Folate in Health and Disease: Determination of Folate Vitamers in Serum, Whole Blood, and Cerebrospinal Fluid

Figure 1. Folate interconversions and degradation. Stability of aqueous folic acid and folate solutions at different pH and temperature. Shown are the degradation and interconversion reactions of the folate coenzymes. Folate is the least stable of the B vitamins; careful sample handing and use of antioxidants are required to maintain sample integrity. Most importantly, a relatively small number of foods are concentrated sources of naturally occurring folate (i.e., food folate); therefore, sufficient care must be exercised while cooking. Because substantial losses of this vitamin can occur during the cooking process as a result of the combination of thermal degradation and leaching into the cooking water. [FA, folic acid; DHF, dihydrofolate; THF, tetrahydrofolate] Adapted with modifications from de Brouwer et al., 2007 [1].

Folates are susceptible to interconversions and oxidative degradation, and their oxidation and breakdown products can also be found in serum. Some of these products no longer exhibit folate activity, nevertheless one needs to ensure that they do not interfere with the measurement of folate forms (vitamers). This means that understanding folate interconversions and degradations helps to better understand what different methods measure [1] (see Figure 1).

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 whole blood (WB) reflects the long-term folate status in the body. The assay of cerebrospinal fluid (CSF) folate may be essential tool in diagnosing the cerebral folate deficiency (CFD) (cf. previous blog entitled as: ‘Folate in Health and Disease: Folate Concentrations in Serum, Whole Blood, and Cerebrospinal Fluid.’).

The quantification of folate forms from various sample materials 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:

1. large number of folates (i.e., according to oxidation states and number of glutamate residues),
2. the complex sample matrix,
3. the instability and interconversions of folates, and
4. the presence of folate binding proteins (FBPs) in the samples.

Folates are easier to determine in serum and where they are present as monoglutamates. In WB, folylpolyglutamates must be deconjugated to their monoglutamate forms before they can be measured. The release of folates in WB should be performed under strict temperature, pH, and time conditions, otherwise, hemolysis and deconjugation can be incomplete, or folates can be trapped in the hemoglobin molecules.

Stability of folate forms in vitro

Reduced folate forms are known to be sensitive to the following [2], for instance:

• pH,
• heat,
• oxidation,
• pressure, and
• ultraviolet light (UV).

Therefore, samples preparation and measurements must be performed under controlled conditions. The major degradation products are (a) p-aminobenzoyl-L-glutamic acid (pABG) and (b) pteridine fragments that have no coenzyme function.

Tetrahydrofolate (THF), dihydrofolate (DHF), 10-formyl-THF readily undergo oxidative degradation in vitro. Moreover, reduced folates undergo pH-dependent enzymatic and non-enzymatic interconversions. The pH and heat stability of individual folates forms in in vitro experiments are summarized in Figure 1.

Folic acid (FA) and 5-methyl-THF are stable at pH 2–10 and 5-formyl-THF 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 (see Figure 1).

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 (DTT). In addition, artificial FA might be generated from the oxidation of THF during sample preparation, even in the presence of antioxidants. This finding might lead to misinterpretation of studies looking at unmetabolized FA in the samples.

Quantification methods

1. Microbiological assay (gold standard)

The microbiological assay (MBA) was introduced over 50 years ago. This assay is based on the fact that a specific microorganism, for example, Lactobacillus rhamnosus, formerly known as Lactobacillus casei,  grows proportionally to the available folate in the sample [3].
The MBA has received renewed interest during the past decade because of improvements in efficiency and robustness, so that the assay can be reliably used in a high-throughput routine setting such as national health and nutrition examination survey (NHANES) [4]. Important improvements introduced in the 1970s and 1980s included, for example:

1. the development of a chloramphenicol-resistant strain of L. rhamnosus,
2. the ability to cryopreserve the inoculum, and
3. the introduction of automated microtiter plate technology.

2. Protein-binding assays

Protein-binding assays (PBAs) were developed with the clinical laboratory in mind, to enable the diagnosis of folate deficiency. These assays use the highly specific folate binding protein (FBP) (mainly from milk or milk fractions, sometimes from porcine plasma or kidney) to ‘extract’ folate from the sample [5].

The protein-binding assays represent the next assay generation. One example is the automated chemiluminescent immunoassay on the ADVIA Centaur platform (ADVIA Centaur XP System, Siemens Healthcare Diagnostics GMbH, Eschborn, Germany). This method consists of a competitive assay using FBP and depends on the release of the folate from its binding proteins. Before folate can be measured, samples are treated with DTT to release the folate from endogenous binding proteins. The released folate competes against acridinium ester-labeled FA for a limited amount of biotin-labeled FBP. The biotin-labeled FBP binds to avidin, which is covalently bound to paramagnetic particles in the solid phase. The amount of sample folate correlates inversely with the obtained signal.

3. Chromatography-based assays

Methods used for quantifying total folate show large disagreements and are unable to detect various forms of the vitamins. In the 1970s and 1980s, first chromatographic methods have been developed. These methods can distinguish between the different folate forms. Numerous high performance liquid chromatography (HPLC) and gas chromatography (GC) methods have been described for the detection of folate forms in serum and WB (see Figure 2). Recently, liquid chromatography coupled with mass spectrometry (LC-MS) methods and liquid chromatography with tandem mass spectrometry (LC-MS/MS) methods have been developed to quantify folate monoglutamates in biological fluids and foods. LC-MS/MS methods provide higher levels of selectivity (see Figure 2).

Figure 2. Chromatography-based assays. Chromatography based methods provide information on individual folate forms and are based on either measurement of intact folates via high performance liquid chromatography (HPLC) or measurement of folate breakdown products as an indicator for total folate via gas chromatography (GC). Over the past decade, tandem mass spectrometers (MS/MS) have become more affordable and user-friendly and smaller in footprint, and their performance in terms of sensitivity and linear dynamic range has greatly improved. They are now considered to be the preferred detectors for HPLC-based methods, i.e., liquid chromatography with tandem mass spectrometry (LC-MS/MS), at least for specialized laboratories.

Modern LC-MS/MS methods depend on sample extraction and clean-up and are high sensitive in the nanomolar range. Monoglutamate or polyglutamate folate standards and stable isotope-labeled internal standards for most of the monoglutamate forms are available. However, at present it is still not possible to detect all available folate forms with one method.
Recently an ultra-performance LC-MS/MS method for the fast, sensitive, and reliable determination of key folate forms (5-methyl-THF, 5-formyl-THF, 5,10-methenyl-THF, THF, and FA) in serum and of 5-methyl-THF and non-methyl-THF (sum of formyl-THF, 5,10-methenyl-THF, 5,10-methylene-THF, THF, DHF,  and FA) has been described.

For example, the methods in serum shows strong correlation with the immunological method on the ADVIA Centaur platform (R = 0.939, p < 0.001). Despite the good correlation, the results for the quantification of the folates may vary greatly in individuals due to the non-specific detection of the folate forms via the immunological methods. The ultra-performance LC-MS/MS methods are preferred over the immunological or microbiological methods when accurate information about the folate forms distribution is needed [6].