ReviewThe analysis of folate and its metabolic precursors in biological samples
Introduction
Lucy Wills first discovered in 1931 [1] a component of yeast capable of curing “pernicious anaemia of pregnancy.” However, it was another 15 years [2] before the structure of folate was elucidated (Fig. 1). This delay in identifying the structure of the “Wills factor” was due in part to the diversity of folate species resulting from differential substitution of one-carbon units on nitrogens 5 and 10, the length of the glutamyl side chain, and/or the oxidation state of the pterin ring. The diverse biological and chemical characteristics of the various folate forms still complicate the task of assaying endogenous folate. The traditional approach for analyzing folates has been to simplify the analysis by determining either the one-carbon species (after hydrolysis of the poly-γ-glutamyl chain) or the γ-glutamate chain length (normally after removal of the pterin moiety). Methods are now appearing whereby both the one-carbon substituents and the poly-γ-glutamate chain length can be determined simultaneously. We review most of the methods commonly used to assay folate and its precursors, and we discuss the strengths and weaknesses of the various analytical approaches.
It should be noted that the folate glutamate peptide is somewhat unusual in that it is a gamma peptide and not α-linked as with proteins. This means that a distinct set of enzymes is used for folate polyglutamate synthesis and hydrolysis.
The International Union of Pure and Applied Chemistry–International Union of Biochemistry (IUPAC–IUB)1 Commission on Biochemical Nomenclature name [3] for the “factor” discovered by Wills is pteroylglutamic acid. However, the trivial names “folic acid” and “folate” are more commonly used. The name folic acid was coined in 1941 by Mitchell and coworkers [4] after the 4 tons of spinach leaves from which it was purified (Latin folium–leaf). The naming convention [1] for the vitamin (Fig. 1) isUsing this convention, the term folate has in the past been used to describe folic acid/pteroylglutamic acid. Recently, however, use of the name folic acid has increased when referring to the unsubstituted fully reduced vitamin, that is, pteroylglutamic acid. However, the term folic acid is now becoming synonymous with the vitamin, in the public’s mind, due to health promotional campaigns.
A further complication arises when discussing folate glutamate chain length. By definition, a folate molecule contains one glutamate residue. However, the convention has arisen, when discussing glutamate chain length, to count all of the glutamate residues (e.g., folate [pteroylmonoglutamate] is often referred to as folate monoglutamate). This confusion does not arise when using the IUPAC nomenclature.
Although reduced folates are synthesized by plants and most microorganisms [5], [6], [7], folic acid does not occur in nature to any significant extent. Its occurrence is dependent on the chemical oxidation of reduced folates or on commercial synthesis for use in supplements and in food fortification. Similarly, although substituent dihydro forms of folate (e.g., 10-formyldihydrofolate [10-CHO-DHF]) have been detected, they are thought to result from oxidation of the corresponding tetrahydro species during sample preparation and oxidation during food storage or handling.
Folates, as various forms of tetrahydrofolate (THF), are substrates and coenzymes in the acquisition, transport, and enzymatic processing of one-carbon units for amino acid and nucleic acid metabolism and metabolic regulation [8], [9]. Folates also donate one-carbon units for the methylation of homocysteine to methionine, which is used in cellular methylation reactions (as S-adenosylmethionine) and protein synthesis. Folates support nucleic acid synthesis through their roles as carbon donors in the synthesis of purines and thymidylate and through deoxycytosine methylation. They are also involved in interconversion of glycine and serine and breakdown of histidine to form glutamate.
There is strong evidence for a relationship among inadequate folate status, elevated homocysteine concentration, and risk of coronary heart disease, venous thrombosis, carotid artery stenosis, and other forms of vascular disease [9], [10], [11]. Mothers with inadequate folate status are at increased risk for having children with neural tube defects or other forms of birth defects [12]. The risk of certain forms of cancer (e.g., colon, cervical, and breast) also increases when folate intake is inadequate [13], [14]. Folate deficiency can also contribute to depression [15], [16], impaired immune response [15], and neural and neurological damage [15], [17], [18].
The folate synthesis pathways (Fig. 2) in plants and bacteria are similar [5], [6], [7] except that the process is split among three subcellular compartments in plants. The folate biosynthetic pathway is absent in mammals and most other animals [19], [20].
The pterin branch involves conversion of guanosine triphosphate to dihydroneopterin (DHN) triphosphate [21], followed by a two-step dephosphorylation to give DHN [22] and then by aldol cleavage of the trihydroxypropyl side chain to yield 6-hydroxymethyldihydropterin (HMDHP). The DHN aldolase that mediates this cleavage also catalyzes epimerization at the second carbon of the side chain, producing dihydromonapterin (DHM), which also can undergo the cleavage reaction [23], [24]. HMDHP subsequently is pyrophosphorylated [19], [25], [26] before its condensation with p-aminobenzoate (pABA).
Synthesis of pABA from chorismic acid is a two-step process. First, the hydroxyl group of chorismate is replaced with an amino group, yielding 4-amino-4-deoxychorismate (ADC). In bacteria, this reaction is catalyzed by two enzymes [27]; PabA acts as a glutamine amido-transferase, supplying an amino group to PabB, which carries out the amination reaction. In plants, both of these reactions are catalyzed by a single fused enzyme [28]. The final step of pABA synthesis in bacteria [29] and plants [30] entails elimination of pyruvate and aromatization of the ADC ring to give pABA catalyzed by ADC lyase (PabC).
In plants [31], [32], the majority of the pABA is reversibly glucosylated to p-aminobenzoyl β-d-glucopyranoside (pABA-Glc). The role of pABA-Glc is unclear. One possibility is that the glucose moiety is added to prevent the free diffusion of pABA and, thus, may be involved in regulating pABA storage or transportation in plants.
Dihydropteroate is produced by condensation of pABA with the pyrophosphorylated HMDHP [33] in a reaction catalyzed by 7,8-dihydropteroate synthase.
The final two steps of folate synthesis, phosphorylation, and glutamylation, are catalyzed by a single enzyme [26], [34], [35], [36]: dihydrofolate synthase. The phosphorylated dihydropteroate remains enzyme bound [26], [34] prior to glutamate addition and phosphate release. The product of this reaction [36] is dihydrofolate (DHF), which is reduced to THF before entering the one-carbon cycle.
Polyglutamyl folates were once thought to be storage forms of the vitamin [37]; however, polyglutamylation is now known to be a means of maintaining a folate gradient across cell membranes and perhaps of regulating enzyme activity.
Elongation of the glutamate chain is the principal means by which cellular folate accumulation and compartmentalization occurs. Polyglutamylation prevents folate egress across cell membranes by increasing protein binding affinity [37] and by providing α-carboxyl charges given that the γ-carboxyl groups are involved in formation polyglutamate side chain. Chain elongation also decreases affinity for the membrane transporter. The apparent minimum poly-γ-glutamate length required for cellular retention of folate is triglutamate [38], [39]. As the additional (n > 1) glutamates are recycled in mammals [40], [41], plants [42], [43], [44], and possibly bacteria, the irretrievable metabolic cost of adding two glutamates to the monoglutamate of folate is two ATP equivalents [45], [46], [47]. In a similar manner, synthesis of the pentaglutamate, the average cellular length [48], [49], [50], costs four ATP equivalents. Thus, 100-fold concentration gradients can be maintained at a metabolic cost of just two to four ATP equivalents per folate molecule.
Polyglutamated folates (3 ⩽ n ⩽ 6) are the preferred coenzymes for most of the one-carbon cycle enzymes, having a 2- to 70-fold lower Km than the corresponding pteroylmonoglutamates [39], [51], [52]. This is especially important in the case of methionine synthase. Folate in the serum is almost exclusively 5-methyltetrahydrofolate (5-CH3-THF), which is a poor substrate for folate poly-γ-glutamate synthase [53]. For 5-CH3-THF to be retained by the cell, it must be demethylated by methionine synthase before polyglutamylation can occur. However, at physiological concentrations [54], 5-CH3-THF poly-γ-glutamates within the cell inhibit 5-CH3-THF use by methionine synthase, preventing its demethylation and polyglutamylation. Thus, the ratio of 5-CH3-THF to 5-CH3-THF-polyglutamate may play a role in regulating folate retention by the cell.
Folate poly-γ-glutamate synthesis also has been implicated in substrate channeling within multifunctional complexes [52].
Section snippets
Antioxidants
It is almost always necessary to include antioxidants in the extraction buffer regardless of the extraction and deconjugation methods employed. These antioxidants not only help to protect against folate loss, primarily involving oxidative cleavage of the C9–N10 bond, but also help to preserve the level of reduction of the pterin ring. Ascorbate (1–5%, w/v) is widely used, often in conjunction with mercaptoethanol [55], [56] or dithiothreitol [56]. However, as ascorbate absorbs light in the
Microbiological assays
(Note. There have been a number of recent changes made to the nomenclature of the bacteria used in the folate assay. Lactobacillus rhamnosus (ATCC 7469) was formerly known as Lactobacillus casei; Enterococcus hirae (ATCC 8043) was formerly known as Streptococcus faecium, Streptococcus faecalis, or Streptococcus lactis R; and Pediococcus acidilactici (ATCC 8081) was formerly known as Pediococcus cerevisiae, Leuconostoc citrovorum, or Streptococcus citrovorus.)
Liquid chromatographic analysis of folate monoglutamates
The self-packed low-pressure columns originally used for folate chromatography yielded reasonably good separation but were limited by slow speed and poor reproducibility. Although the advent of prepacked HPLC columns improved peak shape and resolution, these early HPLC columns often suffered from poor intercolumn reproducibility as a result of interbatch variability in the packing material. This led to situations whereby methods developed on one column would not work on columns from a different
Analysis of folate poly-γ-glutamates
Note.Folate pteroylpoly-γ-glutamylcarboxypeptidase activity is apparently ubiquitous in mammals [87], [88], [89], [91], birds [88], [90], and plants [43]. Consequently, when performing folate poly-γ-glutamate analyses, the sample should be prepared as quickly as possible, taking care to minimize poly-γ-glutamate hydrolysis.
Extraction
Pterins from plant material (Fig. 5) [81], [205] can be extracted by triturating with a mortar and pestle under liquid nitrogen. The fine powder is resuspended in sample buffer [205] (authors’ unpublished data) or in methanol [81]. The methanol suspension can be lyophilized (authors’ unpublished data) or extracted in methanol–chloroform–water (MCW) to remove lipids and proteins from the sample [81]. In brief, to 600 μl of the methanolic sample (0.1 g tissue/1 ml methanol) are added 250 μl
Acknowledgments
This work was supported by grants DK56274 and GM071382 from the National Institutes of Health and grant MCB-0443709 from the National Science Foundation. This manuscript is Florida Agricultural Experiment Station Journal Series No. R-11009.
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