title: Quantitation of whole-blood total folate within defined MTHFR C677T genotype groups by isotope dilution-liquid chromatography-tandem mass spectrometry differs from microbiologic assay.
abstract: Standardization of folate measurement is needed for accurate assessment of folate status. We compared the measurement of whole-blood folate by isotope dilution-liquid chromatography-tandem MS (ID-LC-MS/MS) with the historical gold standard microbiological assay (MA) using 3 common calibrators within the frame of the methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism. Seventy-three whole-blood samples with an even distribution of MTHFR C677T genotypes (24 CC, 24 CT, 24 TT) were prepared, and total folate was determined by ID-LC-MS/MS and MA using the following calibrators: 5-methyltetrahydrofolate (5-methylTHF) (Merck), folic acid (FA) (Merck), and FA (Sigma). To compare the methods, 5-formyltetrahydrofolate (5-formylTHF) was excluded in the ID-LC-MS/MS summation of total folate, because it is likely that the majority of 5-formylTHF detected is a pyrazino-s-triazine oxidation product of 5-methylTHF. MA whole-blood folate measured by using the FA calibrators was consistently higher than with the 5-methylTHF calibrator. Differences between dilutions and analysis of spiked whole-blood samples showed a nonlinear response, with overrecovery of 5-methylTHF by ~23% toward the higher end of the MA calibration range. Significant proportional biases between ID-LC-MS/MS and MA were found in all comparisons except when the MA was calibrated with 5-methylTHF and a higher sample dilution of 1:1600 (regression slope: 1.05; P = 0.31; intercept-21, P = 0.16). Calibration bias and matrix effects in the MA underscore the need for a formally accepted whole-blood folate reference method. ID-LC-MS/MS procedures have the potential to offer a high degree of accuracy; however, further work is needed to determine the origin of the pyrazino-s-triazine derivative.
authors.affiliation: Department of Human Nutrition, University of Otago, Dunedin, New Zealand.
authors.forename: Rosemary A
authors.initials: RA
authors.lastname: Stamm
authors.forename: Michelle J
authors.initials: MJ
authors.lastname: Harper
authors.forename: Lisa A
authors.initials: LA
authors.lastname: Houghton
mesh_terms: D000328:Adult; D002138:Calibration; D002853:Chromatography, Liquid / Q000379:methods*; D005260:Female; D005492:Folic Acid / Q000097:blood*; D005838:Genotype; D006801:Humans; D007201:Indicator Dilution Techniques; D008297:Male; D042965:Methylenetetrahydrofolate Reductase (NADPH2) / Q000235:genetics*; D008828:Microbiological Techniques; D008875:Middle Aged; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D005492:Folic Acid; D042965:Methylenetetrahydrofolate Reductase (NADPH2)