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Clinical Chemistry and Laboratory Medicine (CCLM)

Published in Association with the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM)

Editor-in-Chief: Plebani, Mario

Ed. by Gillery, Philippe / Lackner, Karl J. / Lippi, Giuseppe / Melichar, Bohuslav / Payne, Deborah A. / Schlattmann, Peter / Tate, Jillian R.

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1437-4331
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Volume 44, Issue 4 (Apr 2006)

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Quantitative determination of erythrocyte folate vitamer distribution by liquid chromatography-tandem mass spectrometry

Desirée E.C. Smith
  • Department of Clinical Chemistry, ICaR-VU, VU University Medical Center, Amsterdam, The Netherlands
/ Robert M. Kok
  • Department of Clinical Chemistry, ICaR-VU, VU University Medical Center, Amsterdam, The Netherlands
/ Tom Teerlink
  • Department of Clinical Chemistry, ICaR-VU, VU University Medical Center, Amsterdam, The Netherlands
/ Cornelis Jakobs
  • Department of Clinical Chemistry, ICaR-VU, VU University Medical Center, Amsterdam, The Netherlands
/ Yvo M. Smulders
  • Department of Internal Medicine, ICaR-VU, VU University Medical Center, Amsterdam, The Netherlands and Institute for Cardiovascular Research, ICaR-VU, VU University Medical Center, Amsterdam, The Netherlands
Published Online: 2011-09-21 | DOI: https://doi.org/10.1515/CCLM.2006.085

Abstract

Background: Given the role of folate in many disorders, intracellular distribution of folate vitamers is of potential clinical importance. In particular, accumulation of non-methyltetrahydrofolates due to altered partitioning of folate metabolism at the level of methylenetetrahydrofolate is of interest.

Methods: We describe a positive-electrospray liquid chromatography tandem mass spectrometry (LC-MS/MS) method that allows determination of erythrocyte folate vitamer distribution by accurately measuring both 5-methyltetrahydrofolate (5-methylTHF) and non-methyl folate vitamers. Whole blood lysates are deconjugated in ascorbic acid solutions, deproteinized, purified using folate-binding protein affinity columns, concentrated by solid-phase extraction (SPE) and evaporation, and separated on a C18 column within 6min.

Results: The limit of quantification for both 5-methylTHF and non-methylTHF was 0.4nmol/L (signal-to-noise >10). Intra- and inter-assay CVs for 5-methylTHF were 1.2% and 2.8%, respectively. Intra- and inter-assay CVs for non-methylTHF as a group were 1.6% and 1.5%, respectively. Recovery results were 97–107%. We measured 8–72% non-methyl folate vitamers in volunteers (n=5) with the methylenetetrahydrofolate reductase (MTHFR) 677 TT genotype. Concentrations ranged from 117 to 327nmol/L and 23 to 363nmol/L for 5-methylTHF and non-methylTHF vitamers, respectively. We measured 0–2% non-methylTHF vitamers in MTHFR 677 CC genotype volunteers. In addition, we found that storage of whole-blood samples in ascorbic acid at low pH resulted in 53–90% loss of the non-methylTHF fraction.

Conclusion: This LC-MS/MS method accurately determines erythrocyte 5-methylTHF and non-methyl folate vitamers.

Keywords: erythrocyte; folate; folate vitamer; liquid chromatography; mass spectrometry

References

  • 1.

    Shaw GM, Schaffer D, Velie EM, Morland K, Harris JA. Periconceptional vitamin use, dietary folate, and the occurrence of neural tube defects. Epidemiology 1995; 6:219–26. [Crossref]

  • 2.

    Zittoun J. [Anemias due to disorder of folate, vitamin B12 and transcobalamin metabolism]. Rev Prat 1993; 43:1358–63.

  • 3.

    Voutilainen S, Rissanen TH, Virtanen J, Lakka TA, Salonen JT. Low dietary folate intake is associated with an excess incidence of acute coronary events: The Kuopio Ischemic Heart Disease Risk Factor Study. Circulation 2001; 103:2674–80. [Crossref]

  • 4.

    Seshadri S, Beiser A, Selhub J, Jacques PF, Rosenberg IH, D'Agostino RB, et al. Plasma homocysteine as a risk factor for dementia and Alzheimer's disease. N Engl J Med 2002; 346:476–83.

  • 5.

    Mason JB. Folate and colonic carcinogenesis: searching for a mechanistic understanding. J Nutr Biochem 1994; 5:170–5. [Crossref]

  • 6.

    Finkelstein JD, Martin JJ. Homocysteine. Int J Biochem Cell Biol 2000; 32:385–9. [Crossref]

  • 7.

    Frosst P, Blom HJ, Milos R, Goyette P, Sheppard CA, Matthews RG, et al. A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase. Nat Genet 1995; 10:111–3. [Crossref]

  • 8.

    Rozen R. Genetic predisposition to hyperhomocysteinemia: deficiency of methylenetetrahydrofolate reductase (MTHFR). Thromb Haemost 1997; 78:523–6.

  • 9.

    Bagley PJ, Selhub J. A common mutation in the methylenetetrahydrofolate reductase gene is associated with an accumulation of formylated tetrahydrofolates in red blood cells. Proc Natl Acad Sci USA 1998; 95:13217–20. [Crossref]

  • 10.

    Levine S. Analytical inaccuracy for folic acid with a popular commercial vitamin B12/folate kit. Clin Chem 1993; 39:2209–10.

  • 11.

    Chanarin I. The megaloblastic anaemias. Oxford, UK: Blackwell Scientific, 1979:187–90.

  • 12.

    Belz S, Nau H. Determination of folate patterns in mouse plasma, erythrocytes, and embryos by HPLC coupled with a microbiological assay. Anal Biochem 1998; 265:157–66.

  • 13.

    Patring JD, Jastrebova JA, Hjortmo SB, Andlid TA, Jägerstad IM. Development of a simplified method for the determination of folates in baker's yeast by HPLC with ultraviolet and fluorescence detection. J Agric Food Chem 2005; 53:2406–11. [Crossref]

  • 14.

    Bagley PJ, Selhub J. Analysis of folate form distribution by affinity followed by reversed-phase chromatography with electrical detection. Clin Chem 2000; 46:404–11.

  • 15.

    Eto I, Krumdieck CL. Determination of three different pools of reduced one-carbon-substituted folates. Anal Biochem 1980; 109:167–84. [Crossref]

  • 16.

    Fazili Z, Pfeiffer CM. 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 2004; 50:2378–81. [Crossref]

  • 17.

    Bertino JR, Coward JK, Cashmore A, Chello P, Panichajakul S, Horvath CG, et al. Polyglutamate forms of folate: natural occurrence and role as substrates in mammalian cells. Biochem Soc Trans 1976; 4:853–6. [Crossref]

  • 18.

    Matthews RG, Ghose C, Green JM, Matthews KD, Dunlap RB. Folylpolyglutamates as substrates and inhibitors of folate-dependent enzymes. Adv Enzyme Regul 1987; 26:157–71. [Crossref]

  • 19.

    Kelly P, McPartlin J, Goggins M, Weir DG, Scott JM. Unmetabolized folic acid in serum: acute studies in subjects consuming fortified food and supplements. Am J Clin Nutr 1997; 65:1790–5.

  • 20.

    Rabinowitz JC. Preparation and properties of 5,10-methenyltetrahydrofolic acid and 10-formyltetrahydrofolic acid. Methods Enzymol 1963; 6:814–5.

  • 21.

    Blakley RL. The biochemistry of folic acid and related pteridines. In: Neuberger A, Tatum EL, editors. Frontiers of biology. London: North-Holland, 1969:92–4.

  • 22.

    Wright AJ, Finglas PM, Southon S. Erythrocyte folate analysis: saponin added during lysis of whole blood can increase apparent folate concentrations, depending on hemolysate pH. Clin Chem 2000; 46:1978–86.

  • 23.

    Pfeiffer CM, Gregory JF III. Enzymatic deconjugation of erythrocyte polyglutamyl folates during preparation for folate assay: investigation with reversed-phase liquid chromatography. Clin Chem 1996; 42:1847–54.

  • 24.

    Kok RM, Smith DE, Dainty JR, Van Den Akker JT, Finglas PM, Smulders YM, et al. 5-Methyltetrahydrofolic acid and folic acid measured in plasma with liquid chromatography tandem mass spectrometry: applications to folate absorption and metabolism. Anal Biochem 2004; 326:129–38.

  • 25.

    Fazili Z, Pfeiffer CM, Zhang M, Jain R. Erythrocyte folate extraction and quantitative determination by LC/MS/MS: comparison of results with microbiologic assay. Clin Chem 2005; 51:2318–25. [Crossref]

  • 26.

    Owens JE, Holstege DM, Clifford AJ. Quantitation of total folate in whole blood using LC-MS/MS. J Agric Food Chem 2005; 53:7390–4. [Web of Science] [Crossref]

  • 27.

    Freisleben A, Schieberle P, Rychlik M. Specific and sensitive quantification of folate vitamers in foods by stable isotope dilution assays using high-performance liquid chromatography-tandem mass spectrometry. Anal Bioanal Chem 2003; 376:149–56.

  • 28.

    Vahteristo LT, Ollilainen V, Koivistoinen PE, Varo P. Improvements in the analysis of reduced folate monoglutamates and folic acid in food by high-performance liquid chromatography. J Agric Food Chem 1996; 44:477–82. [Crossref]

  • 29.

    Zhang G-F, Storozhenko S, Van Der Straeten D, Lambert WE. Investigation of the extraction behaviour of the main monoglutamate folates from spinach by liquid chromatography-electrospray ionisation tandem mass spectrometry. J Chromatogr A 2005; 1078:59–66.

  • 30.

    Gunter EW, Bowman BA, Caudill SP, Twite DB, Adams MJ, Sampson EJ. Results of an international round robin for serum and whole-blood folate. Clin Chem 1996; 42:1689–94.

  • 31.

    Suh JR, Herbig AK, Stover PJ. New perspectives on folate catabolism. Annu Rev Nutr 2001; 21:255–82. [Crossref]

About the article

Corresponding author: Dr. Yvo M. Smulders, Department of Internal Medicine, VU University Medical Center, P.O. Box 7057, 1007 MB Amsterdam, The Netherlands Phone: +31-20-4444307, Fax: +31-20-4444313


Received: 2005-11-08

Accepted: 2006-01-16

Published Online: 2011-09-21

Published in Print: 2006-04-01



Citation Information: Clinical Chemistry and Laboratory Medicine (CCLM), ISSN (Online) 1437-4331, ISSN (Print) 1434-6621, DOI: https://doi.org/10.1515/CCLM.2006.085. Export Citation

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