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Determination of folate forms

The accurate measurement of folate forms is essential for monitoring the folate intake and the evaluation of the folate status. The quantification of folate forms in WB reflects the long-term folate status in the body. The assay of CSF folate may be an essential tool in diagnosing cerebral folate deficiency. The quantification of folate forms from various sample material is challenging. Most methods, including the microbiological and the protein-binding assay only determine the total folate and are unable to distinguish between the different folate forms. Several analytical difficulties delayed the development of methods for measuring the main folate forms. Examples of these factors are: the large number of folates (according to oxidation states and number of glutamate residues), the complex sample matrix, the instability and interconversions of folates, and the presence of folate binding proteins in the samples. Folates are easier to determine in serum where they are present as monoglutamates. In whole blood, folylpolyglutamates must be deconjugated to their monoglutamate forms before they can be measured. The release of folates in whole blood should be performed under strict temperature, pH, and time conditions (1), otherwise, hemolysis can be incomplete, or folates can be trapped in the hemoglobin molecule (2).

Stability of folate forms in vitro (pre analytics)

Reduced folate forms are known to be sensitive to heat, pH, oxidation, pressure, and ultraviolet light. Therefore, sample preparation and measurements must be performed under controlled conditions. The major degradation products are p-aminobenzoyl-L-glutamic acid (pABG) and pteridine fragments that have no coenzyme function (3). THF, DHF, and 10-formylTHF readily undergo oxidative degradation in vitro. Moreover, these reduced folates undergo pH-dependent enzymatic and non-enzymatic interconversions. De Brouwer et al. summarized the pH and heat stability of individual folate forms in in vitro experiments (4). FA and 5-methylTHF are stable at pH 2 – 10 and 5 formylTHF is relatively stable at 37°C and pH 3 – 10. THF is relatively stable below pH < 5, whereas DHF is relatively stable at pH > 8. In contrast to THF, DHF is instable under all pH conditions after heating. Under low pH conditions, THF can be oxidized in vitro to DHF and FA. In order to stabilize the reduced folates during sample preparation, it is decisive to add adequate antioxidant such as ascorbic acid, β-mercaptoethanol, and dithiothreitol. In addition, artificial FA might be generated from the oxidation of THF during sample preparation, even in the presence of antioxidants (5). This finding might lead to misinterpretation of studies looking at unmetabolized FA in the samples.



1.     Pfeiffer CM, Gregory JF, III. Enzymatic deconjugation of erythrocyte polyglutamyl folates during preparation for folate assay: investigation with reversed-phase liquid chromatography. Clin Chem 1996;42:1847-54.
2.     Wright AJ, Finglas PM, Southon S. Erythrocyte folate analysis: saponin added during lysis of whole blood can increase apparent folate concentrations, depending on hemolysate pH. Clin Chem 2000;46:1978-86.
3.     Eitenmiller RR, Lin Ye, Landen WO, Jr. Folate and Folic Acid. Vitamin Analysis for the Health and Food Sciences. CRC Press, 2007:443-506.
4.     De Brouwer V, Zhang GF, Storozhenko S, Straeten DV, Lambert WE. pH stability of individual folates during critical sample preparation steps in prevision of the analysis of plant folates. Phytochem Anal 2007;18:496-508.
5.     Kirsch SH, Herrmann W, Geisel J, Obeid R. Assay of whole blood (6S)-5-CH(3)-H (4)folate using ultra performance liquid chromatography tandem mass spectrometry. Anal Bioanal Chem 2012;404:895-902.