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.
Examined participants aged 1 year and older were eligible.
Similar to the previous survey cycle, in NHANES 2009-2010, population folate status was assessed by measuring serum folate and whole-blood folate by microbiologic assay and by calculating RBC folate using these data. Microbiological assays have been used for many years to estimate the concentration of folate in blood and other tissues. In the 1990s, more robust and reliable procedures that use microtiter plates for higher throughput and a cryopreserved antibiotic resistant microorganism to avoid having to work under aseptic conditions were developed. The described procedure is an adaptation of such a method, and was introduced by the National Center for Environmental Health (NCEH) to NHANES in 2007. Diluted serum or whole blood is added to an assay medium containing Lactobacillus rhamnosus (formerly known as Lactobacillus casei) (NCIB 10463) and all of the nutrients necessary for the growth of L. rhamnosus except folate. The inoculated medium is incubated for 45 hours at 37oC. Since the growth of L. rhamnosus is proportional to the amount of total folate present in serum or whole blood samples; the 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). The assay was calibrated with 5-methyl-tetrahydrofolate from Merck Cie (Eprova).
There were no changes (from the previous 2 years of NHANES) to the lab site, equipment, or method.
Refer to the Laboratory Method Files section for detailed description on the laboratory methods used.
Whole Blood and Serum Folate (October 2011)
Whole blood and blood serum 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 (-20oC) 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).
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:
LBDRBF: The RBC folate value in nmol/L RBC (LBXRBFSI) was converted to ng/mL RBC (LBDRBF) by dividing LBXRBFSI by 2.265 (rounded to 1 decimal).
LBDFOL: The serum folate value in nmol/L (LBXFOLSI) was converted to ng/mL (LBDFOL) by dividing LBXFOLSI by 2.265 (rounded to 1 decimal).
Refer to the 2009-2010 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.
Demographic and Other Related Variables
The analysis of NHANES laboratory data must be conducted using the appropriate survey design and demographic variables. The NHANES 2009-2010 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
An exact lower limit of detection (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).
Serum Folate Regression Equations to Compare 2007-2010 and 1999-2006 Data
Background:
There
was a change in serum folate methods from 1999-2006 to 2007-2010. In 1999-2006,
serum folate was performed using the Bio-Rad (BR) Quantaphase II radioassay. In
2007-2010, serum folate was measured using a microbiological assay (MA). Serum
folate levels were much higher in 2007-2010 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
2007-2010, 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 CDC laboratory as described by Fazili et al. (Fazili Z, et al. 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 2007-2010 MA results
(nmol/L):
Convert the 1999-2006 BR serum folate (FOL, nmol/L)
results to 2007-2010 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 2007-2010 and
1999-2006 Data
Background:
There
was a change in red blood cell (RBC) folate methods from 1999-2006 to 2007-2010.
In 1999-2006, RBC folate measurements were performed using the Bio-Rad (BR)
Quanta Phase II radioassay. In 2007-2010, RBC folate was measured using a
microbiological assay (MA). RBC folate levels were much higher in 2007-2010
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 2007-2010, 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 CDC laboratory as described by Fazili et al. (Fazili Z, et al. 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 1999-2010 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 1999-2010. 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 2007-2010 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 2007-2010 MA WBF:
WBFadjusted = 10**[0.2204 + (1.017 * Log10WBF)]
3) Convert the 1999-2006 BR serum folate (FOL, nmol/L) results to 2007-2010 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
| Code or Value | Value Description | Count | Cumulative | Skip to Item |
|---|---|---|---|---|
| 87.4 to 3125.8 | Range of Values | 8764 | 8764 | |
| . | Missing | 1071 | 9835 |
| Code or Value | Value Description | Count | Cumulative | Skip to Item |
|---|---|---|---|---|
| 198 to 7080 | Range of Values | 8764 | 8764 | |
| . | Missing | 1071 | 9835 |
| Code or Value | Value Description | Count | Cumulative | Skip to Item |
|---|---|---|---|---|
| 1.9 to 253.9 | Range of Values | 8713 | 8713 | |
| . | Missing | 1122 | 9835 |
| Code or Value | Value Description | Count | Cumulative | Skip to Item |
|---|---|---|---|---|
| 4.2 to 575 | Range of Values | 8713 | 8713 | |
| . | Missing | 1122 | 9835 |