BACKGROUND:Folate status is associated with several chronic diseases; thus accurate assessment of folate status has become important in the clinical setting and in epidemiological studies. The diversity of folate forms complicates the task of assaying endogenous folate. We developed and validated an assay that measures various forms of folate in addition to folate catabolites in human serum.

Folate is not stable in serum and plasma. This may impair laboratory diagnostics and distort the outcome of epidemiological studies on folate and chronic diseases. The present study was designed to determine the kinetics of folate loss in human serum and plasma (collected into tubes containing EDTA, heparin, or citrate) at room temperature and the recovery of folate as 4-alpha-hydroxy-5-methyltetrahydrofolate (hmTHF) or p-aminobenzoylglutamate (pABG) equivalents. Different folate species and pABG were determined by liquid chromatography-tandem MS and microbiologically active folate was measured by a Lactobacillus rhamnosus assay. Concentrations of 5mTHF and microbiologically active folate had a parallel and rapid decrease in EDTA plasma to approximately 60% of the initial concentration after 24 h. In serum, heparin plasma, and citrate plasma, folate decreased more slowly to approximately 50% after 192 h. The loss of 5mTHF that occurred within 48 h was totally recovered as hmTHF. Folate measured as pABG equivalents decreased slowly to approximately 80% in 192 h and the decline was essentially matrix independent. In conclusion, the degradation of 5mTHF and microbiologically active folate in serum and plasma at room temperature can largely be corrected for by determining hmTHF or measuring folate as pABG equivalents. Moreover, results obtained using conventional folate assays may be biased by improper sample handling or if samples contained high concentrations of hmTHF.

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