21.2: Biomarkers of folate status (Chapter 22a.2)

In 2015, an expert international panel produced a comprehensive review of folate biology and bio­markers as part of the Biomarkers of Nutrition for Development (BOND) project convened by the U.S. National Institutes of Health (Bailey et al., 2015). This review identified serum folate, red blood cell (RBC) folate and plasma homo­cys­teine as the “Priority Folate Biomarkers” for assessing folate status. Serum folate reflects recent dietary intake. RBC folate, compared with serum folate, is a better indicator of folate intake and status over the previous 3–4mos. Plasma homo­cys­teine provides a functional indicator of folate status and is elevated in folate defi­ciency, on the basis that normal homo­cys­teine meta­bolism requires an adequate supply of folate. Concentrations of folate in erythro­cytes, but not serum, also fall in vitamin B₁₂ defi­ciency. Ideally, both serum and RBC folate concen­trations should be measured.

Cut-off values for assessment of folate status. The sequential stages in the development of folate defi­ciency were originally estab­lished through detailed depletion / repletion experiments conducted during the late 1960's, providing a basis for setting cut-off values for serum and RBC folate for the assess­ment of status (Herbert, 1987); these sequential stages of folate defi­ciency and related cut-off values are summarized in Box 22a.1

Box 22a.1. Cut-off values for assessment of folate status in populations

Historical pers­pec­tive

During the late 1960’s, cut-off values for sequential stages of folate deficiency were estab­lished through depletion/repletion experiments (Herbert, 1987):

• A serum folate value < 7nmol/L (3ng/mL) indicated negative folate balance
• For RBC folate, cut-off values were set at:
  • < 363nmol/L (160ng/mL) to indicate the onset of folate depletion,
  • < 272nmol/L (120ng/mL) to mark the beginning of folate-deficient erythropoiesis, and
  • < 227nmol/L (100ng/mL) to indicate folate-deficient anemia

More recently
Folate deficiency was defined more simply, using a single cut-off value for each biomarker, based on a metabolic indicator (increased plasma homocysteine) (Selhub et al., 2008):

• Serum folate < 10nmol/L to indicate folate deficiency
• RBC folate < 340nmol/L to indicate folate deficiency

These cutoff values have been recommended by the 2005 WHO Technical Consultation on folate and vitamin&bsp; B₁₂deficiencies for the assessment of folate status of populations (de Benoist, 2008). Adapted from: Bailey et al., 2015;Herbert, 1987; Selhub et al., 2008; de Benoist, 2008.

The initial stage of folate defi­ciency, termed negative folate balance, is consistent with serum folate values of < 7nmol/L (Herbert, 1987). If the negative balance persists, tissue folate becomes depleted, as indicated by RBC folate falling below the normal range to < 363nmol/L. At this second stage, there is little evidence that biochemical function is impaired, although plasma homo­cys­teine concen­trations may be slightly elevated. By the third stage, termed folate-deficient erythropoiesis, functional impairment is usually evident, with erythrocyte folate values < 272nmol/L. Tissue folate stores are severely depleted in the fourth and final stage where the classical hematological changes occur, manifested as folate-defi­ciency anemia. These include macro-ovalocytic erythro­cytes in the circulating blood and megaloblasts in the bone marrow. At this stage, hypersegmented neutro­phils in the peripheral blood smear and abnormal RBC indices are also apparent; mean red cell volume and mean cell hemo­globin are elevated and hemo­globin concen­tration is low.
More recently, revised cut-off points for folate defi­ciency (serum folate <10nmol/L and RBC folate < 340nmol/L) have been defined based on the meta­bolic indicator, plasma homo­cys­teine (Selhub et al., 2008), as summarized in Box 22a.1.

22a.2.1 Serum and RBC folate concen­trations

Serum folate represents the sum of several folate forms circulating in blood. The main circulating folate is 5‑methyl‑THF; other reduced forms such as THF and formyl‑THF may also be present, but in very small concen­trations (Bailey et al., 2015). As will be discussed later (section 22a.2.4), unmeta­bolized folic acid can also be present in varying concen­trations in plasma (Bailey et al., 2010).

Red cells contain much higher folate concen­trations than serum. 5‑methyl‑THF poly­glutamates are the main RBC folate forms. The measure­ment of RBC folate is more complex than that of serum folate, because of the need to convert poly­glutamates to mono­glutamates prior to analysis. In individuals homo­zygous for the MTHFR C677>T poly­morphism, a portion of the 5‑methyl‑THF poly­glutamates is replaced by formyl-folates (Bagley & Selhub, 1998).

Serum folate is the earliest indicator of altered folate exposure and reflects recent dietary intake (Pfeiffer et al 2010). In the individual, serum folate concen­trations can increase markedly in response to dietary folate, reaching a peak concen­tration 90min after ingestion.

RBC folate is a sensitive indicator of long-term folate status. RBC folate parallels liver concen­trations, accounting for about 50% of total body folate, and thus reflects tissue folate stores (Wu et al., 1975). RBC folate, compared with serum folate, responds more slowly to changes in dietary folate intake and is a better indicator of folate intake over the previous 3–4mos when circulating folate is incorporated into the maturating red cells during erythropoiesis, thereby reflecting folate status during the preceding 120d, the half‑life of the RBC (Mason, 2003).

Factors affecting serum and RBC folate concen­trations

Dietary folate intakes. Blood folate levels are primarily affected by dietary folate intakes. Serum and RBC folate are highly responsive to intervention with folic acid (Duffy et al., 2014), with natural food folates typically resulting in poorer folate responses compared to folic acid at similar intervention levels (Cuskelly et al., 1996). Like­wise, population data show that both serum and RBC folate concen­trations are highly reflective of exposure to folic acid, with the highest concen­trations observed in people who consume folic acid in both supplements and fortified foods (Yang et al., 2010; Hopkins et al., 2015).

Vitamin B₁₂ status. Because vitamin B₁₂ is required for normal folate recycling and folate retention within cells, vitamin B₁₂ defi­ciency leads to a failure to retain folate within cells (Hoffbrand & Weir, 2001). Consequently, RBC folate concen­trations fall, despite the presence of normal (or sometimes even elevated) serum folate. Thus, low RBC folate may reflect vitamin B₁₂ defi­ciency as well as folate defi­ciency.

Fasting versus non fasting samples. Samples from fasted individuals, on average, have lower concen­trations of serum (by 10%) and RBC folate (by 5%) compared with samples from nonfasted (< 3h) participants, but the small differences generally indicate that fasting is not essential when assessing the folate status of populations. (Bailey et al., 2015).

Important preanalytical factors. Detailed information on preanalytical factors affecting serum and RBC folate is described in a comprehensive review article on analytical approaches by Pfeiffer et al., 2010. Folate is the least stable of the B vitamins and is susceptible to oxidative degradation during preanalytical sample handling and storage. Careful sample handling and use of anti­oxidants are therefore required to maintain sample integrity. Blood for serum folate analysis should be processed and frozen promptly. Although serum is generally preferred over plasma in most labora­tories, both matrices generally produce comparable results for folate, as long as the sample processing is not delayed. If delays are unavoidable, the sample should be protected from light, kept cool and processed within a few days of collection. Folate in serum and hemolysates (but not in whole blood) can withstand a few short freeze / thawing cycles, particularly if the vials are kept closed as much as possible to minimize the exposure of the sample to air. Folate in serum / plasma degrades rapidly at room temperature, particularly in the presence of EDTA.

Analytical methodologies for measure­ment of serum and RBC folate

Over the past 50y, analytical methods to assess serum and RBC folate concen­trations have been continuously improved; however, they have not yet reached the point where they produce sufficiently comparable results across methods or labora­tories. The within-person variability for serum folate is about twice that for RBC folate (CV of 21.5% and 9.1%, respectively, Bailey et al., 2015).

Critical considerations in folate assess­ment. Assessing folate status is complicated by the large number of folate forms that may be readily interconverted. To overcome this problem, micro­bio­logical assays have been used for many decades due to the ability of some bacteria to grow in the presence of many different forms of folate, i.e. L.rhamnosus responds to all active mono­glutamate forms (see below). Subsequently, assays using competitive protein binding became common because of their simplicity. In more recent years, the use of LC‑MS/MS has emerged. Of note, the emergence of LC‑MS/MS enables investigation of relevant research findings related to folate meta­bolism. These include alterations in the relative proportion of different folate forms in red cells owing to the common MTHFR C677>T poly­morphism, along with an overall lower RBC folate (Molloy et al., 1997; Bagley & Selhub, 1998; Shane et al., 2011), and the presence of free (“unmeta­bolized”) folic acid in the blood as a result of high intakes of folic acid (Pfeiffer et al, 2010).

There are three main method types for measure­ment of serum and RBC folate, each with advantages and disad­van­tages (Bailey et al., 2015).

Microbiolog­ical assay. This is widely considered to be the gold standard assay for serum and RBC total folate because it measures all biolog­ically active forms equally and does not measure folate species that lack vitamin activity. The underlying principle of the micro­biolog­ical assay is that a folate-dependent micro­organism, namely Lactobacillus rhamnosus (formerly called Lactobacillus casei), grows proportionally to the amount of folate present in serum or whole blood and the folate concen­tration can be quantified by measuring the turbidity of the inoculated medium after a 2d incubation. A chloramphenicol-resistant strain of L.rhamnosus is used and the assay is performed using automated micro­titer plate technology.

The key advantages of the micro­biolog­ical assay include its excellent sensitivity, low cost and simple instrumentation (thus suited for low-resource settings), and the fact that it can be used with dried blood spot samples. The high sensitivity is a particular advantage when limited volume is available, such as for samples collected from a fingerstick or dried blood spot. The disad­van­tages are that the micro­biolog­ical assay is relatively laborious unless automated liquid handling is introduced, lengthy assay time with limited throughput and limited linear range (thus requiring dilution of samples). It is also prone to contamination issues and potential inter­ference by the presence of antibiotics or anti­folates. The latter limitation is well recognized, but in practice may not be such an issue, in that analysts in the US reported that < 1% of samples from the population-based NHANES cohort exhibited a pattern of inter­ference due to the presence of anti­biotics or anti­folates (Pfeiffer et al, 2010).

Protein-binding assays. These assays were developed primarily for clinical settings, to enable the diagnosis of folate defi­ciency. Protein-binding assays use the highly specific folate-binding protein to extract folate from the sample. The strengths of this approach include the high sample throughput, quick turn­around time, avail­ability in commercial kit form and minimum operator involvement. The disad­van­tages are that the various folate forms have different affinities to folate-binding protein, the ques­tion­able accuracy when mixtures of folate are present, the limited linear range (thus requiring dilution of samples), matrix effects when sample is diluted, and lot-to-lot variability of commercial kits. Also, although not sensitive to anti­biotics, protein-binding assays are influenced by certain anti­folates such as metho­trexate.

Chromatography-based assays (HPLC-FD, LC‑MS/MS). Chromatography-based methods typically provide information on individual folate forms based on measure­ment of intact folates via HPLC. More recently, LC-tandem mass spec­trometry (LC‑MS/MS) is now the preferred detection for HPLC-based methods in specialized labora­tories. Advantages of chromatography-based approaches are that they measure all folate forms, are highly selective and specific, have good sensitivity and precision, enable in-house control of perform­ance and use of stable-isotope-labeled internal standards to compensate for procedural losses. Ensuring accurate calibration is a big task for chrom­atography-based methods, due to the high number of folate forms and also because of their instability. Disadvan­tages include the high costs and require­ment for expensive instru­men­tation, experienced operator and frequent technical service, along with being a relatively laborious approach unless automated liquid handling is introduced, and the requirement for complex sample extraction / cleanup. Also inter­conversions of folate forms during the assay procedure need to be considered in inter­pretation of data.

Cut-off values and Interpretation of Serum and RBC folate

The measure­ment of total folate provides information on the folate status of the individual, either in the short-term through serum folate, or in the long-term through RBC folate. An historical pers­pec­tive on the use of folate cutoffs is provided in Box 22a.1. However, the inconsistent use of cutoff values over time to assess the proportion of populations with deficient or low folate status led to a certain degree of confusion. More recently, revised cut-off points for folate defi­ciency (serum folate < 10nmol/L and RBC folate < 340nmol/L) have been defined based on the meta­bolic indicator, plasma homo­cys­teine (Selhub et al., 2008), as summarized in Box 22a.1. These cutoff values have been recommended by the WHO for assessing folate status of populations (de Benoist, 2008). Of note, the values were derived from data generated using the micro­biolog­ical assay.

As discussed earlier, the risk of NTD is associated with a maternal folate status that would not conventionally be classed as deficient. Although there is no cutoff value estab­lished by international organizations for folate concen­trations to define NTD risk in populations, it is accepted that the number of NTDs that can be prevented in a population is dependent on maternal folate status. Specifically, RBC folate concen­tration in the mother has been shown to be a sensitive bio­marker of NTD risk. In the one and only prospective study that has been conducted to date, Daly et al. (1995) found that the prevalence of NTD in an Irish population was lowest when maternal RBC folate concen­trations were ≥ 906nmol/L (400ng/mL). Subsequently, data modeled from folic acid intervention studies in China by Crider et al. (2014) confirmed the dose response relationship between RBC folate concentrations and NTD risk as shown in the Irish study (Daly et al., 1995).

22a.2.2 Plasma homo­cys­teine concen­trations

Plasma homo­cys­teine provides a sensitive functional indicator of folate status. On the basis that normal homo­cys­teine meta­bolism requires an adequate supply of folate, plasma homo­cys­teine becomes elevated when folate status is low.

Homo­cys­teine is a four-carbon, thiol-containing amino acid found in human plasma, mostly present in the form of various disulfides, such as homo­cys­teine-cysteine disulfide. About 75% of total homo­cys­teine is bound to protein (mainly albumin), whereas the remainder occurs in nonprotein-bound “free” forms. Total homo­cys­teine is defined as the sum of all homo­cys­teine species in serum or plasma, including free and protein-bound forms. Only a very small portion (1–2%) of plasma homo­cys­teine is present as the thiol, however the relative contribution of the thiol to total homo­cys­teine increases to 10–25% in patients with abnormally elevated plasma homo­cys­teine (Bailey et al., 2015).

Homo­cys­teine is derived from the essential amino acid, methionine. It is meta­bolized in two ways: remethyl­ation to methionine (by methionine synthase) or trans­sulfur­ation to cysta­thionine (by cystathionine beta-synthase; CBS) then to cysteine (Figure 22a.2). These pathways require adequate status of folate and the meta­bolically related B‑vitamins. The remethyl­ation pathway, whereby methionine is synthesized from homo­cys­teine by methionine synthase, is dependent on both folate and vitamin B₁₂ as cofactors, whilst the trans­sulfuration pathway that converts homo­cys­teine to cysteine is catalyzed by two vitamin B₆ dependent enzymes. A fourth B vitamin, ribo­flavin, is required in its cofactor form flavin adenine dinucleo­tide (FAD) for the activity of MTHFR, the enzyme that catalyzes the reduction of 5,10 methyl­eneTHF to 5 methylTHF. Once formed, 5 methylTHF is used by methionine synthase for the vitamin B₁₂-dependent conversion of homo­cys­teine to methionine and the formation of THF. Thus, the concen­tration of homo­cys­teine in plasma / serum is regulated by up to four B‑vitamins: folate, vitamin B₁₂, vitamin B₆ and ribo­flavin.

Apart from providing a functional bio­marker of folate, higher plasma homo­cys­teine is associated with a number of chronic diseases of ageing, including an increased risk of cardio­vascular disease (CVD; Graham et al., 1997), cognitive impairment and dementia (Smith & Refsum, 2016). It remains to be estab­lished, however, whether plasma homo­cys­teine per se a risk factor for these or other diseases. In the case of CVD, this issue is particularly contro­versial. Despite strong and consistent evidence from observational studies over many years, several secondary prevention trials published between 2004 and 2014 failed to demon­strate a benefit of homo­cys­teine-lowering therapy against the recurrence of CVD events in patients with existing disease. The evidence is however stronger for the relation­ship of homo­cys­teine with stroke than heart disease, with good evidence from both population data and randomized trials that folic acid intervention and/or homo­cys­teine-lowering can signif­icantly reduce the risk of stroke, and particularly so in people with no previous history of stroke (McNulty et al., 2017). Of note, although the literature in this area focuses on homo­cys­teine as the CVD risk factor, it is possible that folate and related B‑vitamins have roles in CVD that are independent of their homo­cys­teine-lowering effects. Thus, plasma homo­cys­teine may be linked with CVD as a functional marker of low B‑vitamin status which reliably reflects perturbed one-carbon meta­bolism, rather than being causatively related to CVD per se (McNulty et al., 2017).

Factors affecting homo­cys­teine concen­trations

Folate status. On the basis that normal homo­cys­teine meta­bolism requires an adequate supply of folate, plasma homo­cys­teine is first and foremost affected by folate status. Thus, plasma homo­cys­teine was shown to respond within 3–4wks of folate depletion (increase) and subsequent repletion (decrease) in a controlled meta­bolic study in healthy women (Jacob et al., 1998). Like­wise, in observational studies, plasma homo­cys­teine is invariably found to be inversely associated with folate status, whether measured as serum or RBC folate.

Intervention with folic acid. Plasma homo­cys­teine is highly responsive to intervention with folic acid (the synthetic vitamin form), alone or in combination with vitamin B₁₂, vitamin B₆, ribo­flavin and betaine (or choline). Thus, food fortif­ication with folic acid has marked effects on homo­cys­teine concen­trations. Using population-based data from the US, Pfeiffer et al. (2008) reported a 10% decrease in plasma homo­cys­teine when comparing values pre-fortif­ication (1991–1994) to post-fortif­ication (1999–2004) of food with folic acid, as implemented on a mandatory basis in 1996–1998. Folic acid-fortif­ication on a voluntary basis (i.e. added to food at the manufacturer's discretion) also affects plasma homo­cys­teine. In a convenience sample of nearly 500 healthy adults in Northern Ireland aged 18–92y, who were not taking folic acid supplements, homo­cys­teine concen­trations were lower by 2µmol/L in high consumers compared to non-consumers of fortified foods (providing > 100µg/d and 0µg/d folic acid, respectively) (Hoey et al., 2007).

Folate-related B vitamins. Because normal homo­cys­teine meta­bolism is dependent on four B vitamins, homo­cys­teine concen­trations will be elevated with other B vitamin deficiencies apart from folate, notably vitamin B₁₂ (Allen et al., 2018). Thus whilst plasma homo­cys­teine is primarily a folate bio­marker, once folate status is optimized, a much greater dependency on vitamin B₁₂ emerges (Quinlivan et al., 2002). Like­wise, in population groups who consume folic acid-fortified foods or folic acid supplements, homo­cys­teine is considered to be a more reliable bio­marker of vitamin B₁₂ than folate (Refsum et al., 2004).

Disease and lifestyle. Apart from inversely reflecting folate and related B vitamin status, plasma homo­cys­teine is found to be elevated in patients with impaired renal function (Yetley et al., 2011; Allen et al., 2018) and with certain drug treat­ments (Refsum et al., 2004). Alcohol intake, coffee-drinking and smoking are also associated with higher plasma homo­cys­teine.

Age, sex and life­cycle stage. Homo­cys­teine concen­trations increase throughout life and are higher in males compared to females at all ages. Although plasma homo­cys­teine decreases in preg­nancy (by about 50%), concen­trations are reported to normalize within a few days post­partum (Murphy et al., 2004).

Ethnic and genetic effects. Plasma homo­cys­teine may also differ among ethnic groups, but these differences appear to be less important than the influence of B vitamin status. Notably, the most common genetic cause of elevated homo­cys­teine in the general population is homozygosity for the MTHFR C677>T poly­morphism (affecting an estimated 10% of people worldwide but much higher in some populations, including Mexico and Northern China). This folate gene variant also contributes to a higher risk of blood pressure throughout the lifecycle, particularly when combined with deficient status of ribo­flavin, which is required as a cofactor for MTHFR, a key folate meta­bolizing enzyme (Psara et al., 2020; Ward et al., 2020). Notably, both phenotypes associated with this common poly­morphism (high homo­cys­teine and high blood pressure) appear to be modifiable with better ribo­flavin status (McNulty et al., 2020). Most other genetic poly­morphisms in enzymes related to one-carbon meta­bolism have little effect on homo­cys­teine concen­trations (Refsum et al., 2004).

Inborn errors of meta­bolism. Homocystinuria refers to rare inborn errors of meta­bolism which lead to severely increased homo­cys­teine concen­trations in plasma, usually > 100µmol/L, along with large amounts excreted in urine. The most common cause of homo­cystin­uria is defi­ciency of the enzyme cysta­thionine β‑synthase (CBS). CBS defi­ciency, an autosomal recessive condition, has a reported world­wide birth prevalence of 1 in 344,000, while that in Ireland the frequency is much higher, at 1 in 65,000, based on newborn screening and cases detected clinically. These patients have a high risk of premature, frequently fatal, throm­boembolic events. Early diagnosis and treatment with pyridoxine and/or folic acid and betaine, preferably from infancy, can however prevent CVD events and most of the clinical symptoms (Refsum et al., 2004).

Analytical methodologies for measure­ment of plasma homo­cys­teine

Plasma total homo­cys­teine is a very stable analyte as long as the plasma is separated from the red blood cells within 1hr of blood collection (or within 8 hours if the whole blood is kept on ice). As described in detail by Refsum et al. (2004), various method types are available for homo­cys­teine determination. These range from fully automated commercial kits (immunoassay or enzymatic methods) to chrom­atographic assays with mass spec­trometry detection, overall providing comparable results and good assay performance. All methods require the reduction of the disulfide bonds to allow measure­ment of total homo­cys­teine. The reported within-person variability CV for plasma homo­cys­teine is 12.2% (Refsum et al., 2004).

Choice of method. Because the measure­ment of plasma homo­cys­teine produces comparable results across different method types, the choice of method is mainly dependent on available instrumentation and technical expertise. Bailey et al. (2015) put forward the following recommendations to help in making this decision:

• Using commercial kits (either immunoassay or enzymatic assay) on a fully-automated clinical analyzer will provide the highest throughput and quickest turnaround time with the least effort. The main disad­van­tage is the relatively high reagent costs, which can make the measure­ments quite expensive, particularly for a large number of samples.
• If an HPLC system with fluorescence detector is available, setting up a manual assay may be the least expensive approach, particularly in settings with low labor costs. The disad­van­tage of the manual HPLC assay is that the number of samples that can be run is quite limited (typically to about 50–70 unknown samples per analytical run).
• If a labora­tory has access to an existing LC‑MS/MS system and the required technical knowhow, plasma homo­cys­teine can be measured in a high throughput semi-automated manner, with quite low reagent costs. Due to the high initial cost, however, an LC‑MS/MS system is economical only if a large number of samples are measured regularly.
• GC-MS or GC-MS/MS have also been used in high throughput research settings, particularly when the co-determination of methyl­malonic acid and methionine is of interest.
• GC-based methods provide better precision, higher resolution, and longer column life compared to LC-based methods and are not subject to ion suppression issues, which can be a problem in LC‑MS/MS methods.

Cut-off values and Interpretation

In one of the most comprehensive reviews of all relevant aspects of homo­cys­teine measure­ment, it is proposed that reference values for homo­cys­teine are estab­lished for different populations to account for the influence of both nonmodifiable and modifiable factors (Refsum et al., 2004; Table 22a.1).

Table 22a.1. Upper reference limits for plasma homocysteine (µmol/L) in populations. Adapted from: Refsum et al., (2004) with Data drawn from the US, Norway, the UK and Israel and from 9 European countries participating in the European Concerted Action Project (Graham et al., 1997)
¹Individuals eating folic acid-fortified food or taking folic acid-containing supplements.

Population sub-group	Folic acid Supplemented^1	Non-supplemented
Pregnancy	> 8	> 10
Children < 15y	> 8	> 10
Adults 15–65y	> 12	> 15
Adults > 65y	> 16	> 20

The following recommendations were put forward for setting reference ranges:

• Each labora­tory should establish reference limits for its region.
• The reference population should exclude individuals with folate or cobalamin defi­ciency or increased creatinine, and patients with diseases or who are taking drugs that increase homo­cys­teine concen­trations.
• Separate reference limits for children, adults, the elderly, and preg­nant women should be used (Table 22a.1).
• In populations consuming folic acid supplements or folic acid-fortified foods, the upper reference limit is usually 20–25% lower than in non­fortified populations (Table 22a.1).
• The upper homo­cys­teine reference limit should be inter­preted with age, sex, and other determin­ants in mind, particularly prevailing food fortif­ication practices.

22a.2.3 Other bio­markers of folate status

Neutro­phil lobe count

Usually, neutro­phils have three or four segments, but in megalo­blastic anemia (owing to folate or vitamin B₁₂ defi­ciency), this number increases. Neutro­phil hyper­segmentation can be evaluated in smears of peripheral blood or in white blood cells obtained from the buffy coat (i.e. interface between the serum / plasma and the sedimented red cells), and may be the earliest morphological change to appear in the blood in folate and B₁₂ defi­ciency. Neutro­phil hyper­segmentation however is not specific to folate or B₁₂ defi­ciency as it also occurs in other conditions including uremia, myelo­pro­liferative disorders, myelo­fibrosis and as a congenital lesion in approximately 1% of the population, even when the status of folate and vitamin B₁₂ is adequate. The observed greater incidence of neutro­phil hyper­segmentation in iron defi­ciency anemia concurrent with folate and/or vitamin B₁₂ defi­ciency implies interactive effects on the erythro­poietic process (Westerman et al., 1999; Metz, 2008).

Serum folic acid

Unmeta­bolized (or “free”) folic acid can be present in varying concen­trations in plasma and is typically associated with higher folic acid intake via fortified foods, supplements, or a combination of both (Bailey et al., 2010). Whilst greater concen­trations of unmeta­bolized folic acid are generally associated with higher serum folate concen­trations, there is large variation in reported values and no clear dose response relation­ship exists between folic acid intake and unmeta­bolized folic acid in plasma (Pfeiffer et al., 2004; Bailey et al., 2010; Obeid et al., 2011). Thus, cut-off values or desirable ranges have yet to be identified. One randomized trial addressed concerns relating to the potential for folic acid to exert adverse biological effects in pregnancy, and demonstrated that folic acid supplements taken at recommended levels throughout pregnancy do not lead to increased circulating unmetabolized folic acid in mothers or their babies (Pentieva et al., 2016). However, the biological impacts and the significance for health in general of unmetabolized folic acid in blood remain to be elucidated.

Genomic bio­markers of folate status

DNA cytosine methy­lation. Apart from its role in DNA synthesis, folate plays an important role in DNA meta­bolism as it is required for the synthesis of methionine and thus SAM, which in turn is required as a methyl donor for the maintenance of cytosine methy­lation, essential for silencing of genes or structural integrity of specific regions of chrom­osomes (Fenech, 2012). When SAM is depleted, the maintenance of DNA methy­lation may become compromised, leading to hypo­methy­lation of cytosine and structural changes in chrom­atin. Some studies suggest that global DNA methy­lation status is reduced when folate is deficient (Kim, 2005; Crider et al., 2012), but findings in this regard are somewhat inconsistent.

Uracil misincorporation into DNA. Measurement of uracil content in DNA may also provide a bio­marker of folate status, on the basis that adequate amounts of folate as 5,10‑methyl­eneTHF are required by thymidylate synthase to convert deoxy­uridine mono­phosphate (dUMP) to thymidylate mono­phosphate (dTMP) in pyrimidine synthesis and thus DNA biosynthesis. If 5,10‑methyl­eneTHF is limiting as a substrate of thymidylate synthase, dUMP accumulates and it becomes more probable that uracil is incorporated into DNA instead of thymidine during DNA synthesis (Stover, 2009; Fenech, 2012).

Micronuclei. Micronuclei have the same morphological features as normal nuclei but are much smaller. Excessive uracil incorporation into DNA and hypo­methy­lation of DNA can lead to the formation of micro­nuclei from chrom­osome fragments which can be measured in lympho­cytes or in erythro­cytes (Blount et al., 1997; Fenech, 2012). Observational studies show that micro­nuclei in lympho­cytes or erythro­cytes are inversely associated with dietary folate intake and RBC folate (Blount et al., 1997), whilst intervention studies have reported a signif­icant reduction in micro­nuclei frequency in lympho­cytes with folic acid supple­men­tation (Fenech et al., 1997). It is now possible to score micro­nuclei automatically and reliably using a wide range of image cytometry platforms making this technique amenable to mass screening. Given its sensitivity to folate defi­ciency, micro­nuclei measure­ment, in combination with uracil and DNA methy­lation measure­ments, can potentially provide a reliable assess­ment of genome pathology resulting from deficient folate status (Bailey et al., 2015).

22a.2.4 Assessment of folate status in children

Most studies assessing folate status have focused on adults. Far fewer studies provide folate bio­marker data for children, with notable exceptions being published reports using data from population-based surveys conducted in the United States (Pfeiffer et al., 2012) and the United Kingdom (Kerr et al., 2009;. Figure 22a.3) The latter report proposes normal ranges for folate bio­markers for use in clinical pediatric settings

Figure 22a.3 Plasma homocysteine and RBC folate concentrations in a representative sample of British children aged 4–18y. Differences between groups were assessed by using one-factor analysis of covariance (with Tukey’s post-hoc test), adjusting for sex, smoking, fortified breakfast cereal consumption, and supplement use. Bars not sharing a common letter differ, P < 0.05. Adapted from Kerr et al (2009).

Population-based data in British and American children, both show progressive declines in folate (and corresponding, increases in homo­cys­teine) concen­trations with age from childhood to adolescence. Consistent with these reports, are the findings from convenience cohorts of Belgian, Dutch, and Greek children, also showing age-related decreases in folate concen­trations in childhood (De Laet et al., 1999; van Beynum et al., 2005; Papandreou et al., 2006). Of note, where dietary intakes were also measured, the data showed that folate intakes generally compared favorably with dietary reference values across all age groups and were not lower in the older children (Papandreou et al., 2006; Kerr et al., 2009; Pfeiffer et al., 2012) The explanation for the decline in folate status bio­markers with age in children, despite no corresponding decline in dietary folate intakes, is not entirely clear but likely reflects the higher folate requirements of older children related to increased meta­bolic demands for growth from childhood to adolescence (Bailey et al., 2015).