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

All examined female participants aged 12-49 years, and other examined participants (all males as well as females 1-11 years and 50+ years) one year and older from a one-half subsample were eligible.

Description of Laboratory Methodology

Five folate forms, 5-methyltetrahydrofolate, folic acid, tetrahydrofolate, 5-formyltetrahydrofolate, 5,10-methenyltetrahydrofolate, and an oxidation product of 5-methyltetrahydrofolate called MeFox (pyrazino-s-triazine derivative of 4-α-hydroxy-5-methyltetrahydrofolate) 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 an 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.

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

There were no changes to the lab method, lab equipment, or lab site for this component in the NHANES 2017-2018 cycle.

Laboratory Method Files

Folate Forms (July 2020)

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 (–30°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 QA evaluation during unscheduled 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 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 QC 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’ 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.

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

LBDFOT: The serum 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 2017-2018 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 2017-2018, approximately 80% 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.

Serum Folate Forms for NHANES 2017–2018

In NHANES 2017–2018, 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.

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 2011–2018 and the previous survey cycles.

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

Analyte

Abbreviation

Variable Name

5-Methyltetrahydrofolate

5-methylTHF

LBXSF1SI

Pteroylglutamic acid (Folic Acid)

Folic acid

LBXSF2SI

5-Formyltetrahydrofolate

5-formylTHF

LBXSF3SI

Tetrahydrofolic acid

THF

LBXSF4SI

5,10-Methenyltetrahydrofolate

5,10-methenylTHF

LBXSF5SI

Pyrazino-s-triazine derivative of 4-α-hydroxy-5-methyltetrahydrofolate

MeFox

LBXSF6SI

Serum total folate (sum of folate forms)

tFOL

LBDFOTSI

 

Subsample Weights

The analytes included in this dataset were measured in all examined female participants aged 12-49 years, and in a one-half subsample of other examined participants one year and older. Special sample weights are required to analyze these data properly. Variable (WTFOL2YR) encoding of the specific sample weights for this subsample is included in this data file and should be used when analyzing these data. These special sample weights were created to account for the subsample selection probability, as well as the additional nonresponse to these lab tests. Therefore, if participants were eligible for the subsample, but did not provide a blood specimen, they would have the sample weight value assigned as “0” in their records.

Demographic and Other Related Variables

The analysis of NHANES laboratory data must be conducted using the appropriate survey design and demographic variables. The 2017-2018 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

Analyte Description

LLOD

LBXSF1SI

5-Methyltetrahydrofolate

0.13

LBXSF2SI

Folic acid

0.14

LBXSF3SI

5-Formyltetrahydrofolate

0.20

LBXSF4SI

Tetrahydrofolate

0.25

LBXSF5SI

5,10-Methenyltetrahydrofolate

0.20

LBXSF6SI

MeFox

0.10

LBDFOTSI

Total folate, serum

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

WTFOL2YR - Folate and Folate Forms Weight

Variable Name:
WTFOL2YR
SAS Label:
Folate and Folate Forms Weight
English Text:
Folate and Folate Forms Weight
Target:
Both males and females 1 YEARS - 150 YEARS
Code or Value Value Description Count Cumulative Skip to Item
3748.376146 to 903457.15327 Range of Values 4620 4620
0 No Lab Result 472 5092
. Missing 0 5092

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
3.27 to 573 Range of Values 4571 4571
. Missing 521 5092

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
1.44 to 253 Range of Values 4571 4571
. Missing 521 5092

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
2.28 to 331 Range of Values 4571 4571
. Missing 521 5092

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 4571 4571
1 Below lower detection limit 0 4571
. Missing 521 5092

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 368 Range of Values 4571 4571
. Missing 521 5092

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 4562 4562
1 Below lower detection limit 9 4571
. Missing 521 5092

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 345 Range of Values 4571 4571
. Missing 521 5092

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 42 42
1 Below lower detection limit 4529 4571
. Missing 521 5092

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 19.8 Range of Values 4571 4571
. Missing 521 5092

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 4183 4183
1 Below lower detection limit 388 4571
. Missing 521 5092

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 43.7 Range of Values 4571 4571
. Missing 521 5092

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 531 531
1 Below lower detection limit 4040 4571
. Missing 521 5092

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
0.113 to 48.5 Range of Values 4571 4571
. Missing 521 5092

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 4571 4571
1 Below lower detection limit 0 4571
. Missing 521 5092