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Nutrition |
1 Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, GA 30341.
aAddress correspondence to this author at: 4770 Buford Hwy., NE, MS F18, Atlanta, GA 30341. Fax 770-488-4139; e-mail CPfeiffer{at}cdc.gov.
Background: Current clinical methods for folate give different results and cannot measure the various forms of folate. We developed an isotope-dilution tandem mass spectrometric method coupled to liquid chromatography (LC/MS/MS) as a candidate reference method for 5-methyltetrahydrofolic acid (5MeTHF), 5-formyltetrahydrofolic acid (5FoTHF), and folic acid (FA) in human serum.
Methods: We quantitatively isolated folates from 275 µL of serum with a phenyl solid-phase extraction cartridge, then detected and quantified them in stabilized serum extracts by positive-ion electrospray ionization LC/MS/MS. We used an isocratic mobile phase of acetic acid in organic solvent on a C8 analytical column. 13C-labeled folates were used as internal standards.
Results: Limits of detection in serum were 0.13 (5MeTHF), 0.05 (5FoTHF), and 0.07 (FA) nmol/L. Within- and between-run imprecision (CV) was <7% for 5MeTHF and <10% for 5FoTHF at concentrations >0.5 nmol/L, and <10% for FA at concentrations >2.0 nmol/L. Total folate (TFOL) concentrations determined by competitive protein binding radioassay were 
9% lower than results obtained with LC/MS/MS. The microbiologic assay gave 15% higher TFOL results with FA calibrator and no difference with 5MeTHF calibrator. The mean (SD) [range] TFOL in 42 sera was 35.5 (17.8) [6.5–75.6] nmol/L. Thirty-two samples with TFOL <50 nmol/L had, on average, 93.3% 5MeTHF, 2.3% FA, and 4.4% 5FoTHF. Ten samples with TFOL >50 nmol/L had, on average, 81.7% 5MeTHF, 15.7% FA, and 2.5% 5FoTHF.
Conclusions: This stable-isotope-dilution LC/MS/MS method can quantify 5MeTHF, 5FoTHF, and FA in serum. Currently used clinical assays agree with this candidate reference method.
The following articles in journals at HighWire Press have cited this article:
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  S. Aufreiter, J. F Gregory III, C. M Pfeiffer, Z. Fazili, Y.-I. Kim, N. Marcon, P. Kamalaporn, P. B Pencharz, and D. L O'Connor Folate is absorbed across the colon of adults: evidence from cecal infusion of 13C-labeled [6S]-5-formyltetrahydrofolic acid Am. J. Clinical Nutrition, July 1, 2009; 90(1): 116 - 123. [Abstract] [Full Text] [PDF]
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 R. Hannisdal, P. M. Ueland, S. J. P. M. Eussen, A. Svardal, and S. Hustad Analytical Recovery of Folate Degradation Products Formed in Human Serum and Plasma at Room Temperature J. Nutr., July 1, 2009; 139(7): 1415 - 1418. [Abstract] [Full Text] [PDF]
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 R. Hannisdal, P. M. Ueland, and A. Svardal Liquid Chromatography-Tandem Mass Spectrometry Analysis of Folate and Folate Catabolites in Human Serum Clin. Chem., June 1, 2009; 55(6): 1147 - 1154. [Abstract] [Full Text] [PDF]
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L. A Houghton, J. Yang, and D. L O'Connor Unmetabolized folic acid and total folate concentrations in breast milk are unaffected by low-dose folate supplements Am. J. Clinical Nutrition, January 1, 2009; 89(1): 216 - 220. [Abstract] [Full Text] [PDF]
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R. D Kalmbach, S. F Choumenkovitch, A. M Troen, R. D'Agostino, P. F Jacques, and J. Selhub Circulating folic acid in plasma: relation to folic acid fortification Am. J. Clinical Nutrition, September 1, 2008; 88(3): 763 - 768. [Abstract] [Full Text] [PDF]
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H. Refsum and A D. Smith Are we ready for mandatory fortification with vitamin B-12? Am. J. Clinical Nutrition, August 1, 2008; 88(2): 253 - 254. [Full Text] [PDF]
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R. Hannisdal, A. Svardal, and P. M. Ueland Measurement of Folate in Fresh and Archival Serum Samples as p-Aminobenzoylglutamate Equivalents Clin. Chem., April 1, 2008; 54(4): 665 - 672. [Abstract] [Full Text] [PDF]
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A D. Smith, Y.-I. Kim, and H. Refsum Is folic acid good for everyone? Am. J. Clinical Nutrition, March 1, 2008; 87(3): 517 - 533. [Abstract] [Full Text] [PDF]
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Z. Fazili, C. M. Pfeiffer, M. Zhang, R. B. Jain, and D. Koontz 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., January 1, 2008; 54(1): 197 - 201. [Abstract] [Full Text] [PDF]
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 T. M. Welzel, H. A. Katki, L. C. Sakoda, A. A. Evans, W. T. London, G. Chen, S. O'Broin, F.-M. Shen, W.-Y. Lin, and K. A. McGlynn Blood Folate Levels and Risk of Liver Damage and Hepatocellular Carcinoma in a Prospective High-Risk Cohort Cancer Epidemiol. Biomarkers Prev., June 1, 2007; 16(6): 1279 - 1282. [Abstract] [Full Text] [PDF]
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 P. I. Holm, S. Hustad, P. M. Ueland, S. E. Vollset, T. Grotmol, and J. Schneede Modulation of the Homocysteine-Betaine Relationship by Methylenetetrahydrofolate Reductase 677 C->T Genotypes and B-Vitamin Status in a Large-Scale Epidemiological Study J. Clin. Endocrinol. Metab., April 1, 2007; 92(4): 1535 - 1541. [Abstract] [Full Text] [PDF]
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Z. Fazili, C. M. Pfeiffer, and M. Zhang Comparison of Serum Folate Species Analyzed by LC-MS/MS with Total Folate Measured by Microbiologic Assay and Bio-Rad Radioassay Clin. Chem., April 1, 2007; 53(4): 781 - 784. [Abstract] [Full Text] [PDF]
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K. D Stark, R. J Pawlosky, R. J Sokol, J. H Hannigan, and N. Salem Jr Maternal smoking is associated with decreased 5-methyltetrahydrofolate in cord plasma Am. J. Clinical Nutrition, March 1, 2007; 85(3): 796 - 802. [Abstract] [Full Text] [PDF]
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A D. Smith Folic acid fortification: the good, the bad, and the puzzle of vitamin B-12 Am. J. Clinical Nutrition, January 1, 2007; 85(1): 3 - 5. [Full Text] [PDF]
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Z. Fazili, C. M. Pfeiffer, M. Zhang, and R. Jain Erythrocyte Folate Extraction and Quantitative Determination by Liquid Chromatography-Tandem Mass Spectrometry: Comparison of Results with Microbiologic Assay Clin. Chem., December 1, 2005; 51(12): 2318 - 2325. [Abstract] [Full Text] [PDF]
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D. H. Wilson, G. Williams, R. Herrmann, D. Wiesner, and P. Brookhart Issues in Immunoassay Standardization: The ARCHITECT Folate Model for Intermethod Harmonization Clin. Chem., April 1, 2005; 51(4): 684 - 687. [Full Text] [PDF]
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 A.M.W. Spijkerman, Y.M. Smulders, P.J. Kostense, R.M.A. Henry, A. Becker, T. Teerlink, C. Jakobs, J.M. Dekker, G. Nijpels, R.J. Heine, et al. S-Adenosylmethionine and 5-Methyltetrahydrofolate Are Associated With Endothelial Function After Controlling for Confounding by Homocysteine: The Hoorn Study Arterioscler. Thromb. Vasc. Biol., April 1, 2005; 25(4): 778 - 784. [Abstract] [Full Text] [PDF]
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A. J. Clifford, E. M. Noceti, A. Block-Joy, T. Block, and G. Block Erythrocyte Folate and Its Response to Folic Acid Supplementation Is Assay Dependent in Women J. Nutr., January 1, 2005; 135(1): 137 - 143. [Abstract] [Full Text] [PDF]
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Z. Fazili and C. M. Pfeiffer Measurement of Folates in Serum and Conventionally Prepared Whole Blood Lysates: Application of an Automated 96-Well Plate Isotope-Dilution Tandem Mass Spectrometry Method Clin. Chem., December 1, 2004; 50(12): 2378 - 2381. [Full Text] [PDF]
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