• Component Description
• Eligible Sample
• Description of Laboratory Methodology
• Data Processing and Editing
• Laboratory Quality Assurance and Monitoring
• Analytic Notes
• Codebook
  • SEQN - Respondent sequence number
  • LBX4PA - 4-pyridoxic acid (nmol/L)
  • LBXPLP - Pyridoxal 5'-phosphate (nmol/L)

National Health and Nutrition Examination Survey

2005-2006 Data Documentation, Codebook, and Frequencies

Vitamin B6 (VIT_B6_D)

Data File: VIT_B6_D.xpt

First Published: September 2010

Last Revised: NA

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 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

Survey participants aged 1 year and older were tested.

Description of Laboratory Methodology

Serum is prepared for analysis by first combining the specimen 1:1 with a 5% solution of metaphosphoric acid to precipitate proteins out of the sample.  After vortex mixing and centrifuging, the sample supernatant is combined with an approximately equivalent amount of dichloromethane to extract lipids from the sample, and vortex mixed and centrifuged a second time.  The top (aqueous) layer from the sample is then filtered through a syringe and ready for high-performance liquid chromatographic (HPLC) HPLC analysis.

Vitamin B6, in the form of PLP, and the metabolite 4-PA are measured by reversed-phase HPLC using fluorometric detection at 325 nm excitation and 425 nm emissions. Post-column introduction of a sodium chlorite derivatization reagent is incorporated into the HPLC system to improve the PLP signal. Quantitation is based on analyte peak area interpolated against a five-point calibration curve obtained from aqueous standards.

Data Processing and Editing

Specimens were processed, stored and shipped to the Division of Environmental Health Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention in Atlanta, Georgia. Detailed specimen collection and processing instructions was discussed in the NHANES LPM. Read the General Documentation of Laboratory Data file for detailed data processing and editing protocols. The analytical methods were described in the Description of the Laboratory Methodology section.

There are no derived variables, fill values, or recoding of data.

Detailed instructions on specimen collection and processing can be found at NHANES web site.

Laboratory Quality Assurance and Monitoring

The NHANES quality control and quality assurance protocols (QA/QC) meet the 1988 Clinical Laboratory Improvement Act mandates. A detailed quality control and quality assurance instruction was discussed in the NHANES Laboratory/Medical Technologists Procedures Manual (LPM). Read the General Documentation of Laboratory Data file for detailed QA/QC protocols.

A detailed description of the quality assurance and quality control procedures can be found at the NHANES web site.

Analytic Notes

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. They also contain all survey design variables and sample weights for these age groups. The phlebotomy file includes auxiliary information such as the conditions precluding venipuncture. The household questionnaire and phlebotomy files may be linked to the laboratory data file using the unique survey participant identifier SEQN.

Please refer to the Analytic Guidelines for further details on the use of sample weights and other analytic issues. The Analytic Guidelines are available on the NHANES website.

Pyridoxal 5’-phosphate (Vitamin B6) for 2005-2006 and 2003-2004:

There was a change in pyridoxal 5’-phosphate (Vitamin B6) methods from 2003-2004 to 2005-2006. In 2003-2004, plasma pyridoxal 5'-phosphate (PLP) was performed using a homogeneous, enzymatic assay by A/C Diagnostics. In 2005-2006, serum PLP and 4-pyridoxic acid (4PA) were measured using a high-performance liquid chromatographic (HPLC) method by NCEH/CDC. 

The two methods differed in their lower limit of detection (LLOD): 10 nmol/L for the enzymatic assay and 0.3 nmol/L for the HPLC method. The weighted distributions of the two datasets were different (Table 1), particularly at the lower percentiles. Using weighted analysis, 14.2% of the PLP results by the enzyme assay compared to only 2.7% of the PLP results by the HPLC method were = 10 nmol/L. Similarly, 23.2% of the PLP results by the enzyme assay compared to only 11.4% of the PLP results by the HPLC method were = 20 nmol/L, a value commonly viewed as a cutoff for inadequate vitamin B6 status. These large differences suggest that the datasets from these two survey periods are not directly comparable and an adjustment is required to account for method differences.

Table 1. Weighted mean and percentiles for PLP (nmol/L) in 1-85 year old NHANES participants

Years	N	Mean	5th	25th	50th	75th	95th
2003-2004	7822	65.9	7.1	24.2	45.8	77.1	195
2005-2006	8311	74.1	14.3	30.1	49.3	83.7	214

To assess method differences, a crossover study was performed between these assays on 236 specimens. The distributions of PLP were positively skewed. The HPLC values were higher (mean: 82.1, median: 45.9 nmol/L) compared to the enzymatic assay (mean: 49.0, median: 23.3 nmol/L). The PLP detection rate (values greater or equal to the method-specific LLOD) was 100% for the HPLC method, but only 71% for the enzymatic assay. The HPLC method measured PLP values between 6.9 and 123 nmol/L for the 68 specimens in which the enzymatic assay did not detect PLP (<LLOD of 10 nmol/L). A linear regression analysis on log-transformed PLP values for specimens with enzymatic assay results <= 20 nmol/L (n = 109) showed a Pearson correlation coefficient of 0.21. Hence, the two assays were poorly correlated at low PLP values that represent inadequate B6 status. In addition, a linear regression analysis on log-transformed PLP values for specimens that produced a PLP result greater than the LLOD by the enzymatic assay (n = 167) showed a Pearson correlation coefficient of 0.85.

Applying the linear regression equation obtained from the crossover study to the 2003-2004 PLP data to forward regress the enzymatic assay to make it comparable to the 2005-2006 HPLC method, resulted in over-adjusting the data and making it even less comparable. Regression analysis based on the crossover study was therefore not able to correct for method differences, likely due to the different distributions of the two methods at the lower concentrations of PLP

The data user is cautioned against combining the 2003-2004 PLP data generated with the enzymatic assay with the 2005-2006 PLP data generated with the HPLC method. NIST certified reference materials and higher-order reference methods for measuring serum or plasma PLP are under development and will be available shortly. This will help with an independent evaluation of methods in the future.

Codebook and Frequencies