Table of Contents

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

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 2015-2016 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.

Serum Folate Forms for NHANES 2015–2016

In NHANES 2015–2016, a comprehensive list of serum folate forms was 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 2011–2016 and the previous survey cycles.

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

Analyte

Abbreviation

Variable Name

5-Methyl-tetrahydrofolate

5-methylTHF

LBXSF1SI

Pteroylglutamic acid

Folic acid

LBXSF2SI

5-Formyl-tetrahydrofolate

5-formylTHF

LBXSF3SI

Tetrahydrofolate

THF

LBXSF4SI

5,10-Methenyl-tetrahydrofolate

5,10-methenylTHF

LBXSF5SI

Pyrazino-s-triazine derivative of 4-α-hydroxy-5-methyl-tetrahydrofolate

MeFox

LBXSF6SI

Serum total folate (sum of folate forms)

tFOL

LBDFOTSI

Demographic and Other Related Variables

The analysis of NHANES laboratory data must be conducted using the appropriate survey design and demographic variables. The 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. 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.

Variable Name

SAS Label

LLOD

LBXSF1SI

5-Methyl-tetrahydrofolate

0.13

LBXSF2SI

Folic acid

0.14

LBXSF3SI

5-Formyl-tetrahydrofolate

0.20

LBXSF4SI

Tetrahydrofolate

0.25

LBXSF5SI

5,10-Methenyl-tetrahydrofolate

0.20

LBXSF6SI

Mefox oxidation product

0.10

LBDFOTSI

Serum total folate

       n/a

 

References

Codebook and Frequencies

SEQN - Respondent sequence number

Variable Name:
SEQN
SAS Label:
Respondent sequence number
English Text:
Respondent sequence number
Target:
Both males and females 1 YEARS - 150 YEARS

LBDFOTSI - Serum total folate (nmol/L)

Variable Name:
LBDFOTSI
SAS Label:
Serum total folate (nmol/L)
English Text:
Serum total folate (nmol/L)
Target:
Both males and females 1 YEARS - 150 YEARS
Code or Value Value Description Count Cumulative Skip to Item
4.07 to 1390 Range of Values 8047 8047
. Missing 1118 9165

LBDFOT - Serum total folate (ng/mL)

Variable Name:
LBDFOT
SAS Label:
Serum total folate (ng/mL)
English Text:
Serum total folate (ng/mL)
Target:
Both males and females 1 YEARS - 150 YEARS
Code or Value Value Description Count Cumulative Skip to Item
1.8 to 614 Range of Values 8047 8047
. Missing 1118 9165

LBXSF1SI - 5-Methyl-tetrahydrofolate (nmol/L)

Variable Name:
LBXSF1SI
SAS Label:
5-Methyl-tetrahydrofolate (nmol/L)
English Text:
5-Methyl-tetrahydrofolate (nmol/L)
Target:
Both males and females 1 YEARS - 150 YEARS
Code or Value Value Description Count Cumulative Skip to Item
3.16 to 1370 Range of Values 8057 8057
. Missing 1108 9165

LBDSF1LC - 5-Methyl-tetrahydrofolate cmt

Variable Name:
LBDSF1LC
SAS Label:
5-Methyl-tetrahydrofolate cmt
English Text:
5-Methyl-tetrahydrofolic comment code
Target:
Both males and females 1 YEARS - 150 YEARS
Code or Value Value Description Count Cumulative Skip to Item
0 At or above the detection limit 8057 8057
1 Below lower detection limit 0 8057
. Missing 1108 9165

LBXSF2SI - Folic acid (nmol/L)

Variable Name:
LBXSF2SI
SAS Label:
Folic acid (nmol/L)
English Text:
Folic acid (nmol/L)
Target:
Both males and females 1 YEARS - 150 YEARS
Code or Value Value Description Count Cumulative Skip to Item
0.099 to 418 Range of Values 8047 8047
. Missing 1118 9165

LBDSF2LC - Folic acid cmt

Variable Name:
LBDSF2LC
SAS Label:
Folic acid cmt
English Text:
Folic acid comment code
Target:
Both males and females 1 YEARS - 150 YEARS
Code or Value Value Description Count Cumulative Skip to Item
0 At or above the detection limit 8006 8006
1 Below lower detection limit 41 8047
. Missing 1118 9165

LBXSF3SI - 5-Formyl-tetrahydrofolate (nmol/L)

Variable Name:
LBXSF3SI
SAS Label:
5-Formyl-tetrahydrofolate (nmol/L)
English Text:
5-Formyl-tetrahydrofolate (nmol/L)
Target:
Both males and females 1 YEARS - 150 YEARS
Code or Value Value Description Count Cumulative Skip to Item
0.141 to 38.9 Range of Values 8057 8057
. Missing 1108 9165

LBDSF3LC - 5-Formyl-tetrahydrofolate cmt

Variable Name:
LBDSF3LC
SAS Label:
5-Formyl-tetrahydrofolate cmt
English Text:
5-Formyl-tetrahydrofolate comment code
Target:
Both males and females 1 YEARS - 150 YEARS
Code or Value Value Description Count Cumulative Skip to Item
0 At or above the detection limit 185 185
1 Below lower detection limit 7872 8057
. Missing 1108 9165

LBXSF4SI - Tetrahydrofolate (nmol/L)

Variable Name:
LBXSF4SI
SAS Label:
Tetrahydrofolate (nmol/L)
English Text:
Tetrahydrofolate (nmol/L)
Target:
Both males and females 1 YEARS - 150 YEARS
Code or Value Value Description Count Cumulative Skip to Item
0.177 to 23.1 Range of Values 8057 8057
. Missing 1108 9165

LBDSF4LC - Tetrahydrofolate cmt

Variable Name:
LBDSF4LC
SAS Label:
Tetrahydrofolate cmt
English Text:
Tetrahydrofolate comment code
Target:
Both males and females 1 YEARS - 150 YEARS
Code or Value Value Description Count Cumulative Skip to Item
0 At or above the detection limit 7642 7642
1 Below lower detection limit 415 8057
. Missing 1108 9165

LBXSF5SI - 5,10-Methenyl-tetrahydrofolate (nmol/L)

Variable Name:
LBXSF5SI
SAS Label:
5,10-Methenyl-tetrahydrofolate (nmol/L)
English Text:
5,10-Methenyl-tetrahydrofolate (nmol/L)
Target:
Both males and females 1 YEARS - 150 YEARS
Code or Value Value Description Count Cumulative Skip to Item
0.141 to 7.13 Range of Values 8057 8057
. Missing 1108 9165

LBDSF5LC - 5,10-Methenyl-tetrahydrofolate cmt

Variable Name:
LBDSF5LC
SAS Label:
5,10-Methenyl-tetrahydrofolate cmt
English Text:
5,10-Methenyl-tetrahydrofolate comment code
Target:
Both males and females 1 YEARS - 150 YEARS
Code or Value Value Description Count Cumulative Skip to Item
0 At or above the detection limit 875 875
1 Below lower detection limit 7182 8057
. Missing 1108 9165

LBXSF6SI - Mefox oxidation product (nmol/L)

Variable Name:
LBXSF6SI
SAS Label:
Mefox oxidation product (nmol/L)
English Text:
Mefox oxidation product (nmol/L)
Target:
Both males and females 1 YEARS - 150 YEARS
Code or Value Value Description Count Cumulative Skip to Item
0.0707 to 59.7 Range of Values 8055 8055
. Missing 1110 9165

LBDSF6LC - Mefox oxidation product cmt

Variable Name:
LBDSF6LC
SAS Label:
Mefox oxidation product cmt
English Text:
Mefox oxidation product comment code
Target:
Both males and females 1 YEARS - 150 YEARS
Code or Value Value Description Count Cumulative Skip to Item
0 At or above the detection limit 8053 8053
1 Below lower detection limit 2 8055
. Missing 1110 9165