Table of Contents

Component Description

The objectives of this component are:

1. To provide data for monitoring secular trends in measures of nutritional status in the U.S. population;

2. 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; and

3. 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 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 year and older from a one-third sample.

Description of Laboratory Methodology

Different folate forms (5-methytetrahydrofolic acid [5-methylTHF], folic acid, 5-formyltetrahydrofolic acid [5-formylTHF], tetrahydrofolic acid [THF], and 5,10-methenyltetrahydrofolic acid [5,10-methenylTHF]) were measured by the CDC Nutritional Biomarkers Laboratory using an isotope-dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) method (Pfeiffer et al., 2004; Fazili and Pfeiffer, 2004). Serum was combined with buffer and an internal standard mixture and sample clean-up was performed using automated solid phase extraction on phenyl cartridges in the 96-well plate format. Folates were eluted from the cartridge and analyzed by LC-MS/MS. Quantitation was by peak area ratio (analyte to internal standard) and was based on a six-point calibration curve in an aqueous medium. A detailed description of the laboratory method used can be found on the NHANES website in the Laboratory Procedures Manual.

The method performance was described in the proceedings of the 2010 Roundtable on “NHANES monitoring of biomarkers of folate and vitamin B-12 status” (Yetley et al., 2011). Results were reported for two folate forms: 5-methylTHF, the major folate vitamer circulating in serum, and folic acid, which may appear in serum if intake of folic acid from fortified food or supplements exceeds 200 micrograms per meal. Results were not reported for the other three minor folate forms (5-formylTHF, THF, and 5,10-methenylTHF). What was measured as 5-formylTHF was in fact the sum of 5-formylTHF and an oxidation product of 5-methylTHF known as MeFox (pyrazino-s-triazine derivative of 4α-hydroxy-5-methylTHF) (Fazili and Pfeiffer, 2013). More recently, the LC-MS/MS method has been improved to separate and accurately measure 5-formylTHF and MeFox in addition to the other folate forms (Fazili and Pfeiffer, 2013). This improved method has been used to generate data for all folate forms in a full sample of NHANES 2011–2012. Data for 5-methylTHF and folic acid in the current NHANES 2007–2008 one-third sample are comparable to the data in NHANES 2011–2012.

Refer to the Laboratory Method Files section for detailed laboratory procedure manuals of the methods used.

There were no changes to the lab method, lab equipment or lab site for this component in the NHANES 2007-2008 cycle.

Laboratory Quality Assurance and Monitoring

Serum blood are processed, stored, and shipped to the Division of Laboratory Science, 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 Procedure Manual (LPM). Vials are stored under appropriate frozen (-20oC) 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 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 NHANES 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 NHANES 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 Environmental Health 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.

Analytic Notes

Refer to the 2007-2008 Laboratory Data Overview for general information on NHANES laboratory data.

Subsample Weights

Serum folate forms were measured in a one-third subsample of persons 1 year and older. Special sample weights are required to analyze these data properly. Specific sample weights for this subsample are included in this data file and should be used when analyzing these data.

NHANES Demographic and Other Related Variables

The analysis of NHANES laboratory data must be conducted using the appropriate survey design and demographic variables. The NHANES 2007-2008 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 on the data set. Two variables are provided for each of these analytes. The variable named ended in “LC” 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., 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 other variable prefixed LBX (ex., LBXSF1) provides the analytic result for that analyte.
The lower limit of detection (LLOD in nmol/L) for LBXSF1 and LBXSF2 are:

Serum folate forms: Limit of Detection in nanomole/liter

 Analyte

 SAS Variable

 LOD (nmol/L)

5-Methyltetrahydrofolic acid

 LBXSF1

 0.5

 Unmetabolized folic acid

 LBXSF2

 0.3

 

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.

Comparability of serum 5-methylTHF and folic acid concentrations in NHANES 2007-2008 versus NHANES 1999-2002

Serum 5-methylTHF and folic acid concentrations were measured by Tufts University using HPLC with electrochemical detection in American seniors (≥60 y) in NHANES 1999–2002 as part of a surplus specimen project to assess the relationship to anemia, macrocytosis and cognitive test performance (Bailey et al., 2010; Morris et al., 2010). To assess method differences between Tufts and CDC, a method comparison study was performed in 2013 on 311 surplus serum specimens from American seniors (≥60 y) in NHANES 2001–2002. Results for serum 5-methylTHF compared well between the two methods: mean (95% CI, nmol/L): 47.7 (44.5–50.9) [CDC] and 48.6 (45.8–51.4) [Tufts]; median (95% CI, nmol/L): 42.9 (38.2–45.4) [CDC] and 45.0 (41.3–48.0) [Tufts]; Pearson r: 0.89; no bias based on Deming regression (using log-transformed data) and Bland Altman relative difference analysis. The CDC folic acid results had to be corrected for the above mentioned calibration bias which occurred after 2011 and resulted in overestimation of folic acid concentrations by about 25% due to solubility issues with the calibrator (see analytic note for “Folate Forms – Serum” in NHANES 2011–2012). The equation used to correct the CDC folic acid concentrations was: New folic acid = 0.7586 * Original folic acid – 0.00016. The CDC method detected folic acid in all samples (LOD = 0.09 nmol/L), while the detection rate of the Tufts method (LOD = 0.18 nmol/L) was ~35% in the full sample set (n = 1330; See the 2001-2002 Surplus Sera Folate data). Overall, the correlation was low in the surplus sample set (Pearson r: 0.64, n = 311). In the subset of samples that had detectable folic acid results by the Tufts method (n = 163) and that did not include extreme outliers (n = 156; difference between the two results was <20 nmol/L), the CDC folic acid results were on average comparable to the Tufts results: mean (95% CI, nmol/L): 3.71 (2.40–5.02) [CDC] and 3.11 (2.19–4.02) [Tufts]; median (95% CI, nmol/L): 1.10 (0.99–1.37) [CDC] and 1.20 (0.88–1.62) [Tufts]; Pearson r: 0.92; Deming slope using log-transformed data (95%CI): 0.89 (0.76 to 1.02); Deming intercept using log-transformed data (95% CI): 0.06 (0.00 to 0.12); Bland Altman relative difference (95% CI): 10.3% (-1.9% to 22.5%). Samples with sufficient sample volume (n = 17) were repeated by the CDC method; the repeat results confirmed the original results and were on average (SD) 5% (7%) higher for UMFA and 2% (5%) higher for 5-methylTHF. The findings from this method comparison study suggest that the NHANES 1999–2002 Tufts surplus results can be compared for 5-methylTHF to the NHANES 2007–2008 CDC results but the comparability for folic acid is limited due to the different detection rates for these two methods.


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

LBXSF1 - 5-Methyl THF(nmol/L)

Variable Name:
LBXSF1
SAS Label:
5-Methyl THF(nmol/L)
English Text:
5-Methyl THF(nmol/L)
Target:
Both males and females 1 YEARS - 150 YEARS
Code or Value Value Description Count Cumulative Skip to Item
5.37 to 190 Range of Values 2734 2734
. Missing 388 3122

LBXSF2 - Folic acid, serum (nmol/L)

Variable Name:
LBXSF2
SAS Label:
Folic acid, serum (nmol/L)
English Text:
Folic acid, serum (nmol/L)
Target:
Both males and females 1 YEARS - 150 YEARS
Code or Value Value Description Count Cumulative Skip to Item
0.21 to 397 Range of Values 2707 2707
. Missing 415 3122

WTSA2YR - Environmental A two year weights

Variable Name:
WTSA2YR
SAS Label:
Environmental A two year weights
English Text:
Environmental A two year weights
Target:
Both males and females 1 YEARS - 150 YEARS
Code or Value Value Description Count Cumulative Skip to Item
0 to 604864.71127 Range of Values 3122 3122
. Missing 0 3122