Folate

Folate is a small water-soluble B-vitamin that is easily absorbed from the diet. Folate deficiency arises from poor diet, malabsorption or alcoholism, or from the use of certain drugs. It is common at all ages, including in childhood and during pregnancy. The prevalence of folate deficiency has declined markedly in populations with folate fortification. Folate is a cofactor for several different enzymes that enables them to transfer CH₃ groups required for the synthesis of DNA and proteins. Folate exists in different chemical forms distinguished by the oxidation state of the pteridine ring, the C₁ substitution at the N-5 and N-10 position and the number of conjugated glutamic acid residues attached to the molecule⁽Reference Smulders and Stehouwer²³⁾. The attached groups differ according to the particular pathway to which the C group is going to be donated either as CH₃, formyl (-CHO) and methylene (-CH₂-), which are collectively termed ‘one-carbon’ (C₁) groups. The derivatives of folate present in the human body are chiefly reduced folates, tetrahydrofolates and dihydrofolate, whereas folic acid is the oxidised form. Folic acid is chemically stable and used as a nutritional supplement, alone or as a component of B-vitamin or multivitamin preparations and for fortification. Folic acid itself, however, is not biologically active, since it cannot bind and pass on C₁ groups. After entry into the small intestinal enterocyte, dihydrofolate reductase acts on folic acid to convert it, via dihydrofolate, to tetrahydrofolate. Subsequently, the tetrahydrofolate requires conversion to 5-methyltetrahydrofolate (5-MTHF) for it to enter the circulation in the same form as natural food folates. It has been shown that folic acid in doses >200–400 μg/d exceeds the potential for intestinal and hepatic reduction and methylation, causing unmetabolised folic acid to appear in the systemic circulation. 5-MTHF is the predominant form of circulating folate, and it is taken up by cells by a carrier protein and folate receptor. After cell entry 5-MTHF-monoglutamate is available for Hcy remethylation, catalysed by methionine synthase. Deficiency of vitamin B₁₂ impairs the activity of methionine synthase and, as a result, 5-MTHF cannot donate its methyl group to Hcy, and Hcy levels rise. Since the conversion of methylenetetrahydrofolate to 5-MTHF is irreversible, there is no way back for 5-MTHF, and it becomes ‘trapped’ in its own metabolism. This ‘folate trap’ explains why vitamin B₁₂ deficiency and folate deficiency share many features. The ‘methylfolate trap’ results in less recycling of folates to tetrahydrofolate, which eventually leads to impaired DNA synthesis (i.e. megaloblastic anaemia). High-dose folic acid supplementation may resolve the problem, since unmetabolised folic acid entering the cell can be reduced to tetrahydrofolate and can subsequently be used for DNA synthesis. Such folic acid will eventually also get trapped as 5-MTHF, but if it is supplied continuously it may reverse the DNA synthesis defect (i.e. correct the megaloblastic anaemia) without correcting the impaired methylation cycle⁽Reference Smulders and Stehouwer²³⁾.

Vitamin B₁₂

Vitamin B₁₂ deficiency chiefly occurs in the elderly, in whom it is predominantly the result of malabsorption caused by lack of intrinsic factor (pernicious anaemia), gastric atrophy or ileal disease⁽Reference Schneede and Ueland²⁴–Reference Clarke, Sherliker and Hin²⁷⁾. Age-independent causes include inadequate intake (e.g. vegetarians) or use of certain drugs. Severe folate and vitamin B₁₂ deficiency often present with the identical haematological abnormality of macrocytic anaemia and megaloblastic bone marrow. Vitamin B₁₂ deficiency also causes a demyelinating neurological disease, usually presenting as a peripheral neuropathy. The neurological symptoms of vitamin B₁₂ deficiency may occur without anaemia, and early intervention is important in order to avoid irreversible damage. In man vitamin B₁₂ serves as a cofactor in only two enzyme reactions: methionine synthase, which is responsible for the methylation of Hcy into methionine; methylmalonyl-CoA mutase, which transforms methylmalonyl-CoA into succinyl-CoA. Deficiency of vitamin B₁₂ results in an accumulation of blood concentrations of Hcy and methylmalonic acid⁽Reference Schneede and Ueland²⁴⁾. Low serum vitamin B₁₂ concentrations have been reported in about 10% of older adults and the prevalence increases with age from about 5% at age 65 years to 20% at age 85 years⁽Reference Clarke, Refsum and Birks^19, Reference Clarke, Grimley Evans and Schneede²⁵⁾. In older adults individuals presenting with low vitamin B₁₂ concentrations rarely have the classical features of macrocytic anaemia and neuropathy. More commonly, such individuals present with non-specific symptoms of fatigue and cognitive impairment that can be attributed to ‘old age’. Some of the uncertainty about the importance of vitamin B₁₂ deficiency relates to the limitations of the standard vitamin B₁₂ assays. Low serum vitamin B₁₂ concentrations do not accurately reflect intracellular vitamin B₁₂ concentrations, and blood levels of Hcy or methylmalonic acid are believed to be more reliable indicators of intracellular vitamin B₁₂ status. About 80% of vitamin B₁₂ circulating in blood is biologically unavailable for most cells; the rest comprises holotranscobalamin, which is the part of serum vitamin B₁₂ bound to transcobalamin, the protein that delivers the vitamin to cells in the body, and is easily measured⁽Reference Clarke, Sherliker and Hin²⁷⁾.

The clinical symptoms of ‘classical’ vitamin B₁₂ deficiency, i.e. severe megaloblastic anaemia combined with neuropsychiatric symptoms (‘megaloblastic madness’) are rarely seen today. Vitamin B₁₂ deficiency is a slowly-progressive process that can take many years to develop. Nowadays, most cases are detected at an earlier stage, when clinical manifestations are often subtle and highly variable, and neuropsychiatric symptoms may occur in the absence of haematological signs. Thus, in clinical practice many patients may present with diffuse non-specific symptoms and vitamin B₁₂ deficiency is only one of many differential diagnoses⁽Reference Hin, Clarke and Sherliker²⁶⁾. As a consequence, the diagnostic value of most of these symptoms and signs is low.

Homocysteine

Hcy is a S-containing amino acid derived from methionine (following the loss of a CH₃ group) that is present in all cells⁽Reference Smulders and Stehouwer²³⁾. Hcy lies at a junction in C₁ metabolism between two metabolic cycles (remethylation and transulfuration) in all cells. In the remethylation pathway Hcy accepts a CH₃ group from 5-MTHF to form methionine. Vitamin B₁₂ is a cofactor and 5-MTHF a substrate for this remethylation reaction that is catalysed by methionine synthase. In the transulfuration pathway Hcy condenses with serine to form cystathionine in an irreversible reaction catalysed by the vitamin B₆-dependent cystathionine β-synthase enzyme. The intracellular levels of Hcy are highly regulated and any increased production is met by export from cells. Consequently, plasma concentrations of Hcy reflect intracellular concentrations of Hcy and homeostasis of the enzymes involved in methionine metabolism to ensure a supply of CH₃ groups for essential reactions in all cells. Hence, elevated Hcy levels reflect both low levels of folate and of vitamin B₁₂.

Dementia

Dementia is characterised by an insidious slowly-progressive memory loss with alteration of higher intellectual function and cognitive abilities⁽Reference Cummings²⁸⁾. The term ‘dementia’ is used to describe a collection of symptoms, including a decline in memory, reasoning and communication skills, and a gradual loss of skills needed to carry out daily activities⁽Reference Cummings²⁸⁾. These symptoms are caused by structural and chemical changes in the brain as a result of physical diseases. Different types of dementia now distinguished include Alzheimer's disease, vascular dementia and dementia with Lewy bodies. Alzheimer's disease is the most common cause of dementia. Alzheimer's disease and vascular dementia have distinct pathological features, but these two disorders frequently co-exist and the combination is associated with a greater severity of cognitive impairment. While clinicians have placed much emphasis on the distinction between dementia and cognitive impairment, the distinction may be viewed as arbitrary. Cognitive impairment is a quantitative disorder and its distribution in the population shows a continuum of severity with dementia at the tail of the distribution. About one in five adults aged >80 years and one in twenty of those aged >65 years have some form of dementia. The fact that cognitive impairment is common in the population does not imply that it is intrinsic to ageing. The distribution of cognitive impairment is shifted downwards with increasing age, such that the mean scores decrease and the prevalence of cognitive impairment increases. The prevalence of dementia among individuals in institutions varies little by age or gender, increasing from about 55% among those aged 65–69 years to 65% in those aged ≥95 years. It has been estimated that 700 000 individuals have dementia in the UK or 1·1% of the entire UK population, of which about 62% have Alzheimer's disease and 27% have vascular dementia and mixed dementia. About two-thirds of individuals with late-onset dementia live in private households and one-third live in care homes. The proportion of those with dementia living in care homes rises steadily with age, from one-quarter of those aged 65–74 years to two-thirds of those aged ≥90 years.

While the aetiology of Alzheimer's disease is unknown, some experts have speculated that the accumulation of β-amyloid peptide in the brain is central to the pathogenesis of Alzheimer's disease⁽Reference Cummings²⁸⁾. Mutations in the amyloid precursor proteins that lead to pre-senile dementia and overexpression of β-amyloid protein in Down's syndrome and mouse knock-out models have provided support for this hypothesis. Alternative hypotheses for the aetiology of Alzheimer's disease have placed greater emphasis on the role of vascular factors and neuronal cell death. The onset of dementia is insidious and the underlying disease is believed to begin many years before the manifestation of symptoms of dementia.

Vitamin B₁₂ and folate and risk of cognitive impairment and dementia

The hypothesis that elevated serum total Hcy may also be a risk factor for Alzheimer's disease was prompted by the observation in a retrospective case–control study that patients with histologically-confirmed Alzheimer's disease had higher concentrations of Hcy in blood samples collected before death than age-matched controls (Fig. 1)⁽Reference Clarke, Smith, Jobst, Refsum, Sutton and Ueland¹⁾. This longitudinal study compared Hcy levels taken during life from seventy-six cases with a histological diagnosis of ‘Alzheimer's disease’ made at post mortem with 108 controls without cognitive impairment. The results showed a 4·5 (95% CI 2·2, 9·2)-fold risk for histologically-confirmed Alzheimer's disease associated with Hcy levels in the upper, compared with the lower, third after controlling for age, gender, smoking, social class and apoE genotype. The Hcy measurements were carried out on blood samples that had been collected yearly for three successive years and were stable over this period and independent of the duration and severity of symptoms of dementia before enrolment.

Fig. 1. Cumulative frequency of serum folate, vitamin B₁₂ and homocysteine (Hcy) in patients with a histological diagnosis of Alzheimer's disease (—) and in controls (- -). (From Clarke et al.⁽Reference Clarke, Smith, Jobst, Refsum, Sutton and Ueland¹⁾.)

Subsequently, several prospective studies have confirmed these findings⁽Reference Seshadri, Beiser, Selhub, Jacques, Rosenberg, D'Agostino, Wilson and Wolf^16, Reference Ravaglia, Forti, Maioli, Martelli, Servadei, Brunetti, Porcellini and Licastro¹⁷⁾ but some studies have been unable to confirm such associations⁽Reference Luchsinger, Tang, Shea, Miller, Green and Mayeux¹⁸⁾. The most reliable evidence for the relevance of Hcy to risk of dementia comes from an 8-year follow-up prospective study of 1092 dementia-free elderly individuals that reported that elevated Hcy levels were associated with a 2-fold higher risk of dementia and of Alzheimer's disease (Fig. 2)⁽Reference Seshadri, Beiser, Selhub, Jacques, Rosenberg, D'Agostino, Wilson and Wolf¹⁶⁾. After adjustment for age, gender, apo-E genotype and vascular risk factors excluding Hcy and plasma levels of folate and vitamins B₁₂ and B₆, the relative risk for dementia was 1·4 (95% CI 1·1, 1·8) for a 1 sd increase in plasma Hcy concentrations⁽Reference Seshadri, Beiser, Selhub, Jacques, Rosenberg, D'Agostino, Wilson and Wolf¹⁶⁾.

Fig. 2. Risk of dementia in relation to homocysteine concentrations. (■–■) Subjects in the highest quartile of plasma homocysteine at baseline; (◆- -◆), all other subjects. (From Seshadri et al.⁽Reference Seshadri, Beiser, Selhub, Jacques, Rosenberg, D'Agostino, Wilson and Wolf¹⁶⁾.)

Several prospective studies of individuals without dementia have reported an association between baseline Hcy and subsequent cognitive decline. For example, the MacArthur Study of Successful Aging involving 499 men aged 70–79 years has reported that elevated Hcy and low folate, vitamin B₁₂ or vitamin B₆ status are each associated with poor cognitive function⁽Reference Kado, Bucur, Selhub, Rowe and Seeman¹²⁾. Brain-imaging studies have provided important information on the associations between Hcy and cognitive impairment and the underlying cerebrovascular and neurodegenerative changes. The initial case–control study of Hcy and Alzheimer's disease had shown that atrophy of the medial temporal lobe on computerised tomography scan of the brain of cases with Alzheimer's disease is more rapid in individuals with elevated Hcy concentrations⁽Reference Clarke, Smith, Jobst, Refsum, Sutton and Ueland¹⁾. In the Rotterdam Brain Scan Study of 1077 men and women aged 60–90 years plasma Hcy concentrations were found to be associated with increased risk of severe deep and periventricular white matter lesions and of silent brain infarcts in a cross-sectional analysis of MRI scans⁽Reference Vermeer, van Dijk and Koudstaal²⁹⁾. These MRI lesions were found to be three times more common in individuals in the top quintile of Hcy values compared with the bottom four quintiles. The severity of the white matter lesions was found to increase with increasing Hcy levels and the association remained significant even after adjustment for atherosclerotic disease and the presence of silent infarcts⁽Reference Vermeer, van Dijk and Koudstaal²⁹⁾. A subsequent analysis from the same study has reported that atrophy in the cerebral cortex and hippocampus is associated with elevated Hcy levels⁽Reference den Heijer, Vermeer and Clarke³⁰⁾. More recent evidence from a cross-sectional study of 1000 older adults in Banbury, Oxon., UK has demonstrated an association between cognitive impairment and low plasma levels of holotranscobalamin (the active fraction of vitamin B₁₂) and with high levels of methylmalonic acid (a metabolic marker of vitamin B₁₂ deficiency) in addition to elevated Hcy concentrations⁽Reference Hin, Clarke and Sherliker²⁶⁾.

It is possible that low vitamin B₁₂ may have an effect on risk of dementia that is independent of differences in plasma Hcy. Many of the Hcy-lowering trials designed for the prevention of CHD and stroke will include some assessment of cognitive function and may provide evidence about whether lowering Hcy concentrations (and administration of high-dose vitamin B₁₂) could slow the rate of cognitive decline.

Possible hazards of folic acid

Concerns that folic acid fortification could delay the diagnosis of vitamin B₁₂ deficiency or exacerbate the neurological or neuropsychiatric complications of vitamin B₁₂ deficiency has delayed the introduction of folic acid fortification in the UK⁽³¹⁾. Both case studies and epidemiological studies have reported that excessive intakes of folic acid among older adults with vitamin B₁₂ deficiency are associated with a more rapid progression of neuropathy or cognitive impairment⁽Reference Morris, Evans, Bienias, Tangney, Hebert, Scherr and Schneider²⁰,21,32–Reference Dhar, Bellevue and Carmel³⁴⁾. About 10–25% of older adults have biochemical evidence of low vitamin B₁₂ status, defined by a low serum concentration (<45 pmol/l) of holotranscobalamin, which is a more sensitive test of vitamin B₁₂ deficiency than conventional vitamin B₁₂ testing⁽Reference Clarke, Sherliker and Hin²⁷⁾. There have been reports that patients with pernicious anaemia who are treated with folic acid have an accelerated decline in neurological function⁽Reference Mills, Von Kohorn and Conley³²–Reference Dhar, Bellevue and Carmel³⁴⁾. Consequently, the amount of folic acid is routinely limited to a maximum of 1000 μg/d because of concerns about the adverse effects of high-dose folic acid in individuals with vitamin B₁₂ deficiency. In 1998 the USA introduced mandatory folic acid fortification of all grain products at a dose of 140 μg/100 g grain. It was believed that this level of fortification would increase the average daily intake by 100 μg/d. The prevalence of low serum folate has decreased from 16–22% pre-fortification to 0·5–1·7% post-fortification⁽Reference Pfeiffer, Caudill, Gunter, Osterloh and Sampson³⁵⁾. The required level of fortification was considered generally safe. However, concern persists about the safety of folic acid fortification in older adults with vitamin B₁₂ deficiency. In the USA the introduction of folic acid fortification has resulted in 200–300% increases in serum folate concentrations⁽Reference Pfeiffer, Caudill, Gunter, Osterloh and Sampson³⁵⁾ and voluntary fortification in the UK has resulted in substantial changes in serum folate concentrations⁽³¹⁾.

Elevated Hcy levels in older adults may reflect impaired status of vitamin B₁₂, folate or a combination. However, the relative importance of vitamin B₁₂ deficiency as a determinant of Hcy concentrations and cognitive impairment is probably greater than that of folate deficiency in older adults⁽Reference Clarke, Sherliker and Hin²⁷⁾. Cross-sectional studies of older adults have shown that a high proportion of older adults have biochemical evidence of low vitamin B₁₂ status, and the prevalence of low vitamin B₁₂ status increases from 5% at age 65 years to 20% at age 80 years⁽Reference Clarke, Grimley Evans and Schneede²⁵⁾. The extent to which the associations between low vitamin B₁₂ status and risk of dementia are causal is unclear⁽Reference Clarke, Smith, Jobst, Refsum, Sutton and Ueland^1, Reference Clarke²⁾. Moreover, low vitamin B₁₂ status may be more relevant in the setting of mandatory folic acid fortification. Consequently, there is some concern, particularly in countries with mandatory folic acid fortification, that individuals with low vitamin B₁₂ status may have more rapid deterioration of neurological function in the context of a high intake of folate. A recent cross-sectional study of 1459 older adults in the USA carried out after the introduction of mandatory fortification has reported that low vitamin B₁₂ (<150 pmol/l) and high serum folate (>60 nmol/l) is associated with a 5-fold increased risk of cognitive impairment⁽Reference Morris, Jacques, Rosenberg and Selhub²¹⁾ compared with with normal levels, providing some evidence of a possible hazard of high levels of folic acid fortification.

It is important to ascertain the relevance, if any, of vitamin B₁₂ for risk of brain disease in older adults by carrying out randomised trials of vitamin B₁₂ supplements in older adults. Table 1 shows several completed and ongoing randomised trials that have assessed, or are assessing, the effects of Hcy-lowering vitamin supplements on vascular disease⁽Reference Clarke^{2, 36)}. It is unclear whether any of these trials will be able to determine the independent relevance of vitamin B₁₂ to folic acid use for prevention of cognitive impairment (Table 2).

Table 1. Characteristics of the homocysteine-lowering trials for prevention of CVD

CHAOS-2, Second Cambridge Anti-Oxidant Heart Study; SU.FOL.OM3, Supplementation en Folate et Omega-3; WENBIT, West of Norway Vitamin Intervention Trial; NORVIT, Norwegian Vitamin Intervention Trial; SEARCH, Study of Additional Reductions in Cholesterol and Homocysteine; VISP, Vitamin Intervention for Stroke Prevention; HOPE-2, Heart Outcomes Prevention Evaluation-2; WACS, Women's Antioxidant Cardiovascular Study; VITATOPS, The Vitamin Intervention to Prevent Strokes Trial; FAVORIT, Folic Acid for Vascular Outcome Reduction In Transplantation; HOST, Homocysteine Study Veteran Affairs Cooperative Study.

* Trial terminated early after a median duration of treatment of 1·7 years; 187 participants experienced a vascular event.

† Trial scheduled to be completed in 2009.

‡ Trial terminated early after the publication of the null findings of NORVIT; results scheduled to be published in 2007.

§ Trial scheduled to be completed in 2008.

∥ Trial was carried out mainly in Canada and USA (both populations with mandatory folic acid fortification), but also included some participants from Brazil, Slovakia and Western Europe.

¶ Trial treatment completed; results scheduled to be published in 2007.

** Participants recruited from twenty countries (Australia, Belgium, Brazil, Hong Kong, India, Italy, Malaysia, Moldova, Netherlands, New Zealand, Pakistan, Philippines, Portugal, Republic of Georgia, Serbia, Monte Negro, Singapore, Sri Lanka, UK and USA) and scheduled to be completed in 2008.

†† The trial terminated early; no significant effect on the risk of recurrent stroke during the 2 years of follow-up.

‡‡ Trial scheduled to be completed in 2011.

§§ Scheduled trial treatment period now completed; results scheduled to be published in 2007.

Table 2. Estimated power of the individual homocysteine-lowering trials and combination of the large trials in individuals with previous CHD, stroke or renal disease to detect differences in risk of 10% or 20% for major coronary events (MCE; non-fatal MI+fatal CHD), stroke (non-fatal or fatal stroke) and major vascular events (MVE; non-fatal MI+fatal CHD+non-fatal stroke+revascularisation)Footnote *

CHAOS-2, Second Cambridge Anti-Oxidant Heart Study; SU.FOL.OM3, Supplementation en Folate et Omega-3; WENBIT, West of Norway Vitamin Intervention Trial; NORVIT, Norwegian Vitamin Intervention Trial; SEARCH, Study of Additional Reductions in Cholesterol and Homocysteine; VISP, Vitamin Intervention for Stroke Prevention; HOPE-2, Heart Outcomes Prevention Evaluation-2; WACS, Women's Antioxidant Cardiovascular Study; VITATOPS, The Vitamin Intervention to Prevent Strokes Trial; FAVORIT, Folic Acid for Vascular Outcome Reduction In Transplantation; HOST, Homocysteine Study Veteran Affairs Cooperative Study; approx, approximate.

* For details of trials, see Table 1.

† No. of subjects scheduled to be randomised.

Cumulative meta-analysis of all randomised trials will assess the effects of lowering Hcy levels with B-vitamins on risk of CVD⁽³⁶⁾. An individual-patient-data meta-analysis of all randomised trials of the effects on vascular risk of lowering Hcy with B-vitamins will maximise the power to assess the epidemiologically-predicted differences in risk (Table 2). Among the twelve randomised Hcy-lowering trials for prevention of CVD involving >1000 participants, data should be available on about 52 000 participants (32 000 with previous CVD in unfortified populations; 14 000 with previous CVD and 6000 with renal disease in fortified populations). In order to minimise bias the design and primary analyses to be carried out have been pre-specified. The analyses will include assessment of effects on major vascular events, stroke and major coronary events, in addition to venous thrombosis, cancer and cognitive function. Additional analyses will assess effects on vascular outcomes in subgroups defined by population, previous disease, the per 3 μmol/l difference in Hcy levels achieved by treatment, pre-treatment vitamin status, duration, age, gender and vascular events excluding revascularisations and, separately, excluding vascular events occurring during the first year of treatment. This meta-analysis of the Hcy-lowering trials should ensure that reliable evidence emerges about the effects of lowering Hcy on risk of vascular and non-vascular outcomes, including cognitive function.

Further trials of vitamin B₁₂ supplementation or placebo involving a large number of elderly participants who are high risk are required in order to assess the relevance of vitamin B₁₂ supplements or placebo for the prevention of cognitive impairment and dementia. In the Folic Acid and Carotid Intima-media Thickness Trial 818 healthy middle-aged adults (age 60 years) were randomised to folic acid (0·8 mg) for 3 years, resulting in a 26% lowering of Hcy concentration and a modest improvement in some domains of cognitive function⁽Reference Durga, van Boxtel, Schouten, Kok, Jolles, Katan and Verhoef³⁷⁾. A systematic review of fourteen randomised trials of vitamin B₆, vitamin B₁₂ or folic acid supplementation and cognitive function has concluded that there is insufficient evidence of beneficial effects of these vitamins on cognitive function⁽Reference Balk, Raman, Tatsioni, Chung, Lau and Rosenberg³⁸⁾.

The results of these ongoing trials of B-vitamins are required before B-vitamin supplementation can be recommended for the prevention of dementia⁽Reference Pfeiffer, Caudill, Gunter, Osterloh and Sampson³⁵⁾. Nevertheless, the available evidence suggests that the benefits of folic acid fortification for the prevention of neural-tube defects are likely to outweigh any possible hazards of folic acid fortification for older adults provided public health strategies avoid an excessive intake of folic acid in older adults with vitamin B₁₂ deficiency. Thus, if mandatory fortification with folic acid is introduced in the UK it will be important to control voluntary fortification of breakfast cereals and spreads (which have already had a substantial effect on increasing the population mean folate levels) to avoid any potential hazard in older adults associated with excessive intakes of folic acid in the setting of vitamin B₁₂ deficiency.