Measurement of unmetabolized folic acid from stored sera specimens from NHANES 1999-2000 in participants >= 60 years of age.
Participants >= 60 years of age from NHANES 1999-2000 with stored sera.
Description of Laboratory Methodology
Measurement of Unmetabolized Folic Acid in Serum. Folic acid and 5MeTHF concentrations were determined using the affinity/HPLC method with electrochemical (coulometric) detection method Which was developed by us at the USDA Human Nutrition Research Center at Tufts University (Bagley et al., 1997; Kalmbach et al., 2008) with the following modifications to increase throughput and precision. Serum samples (0.2 mL) were diluted with 1.2 mL of 50mM potassium tetraborate in 1% sodium ascorbate and vortexed. Twenty picomoles of ethyltetrahydrofolate (eTHF) was added as an internal standard. The mixture was boiled for 30 minutes and then cooled on ice and kept covered overnight at 4° C. The mixture was centrifuged for 20 minutes at 4° C at 36,000xg and filtered. An aliquot (0.9ml) of the supernatant fraction was injected onto the affinity/HPLC system. The revised affinity/HPLC system consisted of two affinity columns, containing immobilized folate binding protein (FBP), that were connected via a 10 port, two position switching valve to an analytical column (Betasil Phenyl 250 x 4.6 mm; Keystone Scientific). This allows one affinity column to be loaded with the sample and subsequently washed with a pH 7.0 phosphate buffer, while the second is eluted into the analytical column with an acetonitrile gradient at acid pH and into the electrochemical detector. At the end of the run, a signal is sent to switch the direction of the mobile phases to allow the elution of the loaded column for folate analysis and simultaneous loading of the second column with the new sample. Folate activity was determined by using an ESA Four Channel Coularray Detector set at 0, 300, 500 and 600mV. Quantification and identification of individual folates was done by comparison to external and internal folate standards of known concentration. Limit of detection was 0.18 pmol/ml for 5-methyltetrahydrofolate and folic acid. Recovery was 92.2% (5-methyltetrahydrofolate) and 98.9% (folic acid). CV’s for samples ran alone or spiked with 5, 10, and 20 pmol of folate were 5.2-8.6% for 5-methyltetrahydrofolate (within day) and 3.2-6.3 (between days) and for folic acid 5.3-6.4% (within day) and 3.5-7.3 (between days). Run time for the HPLC was reduced to 30 min per sample (40-45 samples analyzed per 24 hours).
Total folate determined by this method highly correlated (r2=0.93, p< 0.001) with values obtained by the microbial assay
Data Processing and Editing
Data was received after all the laboratory testing was complete. The data were not edited.
Data Access: All data are publicly available.
Laboratory Quality Assurance and Monitoring
There are 7 variables:
SEQN=Respondent sequence number
Value Range: (1.57-291.03)
SSFOLSI=Folic Acid (pmol/ml)
Value Range: 0=undetectable,
Value Range: (0.185 -272.99)
SSFOLTSI=Total Folate (pmol/ml)
Value Range: (1.57-432.61)
Value Range: (0.69-128.34)
SSFOL=Folic Acid (ng/ml)
Value Range: 0=undetectable,
Value Range: (0.082 -120.39)
SSFOLTOT=Total Folate (ng/ml)
Value Range: (0.69-190.78)