Abstract
Volumetric absorptive microsamplers (VAMS) have been developed recently as a promising tool for clinical blood sampling. Compared to dried blood spot samples analyzed by accurate stable isotope dilution assays (SIDAs), the new technique could provide further substantial miniaturizing of folate assays by eliminating hematocrit effects and uneven analyte distribution within the sample. Herein, we present a miniaturized SIDA coupled with LC-MS/MS measurement of 5-methyltetrahydrofolic acid as main folate vitamer in whole blood (WB) using [13C5]-5-methyltetrahydrofolic acid as internal standard. Elution and extraction of only 10.8 μL-dried WB were carried out by centrifugation followed by enzymatic treatment for polyglutamate deconjugation. Matrix separation was achieved by heating and centrifugation. To verify applicability, WB folate status of 11 volunteers was screened. Limits of detection and limits of quantitation were 9 and 26 nmol·L−1, respectively, which is sufficiently low for screening folate status. Recoveries were 97 (±5.8), 99 (±2.8), and 96 (±6.1)% for 800, 400, and 200 nmol L−1 5-methyltetrahydrofolic acid, respectively. Precision of the LC-MS/MS instrument and inter-assay precision trials revealed CVs of 8.1 and 3.5% (294 nmol L−1), respectively, thus confirming reproducible and precise quantitation. Compared to fresh WB, no significant degradation of 5-methyltetrahydrofolate was observed after 2.5 h of drying at room temperature. VAMS 5-CH3-H4folate was stable for at least 3 weeks at −20°C. In our pilot study, accurate and diagnostically conclusive determination of folate status was verified. Nevertheless, blood sampling should be performed by trained individuals to avoid substantial errors concerning the absorbed volume. Endogenous folate in rat serum and chicken pancreas caused a significant background especially at low blood 5-CH3-H4folate levels and, thus, for polyglutamate deconjugation, these background folates or alternative mixtures need to be removed. The superior feasibility of a minimized blood collection with VAMS allows further progress regarding time- and cost-effective methodologies in newborn or population screenings for 5-methyltetrahydrofolate status. Further steps toward minimization could include an automated assay coupled with UPLC-MS/MS.
Highlights
Folates are essential micronutrients, which are directly involved in purine and pyrimidine synthesis (1) and vitamin B12 mediated remethylation of homocysteine to methionine
Red blood cells are not influenced by recent dietary intake and maintain its folate content over 4-month lifespan (14). 5-CH3-H4folate represents the main vitamer in erythrocytes with reduced concentrations in humans with folate deficiency or increased levels of non-5-CH3-H4folate related to MTHFR C677T polymorphism (10)
Analysis of 5-CH3-H4folate in dried blood spot (DBS) by means of stable isotope dilution assay (SIDA) conducted as LC-MS/MS measurements proved to be the method of choice for accurate and precise evaluation of folate status in whole blood (WB), as they compensate for matrix interferences and poor analyte stability (18)
Summary
Folates are essential micronutrients, which are directly involved in purine and pyrimidine synthesis (1) and vitamin B12 mediated remethylation of homocysteine to methionine. The latter ensures maintenance of one-carbon metabolism and methylation of essential molecules like DNA, proteins, phospholipids, or neurotransmitters (2). As a result, reduced transformation of 5,10-methylenetetrahydrofolic acid to 5-methyltetrahydrofolate (5-CH3-H4folate) leads to higher levels of circulating non-methylated folates such as 5,10-methylenetetrahydrofolic acid, 5-formyltetrahydrofolic acid, and tetrahydrofolic acid in erythrocytes (10). 5-CH3-H4folate represents the main vitamer in erythrocytes with reduced concentrations in humans with folate deficiency or increased levels of non-5-CH3-H4folate related to MTHFR C677T polymorphism (10) Red blood cells are not influenced by recent dietary intake and maintain its folate content over 4-month lifespan (14). 5-CH3-H4folate represents the main vitamer in erythrocytes with reduced concentrations in humans with folate deficiency or increased levels of non-5-CH3-H4folate related to MTHFR C677T polymorphism (10)
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