methyl-tetrahydrofolate (nmol/L)
  • LBDSF1LC - 5-Methyl-tetrahydrofolate cmt
  • LBXSF2SI - Folic acid (nmol/L)
  • LBDSF2LC - Folic acid cmt
  • LBXSF3SI - 5-Formyl-tetrahydrofolate (nmol/L)
  • LBDSF3LC - 5-Formyl-tetrahydrofolate cmt
  • LBXSF4SI - Tetrahydrofolate (nmol/L)
  • LBDSF4LC - Tetrahydrofolate cmt
  • LBXSF5SI - 5,10-Methenyl-tetrahydrofolate (nmol/L)
  • LBDSF5LC - 5,10-Methenyl-tetrahydrofolate cmt
  • LBXSF6SI - Mefox oxidation product (nmol/L)
  • LBDSF6LC - Mefox oxidation product cmt

National Health and Nutrition Examination Survey

2013-2014 Data Documentation, Codebook, and Frequencies

Folate Forms - Total & Individual - Serum (FOLFMS_H)

Data File: FOLFMS_H.xpt

First Published: February 2019

Last Revised: NA

Component Description

The objectives of this component are to: 1) provide data for monitoring secular trends in measures of nutritional status in the U.S. population; 2) evaluate the effect of people's habits and behaviors, such as physical activity and the use of alcohol, tobacco, and dietary supplements on nutritional status; and 3) 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 deficiencies and toxicities of specific nutrients in the population and subgroup, 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 in research to further define nutrient requirements as well as optimal levels for disease prevention and health promotion.

Eligible Sample

Examined participants aged 1 year and older were eligible.

Description of Laboratory Methodology

Five folate forms, 5-methyl-tetrahydrofolate, folic acid, 5-formyl-tetrahydrofolate, tetrahydrofolate, 5,10-methenyl-tetrahydrofolate, and an oxidation product of 5-methyl-tetrahydrofolate called MeFox (pyrazino-s-triazine derivative of 4-α-hydroxy-5-methyl-tetrahydrofolate) are measured by isotope-dilution high performance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) (Fazili, et al. 2013). This is a modification of a previously published method (Fazili and Pfeiffer, 2013) to scale down the amount of specimen needed and increase the sample throughput. The assay is performed by combining specimen (150 µL serum) with ammonium formate buffer and an internal standard mixture. Sample extraction and clean-up is performed by automated 96-probe solid phase extraction (SPE) using 96-well phenyl SPE plates and takes ~1 h for a 96-well plate. Folate forms are separated within 6 min using isocratic mobile phase conditions and measured by LC-MS/MS. Quantitation is based on peak area ratios interpolated against a five-point aqueous linear calibration curve using 1/x² weighting.

There were some changes to the lab method (scale down amount of specimen needed and increase sample throughput), but no changes to lab equipment, or laboratory performing the analyses for this component in the NHANES 2013-2014 cycle. On average, the method change resulted in slightly higher serum total folate (5.6%), 5-methyl-tetrahydrofolate (7.3%), and MeFox (3.6%) concentrations and approximately 15% higher folic acid concentrations (Fazili, et al. 2013). However, folic acid concentrations above the limit of quantitation were only slightly higher (8.8%).

Refer to the Laboratory Method Files section for detailed description on the laboratory methods used.

Laboratory Method Files

Folate Vitamers (December 2018)

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 (LPM). Vials are stored under appropriate frozen (–20°C) conditions until they are shipped to National Center for Environmental Health for testing.

The NHANES quality assurance and quality control (QA/QC) protocols meet the 1988 Clinical Laboratory Improvement Act 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 quality assurance evaluation during unscheduled 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 during “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 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’ 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.

One variable was created in this data file. The variable (LBDFOT) was created using the following formula:

LBDFOT: The serum total folate value in nmol/L (LBDFOTSI) was converted to ng/mL (LBDFOT) by dividing LBDFOTSI by 2.265 (rounded to 3 significant figures).

Analytic Notes

Refer to the 2013-2014 Laboratory Data Overview for general information on NHANES laboratory data.

Exam sample weights should be used for analyses. Please refer to the NHANES Analytic Guidelines and the on-line NHANES Tutorial for 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 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 name ending in “LC” (ex., LBDSF1LC) 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., LBXSF1SI) provides the analytic result for that analyte. For analytes with analytic results below the lower limit of detection (ex., LBDSF1LC=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 nmol/L) for the 6 folate forms are shown below. Because total folate is calculated from the sum of folate forms, a lower limit of detection does not apply. The LLOD specified for folic acid is the corrected LLOD after taking the folic acid calibration bias into consideration (see below “Serum Folate Forms for NHANES 2013–2014” session). The original LLOD for folic acid was 0.28 nmol/L.

Variable Name	SAS Label	LLOD
LBXSF1SI	5-Methyl-tetrahydrofolate	0.06
LBXSF2SI	Folic acid	0.20
LBXSF3SI	5-Formyl-tetrahydrofolate	0.20
LBXSF4SI	Tetrahydrofolate	0.20
LBXSF5SI	5,10-Methenyl-tetrahydrofolate	0.31
LBXSF6SI	Mefox oxidation product	0.08
LBDFOTSI	Serum total folate	n/a

Serum Folate Forms for NHANES 2013–2014

The NHANES 2013–2014 is the second survey cycle where a comprehensive list of serum folate forms has been measured by isotope-dilution high performance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) (Table 1). Serum total folate (LBDFOTSI) was calculated by adding LBXSF1SI-LBXSF5SI. LBXSF6SI was not included in the total folate calculation, due to evidence that it may already be present in vivo. (Pfeiffer, et al. 2015). An imputed value of LOD divided by the square root of 2 was used for individual folate forms with results that were < LOD. No LBDFOTSI was calculated if the result for one or more of the folate forms was missing.

Please refer to the Analytic Notes for the 2011-2012 Folate Forms – Serum (FOLFMS_G) file for additional details on the comparability in serum total folate and folate forms measured between NHANES 2011–2014 and the previous survey cycles.

Table 1. Folate forms measured by LC-MS/MS

After the completion of the LC-MS/MS analyses, the CDC Nutritional Biomarkers Laboratory discovered a calibration bias in the folic acid determination that resulted in overestimation of folic acid concentrations by about 25% as a result of solubility issues with the calibrator (Fazili, et al. 2017). The laboratory corrected the assay and conducted a crossover study to adjust the incorrect folic acid results. Using 23 serum quality control (QC) materials analyzed over 10 days (concentration range 0.60–12 nmol/L), the Pearson correlation for new vs. original folic acid results was r = 1.0.

The weighted Deming regression was (nmol/L):

LBXSF2SI_new = 0.7586 * LBXSF2SI_original – 0.016; 95% CI of slope (0.7448 to 0.7724) and intercept (-0.03367 to 0.001678), or

LBXSF2SI_original = 1.318 * LBXSF2SI_new + 0.02109; 95% CI of slope (1.294 to 1.342) and intercept (-0.001921 to 0.04409).

The laboratory also randomly selected approximately 10% of the NHANES 2013–2014 serum samples stratified by 4 time periods based on date of analysis (n = 800) and reanalyzed 25% of samples from each time period by random selection based on available sample volume (n = 196; concentration range 0.36–47 nmol/L). The Pearson correlation for new vs. original folic acid results was r = 0.997 and the weighted Deming regression equation was not significantly different from the equation obtained with 23 serum QC materials (overlapping 95% confidence intervals for slope [0.7282 to 0.8184] and intercept [-0.04349 to 0.03810]).  

The original folic acid results were adjusted using the weighted Deming regression obtained with 23 serum QC materials and released in the present file. The original limit of detection (LOD) of 0.28 nmol/L was also adjusted using the same regression equation (new LOD 0.20 nmol/L). If the original folic acid result was less than the original LOD, which was the case for 81 samples in NHANES 2013–2014, the new folic acid result was also adjusted to less than the new LOD.

Folic acid (LBXSF2SI) is a minor contributor to serum total folate (LBDFOTSI) (about 5%) and is part of the formula to calculate serum total folate. The original serum total folate results were revised using the new folic acid results and released in the present file.

LBDFOTSI_new = LBDFOTSI_original – LBXSF2SI_original + LBXSF2SI_new

The folic acid correction resulted in an approximately 25% lowering of concentrations across the entire folic acid distribution, while serum total folate concentrations decreased by about 1–2% (Table 2).

Table 2. Comparison of new and original serum folic acid and total folate results

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.
• Fazili Z and Pfeiffer CM. Accounting for an isobaric interference allow correct determination of folate vitamers in serum by isotope dilution-liquid chromatography-tandem mass spectrometry. J Nutr. 2013; 143:108-13.
• Fazili Z, Sternberg M, Paladugula N, Pfeiffer C. Two International Round-Robin Studies Showed Good Comparability of 5-Methyltetrahydrofolate but Poor Comparability of Folic Acid Measured in Serum by Different High-Performance Liquid Chromatography–Tandem Mass Spectrometry Methods. J Nutr. 2017;147(9): 1815–1825.
• Fazili Z, Whitehead RD Jr, Paladugula N, Pfeiffer CM. A high-throughput LC-MS/MS method suitable for population biomonitoring measures five serum folate vitamers and one oxidation product. Anal Bioanal Chem.
  2013;405:4549–60.
  
• Pfeiffer C, Sternberg M, Fazili M, Lacher D, Zhang M, Johnson C, Hammer H, Baily R, Rader J, Yamini S, Berry RJ, Yetley E. British Journal of Nutrition (2015) 113:1965:1977.
• 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