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 6 min. RESULTS: The limit of quantification for both 5-methylTHF and non-methylTHF was 0.4 nmol/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 327 nmol/L and 23 to 363 nmol/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.
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 6 min. RESULTS: The limit of quantification for both 5-methylTHF and non-methylTHF was 0.4 nmol/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 327 nmol/L and 23 to 363 nmol/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.
Authors: Siddharth Banka; Henk J Blom; John Walter; Majid Aziz; Jill Urquhart; Christopher M Clouthier; Gillian I Rice; Arjan P M de Brouwer; Emma Hilton; Grace Vassallo; Andrew Will; Desirée E C Smith; Yvo M Smulders; Ron A Wevers; Robert Steinfeld; Simon Heales; Yanick J Crow; Joelle N Pelletier; Simon Jones; William G Newman Journal: Am J Hum Genet Date: 2011-02-11 Impact factor: 11.025
Authors: Yuehua Huang; Stefanie Khartulyari; Megan E Morales; Anna Stanislawska-Sachadyn; Joan M Von Feldt; Alexander S Whitehead; Ian A Blair Journal: Rapid Commun Mass Spectrom Date: 2008-08 Impact factor: 2.419
Authors: Nisha Padmanabhan; Dongxin Jia; Colleen Geary-Joo; Xuchu Wu; Anne C Ferguson-Smith; Ernest Fung; Mark C Bieda; Floyd F Snyder; Roy A Gravel; James C Cross; Erica D Watson Journal: Cell Date: 2013-09-26 Impact factor: 41.582