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.
Examined participants aged 1 year and older.
Microbiological assays have been used for many years to estimate the concentration of folate in blood and other tissues. In the 1990's robust and reliable procedures were introduced that use microtitre plates for higher throughput and a cryopreserved antibiotic resistant microorganism to avoid having to work under aseptic conditions. The herein described procedure is an adaptation of such a method. Diluted serum or whole blood is added to an assay medium containing Lactobacillus casei (NCIB 10463) and all of the nutrients necessary for the growth of L. casei except folate. The inoculated medium is incubated for 45 hours at 37oC. Since the growth of L. casei is proportional to the amount of total folate present in serum or whole blood samples, the total folate level can be assessed by measuring the turbidity of the inoculated medium at 590 nm in a PowerWave X340 Microplate reader (Bio-Tek Instrument). We calibrate the assay with 5-methyltetrahydrofolic acid (5MeTHF) from Epro.
There were no changes to the lab method, lab equipment or lab site for this component in the NHANES 2007-2008 cycle. However, there were changes (from the previous 2 years of NHANES) to equipment, and lab method. In 2005-2006, RBC folates and serum folates were measured using the Bio-Rad Laboratories "Quantaphase II Folate" radioassay kit. In 2007-2008, RBC folates and serum folates were measured using the microbiologic assay.
Refer to the Laboratory Method Files section for detailed laboratory procedure manuals of the methods used.
Whole blood and blood serum 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 Amendments 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).
The data were reviewed. Incomplete data or improbable values were sent to the performing laboratory for confirmation.
Two derived variables were created in this data file. The variables were created using the following formula:
Convert RBC folate results by dividing (LBXRBF) in nmol/L RBC by 2.265 into ng/mL RBC (LBDRBF) (round, 0 decimal).
Convert serum folate results by dividing (LBXFOL) in nmol/L by 2.265 into ng/mL (LBDFOL) (round, 1 decimal).
Refer to the 2007-2008 Laboratory Data Overview for general information on NHANES laboratory 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.
The demographics and fasting questionnaire files may be linked to the laboratory data file using the unique survey participant identifier (i.e., SEQN).
The laboratory data file can be linked to the other NHANES data files using the unique survey participant identifier (i.e., SEQN).
Detection Limits
An exact LLOD for RBC folate cannot be calculated because the value is a composite of whole blood folate, serum folate and hematocrit. Therefore, there is no LLOD for the calculated value of RBC folate. Furthermore, the LOD of this method for serum folate and whole blood folate depends on the dilution factor (i.e., LOD = 1 nmol/L if serum is only diluted 1/20).
Examined sample weights should be used for analyses. 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.
No correction needed for serum folic acid results for NHANES 2007–2008:
The CDC Nutritional Biomarkers Laboratory measured serum 5-methyltetrahydrofolate and 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. Recently, 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 (see analytic note for “Folate Forms – Serum” in NHANES 2011–2012 ). The laboratory corrected the assay and conducted a crossover study to assess whether folic acid and serum total folate data from NHANES 2007–2008, which were analyzed a few years prior to the NHANES 2011–2012, needed to be adjusted.
The CDC laboratory randomly selected approximately 10% of the NHANES 2007–2008 serum samples stratified by 4 time periods based on date of analysis (n = 300) and reanalyzed 25% of samples from each time period by random selection based on available sample volume (n = 75; concentration range 0.34–14.1 nmol/L). The Pearson correlation for new vs. original folic acid results was r = 0.989 and the weighted Deming regression equation did not show a significant slope or intercept (95% confidence interval for slope included 1 and for intercept included 0) (nmol/L):
New folic acid = 0.9570 * Original folic acid – 0.01359; 95% CI of slope (0.85 to 1.06) and intercept (-0.1016 to 0.07445).
Therefore, the NHANES 2007–2008 serum folic acid results did not have to be adjusted.
Serum folate regression equations to compare 2007-2008 and 1999-2006 data:
Background:
There was a change in serum folate methods from 1999-2006 to 2007-2008. In 1999-2006, serum folate was performed using the Bio-Rad (BR) Quantaphase II radioassay. In 2007-2008, serum folate was measured using a microbiological assay (MA). Serum folate levels were much higher in 2007-2008 using the MA method compared with previous data (1999-2006) using the BR method. There are two variables for serum folate: one expressed in conventional units (ng/mL) and one in SI units (nmol/L). For 1999-2006, they were LBXFOL (ng/mL) and LBDFOLSI (nmol/L), and for 2007-2008, they were LBDFOL (ng/mL) and LBXFOLSI (nmol/L). To convert conventional units (ng/mL) to SI units (nmol/L), multiply by 2.265.
A crossover study was performed between the BR and MA methods by the NCEH/CDC laboratory as described by Fazili et al. (Fazili Z, Pfeiffer CM, Zhang M. Comparison of serum folate species analyzed by LC-MS/MS with total folate measured by microbiological assay and Bio-Rad assay. Clin. Chem. 53(4):781-4, 2007). Several regression methods were examined to relate the BR assay and the MA including linear, Deming, piecewise linear, and fractional polynomial. The fractional polynomial regression method best fit the Fazili crossover data. The forward fractional polynomial regression equation is recommended to trend the serum folate data since the MA method is a more accurate “gold standard” method and the BR method is no longer available.
Forward regression of 1999-2006 BR results (nmol/L) to match 2007-2008 MA results (nmol/L):
Convert the 1999-2006 BR serum folate (FOL, nmol/L) results to 2007-2008 equivalent values to match the MA serum folate (FOLadjusted, nmol/L) results using the following fractional polynomial regression equation:
FOLadjusted = 10**(0.0188 * x3 - 2.7109 * x-1/2 + 3.8276), where X = Log10FOL
Example calculation:
For NHANES 2005-2006, sequence number (SEQN) = 31128, serum folate (LBDFOLSI) = 30.4 nmol/L
FOLadjusted = 10**[0.0188 * (Log1030.4)3 – 2.7109 * (Log1030.4)-1/2 + 3.8276]
= 10**(0.0188 * 1.482873 – 2.7109 * 1.48287-1/2 + 3.8276) = 45.9955
= 46 nmol/L
Red blood cell folate - Comparison of 2007-2008 and 1999-2006 data:
Background:
There was a change in red blood cell (RBC) folate methods from 1999-2006 to 2007-2008. In 1999-2006, RBC folate measurements were performed using the Bio-Rad (BR) Quanta Phase II radioassay. In 2007-2008, RBC folate was measured using a microbiological assay (MA). RBC folate levels were much higher in 2007-2008 using the MA method compared with previous data (1999-2006) using the BR method. There are two variables for RBC folate: one expressed in conventional units (ng/mL) and one in SI units (nmol/L). For 1999-2006, they were LBXRBF (ng/mL) and LBDRBFSI (nmol/L), and for 2007-2008, they were LBDRBF (ng/mL) and LBXRBFSI (nmol/L). To convert conventional units (ng/mL) to SI units (nmol/L), multiply by 2.265.
A crossover study for whole blood folate (WBF) was performed between the BR and MA assays by the NCEH/CDC laboratory as described by Fazili et al. (Fazili Z, Pfeiffer CM, Zhang M, Jain RB, Koontz D. Influence of 5,10-methylenetetrahydrofolate reductase polymorphism on whole-blood folate concentrations measured by LC-MS/MS, microbiologic assay, and Bio-Rad radioassay. Clin. Chem. 54(1):197-201, 2008). The authors showed that while the WBF values for the BR assay measured lower than the MA (average 45%), there was a different relationship between the two assays depending on the 5,10-methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism. Whole blood samples from persons with the T/T genotype showed a smaller difference between the two assays (average 31%) compared to whole blood samples from persons with the C/C and C/T genotype (average 48%). The reason for this was that the BR assay recovered the various folate forms differently compared to the MA assay.
The MTHFR genotype information is not available for NHANES 1999-2008 and the genotype-specific regression equations reported by Fazili et al. cannot be used. Using the crossover data for all genotypes presented in the Fazili et al. paper, several regression methods were evaluated to relate the BR assay and the MA method for the WBF data for 1999-2008. These regression methods included the linear, Deming, piecewise linear, and fractional polynomial models. The linear regression model for WBF best fit the Fazili crossover data. As presented in the analytic note on serum folate, the fractional polynomial regression model was applied to adjust the serum folate data. This was necessary because serum folate is part of the equation to calculate RBC folate. A forward regression model is recommended to trend the RBC folate data since the MA method is considered a more accurate “gold standard” method and the BR method is no longer available.
Forward regression of 1999-2006 BR results (nmol/L) to match 2007-2008 MA results (nmol/L):
Since the Fazili crossover study presents WBF data rather than RBC folate data, it is necessary to first convert RBC folate data to WBF data before applying any regression equation to make the data sets comparable.
The following algorithm is recommended:
1) Convert the 1999-2006 BR RBC folate (RBF, nmol/L) to whole-blood folate (WBF, nmol/L) using the hematocrit (HCT, %) and the BR serum folate (FOL, nmol/L) in the following equation:
WBF = (RBF * HCT/100) + FOL * [1.0 - (HCT/100)]
The hematocrit can be obtained from the NHANES hematology files containing the complete blood count.
2) Apply the following forward linear regression to obtain an adjusted WBF (WBFadjusted) using WBF from step 1 to match the 2007-2008 MA WBF:
WBFadjusted = 10**[0.2204 + (1.017 * Log10WBF)]
3) Convert the 1999-2006 BR serum folate (FOL, nmol/L) results to 2007-2008 equivalent values to match the MA serum folate (FOLadjusted, nmol/L) results using the forward fractional polynomial regression equation specified in the analytic note on serum folate:
FOLadjusted = 10**(0.0188 * X3 – 2.7109 * X-1/2 + 3.8276), where X = Log10FOL
4) Calculate RBFadjusted by using WBFadjusted (from step 2) and FOLadjusted (from step 3):
RBFadjusted = {WBFadjusted – [FOLadjusted * (1.0 - (HCT/100))]} / (HCT/100)
The analyst is cautioned that trending RBC folate by this procedure does not use genotype-specific equations as the MTHFR genotype was not determined in NHANES. This may slightly underestimate RBC folate concentrations for some participants and overestimate concentrations for others.
Example calculation:
1) WBF = (378.3* 41.4/100) + 30.4 * [1.0 - (41.4/100)]
= 174.4306 nmol/L
2) WBFadjusted = 10**(0.2204 + 1.017 * Log10174.4306)
= 10**(0.2204 + 1.017 * 2.24162)
= 316.323 nmol/L
3) FOLadjusted = 10**[0.0188 * (Log1030.4)3 – 2.7109 * (Log1030.4)-1/2 + 3.8276]
= 10**(0.0188 * 1.482873 – 2.7109 * 1.48287-1/2 + 3.8276)
= 45.9955 nmol/L
4) RBFadjusted = {316.323 – [45.9955 * (1.0 - (41.4/100))]} / (41.4/100)
≈ 699 nmol/L
The analysis of NHANES laboratory data must be conducted with the key survey design and basic demographic variables. The NHANES Household Questionnaire Data Files contain demographic data, health indicators, and other related information collected during household interviews. The Household Questionnaire Data Files also contain all survey design variables and sample weights required to analyze these data. The Phlebotomy Examination file includes auxiliary information on duration of fasting, the time of day of the venipuncture, and the conditions precluding venipuncture. The Household Questionnaire and Phlebotomy Exam files may be linked to the laboratory data file using the unique survey participant identifier SEQN.
| Code or Value | Value Description | Count | Cumulative | Skip to Item |
|---|---|---|---|---|
| 85.7 to 2984.5 | Range of Values | 8243 | 8243 | |
| . | Missing | 1064 | 9307 |
| Code or Value | Value Description | Count | Cumulative | Skip to Item |
|---|---|---|---|---|
| 194 to 6760 | Range of Values | 8243 | 8243 | |
| . | Missing | 1064 | 9307 |
| Code or Value | Value Description | Count | Cumulative | Skip to Item |
|---|---|---|---|---|
| 2.2 to 209.3 | Range of Values | 8205 | 8205 | |
| . | Missing | 1102 | 9307 |
| Code or Value | Value Description | Count | Cumulative | Skip to Item |
|---|---|---|---|---|
| 5 to 474 | Range of Values | 8205 | 8205 | |
| . | Missing | 1102 | 9307 |