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Challenges in determination of unsubstituted food folates: impact of stabilities and conversions on analytical results Hanna Sara Strandler, Johan J. Patring, Margaretha Jägerstad, and Jelena Jastrebova J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf504987n • Publication Date (Web): 02 Feb 2015 Downloaded from http://pubs.acs.org on February 18, 2015

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Journal of Agricultural and Food Chemistry

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Revised manuscript of jf2014-04978n

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Challenges in determination of unsubstituted food folates: impact of

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stabilities and conversions on analytical results

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Hanna Sara Strandler, ‡Johan Patring, J, ‡Margaretha Jägerstad, and ‡Jelena Jastrebova

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The National Food Agency Box 622, SE-75126 Uppsala, Sweden (corresponding author)

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Phone +4618175757, Fax +4618105848, e-mail [email protected]

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Sciences (SLU), P.O. Box 7051, SE-750 07 Uppsala, Sweden

Department of Food Science, Uppsala BioCenter, Swedish University of Agricultural

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ACS Paragon Plus Environment


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ABSTRACT

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Tetrahydrofolate is the parent molecule of the folate coenzymes required for one carbon

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metabolism. Together with other unsubstituted folates such as dihydrofolate and folic acid,

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tetrahydrofolate represent the third pool of dietary folates following 5-methyl-

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tetrahydrofolate and formyl folates. Low intake of dietary folates and poor folate status are

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common problem in many countries. There is a critical need for reliable methods to

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determine folate in foods to accurately estimate folate intakes in populations. However,

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current values for folates in foods in databanks are often underestimated due to high

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instability of several folate forms, especially tetrahydrofolate. The present review highlights

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occurrence of unsubstituted folates in foods, their oxidation mechanisms and chemical

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behavior as well as interconversion reaction between tetrahydrofolate and 5,10-methylene-

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tetrahydrofolate. The review shows also the important role of antioxidants in protecting

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folates during analysis and describes strategies to stabilize unsubstituted folates throughout

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all steps of the analytical procedure.

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Keywords: folic acid, tetrahydrofolate, dihydrofolate, 5,10-methylene-tetrahydrofolate, folate

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oxidation, folate stabilization

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INTRODUCTION

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Folate belongs to the B vitamin group and it is essential for cell growth, particularly during

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pregnancy when a low folate status (intake) increases the risk for neural tube defects (NTD)

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early in the pregnancy.1 The classical human folate deficiency disease is megaloblastic

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anemia.2 Other clinical aspects link a good folate status to protection against coronary heart

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disease, malformations, cancer and cognitive functions, but the results from these studies are

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still not conclusive.3-6 High intake of synthetic folic acid is investigated for cancer risks, but

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so far the results are not consistent.6-8

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Folate exerts its biological function as transporter of C1-groups. Tetrahydrofolate accepts and

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donates C1 groups (methyl, formyl, formimino, and methylene) by acting as coenzyme.9,10

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The substituted tetrahydrofolates are generally more stable than tetrahydrofolate (H4folate),

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which is one of the most oxygen-sensitive forms among the native folates. If not protected

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from oxidation, it is only stable for up to 30 min in room temp; the main oxidation pathway

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splits the molecule into two biologically inactive parts, para-aminobenzoylglutamic acid

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(pABG) and pterins.11 Lower temperature, neutral to alkaline pH, and antioxidants stabilize

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H4folate and retard its oxidative degradation, thereby forming also other unsubstituted

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folates, e.g. dihydrofolate (H2folate) and folic acid in addition to pABG and pterins.9, 12-15 The

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three unsubstituted folates represent the third pool of food folates following 5-methyl-

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tetrahydrofolate and formyl folates.16 They are most common in yeast, liver, green vegetables

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and cereals or products fortified with folic acid.17

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This review gives a brief background of the chemistry, biological functions, and the

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occurrence of the unsubstituted folates in foods. Their stability, chemical behavior and factors

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affecting them in the food chain and during determination are highlighted. In addition, the

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stability and formation of the even more labile 5,10-methylenetetrahydrofolate (5,10-CH2-

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H4folate) is included in this review because this form easily dissociates back to H4folate at



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neutral and acidic pH and contributes to the H4folate pool.18 Because of this, determination

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could potentially lead to false results by underestimating 5,10-CH2-H4folate or

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overestimating H4folate or both. The purpose of this review is to compile current knowledge

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of the unsubstituted folates in food to achieve a better understanding of their stabilities and

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conversions during food processing, storage, and determination. Only monoglutamate forms

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of the unsubstituted folates are discussed in the review as the native polyglutamate forms

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occurring in foods are deconjugated to monoglutamates prior to analyses.

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BIOCHEMISTRY AND BIOLOGICAL FUNCTIONS

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Plants and microorganisms synthesize folates de novo through a complex metabolic route that

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is now fully elucidated.19-20 In contrast, human and other vertebrates lack a complete

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biosynthetic pathway and thus need dietary folates, of which plants are major sources.9, 20

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Dietary folates are usually linked to a polyglutamic chain, which is hydrolyzed to

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monoglutamyl folate by a brush-border enzyme before absorption. During the passage

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through the intestinal mucosa, dietary folates are reduced and converted to

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5-methyltetrahydrofolic acid (5-CH3-H4folate) which is the major form of folate present in

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the systemic circulation. Synthetic (oxidized) folic acid given as a supplement or through

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fortified foods is reduced enzymatically by folate reductase, partly during the transit through

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the intestinal mucosa and finally intracellularly, especially in the liver, first to H2folate and

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subsequently to H4folate.9, 12

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Immediately on intracellular entrance, H4folate participates in the biosynthesis of

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polyglutamate forms, usually up to 5-7 residues, to be retained within cells and subcellular

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compartments. Polyglutamated folates are the preferred coenzymes involved in the C1

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metabolism. Chain elongation increases the anionic nature of folate coenzymes by providing

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α-carboxyl charges and decreases affinity for membrane carriers, thus impairing folates


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diffusion through hydrophobic barriers.19 The polyglutamated H4folate molecules accept C1

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groups supplied from the catabolism of certain amino acids such as glycine, serine, and

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histidine or from the catabolism of purines. The C1 groups are donated enzymatically to be

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included in the nucleotide synthesis of DNA and RNA and in the methylation of

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homocysteine to methionine. After donation of their C1groups, the substituted folates

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regenerate to polyglutamate forms of H4folate or H2folate and are then ready for accepting

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new C1 groups. 2, 9-10

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OCCURRENCE IN FOOD

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Plant foods are the major source of folate because they can synthesize folate. This

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biosynthesis route has already been explored for biofortification of tomato fruit and rice with

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25-fold to 50-fold increases in folate content, respectively.21-22 Other types of bioprocessing

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to enhance dietary folate are germination, sprouting, malting or fermentation based on yeast

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or lactic acid bacteria (LAB) or both. Bioprocessing is commonly used for manufacture of

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bread, yoghurt, buttermilks, beer and wine.23-26 Good dietary sources of native folate include

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liver, legumes, green leafy vegetables, citrus fruit, and juice, certain berries, cereals, and

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egg.17 Mandatory or voluntary fortification by folic acid is also common mostly as

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prophylaxis against neural tube defects because women of fertile ages throughout the world

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have difficulties meeting the daily folate recommendations during pregnancy and lactation.27

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Most of the folate information in food data basis originates from microbiological assays

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(MA) which only give information on the total folate content determined after deconjugation.

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During the past decades, chromatographic methods have been developed, which can separate

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the different folate vitamers. Such studies have shown 5-CH3-H4folate to be the major native

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food folate form, followed by the formyl folates. Overall, unsubstituted folates are ranked as

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the third folate pool in diet. Table 1 gives an overview of deconjugated native folates in



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monoglutamate forms that have been determined in food using chromatographic methods,

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e.g. high performance liquid chromatography (HPLC) or ultra-high performance liquid

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chromatography (UHPLC). Only data based on HPLC and highly selective detectors such as

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mass spectrometry (MS) are displayed in Table 1.22, 25, 28-34

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Dietary folates are divided into 5-CH3-H4folate, sum of formyl folates and two individual

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unsubstituted folates, i.e. H4folate and folic acid. H2folate is not recorded because it is too

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labile to be determined. Folic acid is included as a stable degradation product of H4folate and

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H2folate.Baker’s dry yeast is one of the richest dietary sources of folate, containing about

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3 mg total folate/100 g of which H4folate constitutes ~25%.28 Another rich dietary folate

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source is liver, where H4folate occurs in similar proportions as 5-CH3-H4folate, and is thus

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being the second most common group there.29 Other good dietary sources of folate and

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H4folate are legumes and green leafy vegetables such as spinach, and cabbage, of which

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H4folate constitutes about 10-20%. 30-33 Cereal fractions also contain H4folate, usually below

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10% (Table 1). Folic acid as a degradation product of H4folate and H2folate is occasionally

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but not regularly found in low concentrations, up to 25% of total folate in cereal fractions

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(whole grain flour, bran).22, 25, 30,34 Apart from the “natural” presence of folic acid in certain

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foods, folic acid can also be added as a mandatory or voluntary food fortificant, especially to

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flours of wheat and corn, bread, fruit juices, and infant foods.

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CHEMICAL PROPERTIES OF UNSUBSTITUTED FOLATES

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Background information about chemical properties is essential to understand the stability and

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conversions of unsubstituted folates during food handling and analysis. All folates comprise a

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pteridine coupled via a carbon-nitrogen (C9-N10) bond to p-amino benzoic acid (pABG),

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which in turn is coupled via an amide bond through its carboxyl moiety to the amino group of

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L-glutamic acid. The pteridine moiety of folates can exist in three oxidation states: fully


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oxidized (folic acid), or as the reduced 7,8-dihydro (H2folate), or 5,6,7,8-tetrahydrofolate

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(H4folate). H4folate is the co-enzymatically active form of the vitamin that accepts, transfers,

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and donates C1 groups, attached either at the N5 or N10 position or by bridging these

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positions.9, 35-36 The C1 groups also differ in their oxidation state, with folates existing as

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derivatives of formate (5-formyl-H4folate, 10-formyl-H4folate, 5,10-methenyl-H4folate, and

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5-forminino-H4folate), methanol (5-methyl-H4folate) or formaldehyde (5,10-methylene-

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H4folate).16 In addition to these structural variables, most naturally occurring folates exist as

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γ-linked polyglutamate conjugates, with chain lengths typically in the range of five to seven

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glutamate residues.9 The chemical structures of unsubstituted folates are displayed in Fig 1.

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Folates are ionogenic and amphoteric molecules, which exist in cationic, anionic or

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zwitterionic form depending on pH. The pH ranges, in which the various ionizable groups of

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unsubstituted folates are predominantly (>50%) charged, are displayed in Fig 2.37-41

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Ionogenic groups of particular significance in the pH values relevant to foods and biological

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systems are the N5 position of H4folate (basic pKa = 4.8) and the glutamate carboxyl groups

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(acidic α pKa=3.5; γ pKa= 4.8). Folates undergo changes in ionic form as a function of pH,

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which in part explains the pH dependency of folate solubility, and stability and behavior

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during chromatographic separation.9, 38-41

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Folates generally have minimum solubility in the mildly acidic pH range (e.g. pH 2-4),

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monocationic and neutral species predominating, whereas solubility generally increases in

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proportion to the pH above this range, mainly zwitterionic and anionic species. The

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solubility of folates in aqueous solutions is low due to the pteridine part. An increase in

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temperature and presence of ions from buffers or salts increases the solubility.9, 35-36

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UV-light, air and heating destroy the molecules by oxidation if not protected by antioxidants.

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Early work shows that the effect of pH on stability of reduced unsubstituted folates is



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U-shaped with highest stability observed between pH 1-2 and pH 8-12, whereas the stability

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is as lowest between pH 4-6.9, 41-43 However, even at these favorable pH intervals, H4folate

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and H2folate are extremely unstable with half-lives of losses in bioactivity below 5 h.9 Folic

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acid, the oxidized form, is much more stable than reduced unsubstituted folates. Its stability

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also depends on pH and is stable under neutral and alkaline conditions, but less stable in

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acidic conditions (pH < 5).44-48 Without light protection all three unsubstituted folates are

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sensitive towards UV-light. For more details on the stability of the unsubstituted folates, see

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Table 2.36, 43,46-54

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DEGRADATION BY AUTO-OXIDATION

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All unsubstituted folates undergoing auto-oxidation are split in their C9-N10 bond into

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biologically inactive products. Figure 3 gives an overview of the pathways and the major

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auto-oxidation products identified in the oxidation carried out in the presence of UV-light, air

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or oxygen and in the absence of antioxidants. 15, 45-7, 55,56 The major products are p-amino

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benzoyl-L-glutamic acid (pABG) and various substituted pterins, ultimately converted into

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pterins under release of formaldehyde. Classical work has shown that the oxidative

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degradation of solutions of H4folate first produces a quinonoid isomer of H2folate as

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intermediate. At acidic pH, this quinonoid is oxidized to pABG and 7,8-dihydropterin under

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release of formaldehyde. This dihydropterin is then further oxidized to pterins. At alkaline

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conditions, the quinonoid-H2folate is converted to H2folate, which is further oxidized to

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pABG and 6-formyltetrapterin, followed by formation of xanthopterin and formaldehyde.15,

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55,56

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carboxyaldehyde, which is converted to 6-carboxylic acid and finally to the decarboxylated

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2-amino-4-hydroxy pteridine.45-48

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MECHANISMS OF AUTO-OXIDATION

Irradiation of folic acid with UV-light first produces pABG and pterine-6-


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The pH dependent oxidation, affecting the rates and type of products, has drawn attention to

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the role of ionization and protonation of the zwitterion folate/folic acid as a mechanism.15, 38

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Akhtar and co-workers studied the effect of pH in the range 2-10 on photodecomposition of

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folic acid.46,47 According to their results folic acid exhibited best stability at highly alkaline

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pH 10.0 whereas its stability was lowest and susceptibility to photolysis was highest in the

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acidic range (pH 2-4). This clearly showed that photolysis of folic acid is pH-dependent

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reaction and occurs faster at low pH when folic acid exists predominantly as neutral species.

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More recently, Martin et al (2009) proposed a mechanism of photolysis of folic acid where

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UV radiation at 350-400 nm caused formation of pterin radicals, which were then oxidized to

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the known photoproduct 6-carboxypterin.57

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According to early studies, steric hindrance is another phenomenon affecting the oxidative

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sensitivity of unsubstituted folates. H4folates with substituents at the N5 position, i.e. 5-CH3-

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H4folate and 5-HCO-H4folate, exhibit much higher stability than unsubstituted H4folate. This

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suggests that the stabilization effect of the N5 methyl group or the N5 formyl group is, at least

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in part, due to steric hindrance in restricting access of oxygen or other oxidants to the

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pteridine ring. The stabilizing effect of the substituent is presumably due to the effect of the

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one-carbon substituent in interfering with the formation of resonance forms involved in

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oxidative degradation.15, 55

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DEGRADATION PRODUCTS IN THE PRESENCE OF ANTIOXIDANTS:

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In the presence of antioxidants and while protected against UV-light, the unsubstituted folates

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are protected against oxidation. Depending on combination and concentration of antioxidants,

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pH, time and temperature, the oxidation of H4folates is either fully or partly prevented.

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Antioxidants decrease the rate of oxidative degradation making it possible to detect even

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early biologically active oxidation products of H4folate, such as H2folate and folic acid.



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Hence, a mixture of these forms appears together with pABG and various pterins in foods

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during processing, cooking, storage or analytical conditions (See Fig 3).13-14, 58

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Thiols, e.g. 2-mercaptoethanol (MCE) and 2,3-dimercaptopropanol (BAL) were the first

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antioxidants to be shown to retard oxidation of H4folate.59,60 In a classical work, Blakley

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(1960)60 found BAL to be a better antioxidant than MCE. Later, the combination of thiols

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with ascorbate was found to be the most effective in protecting H4folate.Wilson and Horne

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(1984)58 was the first group to combine the use of ascorbate and MCE as antioxidants during

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food folate analysis. Today, combinations of 1-2% ascorbate with MCE or BAL are

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commonly used to stabilize H4folate during determination by HPLC.

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Although MCE is the most commonly used and inexpensive thiol in combination with

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ascorbic acid/ascorbate to protect H4folate during food analysis, it is less effective and more

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toxic than other thiols such as BAL,1,4-dithiothreitol (DTT) or 2-thiobarbituric acid

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(TBA).22,61Patring et al (2005) 61 compared the combination of ascorbate (2%) and four

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different antioxidants (MCE, DTT, BAL, TBA), all at 0.1%, on stability of H4folate solutions

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boiled for 1 h. BAL, DTT and TBA were all capable of protecting > 96% of H4folate in three

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different buffers varying between pH 5.1-7.5. DTT was the most effective antioxidant in

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acetate buffer (pH 5), DTT and BAL, in phosphate buffer (pH 6.1) and BAL and TBA in

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HEPES/CHES buffer, pH 7.85, whereas MCE was the least effective in all buffers compared

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to the other thiols (degradation range 7-12%). Recently, De Brouwer and coworkers found

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DTT in concentration of 0.5% to be as efficient as BAL when using phosphate buffer pH 7.2.

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efficient and environment friendly substitutes for MCE in modern analytical methods for

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folate analysis including AOAC methods.

Thereby, these three antioxidants (BAL, DTT and TBA) should be considered as more

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H4folate is easier to stabilize by proper combination of antioxidants than H2folate because the

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initial/primary oxidation product of H4folate is quinoid-H2folate, which can be reduced back

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to H4folate by antioxidants.35 This step is the only one that could be reversible in the

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oxidation chain of H4folate, which means that once H2folate has been formed, it is further

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oxidized to folic acid or to degradation products such as pABG and pterins (See Fig.3).

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Several studies have reported between 12-30% of folic acid formation from H2folate under

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analytic conditions in the pH range 2.6-7.4.13-14, 58,62 As mentioned in a previous section

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(“Occurrence in food”), H2folate is usually not analyzed in foods due to its high instability.

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H2folate seems to require much higher concentrations of antioxidants than H4folate to be

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protected.42, 63

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MECHANISM OF ANTIOXIDANT PROTECTION AGAINST OXIDATION

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Reducing agents such as ascorbic acids and thiols exert multiple protective effects on folates

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through their action as oxygen scavenger, reducing agents, and free radical scavengers. The

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degradation of unsubstituted folates via a free radical mechanism has been proposed by

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several groups.15, 55,56, 64 Thomas et al48 suggested that singlet oxygen, probably produced by

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electronically excited states of folic acid molecule, is the oxidant agent that starts the

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degradation reaction. Further support for free radical mechanisms is the finding that

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riboflavin (vitamin B2) sensitizes the photo degradation of folic acid leading to the

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deactivation of the vitamin.45, 47 In addition, transition metals ions such as Fe3+ or Cu2+

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recognized for their involvement in free radical mechanisms, have been found to catalyze the

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oxidation of H4folate.55

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INTERCONVERSIONS BETWEEN H4FOLATE AND 5,10-METHYLENE-H4FOLATE

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H4folate rapidly and reversibly condensates with formaldehyde (in ~30-fold excess) in

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aqueous solutions at room temperature, forming 5,10 methylene-tetrahydrofolate (5,10-CH2-



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H4folate (See Fig 3). 35, 65 This is an example of interconversion reaction because it includes a

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reversible conversion between two bioactive folate vitamers. The N5-N10 bridged compound

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is found to be more stable against chemical oxidation than H4folate, but its stability depends

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on an excess of formaldehyde.11, 35, 65 Without excess of formaldehyde, the stability of

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5,10-CH2-H4folate is strongly pH dependent; 5,10-CH2-H4folate appears to be completely

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stable at pH 9.5 or above, either in the presence or absence of antioxidants. As soon as pH

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decreases to neutral pH, 5,10-CH2-H4folate dissociates rapidly, within 15 min, back to

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H4folate and formaldehyde, and heating enhances the rate of the dissociation.65,66

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5,10-CH2-H4folate is the one of the C1-derivatives of H4folate, used for enzymatic

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methylation of uridinylate to thymidinylate, a key step for DNA synthesis. Hence 5,10-CH2-

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H4folate is a native folate form that could be present in intact living cells. Because 5,10-CH2-

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H4folate requires either alkaline pH ( > 8) or excess of formaldehyde to remain stable, this

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form is not likely to occur in foods because the pH of food is generally neutral or slightly

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acidic. Hence 5,10-CH2-H4folate most probably converts to H4folate or its degradation

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products in food samples or both.

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There is only one recorded condition in food analysis where formaldehyde can be formed in

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excess with potential to interconvert H4folate into 5,10-CH2-H4folate. When Wilson and

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Horne (1983)14 boiled aqueous solutions of H4folate for 10 min in a buffer (pH 7.8)

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containing 2% sodium ascorbate, 5,10-CH2-H4folate was formed in a yield of 25%. The

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authors were able to show that heating of ascorbate led to its thermal degradation followed by

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formation of formaldehyde which converted H4folate into 5,10-CH2-H4folate. This

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conversion did not occur without heating, neither did formation of formaldehyde. More

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recently, this interconversion was confirmed by de Brouwer et al (2007), using HPLC and

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MS detection.62 This emphasizes the need to combine thiols and ascorbate during heat

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extraction, which is also common practice today in folate determination. The inhibitory effect


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of adding thiols is a formation of hemithioacetal with formaldehyde, thereby preventing the

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reaction between formaldehyde and H4folate.58,66,67

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STABILITY OF UNSUBSTITUTED FOLATE IN THE FOOD CHAIN

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Because H4folate is the most vulnerable folate form, one could expect high losses of H4folate

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in the food chain, especially after heat treatment such as thermal processing or cooking.

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Hence, the richest dietary sources of H4folate should occur in raw foods with intact cell

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structure, where it may be bound and stabilized by proteins. A folate binding protein (FBP) is

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present in milk. By adding FBP to labile folate forms in model systems, H4folate has been

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shown to become significantly stabilized.68

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As soon as the cell integrity is compromised by freezing and thawing, mechanical stress or

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thermal processing, H4folate starts to be oxidized unless protected by native antioxidants such

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as ascorbic acid or being bound to proteins and other cell constituents. Note that iron and

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copper ions leaking from process equipment or process water are pro-oxidative.41

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Baker’s yeast, in compressed or dried form, still contains viable cells and is therefore an

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example of a food ingredient with intact cell structure. It contains about 25% of its total folate

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as H4folate (Table 1). When dry baker’s yeast was stored in room temperature during 45

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days, H4folate was stable. In contrast, compressed baker’s yeast stored in refrigerator (+5 °C)

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lost 40% of H4folate after 17 days. Drying of yeast reduced the content of H4folate because

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the content of H4folate was considerably higher in compressed baker’s yeast prior to storage,

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but these differences reduced progressively with storage time.28

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Cereal fractions contain about 10% of its folate as H4folate. During bread making, yeast and

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fermentation could add further H4folate. However, several studies indicate undetectable

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levels of H4folate in bread; instead they often report occurrence of folic acid ranging from a



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few percent up to 50 percent of total folate.25, 69,70 The breads in these studies were not made

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from folic acid fortified flours. Hence the folic acid most probably originates from oxidation

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of unsubstituted reduced folates as well as from 5,10-CH2-H4folate during fermentation and

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oven baking.

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Green vegetables are mostly blanched and frozen before being cooked and served. Their

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content of ascorbate and possibly other antioxidants could protect the H4folate from being

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oxidized, but losses by leakage could occur. In one study by Stea and co-workers broccoli

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and green peas were investigated after different thermal processes.71 Blanching did not

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reduce H4folate, but in general, these vegetables retained only up to half of their H4folate

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after other thermal treatments, e.g. steam cooking, microwaving, boiling and reheating. In a

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recent study on effects of industrial processing on folate content in green vegetables, Delchier

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et al 2013 found leakage to be a major cause behind folate losses. 72

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Some losses of dietary folates by leakage and/or degradation seem inevitable in the food

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chain. 71, 72 Therefore it is of utmost importance to avoid further degradation and conversions

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of unsubstituted folates during the analytical procedure as outlined in the following section.

321

STABILIZATION DURING DIFFERENT STEPS OF THE ANALYTICAL PROCEDURE

322

Analytical techniques used to determine folate content are biological, such as microbiological

323

and competitive-binding methods or chemical, such as HPLC methods. 9,12,18,73 The

324

microbiological assay (MA) utilizes the nutritional requirement of a microorganism (L.

325

rhamnosus), which exhibited high response to all bioactive folate forms in monoglutamic or

326

diglutamic forms including biologically active interconversion products. Hence MA gives the

327

sum of all bioactive forms, i.e. total folate.9 There are standard methods assessed in

328

collaborative studies established for microbiological assays, for fortified infant formula,

329

cereals, and for foodstuffs. 74-76 A disadvantage of the microbiological assay is the overall


Page 14 of 45

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330

time needed for analysis, almost two days, where the samples are kept at 37 °C, which

331

emphasizes the need of optimal protection by antioxidants.

332

The competitive binding assay, using a folate-binding protein and a radiolabeled folate to

333

compete with the folate to be determined, also gives total folate content. The Biacore method,

334

certified by AOAC for folic acid determinations, uses a folate binding protein isolated from

335

bovine milk and measures the amount of vitamin with plasma surface resonance technique.77

336

However, the folate binding protein does not have the same affinity to the various folate

337

forms. 78,79 Therefore these methods are appropriate only if it is possible to calibrate against

338

the same form of folate as the one present in the sample, such as folic acid in fortified foods.

339

They are less useful for food analysis with more complex matrices and several combinations

340

of folate forms.

341

However, if the focus of interest is on the individual folate forms, chromatographic methods

342

such as an HPLC, UHPLC or LC-MS, which separates and detects the different forms based

343

on their chemical properties, have to be used. Fig 4 gives a schematic overview of the

344

analytical methods for folate determination in foods. All methods include the following steps:

345

extraction, deconjugation, clean-up by filtration and centrifugation, and

346

detection/quantification. 80 The chromatographic methods often include additional clean-up

347

by affinity chromatography or solid phase extraction. It is beyond the scope of this paper to

348

go into all analytical details about chromatography-based methods for determination of food

349

folates. Instead, we refer to recent reviews.12,18,30,62,73,81-83

350

To obtain reliable analytical results, the stabilization of unsubstituted folates, especially

351

H4folate, is of great importance. In Table 3 we summarize the most crucial factors in

352

stabilization of unsubstituted folates during analytical procedure. As seen from Table 3, it is

353

possible to efficiently stabilize unsubstituted folates by using following protection strategies:



354

-

copper ions throughout the analytical procedure;

355 356

-

-

361

choice of lower temperature and shorter time for each analytical step everywhere it does not compromise the yield of folates;

359 360

avoidance of repeated freeze-thaw cycles of food samples, food extracts and stock solutions;

357 358

protection from UV light, oxygen and catalysts of free radical reactions such as iron and

-

the use of combination of two antioxidants - ascorbic acid/ascorbate and a thiol (BAL, DTT or TBA)

362

Sampling, storage, and homogenization. To obtain reliable analytical results, good

363

laboratory practice of sampling and storage of food samples is important, by protecting them

364

from UV-light, air and high temperatures. Storage should preferably be carried out at -80 °C.

365

Once the samples are homogenized they should be dissolved in buffers of neutral pH

366

containing two antioxidants, usually 1-2% ascorbic acid/ascorbate and a thiol to protect

367

H4folate from oxidative degradation.58-61Further storage after homogenization should be

368

avoided because repeated freezing and thawing results in degradation of H4folate. 61

369

Extraction and deconjugation. Three major procedures are used to extract folate from the

370

food matrix, heat treatment, enzymatic extraction or a combination of heat- and the

371

enzymatic treatment. Most studies use neutral pH during extraction and other enzyme

372

treatments. Heat treatment is usually done by boiling in water bath (100 ° C for10 min)73,81 or

373

heating (75 °C for 1 h),80,85 when using HPLC methods, or by autoclave (120 °C/15 min),

374

when applying MA. 74-76 The enzymatic extraction is commonly performed at 37 °C for 1-4 h

375

by adding protease and/or amylase to the sample and terminate each enzyme treatment by

376

boiling (5 min).73,81 To combine two antioxidants - ascorbic acid and thiol - is crucial during

377

sample extraction to protect the labile H4folate from oxidative degradation as well as to

378

prevent interconversion of H4folate into 5,10-CH2-H4folate caused by formaldehyde formed


Page 16 of 45

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379

from ascorbic acid when heated.14 Three thiols, BAL, TBA and DTT, have been shown to be

380

the highly efficient for these purposes, whereas the most commonly used MCE appeared to

381

be the least efficient.61 Besides efficiency, the toxicity and environmental risks should also be

382

considered when choosing thiol to stabilize H4folate. From this point of view, the nontoxic

383

TBA is the best choice followed by low-toxic DTT and BAL.22, 61

384

To limit the amount of forms for determination, mono or diglutamates are achieved by

385

enzymatic deconjugation of the glutamate residues. Choice of conjugase (γ-glutamyl

386

hydrolase) source depends on the method for determination used. For HPLC it is necessary to

387

use a conjugase source, usually rat plasma, human plasma or hog kidney conjugase with

388

monoglutamates as the end product, while the bacteria in the MA can use the diglutamates

389

produced by chicken pancreas as well.86 The duration of deconjugation procedure usually

390

varies from 2-3 h to overnight incubation at 37° C, followed by inactivation by 5 min of

391

boiling.81 In addition to the mammal-based conjugase sources hitherto used, de la Garzia’s

392

group recently evaluated two recombinant γ- glutamyl hydrolases from Arabidopsis

393

thaliana.87 These enzymes were far more efficient in hydrolysing folate polyglutamates in

394

plant food samples compared with rat plasma; in fact the deconjugation time could be

395

shortened to 0.5-1h. Moreover, the recombinants also seemed to be less sensitive towards

396

conjugase inhibitors present in certain plant extracts.87 The repeated heating and relatively

397

long-time incubation during deconjugation, (overnight incubation is most common for MA),

398

emphasizes the need to optimize the antioxidative protection of H4folate, as mentioned above

399

for the extraction enzymes. Moreover, blanks need to be included for all enzymes added in

400

the assay to subtract their endogenous folate content.

401

Storage of food extracts, after heating and deconjugation should be avoided. Tamura and co-

402

workers showed that storage at -70 °C for a few months caused considerable degradation of

403

H4folate (~60%) in food extracts.88 Patring and co-workers also observed losses of native



404

H4folate in yeast extract stored at -22 °C for 4 weeks.61 With 2 % sodium ascorbate and

405

0.1 % MCE the losses amounted to 53%, whereas the replacement of MCE with BAL

406

reduced losses to only 19%. De Brouwer and co-workers reported stability of 63.7-77.7% for

407

native H4folate in rice extract stored at +4 °C for 36 h, whereas the stability was above 95%

408

when stored at -20 °C or -80 °C with one freeze/thaw

409

cycle. 22, 31 Daele and co-workers used 1% ascorbic acid and 0.5% DTT for stabilization of

410

folates in standard solutions and potato extracts. They reported stability above 90 % for

411

H4folate solution when stored at +4 °C for 24 h in the autosampler or at -20 °C with three

412

freeze/thaw cycles.89

413 414

Clean-up by SPE or affinity chromatography.

415

Chromatographic methods are more sensitive to interfering substances and have higher

416

demands on sample clean-up than MA. Solid phase extraction (SPE) can be used both to

417

remove interfering substances and to concentrate the analyte. Affinity chromatography clean-

418

up using folate binding protein applied on columns can be used to increase sensitivity.

419

However, different affinity to the various folate forms may easily lead to considerable losses

420

of folate forms with low affinity.18

421

Folates are eluted by acidic buffers and affinity chromatography causes the most pronounced

422

acidification by operating close to pH 2 during the 10 to 20 min the elution takes. SPE-based

423

clean-up procedures using strong-anion exchange (SAX) cartridges elute the folates by

424

sodium acetate solution containing sodium chloride salt (5 % or 10 %) and ascorbic acid

425

(1 %), which keeps the pH close to 5. However, it is still important to use antioxidants in

426

optimal amounts and UV-light protection to prevent oxidation and degradation of reduced

427

unsubstituted folates, especially if the samples are stored in refrigerator or auto sampler

428

overnight until subjected into HPLC. For instance Kirsch and co-workers 13 studied the


Page 18 of 45

Page 19 of 45


429

stability of individual folate standards stored at 4 °C during 24 h in buffers of different pH

430

(2.6, 3.4 and 7.0) containing 0.1 % ascorbic acid. Main interconversion products were

431

presented for each folate form based on UHPLC-MS/MS analysis. H4folate showed minor

432

interconversion (< 5 %) into H2folate and folic acid, whereas H2folate showed degradation at

433

acidic conditions and interconversion (12.7-16.7 %) to folic acid.

434

Chromatographic separation by HPLC and UHPLC

435

No antioxidants are present in the mobile phase during chromatographic separation, which

436

makes folates unprotected against oxidation. Moreover column temperatures might vary

437

between 20 °C and 35 °C in HPLC28,30 and 60 °C in UHPLC.34,89 The extent to which acid-

438

catalyzed oxidations of unsubstituted folates occur at this stage of analysis has received little

439

attention over the years. Strandler and co-workers found a concentration-dependent stability

440

of H4folate under these conditions, pH around 3 and a temperature of 35 °C.90At

441

concentration of 5 µg/ml the losses of H4folate were 20% after 30 min, whereas at lower

442

concentration of 0.5 µg/ml H4folate was degraded completely after 10 min.90 Usually the

443

concentrations of H4folate in injected standard and food extracts are in the range of 1-50

444

ng/ml, i.e. much lower than those studied by Strandler and co-workers (for references, see

445

Table 1). This indicates a considerable risk for losses of H4folate during the passage of the

446

chromatographic column, which can lead to deviations from linearity in the lower region of

447

calibration curve and false negative results for H4folate. Therefore, special consideration

448

should be taken to the on-column stability of H4folate when developing and optimizing new

449

HPLC or UHPLC methods to determine folates in view of the concentration-dependent

450

stability of H4folate. Shorter retention time and lower column temperature are preferable to

451

improve the on-column stability of H4folate.

452

HPLC or UHPLC with different detectors



453

When using HPLC, the choice of calibrants becomes more crucial because only the forms

454

that are used as calibrants can be identified in the sample. Due to interconversion and stability

455

problems, there are limitations to which folate forms can be found in food extracts and this

456

has been reviewed in previous sections of this review. Determination can be made by using

457

UV, fluorescence, MS or electrochemical detection. 12,73,81,82, The reduced folates have native

458

fluorescence of different intensity and wavelength maximum. 91 Folic acid does not

459

fluorescence and is better determined with UV detection. 81 This means that a combination of

460

UV and fluorescence detection at different excitation/emission wavelengths is necessary to

461

quantify all folate forms. When using both detectors connected in sequence (UV detector

462

followed by fluorescence detector), special attention should be paid to the stability of

463

H4folate during the passage of UV detector flow cell because of the high risk for degradation

464

of H4folate due to UV light and increased temperature in the flow cell. The stability check

465

can be performed by comparing the fluorescence response for H4folate with and without UV

466

detector connected before fluorescence detector. If the fluorescence response for H4folate is

467

lower without UV detector, this indicates that there are losses of H4folate in the UV detector

468

flow cell. In such a case it is preferable to use split and connect detectors in parallel.

469

Higher specificity is achieved by using mass spectrometric detectors. Analytes are ionized,

470

giving a specific mass to charge pattern and folates can be determined in both positive and

471

negative mode28. Quantification is usually performed with external calibration in HPLC

472

methods when using UV and fluorescence detection. In methods with mass spectrometric

473

detection the use of stable isotopes as internal standards, such as deuterium-labeled (2H4) 31or

474

13

475

it cannot affect the absolute recovery. 92 Although the relative recovery of H4folate is about

476

100%, the absolute recovery may be much lower, about 37-70% 22,30,33,34, ,89, which may lead

477

to negative false results for H4folate if concentrations in food samples are close to the limit of

C-labeled (13C5) isotopes of folates 22,89 can considerably improve the relative recovery, but


Page 20 of 45

Page 21 of 45


478

quantification. Moreover, the use of internal standards or external standards will not correct

479

for oxidations or conversions into other bioactive folate forms during analysis as recently

480

discussed by Ringling and Rychlik 30; for instance when native 5,10-CH2-H4folate dissociates

481

to H4folate and H4folate is oxidized to H2folate and folic acid. Hence, conversions and

482

oxidations may cause some overestimations of H4folate and folic acid because the changes

483

are not exactly the same in the food matrix as in the standards.

484

It is important to consider that the bioactive unsubstituted folates analyzed in foods, H4folate

485

and folic acid, reflect the sum of oxidative and interconversion changes of H4folate, H2folate

486

and 5,10-CH2H4folate originally present in the intact cells of vegetable, animal or

487

microbiological origin such as yeast and LAB. In addition, folic acid in foods either depends

488

on oxidation of H4folate or H2folate or is a consequence of supplementation/fortification or

489

both. Analysts monitoring folic acid content in fortified foods need to consider that parts of

490

the analyzed folic acid could be due to oxidation of unsubstituted reduced folates or

491

conversion from 5,10-CH2-H4folate to H4folate and its further oxidation. Also worth

492

mentioning is that the relatively stable folic acid in fortified foods might be underestimated

493

due to photodecomposition if not protected from UV-light during sample pretreatment. When

494

it comes to UV-light sensitivity both oxidized and reduced folates are sensitive towards

495

degradation.

496 497 498



499

ABBREVIATIONS USED

500

5-CH3-H4folate

5-methyl-tetrahydrofolate

501

5-HCO-H4folate

5-formyl-tetrahydrofolate

502

10-HCO-H4folate

10-formyl-tetrahydrofolate

503

10-HCO-H2folate

10-formyl-dihydrofolate

504

10-HCO-folic acid

10-formyl-folic acid

505

5,10-CH+=H4folate

5,10-methylene-tetrahydrofolate

506

5,10-CH2-H4folate

5,10-methenyl-tetrahydrofolate

507

AOAC

Association of Analytical Communities

508

BAL

2,3-dimercapto-1-propanol

509

C1

one-carbon

510

CHES

2-(N-cyclohexylamino)ethanesulfonic acid

511

CH2O

formaldehyde

512

DAD

diode array detector

513

DTT

1,4-dithiothreitol

514

ECD

electrochemical detection

515

FBP

folate binding protein

516

FW

fresh weight

517

H2folate

dihydrofolate

518

H4folate

tetrahydrofolate

519

HEPES

N-(2-hydroxyethyl)-piperazine-N-2-ethanesulfonic acid

520

IS

internal standard

521

LAB

lactic acid bacteria

522

MA

microbiological assay

523

MCE

2-mercaptoethanol


Page 22 of 45

Page 23 of 45


524

NTD

neural tube defect

525

pABG

para-aminobenzoylglutamic acid

526

SAX

strong anion exchange

527

SPE

solid phase extraction

528

TBA

2-thiobarbituric acid

529

UHPLC

ultra-high performance liquid chromatography

530 531



532

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533

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534 535 536 537

Medical Research Council Vitamin Study. Lancet 1991, 338, 131-137. 2. Lucock, M. Folic acid: Nutritional biochemistry, molecular biology, and role in disease processes. Mol. Gen.Metab. 2000, 71, 121-138. 3. Clarke, R.; Halsey, J.; Bennett, D.; Lewington, S.Homocysteine and vascular disease:

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4. De-Regil, L.; Fernández-Gaxiola, A.; Dowswell, T.; Peña-Rosas, J. Effects and safety of

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7. Qin, X.; Cui, Y.; Shen, L.; Sun, N.; Zhang, Y.; Li, J.; Xu, X.; Wang, B.; Xu, X.; Huo, Y.;

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8. Wien, T. N.; Pike, E.; Wisløff, T.; Staff, A.; Smeland, S.; Klemp, M. Cancer risk with folic

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9. Gregory, J. F. Chemical and nutritional Aspects of Folate Research: analytical procedures,

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32. Zhang, G.F.; Storozhenko, S.; Van der Straeten, D.; Lambert, W. E., Investigation of the

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33.Wang, C.; Riedl, K. M.; Schwartz, S. J. Fate of folates during vegetable juice processing — deglutamylation and interconversion. Food Res. Int. 2013, 53, 440-448. 34 De Brouwer, V.; Storozhenko, S.; Stove, C. P.; Van Daele, J.; Van der Straeten, D.;

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35. Blakley, R. L. The Biochemistry of folic acid and related pteridines. In Frontiers of

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37. Patring, J.; Lanina, S.A.; Jastrebova, J.A. Applicability of alkyl-bonded ultra-pure silica

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38. Kallen, R. G.; Jencks, W. P. The Dissociation Constants of Tetrahydrofolic Acid. J. Biol. Chem. 1966, 241, 5845-5850. 39. Kallen, R. Tetrahydrofolic acid and formaldehyde. In Methods in Enzymology, Wright, D. M. A. L., Ed. Academic Press: New York, 1971; 66, pp 705-716. 40. Poe, M. Acidic dissociation constants of folic acid, dihydrofolic acid, and methotrexate. J.Biol. Chem. 1977, 25, 3724-8. 41.Gregory, J. F.Vitamins. In Food Chemistry, Damodaran, S.; Parkin, K. L.; Fennema, O. R., Eds. Marcel Dekker:New York, 2008; pp 498-503. 42. O'Broin, J.; Temperley, I.; Brown, J.; Scott, J. Nutritional stability of various naturally occurring monoglutamate derivatives of folic acid. Amer. J. Clin. Nutr. 1975, 28, 438-444. 43. Paine-Wilson, B.; Chen, T. S. Thermal destruction of folacin: effect of pH and buffer ions. J. Food Sci. 1979, 44, 717-722. 44. Mnkeni, A. P.; Beveridge, T.Thermal destruction of pteroylglutamic acid in buffer and model food systems. J. Food Sci. 1982, 47, 2038-2041. 45. Lowry, O. H.; Bessey, O. A.; Crawford, E. J. Photolytic and enzymatic transformations of pteroylglutamic acid. J. Biol Chem. 1949, 180, 389-398. 46. Akhtar, M. J.; Khan, M. A.; Ahmad, I. Photodegradation of folic acid in aqueous solution. J. Pharm. Biomed. Anal. 1999, 19, 269-275. 47. Akhtar, J. M.; Khan, A. M.; Ahmad, I. Effect of riboflavin on the photolysis of folic acid in aqueous solution. J. Pharm Biomed. Anal. 2000, 23, 1039-1044.


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49. Poe, M., Dihydrofolate reductase from a methotrexate-resistant strain of Escherichia coli:

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TABLES AND ARTWORK

Fig 1. Structures of folic acid and the native monoglutamate folate forms. Fig 2. Ionized groups present in unsubstituted folates at different pH ranges and their pKa values. pH ranges where they are predominantly charged, 50 % or more, are displayed by color. pKa-values for each group are displayed where their log C-curve cross the x-axis. For pKa-values displayed in table, see Patring et al (2006). 37 Fig 3. Pathways for interconversion between H4folate and 5,10-CH2-H4folate and degradation of unsubstituted folates exposed to air at ambient temperature.15, 55,56 Degradation of folic acid in the presence of light or UV-irradiation. 44-46, 48 Fig. 4. Schematic overview of analytical methods for folate determination in foods modified from 12,18,78,81


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Table 1. Distribution of the three main food folate pools: 5-CH3-H4folate, formyl folatea and unsubstituted folateb in some non-fortified foods expressed in percentage (%) of total folate calculated as folic acid equivalents per100g fresh weight (FW). All data are based on deconjugated folates determined by LC-MS or LC-MS/MS. 5-CH3-

Formyl

Unsubstituted folateb

H4folate

folatea

H4-folate

%

%

Baker’s yeast, dry

61.0

Pig liver (CRM 487)c

Σ of unsubsti-

Folic acid

folic acid

tuted folateb

equivalents

%

%

%

µg/100g FW

18.7

20.3

nd

20.3

3 520 ± 6025

33.4

20.2

27.4

19.1

46.5

1 443 ± 1329

Wheat germs

< 10

81

Challenges in the Determination of Unsubstituted ... - ACS Publications

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