Vitamin B 12 and ageing: current issues and interaction with folate

Abstract

A compromised vitamin B₁₂ status is common in older people despite dietary intakes that typically far exceed current recommendations. The maintenance of an optimal status of vitamin B₁₂ is not only dependent on adequate dietary intake but more critically on effective absorption which diminishes with age. The measurement of vitamin B₁₂ is complicated by the lack of a gold standard assay. There are a number of direct and functional indicators of vitamin B₁₂ status; however, none of these are without limitations and should be used in combination. Vitamin B₁₂ is of public health importance, not only because deficiency leads to megaloblastic anaemia and irreversible nerve damage, but also because emerging evidence links low B₁₂ to an increased risk of a number of age-related diseases, including cardiovascular disease, cognitive dysfunction, dementia and osteoporosis. Furthermore, there are concerns relating to potential adverse effects for older adults with low vitamin B₁₂ status of over-exposure to folic acid in countries where there is mandatory fortification of food with folic acid. The aim of this review is to examine the known and emerging issues related to vitamin B₁₂ in ageing, its assessment and inter-relationship with folate.

Introduction

Major advances in medicine over the last century have resulted in a dramatic increase in life-expectancy. This, however, has been accompanied by a subsequent increase in the prevalence of age-related chronic diseases; with morbidity, disability and poor quality of life frequently present in older age. Poor nutritional status is recognized as a contributing factor and, recently, the role of vitamin B₁₂ in ageing has attracted considerable attention. Apart from the clinical features of B₁₂ deficiency (i.e. megaloblastic anaemia and irreversible neuropathy), emerging evidence indicates that subclinical deficiency (i.e. low biomarker status) of B₁₂ may be implicated in the development of several chronic age-related diseases. The aim of this paper is to consider this evidence and also review its inter-relationship with folate in ageing. The stages of vitamin B₁₂ depletion and the analytical methods used to assess biomarker status will be considered. The scientific evidence linking low/deficient B₁₂ status to adverse health outcomes will be reviewed, and consequences for relevant public health policies will be considered.

Structure and physiological functions of vitamin B₁₂

Vitamin B₁₂ is one of a group of cobalt containing compounds known as cobalamin. There are a number of different forms of the vitamin including the two metabolically active forms methylcobalamin and deoxyadenosylcobalamin and cyanocobalamin, the synthetic form of the vitamin typically used in supplements and fortified foods. Vitamin B₁₂ is required as a co-factor for only two mammalian enzymes, methionine synthase and methylmalonyl CoA mutase. Adenosylcobalamin acts as a co-factor for the latter enzyme which converts methylmalonyl CoA to succinyl CoA, a metabolite in the tricarboxylic acid cycle (Figure 1). This is an important reaction in the metabolism of branched-chain amino acids and odd-chain length fatty acids. ¹ In vitamin B₁₂ depletion, the reduced activity of methylmalonyl-CoA mutase causes the concentrations of its substrate methylmalonyl CoA to build up and form the by-product methylmalonic acid (MMA), which is then excreted into the circulation. Methylcobalamin acts as a co-factor for the enzyme methionine synthase, which catalyses the re-methylation of homocysteine to methionine, the precursor of S-adenosylmethionine (Figure 2). S-adenosylmethionine is a universal methyl donor essential for the methylation of phospholipids, neurotransmitters, amines, DNA, RNA and myelin basic protein. In vitamin B₁₂ depletion, inactivity of the methionine synthase enzyme leads to a reduction in important methylation reactions and elevated homocysteine concentrations in the circulation. ²

Figure 1.

The vitamin B₁₂ (i.e. adenosylcobalamin)-dependent conversion of methylmalonyl-CoA to succinyl-CoA.

Figure 2.

Metabolic pathway involving vitamin B₁₂ of the remethylation of homocysteine to methionine and the recycling of folate. Shows co-factor forms for vitamin B₁₂ (methylcobalamin), vitamin B₆ (pyridoxal-5-phosphate) and riboflavin (vitamin B₂; flavin mononucleotide [FMN]; flavin adenine dinucleotide [FAD]). Also shows the metabolism of betaine and dimethylglycine (DMG).

Vitamin B₁₂ and folate interrelationship

The haematological sign of vitamin B₁₂ deficiency is megaloblastic anaemia characterized by megaloblastic (immature, enlarged) blood cells that occur as a result of impaired DNA synthesis. An identical anaemia arises with folate deficiency because these two vitamins are intrinsically linked via the enzyme methionine synthase. ² Methionine synthase sits at the junction between two major biosynthetic pathways: the remethylation cycle and the folate cycle (Figure 2). Methionine synthase is the only known enzyme to use 5-methyltetrahydrofolate, which in turn is converted to the metabolically active form, tetrahydrofolate, needed in one-carbon metabolism. ² In vitamin B₁₂ depletion, methionine synthase activity is reduced and the formation of tetrahydrofolate is blocked, with folate essentially becoming trapped as 5-methyltetrahydrofolate (i.e. the methyl-trap hypothesis). This is an unusable form of the vitamin as it cannot be converted back to its precursor 5,10-methylenetetrahydrofolate because the reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate (by the enzyme methylenetetrahydrofolate reductase [MTHFR]) is irreversible. S-adenosylmethionine is an allosteric inhibitor of MTHFR, thus reduced S-adenosylmethionine concentration (as seen in vitamin B₁₂ depletion) removes the inhibition of MTHFR, promoting the reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate so that it can be used to re-synthesise methionine (the precursor of S-adenosylmethionine) thus further trapping folate as 5-methyltetrahydrofolate. In this situation, cells suffer from a folate pseudo-deficiency; folate is available but it is not in an active form that can be used by the cell, and consequently, the production of purines and pyrimidines needed for DNA synthesis is disrupted. ³ This affects the rapidly dividing cells of the bone marrow, leading to the production of megaloblastic blood cells and subsequent megaloblastic anaemia. In addition, 5-methyltetrahydrofolate is a poor substrate for folylpolyglutamate synthase, the enzyme responsible for cellular retention of folate. In order for folate to be retained by the cell 5-methyltetrahydrofolate must first be converted to tetrahydrofolate via methionine synthase and thus, intracellular erythrocyte folate concentration has been reported to be lower in individuals with low vitamin B₁₂. ^3–5

Absorption, transport and storage

The absorption of vitamin B₁₂ is a highly complex process that is dependent on normal functioning of the gastrointestinal tract (Figure 3), consequently, it often becomes less efficient with age. Vitamin B₁₂ is tightly bound to protein in food and must be released by hydrochloric acid and pepsin in the stomach in order for B₁₂ to be absorbed. The synthetic form of vitamin B₁₂, found in fortified foods and supplements, is however already free and thus has no gastric acid requirement. Free vitamin B₁₂ immediately binds with R-binding proteins and then with intrinsic factor (IF) in the duodenum forming the IF-B₁₂ complex which travels to the terminal ileum where it is absorbed via specific cubilin receptors by calcium-dependent endocytosis. There is a limit to the amount of vitamin B₁₂ that can be absorbed at any given time via the IF-related pathway; receptors are reported to become saturated with B₁₂ intakes greater than 1.5–2.0 µg of vitamin B₁₂. ⁶ In addition, an alternative, albeit inefficient pathway exists whereby small amounts (1%) of vitamin B₁₂ are absorbed through passive diffusion (i.e. even in the absence of IF). ⁷ Once absorbed, vitamin B₁₂ then binds to one of two transport proteins, transcobalamin or haptocorrin. The majority of vitamin B₁₂ binds to haptocorrin, a glycoprotein with an unknown function, while 20–30% is bound to transcobalamin, forming holo-transcobalamin (holoTC), the metabolically active fraction of the vitamin. ⁸ Total body stores of vitamin B₁₂ can reach 5 mg, with the majority stored in the liver. ⁹ The liver excretes around 1–10 µg/d of vitamin B₁₂ into bile; ¹⁰ however, up to 90% of this is re-absorbed via a highly efficient enterohepatic cycle while the rest is excreted in faeces. The daily requirement for vitamin B₁₂ is so small in comparison to body stores that it is considered to take years for vitamin B₁₂ deficiency to develop, even when there is a complete halt in B₁₂ absorption. ¹¹

Figure 3.

The absorption and transport of vitamin B₁₂ (adapted from Andrès et al. ¹²⁷ ).

Analytical measurement of vitamin B₁₂ status

Serum total vitamin B₁₂

Measurement of serum/plasma total vitamin B₁₂ has been the standard clinical test for vitamin B₁₂ deficiency for many years and a variety of analytical approaches are available such as the traditional microbiological assay, competitive binding assay, radioimmunoassay and chemiluminescence assay. Early versions of the newer techniques lacked sensitivity and led to falsely elevated B₁₂ concentrations; ¹² however, there is now better agreement between the more recently developed protein binding assays and the microbiological assay; generally considered to be the gold standard assay. ¹³ These newer assays have a number of advantages over the microbiological assay, they are generally less time consuming, are not as sensitive to antibiotics or chemotherapy drugs, are widely available and have low running costs. ¹³ It has become apparent, however, that a low serum total vitamin B₁₂ concentration does not always equate with B₁₂ deficiency nor does a normal value necessarily exclude it. ¹⁴ There is no established cut-off value used to define vitamin B₁₂ deficiency, with each laboratory recommended to determine its own reference range depending on its assay method. There is a large variation between laboratories with one review reporting that the lower reference limit of values cited in the literature ranged from 110 to 260 pmol/L. ¹⁵ Also, metabolic and clinical features of deficiency have been reported in individuals with serum total B₁₂ within the ‘normal range’. ¹⁶ Furthermore, 5% of individuals with classical clinical signs of vitamin B₁₂ deficiency were found to have serum total vitamin B₁₂ concentrations within the normal range. ¹⁷

Serum total vitamin B₁₂ does not reflect intracellular vitamin B₁₂ as it is a measure of all forms of the vitamin, the majority of which is bound to haptocorrin. Haptocorrin-bound vitamin B₁₂ is considered to be metabolically inert and has been shown to respond slowly to changes in vitamin B₁₂ status compared with holoTC, which may explain its lack of sensitivity. ¹² Furthermore, liver disorders, myeloproliferative disorders, renal failure, transcobalamin II deficiency, bacterial overgrowth and haemolysis can all lead to falsely elevated serum total B₁₂ concentrations. False low concentrations of serum total vitamin B₁₂ are linked with severe folate or iron deficiency, multiple myeloma, pregnancy, HIV infection and oral contraceptive use. ¹⁸

Plasma total homocysteine

The re-methylation of homocysteine to methionine is disrupted during vitamin B₁₂ depletion (Figure 2) because the necessary enzyme, methionine synthase requires vitamin B₁₂ as cofactor. As a consequence, plasma homocysteine concentrations accumulate with B₁₂ deficiency thus providing a functional biomarker of vitamin B₁₂ status. Although plasma homocysteine is highly sensitive to B₁₂ status, it lacks specificity. This is because plasma concentrations of homocysteine are determined not only by the status of vitamin B₁₂ ¹⁹ but also by folate, ²⁰ and to a lesser extent vitamin B₆ ²¹ and riboflavin. ²² Once folate status is optimised (as in countries with mandatory folic acid fortification) vitamin B₁₂ emerges as the major nutritional determinant of homocysteine. ²³ Apart from B vitamin status, plasma homocysteine concentrations are also influenced by other factors such as renal function, age and gender ²⁴ and genetic polymorphisms, mainly homozygosity (TT genotype) for the 677C → T polymorphism in the gene encoding MTHFR, the enzyme required to generate 5-methyltetrahydrofolate. ²⁵ Blood sample collection and handling can also impact considerably on homocysteine measurement. Homocysteine concentrations will increase if the sample is kept at room temperature, or if separation of the sample is delayed following collection, as homocysteine is released from red blood cells. However, this can be minimized if the sample is separated immediately or placed on ice until centrifugation. ^24,26 After separation, homocysteine is extremely stable and can be stored, once frozen, for many years. ²⁴ Several analytical methods, e.g. high-performance liquid chromatography, gas and liquid chromatography-mass spectrometry, stable-isotope dilution and immunoassay techniques have been developed for measuring plasma homocysteine and there is generally good agreement between the various techniques. ²⁶ The assays are reliable and have become less expensive in recent years ⁵ but reference ranges may vary considerably between laboratories. ²⁴ Although a plasma homocysteine concentration between 5 and 15 µmol/L is generally considered normal, ²⁶ this reference range may not be appropriate for older adults. Despite its lack of specificity, plasma homocysteine is a sensitive and useful indicator of vitamin B₁₂ status in situations where folate is optimized.

Serum MMA

Vitamin B₁₂ acts as a co-factor for the enzyme methylmalonyl-CoA mutase required for the conversion of methylmalonyl-CoA to succinyl CoA. In vitamin B₁₂ deficiency, the activity of this enzyme is reduced leading to the accumulation of the by-product MMA, which can be detected in both blood and urine (Figure 1). ¹⁸ Plasma MMA is therefore a specific, functional marker of vitamin B₁₂ status that reflects the availability of intracellular B₁₂, although it becomes elevated in individuals with renal impairment ²⁷ and therefore may be less useful in older people where renal dysfunction is a common problem. MMA is more concentrated in urine and methods for its measurement have been available since the 1950s. However, it lacks sensitivity and requires a 24-hour urine collection as concentrations are more sensitive to variation in food intake than serum MMA concentrations. ²⁸ More than 95% of patients with vitamin B₁₂ deficiency have significantly elevated serum MMA. ²⁷ A variety of cut-off values have been used to define vitamin B₁₂ status; however, serum MMA value <0.30 µmol/L is generally considered indicative of no deficiency. ²⁹ Although the development of a gas-chromatography-mass spectrometry method has increased sensitivity, the utility of MMA is compromised by high running costs, technical demands and dependence on normal renal function.

Serum holoTC

Serum total vitamin B₁₂ and the functional assays (homocysteine; MMA) lack sensitivity and specificity, thus there has been an ongoing search for a more reliable clinical measure of B₁₂ status. The major limitation of serum vitamin B₁₂ is that it measures the total amount of the vitamin, while holoTC represents the metabolically active fraction of the vitamin that is involved in cellular reactions and thus may be a more reliable indicator of B₁₂ status. It is thought that a decrease in holoTC will indicate the earliest sign of B₁₂ depletion as it has a much shorter half-life than haptocorrin. ³⁰ Low holoTC concentrations have been detected in populations at risk of deficiency such as vegetarians, vegans ³¹ and the elderly. ³² Preliminary results from an ongoing elderly Irish cohort indicate that holoTC is a considerably better indicator of vitamin B₁₂ status than serum total vitamin B₁₂ or MMA in an elderly population. ³³ In addition, several studies have clearly demonstrated that holoTC is diagnostically superior to vitamin B₁₂ in the detection of vitamin B₁₂ deficiency ^31,34,35 and was better correlated with symptoms of vitamin B₁₂ malabsorption than serum total vitamin B₁₂. ³⁶ There is also increasing evidence that holoTC is more strongly correlated with diseases associated with low B₁₂ status, however, this has not been thoroughly evaluated. ³⁷

Measurement of holoTC has been an attractive prospect for more than 20 years; however, until recently, it was hampered by the lack of a good quality assay. ⁸ Currently, there are a number of different methods available for the measurement of holoTC including a microbiological assay, an enzyme-linked immunosorbent assay, a radioimmunoassay and a novel automated holoTC specific monoclonal assay that is suitable for routine use in clinical laboratories. ⁸ There is no general consensus as to what concentration of holoTC is sufficient, with the lower normal limit for holoTC varying from 19 to 42 pmol/L depending on the laboratory method used. ⁸ Several studies have demonstrated that holoTC increases dramatically after vitamin B₁₂ ingestion, although the dose used in these studies was much larger than what would be typically consumed through the diet. ^38,39 HoloTC is highly sensitive to altered renal function with concentrations increasing in renal impairment ³¹ and is influenced by a common genetic polymorphism of the transcobalamin gene (TC776C → G), liver disease and female sex hormones. ⁴⁰ The future of holoTC as a first-line biomarker of vitamin B₁₂ is promising, although further research is warranted to address the issues raised above before it can be used routinely.

Causes of low/deficient vitamin B₁₂ status in older adults

Vitamin B₁₂ absorption is a complex process and a defect at any step could lead to vitamin B₁₂ depletion. Therefore, there are multiple causes of low B₁₂ in the older population as shown in Table 1.

Table 1.

Causes of low/deficient vitamin B₁₂ status in older adults.

Causes of low vitamin B12 status
Inadequate dietary Intake
Low dietary Intake
Vegetarianism
Veganism
Malabsorption
Pernicious anaemia
Atrophic gastritis
Bacterial overgrowth
Helicobacter pylori
Zollinger–Ellison Syndrome
Gastric/intestinal resection
Intestinal diseases
Crohn's disease
Coeliac disease
Drug–nutrient interactions
Proton pump inhibitors
H2 antagonists
Metformin
Nitrous oxide
Pancreatic insufficiency
Alcohol abuse

Inadequate dietary intake

Recommended daily intakes for vitamin B₁₂ vary from country to country. ⁴¹ The UK National Diet and Nutrition Survey found that dietary intakes of vitamin B₁₂ in those aged over 65 were more than three times greater than current dietary recommendations. ⁴² Thus, inadequate intakes of vitamin B₁₂ are not a main concern as diets of older people appear to provide vitamin B₁₂ well in excess of needs. Vitamin B₁₂ is only found in foods of animal origin such as meats, poultry, fish, eggs and dairy products. It can also be obtained from foods such as ready to eat breakfast cereals and spreads fortified with the synthetic form of the vitamin. ¹⁰ In developed countries, inadequate dietary intakes of vitamin B₁₂ are extremely rare, except for strict vegetarians and vegans. ³⁰

Malabsorption

Intrinsic factor deficiency – pernicious anaemia

Pernicious anaemia is the classical form of vitamin B₁₂ deficiency and, although more prevalent in those over 60 years, only accounts for 1–2% of vitamin B₁₂ deficiency found in older populations. ¹¹ It is an autoimmune disease characterized by the destruction of the gastric mucosa and subsequent loss of IF, the essential transport protein required for vitamin B₁₂ absorption. Loss of IF prevents not only the absorption of food-bound and free vitamin B₁₂ but also the re-absorption of vitamin B₁₂ secreted in bile. This creates a significant negative vitamin B₁₂ balance with a progressive and permanent depletion of body stores. ⁴³ Although it may take a number of years for clinical deficiency to develop, pernicious anaemia would ultimately lead to death if left untreated. ⁴⁴ In order to bypass the gastrointestinal tract and the requirement for IF, patients are traditionally treated with intramuscular B₁₂ injections for life. However, there is evidence to suggest that oral vitamin B₁₂ administered at pharmacological doses of the vitamin can be as effective. ⁴⁵

Food-bound malabsorption

Food-bound malabsorption of vitamin B₁₂ is common in older people. It arises mainly as a result of atrophic gastritis, an age-related disorder affecting up to 30% of people ≥60 years. ⁴⁶ Atrophic gastritis is a chronic inflammation of the stomach leading to atrophy of the mucosa which in turn results in reduced gastric acid secretion (hypochlorhydria), and therefore diminished B₁₂ absorption as hydrochloric acid and pepsin are essential for the release of vitamin B₁₂ from food proteins. ¹¹ In addition, hypochlorhydria can promote the overgrowth of bacteria in the stomach and small intestine which then utilize vitamin B₁₂, thereby further reducing the amount of the vitamin available for absorption. ⁴⁷ In particular, Helicobacter pylori (H. pylori) infection has been associated with the development of atrophic gastritis and low vitamin B₁₂ status. ⁴⁸

In theory, individuals with food-bound malabsorption of vitamin B₁₂ should still be able to absorb free B₁₂ because the secretion of IF remains intact and free vitamin B₁₂ has no gastric acid requirement (because it is not intrinsically bound to food proteins). Food-bound malabsorption leads to a more subtle less profound depletion of B₁₂ status and it remains unclear whether this will ultimately progress to clinical vitamin B₁₂ deficiency. ⁴⁴

Drug induced malabsorption – proton pump inhibitors

Proton pump inhibitors (PPIs) are a group of acid suppressant drugs intended to treat gastric acid conditions such as gastro-oesophageal reflux disorder, conditions associated with the use of non-steroidal anti-inflammatory drugs, Barrett's oesophagus and hypersecretory disorders such as Zollinger–Ellison syndrome. PPIs bind to and deactivate the gastric H⁺/K⁺ adenosine triphosphatase pump which reduces gastric acid secretion leading to profound hypochlorhydria; ⁴⁹ thus PPI therapy can induce a state clinically identical to atrophic gastritis, the most common cause of vitamin B₁₂ deficiency. This has led to the concern that long-term acid-suppression medication may lead to food-bound malabsorption and ultimately vitamin B₁₂ deficiency especially in those vulnerable to low status such as older people. ⁵⁰ Chronic acid suppressant use in older people has been associated with the initiation of vitamin B₁₂ therapy. ⁵¹ Short-term studies demonstrated that PPIs reduced the absorption of food-bound vitamin B₁₂. ^52,53 Long-term omeprazole treatment has been associated with a decrease in serum B₁₂ status in both Zollinger–Ellison patients and older adults; ^54,55 however, results have not been entirely consistent with two recent large studies reporting conflicting results. ^55,56 Future studies, using a variety of vitamin B₁₂ biomarkers, are required to more fully investigate the effect of prolonged PPI therapy on vitamin B₁₂ status. The impact of PPIs on B₁₂ status is important because they are frequently misprescribed to treat less serious conditions and are in fact among the most frequently used medications, with millions of users worldwide. ⁵⁷

Gastric/intestinal resection and intestinal disease

As previously described vitamin B₁₂ absorption is dependent upon the normal functioning of the gastrointestinal tract, a function that deteriorates with normal ageing. Diseases that affect either the structure or function of the stomach or part of the small intestine will have a negative impact on the absorption of vitamin B₁₂. Gastric resection destroys the parietal and chief cells in the stomach so the secretion of hydrochloric acid, pepsin and intrinsic factor will be reduced and thus, the absorption of vitamin B₁₂ will be severely disrupted. ⁵ Furthermore, the subsequent hypochlorhydria promotes bacterial overgrowth in the stomach and small intestine which further exacerbates the problem of vitamin B₁₂ malabsorption. ⁴⁷ Ileal resection results in the loss of the IF-B₁₂ receptors in the terminal ileum which is one of the fundamental steps in vitamin B₁₂ absorption. A variety of other intestinal conditions such as Crohn's disease, coeliac disease, diverticulitis, intestinal blind loops and tapeworms have also been associated with vitamin B₁₂ depletion. ⁵

Genetic polymorphisms

Apart from the rare inborn errors of metabolism, a number of polymorphisms in genes involved in B vitamin metabolism have been identified. The most important of these is the 677C → T variant in the gene coding for the folate metabolizing MTHFR enzyme. This polymorphism has been extensively reviewed elsewhere, but in brief, individuals homozygous for the 677C → T polymorphism (TT genotype) in MTHFR have impaired folate metabolism, lower serum and red cell folate and higher plasma homocysteine concentrations and are at increased risk of certain age-related diseases. ⁵⁸ Meta-analyses of genetic studies have also shown that those individuals with the TT genotype are at a 14–21% greater risk of cardiovascular disease (CVD) compared with those without the polymorphism. ^59,60 A more recent meta-analysis reported that individuals with the TT genotype are at increased risk of stroke, however, this association was limited to those with low folate status. ⁶¹ There are also several known polymorphisms of the transcobalamin protein, the most common being the 776C → G polymorphism. Individuals homozygous for this polymorphism (GG genotype) are reported to have lower concentrations of holoTC compared with individuals with the wild-type genotype, hence, may impact the use of holoTC as a routine measurement for vitamin B₁₂. ^40,62 It is thought that this polymorphism decreases the binding affinity and transport of vitamin B₁₂. ⁶³

Vitamin B₁₂ diagnosis and treatment

Vitamin B₁₂ depletion has been described in four stages ranging from mild depletion to clinical deficiency. ³⁰ Stage 1 is early vitamin B₁₂ depletion characterized by a decrease in holoTC concentration. Stage 2 is cellular depletion characterized by a further decrease in holoTC. Serum total B₁₂ takes longer to respond to changes in status and can remain within the normal range in stages 1 and 2. Stage 3 is characterized by metabolic evidence of deficiency, with elevated concentrations of both plasma homocysteine and serum MMA, low holoTC and normal or below normal serum total B₁₂ concentrations. Stage 4 is clinical deficiency where both serum total B₁₂ and holoTC are low, the metabolic indicators are elevated and there are clinical signs of deficiency including haematological and neurological manifestations. Haematological features, which occur as a result of impaired DNA synthesis, include megaloblastic anaemia characterized by megaloblastic (large immature blood cells with low concentrations of haemoglobin) blood cells, hypersegmentation of neutrophils, leukopaenia and thrombocytopaenia. Neurological symptoms are diverse and irreversible if left untreated. The most severe form manifests as a sub-acute combined degeneration of the spinal cord and is characterized by degeneration of the posterior and lateral columns of the spinal cord. ⁶⁴

Currently, there is no consensus on how best to diagnose vitamin B₁₂ deficiency. Traditionally, diagnosis of vitamin B₁₂ deficiency relied upon the symptoms associated with megaloblastic anaemia such as muscle weakness, fatigue and shortness of breath and the associated haematological signs. ⁶⁵ However, the clinical signs of anaemia only occur in the late stages of depletion and furthermore it is now clear that there is not always a continuous progression from mild vitamin B₁₂ depletion to clinical deficiency. ^30,44 Clinical deficiency is caused by IF deficiency (i.e. pernicious anaemia) and leads to a progressive and permanent depletion of vitamin B₁₂, but this accounts for only 1–2% of B₁₂ deficiency. ¹¹ Conversely, subclinical vitamin B₁₂ deficiency is much more common and is usually as a result of food-bound malabsorption of B₁₂. ¹¹ Diagnosis of pernicious anaemia is relatively straightforward, patients have very low vitamin B₁₂ concentrations and present with the typical haematological and neurological symptoms. However, diagnosis of subclinical deficiency is much more problematic as it relies on metabolic evidence of deficiency. Thus, it is recommended that the vitamin should be measured by more than one biomarker, both a direct (i.e. serum total vitamin B₁₂ or holoTC) and indirect functional marker (i.e. plasma homocysteine or serum MMA) should be used in combination. ⁴⁴

Treatment for IF deficiency is well established with patients receiving monthly intramuscular vitamin B₁₂ injections for life. However, some studies demonstrate that oral supplementation with very high doses (i.e. 1000 µg/day) B₁₂ may be equally effective because 1% of the vitamin can be passively absorbed without the need for IF. ^66,67 Although this is an attractive prospect for clinicians, caution must be exercised as patients may not be fully compliant in the long-term. ⁶⁸ Conversely, there is no consensus on how to best treat (if at all) subclinical deficiency as its clinical significance remains unclear. In the USA (but not in Europe or elsewhere), specific age-related dietary recommendations for the population have been issued by the Institute of Medicine, ⁶⁹ recommending that people aged ≥50 years should consume most of their vitamin B₁₂ from crystalline sources, i.e. fortified food and supplements. A regular consumption of foods fortified with B₁₂ or low-dose supplements will be more than sufficient for addressing food-bound malabsorption. ^70,71

Emerging roles in health of low/deficient vitamin B₁₂ status in the older population

The potential role of homocysteine and the metabolically related B-vitamins in the development of chronic disease has attracted much interest within the scientific community. This interest was initially sparked from observations that individuals with homocystinuria, an inborn error of metabolism characterized by excessive concentrations of homocysteine, had premature vascular disease and mental retardation. ⁷² Since then, much scientific literature has emerged focusing mainly on homocysteine, but also on the metabolically related B vitamins, most notably folate and vitamin B₁₂, in relation not only to CVD, ^73,74 and stroke, ⁷⁵ but also cognitive function, ⁷⁶ fracture risk ⁷⁷ and osteoporosis. ⁷⁸ The possibility that B vitamin supplementation might help delay or prevent chronic disease (via correction of suboptimal status and/or lowering homocysteine) is of major interest and could have important public health implications. ⁷⁹

Cardiovascular disease

CVD is the leading cause of death worldwide. Two major meta-analyses of epidemiological evidence published in 2002 predicted that a lowering of plasma homocysteine by 3 µmol/L (or by about 25% based on typical homocysteine concentrations of 12 µmol/L) would reduce the risk of heart disease by 11–16% and stroke by up to 19–24%. ^73,74 As recently reviewed, the evidence from the secondary prevention trials in at-risk patients has failed to demonstrate a benefit of homocysteine-lowering therapy with B vitamins (usually using folic acid in combination with vitamin B₁₂) on CVD events. ⁵⁸ Currently, the evidence is stronger for stroke, with meta-analyses of randomized controlled trials (RCTs) showing that homocysteine lowering reduces the risk of stroke. ^75,80 Few studies have investigated the independent effects of vitamin B₁₂ on CVD risk. Subgroup analysis of the Vitamin Intervention for Stroke Prevention (VISP) Trial confined to those individuals considered to benefit from supplementation (i.e. non-supplement users and those without pernicious anaemia) suggests that higher doses of vitamin B₁₂ may be required as they reported that individuals with higher baseline vitamin B₁₂ and taking the high-dose B vitamin had reduced risk of recurrent vascular events while those with the lowest vitamin B₁₂ status on the low-dose combination had the greatest risk of recurrent vascular events. ⁸¹ In addition, a number of other B vitamin RCTs have detected beneficial effects on a number of cardiac endpoints. Combined B vitamin supplementation including high-dose vitamin B₁₂ has been associated with reduced re-stenosis and cardiovascular events in patients following coronary angioplasty. ^82,83 Combined vitamin B₁₂ and folic acid supplementation was also associated with improved coronary blood flow in patients with coronary artery disease. ⁸⁴ Evidence from meta-analyses also supports a role for vitamin B₁₂ in CVD, with a greater effect on stroke reported when folic acid was used in combination with vitamins B₁₂ and B₆ compared with folic acid alone. ^80,85 Further research is warranted to more fully investigate the potential benefits of B vitamin supplementation on CVD risk, particularly in those with pre-existing vascular disease.

Cognitive function

Dementia and cognitive impairment disorders are a major public health concern in ageing populations. ⁸⁶ Observations from case-control studies, that patients with Alzheimer's disease (the most common form of dementia) had significantly higher homocysteine concentrations than age-matched controls, prompted the theory that B vitamin status might be an important modifiable risk factor for cognitive dysfunction in ageing. ^76,87 Cross-sectional and longitudinal studies provide a wealth of evidence to support an association between homocysteine and/or related B vitamins with cognitive function in both healthy older adults and patients with dementia. ⁸⁸ A recent meta-analysis of epidemiological studies estimated that a 3-µmol/L reduction in homocysteine would reduce the risk of dementia by up to 22%. ⁸⁹ Both folate and vitamin B₁₂ have been extensively investigated in relation to cognitive performance in ageing; however, the evidence is less consistent for vitamin B₁₂. ^90–93 Many of these studies relied on serum total vitamin B₁₂ as the sole biomarker measure of status; however, those that used MMA or holoTC are generally more supportive of a protective role for vitamin B₁₂ in maintaining cognitive function in ageing. ^79,94

A number of RCTs have investigated the potential benefits of B vitamin supplementation on cognitive function; however, many of these studies were of insufficient duration or sample size necessary to reach useful conclusions. ^95–104 One large trial of long-term duration that reported no benefit was inherently flawed as the cognitive arm of the investigation was initiated 1.2 years after commencement of B vitamin treatment; therefore, any potential impact on cognitive function may have been missed. ⁹⁶ Two a priori RCTs, both conducted in healthy older people for a considerable duration, have been published with conflicting results. ^101,102 McMahon et al. ¹⁰¹ found no benefit of combined B vitamin supplementation (1000 µg folate, 500 µg vitamin B₁₂, 10 mg vitamin B₆ daily) on cognitive performance in 276 dementia-free older people (aged ≥65 years). Conversely, a supplementation trial with folic acid (800 µg/day) alone in 818 people aged 50–70 years reported significant improvements in cognition over a-three year intervention period. ¹⁰² This discrepancy is intriguing given that both studies were similar in design, interestingly however, the baseline B vitamin status between the two studies was very different. The study by Durga was conducted in the Netherlands, a country which does not permit folic acid fortification (even on a voluntary basis), while the trial by McMahon showing a null effect was conducted in New Zealand which has had an active voluntary fortification programme since 1996. Hence, the participants in Durga's study had lower baseline concentrations of folate and vitamin B₁₂ compared with McMahon's study and therefore may have been more likely to benefit from folic acid intervention to optimize status. More recently, combined B vitamin supplementation was shown to improve cognitive performance in community-dwelling older adults with depressive symptoms. ¹⁰³ The strongest evidence to date to provide support for a cause and effect relationship between B vitamin status and cognitive function is from the homocysteine and B vitamins in cognitive impairment (VITACOG) study. B vitamin supplementation (0.8 mg folic acid, 0.5 mg vitamin B₁₂, 20 mg vitamin B6) slowed down the rate of brain atrophy (as measured using magnetic resonance imaging) ¹⁰⁰ and improved cognitive performance ¹⁰⁴ in patients with mild cognitive impairment. A number of RCTs conducted to investigate the effect of B vitamin intervention on CVD also include a measurement of cognitive function. While not primarily designed to examine cognitive health, the evidence from these trials should be helpful in contributing to the evidence linking optimal B vitamin status to the maintenance of cognitive function in ageing, although they will be confounded to some extent by the fact that many of the participants will have existing vascular disease.

Bone health

Osteoporosis is characterized by low bone mass and micro-architectural deterioration in bone tissue, resulting in an increased risk of fracture and affects over 75 million people in Europe, USA and Japan. Evidence from epidemiological studies has reported that elevated plasma homocysteine concentrations and/or low B vitamin status are associated with lower bone mineral density (BMD) ^78,105 and a higher risk of osteoporosis ¹⁰⁶ and osteoporotic fracture. ¹⁰⁷ Several studies have linked low vitamin B₁₂ status with osteoporosis and low BMD. ^106,108,109 A retrospective study of postmenopausal women found that those with pernicious anaemia had a more than two-fold risk of fracture compared with those without pernicious anaemia. ¹¹⁰ Moreover, an increase in BMD and a reversal of osteoporosis was demonstrated in a patient with pernicious anaemia, severe osteoporosis and multiple vertebral compression fractures after two years treatment with vitamin B₁₂. ¹¹¹ Low vitamin B₁₂ status has been associated with increased risk of fracture ^77,107 and more rapid bone loss in older women; ¹¹² however, the evidence is not entirely consistent. ¹⁰⁷

Many RCTs investigating the effect of B vitamin supplementation showed no evidence of a beneficial effect; however, many of these interventions were conducted over too short a timeframe and typically measured bone turnover biomarkers, the validity of which has yet to be established. ^113,114 One RCT of sufficient duration and with robust bone outcomes did, however, show that combined folic acid and vitamin B₁₂ supplementation for two years was effective in reducing the risk of hip fractures in Japanese patients following stroke. ¹¹⁵ There is a need for further well-conducted RCTs before any firm conclusions can be made in relation to the role of vitamin B₁₂ and related B vitamins in bone health.

Public health implications

The policy in North America and elsewhere of mandatory fortification of staple foods with folic acid for the prevention of neural tube defect (NTD) pregnancies has been an indisputable success. ¹¹⁶ In addition to lowering NTDs, this measure may also have had a benefit in reducing stroke mortality. ¹¹⁷ The implementation of a similar policy has, however, been delayed in the UK because of concerns that high-dose folic acid could have a detrimental effect on health.

Potential adverse effects of over exposure to folic acid in those with low vitamin B₁₂

Evidence from historic case reports of vitamin B₁₂-deficient patients mistakenly treated with folic acid have led to the concern that high doses of folic acid may exacerbate the neurological symptoms and mask anaemia in individuals with low vitamin B₁₂. ¹¹⁸ Analysis of post folic acid fortification data from the USA and Canada has been useful in assessing the impact of high folic acid exposure on vitamin B₁₂ status. Since the introduction of mandatory fortification there has been a dramatic increase in blood folate concentrations by up to 40%, ¹¹⁹ with unmetabolized folic acid (i.e. not the normal circulating co-factor form) now being detected in the circulation. ¹²⁰ This in turn has led to an intriguing scenario where an increasing number of older adults have very high folate concentrations coupled with low vitamin B₁₂ status. This is a relatively new phenomenon and there are concerns over potential adverse effects on health. The most widely documented concern is that folic acid at high doses may potentially ‘mask’ the anaemia of vitamin B₁₂ deficiency, traditionally diagnosed by macrocytosis (elevated mean corpuscular volume [MCV] >100 fL). ⁵ This could arise as a result of folic acid being converted to tetrahydrofolate (through the activity of dihydrofolate reductase), bypassing the vitamin B₁₂-dependent enzyme methionine synthase, thus promoting DNA synthesis and correcting signs of anaemia (independent of whether it was caused by folate or vitamin B₁₂ deficiency) but not the neurological damage specifically caused by vitamin B₁₂ deficiency. This is of concern in individuals with low B₁₂ status because correction of anaemia will alleviate the symptoms associated with it and thus may delay diagnosis allowing the irreversible neurological damage to progress unabated. ^5,118

An early report concluded, however, that the introduction of mandatory folic acid fortification in the USA has not had an adverse effect on the diagnosis of vitamin B₁₂ deficiency. ¹²¹ Analysis from the National Health and Nutrition Examination Survey (NHANES) in the USA, however, observed another potential adverse interaction between folate and vitamin B₁₂. Low vitamin B₁₂ was, as expected, associated with elevated homocysteine and MMA. ¹²² Unexpectedly, however, homocysteine and MMA were highest in individuals with low vitamin B₁₂ coupled with high folate, and furthermore, these individuals were at greater risk of both anaemia and cognitive impairment. ¹²³ Two subsequent studies conducted in the UK and Ireland failed to detect similar adverse interactions between high folate and low vitamin B₁₂, an inconsistency that might be explained by the absence of mandatory folic acid fortification in both countries resulting in generally lower folate, and particularly lower unmetabolized folic acid concentrations. ^124,125

Apart from concerns regarding potential adverse interactions with vitamin B₁₂, a new concern has emerged in recent years that excessively high intakes of folic acid (the synthetic vitamin form) could have a cancer-promoting effect in segments of ageing populations, but this is rather controversial. On the one hand, there is consistent epidemiological evidence that higher folate intake within the dietary range plays a protective role against cancer at various sites, on the other hand there is evidence suggesting that long-term exposure to high intakes of folic acid (greater than 1 mg/day) may promote colorectal tumorigenesis in patients with pre-existing lesions or even increase cancer risk generally. ¹²⁶ Thus policy-makers worldwide (including the UK government) have delayed decisions to implement population-based folic acid fortification policies similar to those introduced in 1996 in North America.

Given the potentially serious consequences of over-exposure to folic acid some have suggested that vitamin B₁₂ should be included together with folic acid in any new food fortification policy. Not only would this prevent any adverse outcomes in individuals with low vitamin B₁₂ status, but would also address the issue of food-bound malabsorption of vitamin B₁₂, the most common cause of low vitamin B₁₂. Alternatively, a specific age-related dietary recommendation similar to that issued by the Institute of Medicine (IOM) in the USA where people aged ≥50 years are recommended to consume most of their vitamin B₁₂ from crystalline sources (i.e. fortified food and supplements) would also address these concerns. ⁶⁹ If mandatory folic acid fortification proceeds in the UK, with or without the addition of vitamin B₁₂, it will be important to closely monitor any health effects in the older population.

Conclusion

Low vitamin B₁₂ status is highly prevalent in older age and is much more common than generally perceived. There is a wealth of epidemiological evidence to suggest that optimal vitamin B₁₂ status is important for healthy ageing and that low status is associated with an increased risk of chronic diseases of ageing including CVD, dementia, cognitive impairment and osteoporosis. The evidence to date from RCTs examining the effect of B vitamin supplementation on chronic disease has often failed to demonstrate beneficial effects although many of these trials were secondary prevention trials. Thus, there is a need for large scale, well-designed intervention studies to investigate the potential benefits of vitamin B₁₂, alone or in combination with related B vitamins, in preventing disease in those without existing disease pathology and particularly in those with lower vitamin B₁₂ status. The importance of detecting low vitamin B₁₂ status has been highlighted in this review; however, conventional vitamin B₁₂ assays may lack sensitivity and could mean that low/deficient B₁₂ status remains undetected in a significant proportion of the older population. HoloTC shows promise as a sensitive biomarker of vitamin B₁₂ status; however, further investigations are needed to determine its sensitivity (and response to vitamin B₁₂ intervention) before its usefulness as the front-line tool in the diagnosis of vitamin B₁₂ deficiency can be confirmed. The issue of food-bound malabsorption of vitamin B₁₂ and consequences of mild subclinical deficiency have still not been fully explored and require further investigation. The possible adverse consequences of high folic acid exposure in older people with low vitamin B₁₂ status will require close monitoring over the coming years. Optimization of vitamin B₁₂ in older people should be an urgent public health priority.