• 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

2011-2012 Data Documentation, Codebook, and Frequencies

Folate Forms - Total & Individual - Serum (FOLFMS_G)

Data File: FOLFMS_G.xpt

First Published: May 2014

Last Revised: February 2019

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 and Pfeiffer, 2013). The assay is performed by combining specimen (275 μL serum) with ammonium formate buffer and an internal standard mixture. Sample extraction and clean-up is performed by automated solid phase extraction (SPE) using 96-well phenyl SPE plates and takes ~5 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 calibration curve.

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

Laboratory Method Files

Folate Vitamers (May 2014)

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 Procedure 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 2011 - 2012 Laboratory Data Overview for general information on NHANES laboratory data.

Examined 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 2011–2012

The NHANES 2011–2012 is the first 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.

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

 

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 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. 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 “Correction of Serum Folic Acid and Total Folate Results for NHANES 2011-2012” session). The original LLOD for folic acid was 0.14 nmol/L.

Variable Name	SAS Label	LLOD
LBXSF1SI	5-Methyl-tetrahydrofolate	0.31
LBXSF2SI	Folic acid	0.09
LBXSF3SI	5-Formyl-tetrahydrofolate	0.30
LBXSF4SI	Tetrahydrofolate	0.37
LBXSF5SI	5,10-Methenyl-tetrahydrofolate	0.34
LBXSF6SI	Mefox oxidation product	0.34
LBDFOTSI	Serum total folate	n/a

 

Comparability of Serum Folate Forms in NHANES 2011–2012 versus Previous Survey Cycles

For serum total folate, the weighted distributions of the NHANES 2011–2012 data were similar to the NHANES 2007–2008 and 2009–2010 data measured by microbiologic assay (MBA) (Table 2).

Table 2. Weighted mean (95% confidence interval) and selected percentiles for serum total folate (nmol/L) in 1 year and older NHANES participants

Survey cycle	Variable name	Method	n	Mean (95% CI)	Percentiles
					5th	25th	50th	75th	95th
2007–2008	LBXFOLSI	MBA	8205	45.8 (44.3 – 47.3)	14.8	27.1	40.5	57.9	92.6
2009–2010	LBXFOLSI	MBA	8713	44.9 (43.7 – 46.0)	14.5	26.2	39.1	56.3	90.1
2011–2012	LBDFOTSI	LC-MS/MS	7459	46.9 (45.6 - 48.2)	17.3	28.6	41.3	58.7	92.5

 

There is a difference between NHANES 2011–2012 and previous survey cycles as to how the serum was treated prior to placing it into frozen storage. During NHANES 2011–2012, ascorbic acid (0.5% w/v) was added to freshly prepared serum samples except during the beginning of 2011 when ascorbic acid was inadvertently not added to the serum for 530 samples. We obtained similar weighted distributions for serum total folate and folate forms whether we included (“All”) or excluded (“With AA”) the 530 samples without additional ascorbic acid (Table 3). Results from these 530 samples were included in the NHANES 2011–2012 data set.

Table 3. Weighted mean (95% confidence interval) and selected percentiles for serum total folate (nmol/L) and folate forms (nmol/L) in 1 year and older NHANES participants, 2011–2012

Analyte	Samples	n	Mean (95% CI)	Weighted percentiles
				5th	25th	50th	75th	95th
tFOL	All	7459	46.9 (45.6 - 48.2)	17.3	28.6	41.3	58.7	92.5
	With AA	6931	47.1 (45.7 - 48.4)	17.3	28.7	41.6	59.0	92.9
5-methylTHF	All	7471	42.9 (41.5 - 44.3)	14.7	26.1	38.4	54.7	84.7
	With AA	6943	43.3 (41.9 - 44.7)	14.9	26.2	38.7	55.1	85.2
Folic acid	All	7479	2.01 (1.83 - 2.20)	0.353	0.552	0.735	1.07	5.89
	With AA	6951	2.04 (1.85 - 2.23)	0.363	0.562	0.743	1.08	6.12
5-formylTHF	All	7483	0.238 (0.216 - 0.260)	0.212	0.212	0.212	0.212	0.212
	With AA	6955	0.239 (0.216 - 0.263)	0.212	0.212	0.212	0.212	0.212
THF	All	7478	1.46 (1.08 - 1.85)	0.262	0.621	0.960	1.55	4.65
	With AA	6950	1.35 (1.01 - 1.68)	0.262	0.592	0.919	1.37	4.46
5,10-methenylTHF	All	7483	0.259 (0.255 - 0.263)	0.240	0.240	0.240	0.240	0.240
	With AA	6955	0.260 (0.256 - 0.265)	0.240	0.240	0.240	0.240	0.240
MeFox	All	7486	1.95 (1.87 - 2.03)	0.530	0.934	1.49	2.43	4.81
	With AA	6958	1.96 (1.88 - 2.04)	0.530	0.942	1.51	2.45	4.81

 

In addition to the five biologically active folate vitamers, MeFox (LBXSF6SI), an oxidation product of 5-methylTHF, was also measured by the LC-MS/MS method as part of the folate forms profile in the 2011-2012 survey cycle. As mentioned above, MeFox was not included in the total folate calculation in the current dataset.

No conclusive evidence exists on whether to include MeFox as part of the total folate. However, there is clear evidence that the total folate concentration will be slightly underestimated if MeFox is not accounted for. MeFox appears to be formed during the pre-analytical phase (blood collection and processing) (Hannisdal, et al. 2009). Therefore, a small portion of “active” folate may be lost after the blood has been collected. The analytical phase of the CDC method (extraction of serum and LC-MS/MS measurement) does not generate additional MeFox (Fazili and Pfeiffer, 2013). It is not clear, whether MeFox is exclusively formed in vitro or may already be present in vivo. In rats, administration of radio-labeled folic acid resulted in the excretion of several labeled products: a precursor of MeFox (4-α-hydroxy-5-methylTHF), 5-methylTHF, 10-formyl-folic acid, and p-aminobenzoyl-L-glutamate, a folate breakdown product (Barford, et al. 1978). It is therefore conceivable that a small portion of the MeFox measured in serum may already be in circulation when the blood is collected. By not including MeFox in the total folate calculation, we err on the side of caution with the interpretation of population folate status by slightly underestimating it by less than 5% in average (Pfeiffer, et al. 2015). In addition, the microbiologic assay method used in the two previous survey cycles (NHANES 2007–2008 and 2009–2010) to measure serum total folate does not recognize the biologically inactive MeFox. Therefore, using serum total folate calculated without MeFox makes it comparable to the data from these previous cycles.

Previous survey cycles provided limited information on selected folate forms. 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 (Bailey, et al. 2010; Morris, et al. 2010). Please see the NHANES web site for descriptions of the 1990-2000 surplus specimen folate data. See also the 2001-2002 surplus specimen folate data.

Furthermore, serum 5-methylTHF and folic acid concentrations were measured by CDC using LC-MS/MS in a random one-third subsample of the entire population 1 year and older in NHANES 2007–2008. Please see the Documentation for this 2007-2008 folate forms data on the NHANES web site.

Correction of Serum Folic Acid and Total Folate Results for NHANES 2011-2012

In the 2011-2012 survey cycle, the CDC Nutritional Biomarkers Laboratory measured serum folate vitamers, including folic acid, by isotope-dilution high performance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and the NHANES data were first published in May 2014. In 2015, the CDC 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 2011–2012 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 0.27–45.7 nmol/L). The Pearson correlation for new vs. original folic acid results was r = 0.994 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 [0.7179 to 0.8097] and intercept [-0.04707 to 0.03792]).

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 0.14 nmol/L was also adjusted using the same regression equation (new LOD 0.09 nmol/L). If the original folic acid result was less than the original LOD, which was the case for 5 samples in NHANES 2011–2012, 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 4).

Table 4. Comparison of new and original serum folic acid and total folate results

 

References

• Bailey RL, Mills JL, Yetley EA, Gahche JJ, Pfeiffer CM, Dwyer JT, Dodd KW, et al. Unmetabolized serum folic acid and its relation to folic acid intake from diet and supplements in a nationally representative sample of adults aged ≥60 y in the United States. Am J Clin Nutr. 2010;92:383–9.
• Barford PA, Staff FJ, Blair JA. The metabolic fate of [2-14C]folic acid and a mixture of [2-14C]-and [3’,5’,9-3H]-folic acid in the rat. Biochem J. 1978;174:579–83.
• 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 allows 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.
• Hannisdal R, Ueland PM, Eussen SJ, Svardal A, Hustad S. Analytical recovery of folate degradation products formed in human serum and plasma at room temperature. J Nutr, 2009;
  139:1415–8.
  
• Morris MS, Jacques PF, Rosenbert IH, Selhub J. Circulating unmetabolized folic acid and 5-methyltetrahydrofolate in relation to anemia, macrocytosis, and cognitive test performance in American seniors. Am J Clin Nutr. 2010;91:1733–44.
• 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