Quantitative determination of erythrocyte folate vitamer distribution by liquid chromatography-tandem mass spectrometry
-
Desirée E.C. Smith
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.
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; 6:219–26.10.1097/00001648-199505000-00005Search in Google Scholar
2. Zittoun J. [Anemias due to disorder of folate, vitamin B12 and transcobalamin metabolism]. Rev Prat 1993; 43:1358–63.Search in Google Scholar
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; 103:2674–80.10.1161/01.CIR.103.22.2674Search in Google Scholar
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; 346:476–83.10.1056/NEJMoa011613Search in Google Scholar
5. Mason JB. Folate and colonic carcinogenesis: searching for a mechanistic understanding. J Nutr Biochem 1994; 5:170–5.10.1016/0955-2863(94)90068-XSearch in Google Scholar
6. Finkelstein JD, Martin JJ. Homocysteine. Int J Biochem Cell Biol 2000; 32:385–9.10.1016/S1357-2725(99)00138-7Search in Google Scholar
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; 10:111–3.10.1038/ng0595-111Search in Google Scholar PubMed
8. Rozen R. Genetic predisposition to hyperhomocysteinemia: deficiency of methylenetetrahydrofolate reductase (MTHFR). Thromb Haemost 1997; 78:523–6.10.1055/s-0038-1657581Search in Google Scholar
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; 95:13217–20.10.1073/pnas.95.22.13217Search in Google Scholar PubMed PubMed Central
10. Levine S. Analytical inaccuracy for folic acid with a popular commercial vitamin B12/folate kit. Clin Chem 1993; 39:2209–10.10.1093/clinchem/39.10.2209Search in Google Scholar
11. Chanarin I. The megaloblastic anaemias. Oxford, UK: Blackwell Scientific, 1979:187–90.Search in Google Scholar
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; 265:157–66.10.1006/abio.1998.2865Search in Google Scholar PubMed
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; 53:2406–11.10.1021/jf048083gSearch in Google Scholar
14. Bagley PJ, Selhub J. Analysis of folate form distribution by affinity followed by reversed-phase chromatography with electrical detection. Clin Chem 2000; 46:404–11.10.1093/clinchem/46.3.404Search in Google Scholar
15. Eto I, Krumdieck CL. Determination of three different pools of reduced one-carbon-substituted folates. Anal Biochem 1980; 109:167–84.10.1016/0003-2697(80)90026-3Search in Google Scholar
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; 50:2378–81.10.1373/clinchem.2004.036541Search in Google Scholar
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; 4:853–6.10.1042/bst0040853Search in Google Scholar
18. Matthews RG, Ghose C, Green JM, Matthews KD, Dunlap RB. Folylpolyglutamates as substrates and inhibitors of folate-dependent enzymes. Adv Enzyme Regul 1987; 26:157–71.10.1016/0065-2571(87)90012-4Search in Google Scholar
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; 65:1790–5.10.1093/ajcn/65.6.1790Search in Google Scholar
20. Rabinowitz JC. Preparation and properties of 5,10-methenyltetrahydrofolic acid and 10-formyltetrahydrofolic acid. Methods Enzymol 1963; 6:814–5.10.1016/0076-6879(63)06256-XSearch in Google Scholar
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.Search in Google Scholar
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; 46:1978–86.10.1093/clinchem/46.12.1978Search in Google Scholar
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; 42:1847–54.10.1093/clinchem/42.11.1847Search in Google Scholar
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; 326:129–38.10.1016/j.ab.2003.12.003Search in Google Scholar PubMed
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; 51:2318–25.10.1373/clinchem.2005.053801Search in Google Scholar PubMed
26. Owens JE, Holstege DM, Clifford AJ. Quantitation of total folate in whole blood using LC-MS/MS. J Agric Food Chem 2005; 53:7390–4.10.1021/jf0510485Search in Google Scholar PubMed
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; 376:149–56.10.1007/s00216-003-1844-ySearch in Google Scholar PubMed
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; 44:477–82.10.1021/jf9503467Search in Google Scholar
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; 1078:59–66.10.1016/j.chroma.2005.04.085Search in Google Scholar PubMed
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; 42:1689–94.10.1093/clinchem/42.10.1689Search in Google Scholar
31. Suh JR, Herbig AK, Stover PJ. New perspectives on folate catabolism. Annu Rev Nutr 2001; 21:255–82.10.1146/annurev.nutr.21.1.255Search in Google Scholar PubMed
©2006 by Walter de Gruyter Berlin New York
Articles in the same Issue
- Natriuretic peptides and evidence-based quality specifications
- Preanalytical variability: the dark side of the moon in laboratory testing
- Clinical relevance of biological variation: the lesson of brain natriuretic peptide (BNP) and NT-proBNP assay
- Hepatorenal syndrome
- Modified Levey-Jennings charts for calculated laboratory tests
- Increased free malondialdehyde concentrations in smokers normalise with a mixed fruit and vegetable juice concentrate: a pilot study
- The exponentially weighted moving average (EWMA) rule compared with traditionally used quality control rules
- Intermethod calibration of alanine aminotransferase (ALT) and γ-glutamyltransferase (GGT) results: application to Fibrotest® and Actitest® scores
- Comparison of TEST 1 with SRS 100 and ICSH reference method for the measurement of the length of sedimentation reaction in blood
- Multicenter evaluation of the interference of hemoglobin, bilirubin and lipids on Synchron LX-20 assays
- Technical evaluation of the Beckman Coulter OV-Monitor (CA 125 antigen) immunoassay
- Erythrocyte membrane Na+,K+-ATPase and Mg2+-ATPase activities in subjects with methylenetetrahydrofolate reductase (MTHFR) 677 C→T genotype and moderate hyperhomocysteinaemia. The role of L-phenylalanine and L-alanine
- Matrix metalloproteinases and their inhibitors in different acute stroke subtypes
- Pyrosequencing protocol requiring a unique biotinylated primer
- Detection of antibodies against 60-, 65- and 70-kDa heat shock proteins in paediatric patients with various disorders using Western blotting and ELISA
- Quantitative determination of erythrocyte folate vitamer distribution by liquid chromatography-tandem mass spectrometry
- Time-level relationship between indicators of oxidative stress and Glasgow Coma Scale scores of severe head injury patients
- Stepwise strategies in analysing haematuria and leukocyturia in screening
- Elevation of serum cerebral injury markers correlates with serum choline decline after coronary artery bypass grafting surgery
- Drug screening in urine by cloned enzyme donor immunoassay (CEDIA) and kinetic interaction of microparticles in solution (KIMS): a comparative study
- Release of anandamide from blood cells
- Rapid decrease in plasma D-lactate as an early potential predictor of diminished 28-day mortality in critically ill septic shock patients
- Evaluation of an immunoassay of whole blood sirolimus in pediatric transplant patients in comparison with high-performance liquid chromatography/tandem mass spectrometry
- Sample processing and its preanalytical impact on the measurement of circulating matrix metalloproteinases
- Physiological matrix metalloproteinase (MMP) concentrations: comparison of serum and plasma specimens
- Importance of the functional sensitivity determination of a serum hyaluronic acid assay for the prediction of liver fibrosis in patients with features of the metabolic syndrome
- The dilemma of invasive and non-invasive investigations for adult and paediatric non-alcoholic fatty liver disease: has the time come for a new biochemical marker?
- Is cystatin C a reliable renal marker in trauma?
- On the independence of intraindividual reference values
- Sixth International Symposium on Molecular Diagnostics, Graz, Austria, May 25-27, 2006
Articles in the same Issue
- Natriuretic peptides and evidence-based quality specifications
- Preanalytical variability: the dark side of the moon in laboratory testing
- Clinical relevance of biological variation: the lesson of brain natriuretic peptide (BNP) and NT-proBNP assay
- Hepatorenal syndrome
- Modified Levey-Jennings charts for calculated laboratory tests
- Increased free malondialdehyde concentrations in smokers normalise with a mixed fruit and vegetable juice concentrate: a pilot study
- The exponentially weighted moving average (EWMA) rule compared with traditionally used quality control rules
- Intermethod calibration of alanine aminotransferase (ALT) and γ-glutamyltransferase (GGT) results: application to Fibrotest® and Actitest® scores
- Comparison of TEST 1 with SRS 100 and ICSH reference method for the measurement of the length of sedimentation reaction in blood
- Multicenter evaluation of the interference of hemoglobin, bilirubin and lipids on Synchron LX-20 assays
- Technical evaluation of the Beckman Coulter OV-Monitor (CA 125 antigen) immunoassay
- Erythrocyte membrane Na+,K+-ATPase and Mg2+-ATPase activities in subjects with methylenetetrahydrofolate reductase (MTHFR) 677 C→T genotype and moderate hyperhomocysteinaemia. The role of L-phenylalanine and L-alanine
- Matrix metalloproteinases and their inhibitors in different acute stroke subtypes
- Pyrosequencing protocol requiring a unique biotinylated primer
- Detection of antibodies against 60-, 65- and 70-kDa heat shock proteins in paediatric patients with various disorders using Western blotting and ELISA
- Quantitative determination of erythrocyte folate vitamer distribution by liquid chromatography-tandem mass spectrometry
- Time-level relationship between indicators of oxidative stress and Glasgow Coma Scale scores of severe head injury patients
- Stepwise strategies in analysing haematuria and leukocyturia in screening
- Elevation of serum cerebral injury markers correlates with serum choline decline after coronary artery bypass grafting surgery
- Drug screening in urine by cloned enzyme donor immunoassay (CEDIA) and kinetic interaction of microparticles in solution (KIMS): a comparative study
- Release of anandamide from blood cells
- Rapid decrease in plasma D-lactate as an early potential predictor of diminished 28-day mortality in critically ill septic shock patients
- Evaluation of an immunoassay of whole blood sirolimus in pediatric transplant patients in comparison with high-performance liquid chromatography/tandem mass spectrometry
- Sample processing and its preanalytical impact on the measurement of circulating matrix metalloproteinases
- Physiological matrix metalloproteinase (MMP) concentrations: comparison of serum and plasma specimens
- Importance of the functional sensitivity determination of a serum hyaluronic acid assay for the prediction of liver fibrosis in patients with features of the metabolic syndrome
- The dilemma of invasive and non-invasive investigations for adult and paediatric non-alcoholic fatty liver disease: has the time come for a new biochemical marker?
- Is cystatin C a reliable renal marker in trauma?
- On the independence of intraindividual reference values
- Sixth International Symposium on Molecular Diagnostics, Graz, Austria, May 25-27, 2006
