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Quantification of key red blood cell folates from subjects with defined MTHFR 677C>T genotypes using stable isotope dilution liquid chromatography/mass spectrometry
Author(s) -
Huang Yuehua,
Khartulyari Stefanie,
Morales Megan E.,
StanislawskaSachadyn Anna,
Von Feldt Joan M.,
Whitehead Alexander S.,
Blair Ian A.
Publication year - 2008
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.3624
Subject(s) - chemistry , methylenetetrahydrofolate reductase , isotope dilution , chromatography , ascorbic acid , red blood cell , mass spectrometry , biochemistry , genotype , food science , gene
Red blood cell (RBC) folate levels are established at the time of erythropoiesis and therefore provide a surrogate biomarker for the average folate status of an individual over the preceding four months. Folates are present as folylpolyglutamates, highly polar molecules that cannot be secreted from the RBCs, and must be converted into their monoglutamate forms prior to analysis. This was accomplished using an individual's plasma pteroylpolyglutamate hydrolase by lysing the RBCs in whole blood at pH 5 in the presence of ascorbic acid. Quantitative conversion of formylated tetrahydrofolate derivatives into the stable 5,10‐methenyltetrahydrofolate (5,10‐MTHF) form was conducted at pH 1.5 in the presence of [ 13 C 5 ]‐5‐formyltetrahydrofolate. The resulting [ 13 C 5 ]‐5,10‐MTHF was then used as an internal standard for the formylated forms of tetrahydrofolate that had been converted into 5,10‐MTHF as well any 5,10‐MTHF that had been present in the original sample. A stable isotope dilution liquid chromatography‐multiple reaction monitoring/mass spectrometry method was validated and then used for the accurate and precise quantification of RBC folic acid, 5‐methyltetrahydrofolate (5‐MTHF), tetrahydrofolate (THF), and 5,10‐MTHF. The method was sensitive and robust and was used to assess the relationship between different methylenetetrahydrofolate reductase ( MTHFR ) 677C>T genotypes and RBC folate phenotypes. Four distinct RBC folate phenotypes could be identified. These were classified according to the relative amounts of individual RBC folates as type I (5‐MTHF >95%; THF <5%; 5,10‐MTHF <5%), type II (5‐MTHF <95%; THF 5% to 20%; 5,10‐MTHF <5%), type III (5‐MTHF >55%; THF >20%; 5,10‐MTHF >5%), and type IV (5‐MTHF <55%; THF >20%; 5,10‐MTHF >5%). Copyright © 2008 John Wiley & Sons, Ltd.