1. Background
Surprisingly, given their pivotal physiological significance, our understanding of the role of the B group of vitamins (thiamine (B1), riboflavin (B2), niacin (B3), pantothenic acid (B5), vitamin B6, folate (B9) and vitamin B12) in health and brain function is limited in several respects. As an example, the major human epidemiological and controlled trial research effort in this area has concentrated almost exclusively on that small sub-set of B vitamins (folate, vitamin B12 and, to a lesser extent vitamin B6) that play the most obvious roles in homocysteine metabolism. The multifarious inter-related roles of the remaining five B vitamins have been largely overlooked. Possibly as a result of this, the many intervention studies that have involved administering just folic acid ± vitamins B12 and/or B6, have generated equivocal results. Similarly, whilst we have some knowledge of the minimum levels of each B vitamin required in order to prevent explicit deficiency related diseases, we have a poor understanding of the negative effects of levels of consumption that lie above the minimum, but under the optimal level of consumption for these vitamins. Indeed, we have no clear idea of where the optimal level of consumption may lie.
What Are Vitamins?
Vitamins are a group of organic compounds which are essential for normal physiological functioning but which are not synthesised endogenously by the body and therefore have to be sequestered in small quantities from the diet. In total, humans require adequate amounts of 13 vitamins: four fat soluble vitamins (A, D, E, K) and nine water soluble vitamins, which comprise vitamin C and the eight B vitamins: thiamine (B1), riboflavin (B2), niacin (B3), pantothenic acid (B5), vitamin B6, folate (B9) and vitamin B12. The B vitamins themselves are not grouped on the basis of any chemical structural similarity, but rather with regards to their water solubility and the inter-related, cellular coenzyme functions that they play (see Section 2).
One key point is that we, and other animals, have generally lost the ability to synthesise a clade-specific palette of vitamins during our evolution. The apparent evolutionary paradox of why an organism would benefit from losing the ability to synthesise a compound required for its survival is resolved by the fact that, during the course of evolution, vitamins have been in ubiquitous and plentiful supply within the food chain.
With regards to human vitamin requirements, the clearest example of this process is the monosaccharide “vitamin C”, which is produced endogenously during normal metabolism by most other animals. The only exceptions to this are guinea pigs, bats, a few passerine birds and the anthropoidea (tarsiers, monkeys and apes, including humans). In the case of humans and our close primate relatives, our inability to synthesise vitamin C is due to a mutation in the gene for l-gulonolactone, an enzyme in the synthetic pathway of ascorbate, which was lost by our common ancestor some 35–55 million years ago [5].
2. Mechanisms of Action and Functions of B Vitamins
B vitamins act as coenzymes in a substantial proportion of the enzymatic processes that underpin every aspect of cellular physiological functioning. As a coenzyme the biologically active form of the vitamin binds within a protein “apoenzyme” creating a “holoenzyme”, thereby increasing the resultant enzyme’s competence in terms of the diversity of reactions that it can catalyse [8]. In this role, the B vitamins play key interacting roles in the majority of cellular functions. As an example of their ubiquity, the primary bioactive form of vitamin B6, pyridoxal 5′-phosphate, is an essential cofactor in the functioning of over 140 separate ubiquitous enzymes required for the synthesis, degradation, and interconversion of amino acids [15], whereas the active coenzyme form of pantothenic acid, coenzyme A (CoA), is an obligatory co-factor for approximately 4% of all mammalian enzymes [31]. Less often B vitamins also function as direct precursors for metabolic substrates; for example, CoA is also acetylated to form acetyl-CoA, an intermediate compound in both the generation of cellular energy and the synthesis of multiple bioactive compounds. Similarly, niacin is a precursor for ADP-ribose, which functions in multiple non-enzymatic cellular roles.
Catabolic energy production: One or more of the B vitamins are involved in every aspect of the absolutely essential catabolic process of generating energy within cells [17], and deficiency in any one B vitamin will have negative consequences for this process. Of particular relevance here, the active forms of thiamine, riboflavin, niacin, and pantothenic acid are essential co-enzymes in mitochondrial aerobic respiration and cellular energy production via their direct roles in the citric acid cycle, the electron transport chain and the resultant formation of adenosine triphosphate (ATP), the cell’s energy currency. Acetyl-CoA (incorporating pantothenic acid) provides the main substrate for this cycle [9,11,14,32,33,34]. In addition, thiamine and biotin/vitamin B12 play unique, intersecting, essential roles in the mitochondrial metabolism of glucose [9] and fatty acids and amino acids, respectively [11], thereby contributing substrates to the citric acid cycle. The inter-related contribution of the B vitamins to the citric acid cycle and electron transport chain, the central catabolic process in mitochondria, is illustrated in Figure 1.
Anabolic processes: The vitamin-dependent, citric acid cycle furnishes not only energy, but also the intermediaries for the biosynthesis of numerous key compounds, including amino acids, fatty acids and pyrimidines. A number of B vitamins also play essential roles in all aspects of one-carbon metabolism [32,33,34,35], the process by which functional compounds, such as amino acids, purines, and pyrimidines, as well as methyl groups required by molecules in order for them to take part in biochemical reactions, are created within cells by the addition of single units of carbon. Of particular relevance, several B vitamin coenzymes are intrinsic contributors to two ubiquitous inter-related cellular processes: the “folate cycle”, during which tetrahydrofolate (one active form of folate) from the diet cycles through several enzymatic modifications which ultimately provide the one-carbon units required for one carbon metabolism, and the “methionine cycle” during which the amino acids methionine and homocysteine are interconverted, resulting in the synthesis of the methyl groups required for all genomic and non-genomic methylation reactions in the form of S-adenosyl methionine (SAM). These two enzymatic cycles are essential to cellular function, including via interactions with other pathways. As an example of the latter, the re-salvaging from dihydrobiopterin of tetrahydrobiopterin, an essential cofactor in trace amine and catecholamine neurotransmitter synthesis and nitric oxide production, is rate limited by provision of the enzyme dihydrofolate reductase produced by the folate cycle [36,37]. Similarly, the trans-sulfuration pathway that converts homocysteine to cysteine, ultimately leading to the synthesis of the potent endogenous antioxidant glutathione and the generation of substrates for the citric acid cycle, is a direct product of the methionine cycle. Whilst the roles of folate and vitamins B6 and B12 are well recognised in these intersecting cycles (see “The homocysteine hypothesis” below), the contribution of other B vitamins is rarely acknowledged. In this regard, the active form of riboflavin is a coenzyme with methyltetrahydrofolate reductase (MTHFR) in the folate cycle, and rate limits the recycling of methionine synthase in the methionine cycle [22]. Similarly, niacin, in the form of NAD, is a necessary co-factor for the enzymes dihydrofolate reductase in the folate/tetrahydrobiopterin cycles and S-adenosylhomocysteine hydrolase in the methionine cycle. The eventual functional products of these intersecting cellular cycles and the rate-limiting contributions made by the full range of B vitamins are illustrated in Figure 2.
Just one of the many consequences of a deficiency in any of these B vitamins (see Figure 2) is a potential hampering of the natural breakdown and recycling of homocysteine, leading to its accumulation and a number of potential, negative cellular consequences. Alongside this, the observation that homocysteine levels are increased in those suffering a range of pathologies including cardiovascular and neurodegenerative diseases has resulted in the “homocysteine hypothesis” that has driven much of the human research into the effects of B vitamins on brain function. This hypothesis will be described and discussed in more detail below.
2.1. Brain Specific Roles of B Vitamins
Indeed, the importance of the B vitamins for brain function is illustrated by the fact that each vitamin is actively transported across the blood brain barrier and/or choroid plexus by dedicated transport mechanisms. Once in the brain, specific cellular uptake mechanisms dictate distribution, and, whilst the B vitamins all have high turnovers, ranging from 8% to 100% per day, their levels are tightly regulated by multiple homeostatic mechanisms in the brain [39,40].
For example, the concentration of methyltetrahydrofolate (the principal circulating form of folate) in the brain is four times that seen in plasma [39], whereas biotin and pantothenic acid exist in the brain at concentrations of up to 50 times that seen in plasma [41].
2.1.1. Thiamine (Vitamin B1)
Thiamine is a coenzyme in the pentose phosphate pathway, which is a necessary step in the synthesis of fatty acids, steroids, nucleic acids and the aromatic amino acid precursors to a range of neurotransmitters and other bioactive compounds essential for brain function [9]. Thiamine plays a neuro-modulatory role in the acetylcholine neurotransmitter system, distinct from its actions as a cofactor during metabolic processes [42] and contributes to the structure and function of cellular membranes, including neurons and neuroglia [35].
2.1.2. Riboflavin (Vitamin B2)
The two flavoprotein coenzymes derived from riboflavin, FMN and FAD are crucial rate limiting factors in most cellular enzymatic processes. As an example, they are crucial for the synthesis, conversion and recycling of niacin, folate and vitamin B6, and for the synthesis of all heme proteins, including hemeglobin, nitric oxide synthases, P450 enzymes, and proteins involved in electron transfer and oxygen transport and storage [11]. The flavoproteins are also co-factors in the metabolism of essential fatty acids in brain lipids [12], the absorption and utilisation of iron [43], and the regulation of thyroid hormones [11]. Dysregulation of any of these processes by riboflavin deficiency would be associated with its own broad negative consequences for brain function. Riboflavin derivatives also have direct antioxidant properties and increase endogenous antioxidant status as essential cofactors in the glutathione redox cycle [44].
2.1.3. Niacin (Vitamin B3)
A vast array of processes and enzymes involved in every aspect of peripheral and brain cell function are dependent on niacin derived nucleotides such as nicotinamide adenine dinucleotide (NAD) and NAD phosphate (NADP). Beyond energy production, these include oxidative reactions, antioxidant protection, DNA metabolism and repair, cellular signalling events (via intracellular calcium), and the conversion of folate to its tetrahydrofolate derivative [45]. Niacin also binds agonistically at two G protein receptors, the high affinity Niacin receptor 1 (NIACR1), responsible for the skin flush associated with high intake of niacin, and the low affinity NIACR2. Niacin receptors are distributed both peripherally in immune cells and adipose tissue, and throughout the brain. Currently established roles include modulation of inflammatory cascades [46,47] and anti-atherogenic lipolysis in adipose tissue [48,49]. NIACR1 receptor populations have been shown to be down-regulated in the anterior cingulate cortex of schizophrenia sufferers [46] and upregulated in the substantia nigra of Parkinson’s disease sufferers, (a group that have low niacin levels generally) with levels correlating with poorer sleep architecture in this group [50]. A recent case study demonstrated that 250 mg niacin administration modulated peripheral immune cell NIACR1 expression and attenuated the disturbed sleep architecture associated with Parkinson’s disease [51].
2.1.4. Pantothenic Acid (Vitamin B5)
This vitamin is a substrate for the synthesis of the ubiquitous coenzyme A (CoA). Beyond its role in oxidative metabolism, CoA contributes to the structure and function of brain cells via its involvement in the synthesis of cholesterol, amino acids, phospholipids, and fatty acids. Of particular relevance, pantothenic acid, via CoA, is also involved in the synthesis of multiple neurotransmitters and steroid hormones [14].
2.1.5. Vitamin B6 (Pyridoxine, Pyridoxal, Pyridoxamine)
Beyond its role as a necessary cofactor in the folate cycle (see above and folate section below), the role of vitamin B6 in amino acid metabolism makes it a rate limiting cofactor in the synthesis of neurotransmitters such as dopamine, serotonin, γ-aminobutyric acid (GABA), noradrenaline and the hormone melatonin. The synthesis of these neurotransmitters is differentially sensitive to vitamin B6 levels, with even mild deficiency resulting in preferential down-regulation of GABA and serotonin synthesis, leading to the removal of inhibition of neural activity by GABA and disordered sleep, behaviour, and cardiovascular function and a loss of hypothalamus-pituitary control of hormone excretion.
More broadly, levels of pyridoxal-5′-phosphate are associated with increased functional indices and biomarkers of inflammation, and levels of pyridoxal-5′-phosphate are down-regulated as a function of more severe inflammation [53,54], potentially as a consequence of pyridoxal-5′-phosphate’s role either in the metabolism of tryptophan or in one-carbon metabolism [53]. This role is particularly pertinent as inflammatory processes contribute to the aetiology of numerous pathological states including dementia and cognitive decline [55].
2.1.7. Folate (Vitamin B9) and Vitamin B12 (Cobolamin)
The functions of these two vitamins are inextricably linked due to their complementary roles in the “folate” and “methionine” cycles. Indeed, a deficiency in vitamin B12 results in a functional folate deficiency, as folate becomes trapped in the form of methyltetrahydrofolate [11,19]. An actual or functional folate deficiency, with an attendant reduction in purine/pyrimidine synthesis and genomic and non-genomic methylation reactions in brain tissue, leads to decreased DNA stability and repair and gene expression/transcription, which could hamper neuronal differentiation and repair, promote hippocampal atrophy, demyelination and compromise the integrity of membrane phospholipids impairing the propagation of action potentials [45]. Folate related downregulation of the synthesis of proteins and the nucleotides required for DNA/RNA synthesis, has ramifications for rapidly dividing tissue in particular, and therefore underlies the foetal developmental disorders and megaloblastic anaemia (alongside aspects of neuronal dysfunction), associated with either folate or vitamin B12 deficiency [11,19,45]. The efficient functioning of the folate cycle is also necessary for the synthesis and regeneration of tetrahydrobiopterin, an essential cofactor for the enzymes that convert amino acids to both monoamine neurotransmitters (serotonin, melatonin, dopamine, noradrenaline, adrenaline), and nitric oxide [56,57] (see Figure 2).
3. The Homocysteine Hypothesis
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One unfortunate consequence of the “homocysteine hypothesis” is that it has effectively funneled the majority of clinical trial research in this area towards elucidating the effects of folic acid, and to a decreasing extent vitamin B12 followed by vitamin B6. The potential effects and roles of the other five B vitamins have been almost entirely ignored, despite the fact that the entire palette of B vitamins work intricately in concert. As an example, staying with the homocysteine theme, the status of folate and vitamin B6/B12 are themselves dependent on levels of riboflavin derived flavoproteins. Riboflavin is also essential for the metabolism of homocysteine as a cofactor for methylenetetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR) [11,12,22].
The potential limitations of administering a restricted range of B vitamins are illustrated by evidence showing that approximately a third of supplementation studies to date have involved the administration of folic acid alone [65,66]. As noted above, folate and vitamin B12 are intimately interlinked within the folate/methionine cycles, and increasing the level of folate can mask the accrual of permanent neurological damage associated with a specific vitamin B12 deficiency [20]. A striking illustration of this was provided by an epidemiological study by Morris et al. [75] who reported that high folate status was associated with protected cognitive function, but only in those with normal vitamin B12 status, with this relationship reversed in participants with low vitamin B12 status. For this group, high folate status exacerbated the detrimental effect of vitamin B12 deficiency, increasing the risk of cognitive impairment and anaemia by a factor of five, compared to those with normal vitamin status.
4. B Vitamin Deficiencies in Developed Societies
A general assumption tends to be made that the populations of developed countries have adequate nutrition, and are therefore free from deficiencies in essential micronutrients. In order to encourage adequate nutrition, governments typically define a set of “dietary reference intakes” or similar for individual nutrients. These always include something akin to the “recommended dietary allowance”, or RDA. These government figures describe the minimum daily intake of the specific nutrient that is considered to be sufficient to meet the nutritional requirement of the majority of the healthy population. However, “meeting the requirements” in this context typically refers to simply preventing chronic, nutrition related diseases or a disease state related to a specific deficiency of that nutrient (see Table 1).
RDAs are population statistics and they therefore represent rough estimates of the average requirement of individuals within a group/population, with an adjustment for the variations in the need for the nutrient among the individuals that make up the population. However, for most micronutrients some of the information that would be required to accurately calculate the daily requirement is either unknown or incomplete, and the recommendations are therefore made on the basis of a number of assumptions and considerations that could lead to large variations in the eventual RDA [81,82]. These figures have also changed little in the last four decades, despite emerging evidence of striking individual differences in the absorption and excretion of vitamins as a consequence of a wide range of factors, including specific genetic polymorphisms, gender, ethnicity, endocrine dysfunction, thyroid function, the habitual co-consumption of medicines, drugs, alcohol and other dietary factors, obesity, overall energy consumption, vigorous exercise, and age [9,21,45,83,84,85,86]. These gaps in our knowledge question the very existence of a “normal” population [87], and suggest that RDAs are, to some extent, arbitrary figures.
Government figures also show that sizeable minorities of the populations of developed countries fail to consume even the minimum recommended quantity of any given micronutrient. As an example, Troesch et al. [88] presented data showing that a sizeable proportion of the populations of the US and several European countries consume less than the RDA for each of the five B vitamins that they assessed. They note that “a gap exists between vitamin intakes and requirements for a significant proportion of the population”. As a result, studies assessing the blood levels of vitamins show that small but significant proportions of the populations of developed countries have biochemical levels of each of the B vitamins that may well predispose them to deficiency related diseases. For example, UK government figures [89] show adult deficiency levels of 3% for vitamin B12 and 5% for folate, with these figures increasing to 5% and 12%, respectively, in the lower socio-economic portion of the population [90]. In the US, the story is similar. For instance, recent US government data [91] demonstrated that 10.5% of the entire US population were biochemically deficient in vitamin B6. A subsequent independent analysis that excluded the substantial minority taking supplements containing vitamin B6 demonstrated much higher deficiency rates of between 23% and 27% for adults, depending on age [16]. Similarly, larger proportions of “at risk” groups exhibit deficiencies in vitamin B12. As an example, more than 30% of a nationally representative US sample of adults over 60 years of age were below deficiency levels (<148 pmol/L) [75]. This may well be due to an age-related impairment in the absorption of the protein-bound vitamin B12 found in food [23], although it should be noted that deficiency levels in this vitamin are similar for vegetarians and vegans, simply due to a lack of consumption [24,92]. It has also been suggested that the available evidence suggests that the typical cut-off point defining deficiency in B12 is simply set too low, with negative health effects associated with reduced vitamin B12 extending well into “normal” levels of this vitamin [93]. Thiamine deficiency levels are also higher in the elderly, with 16%–18% deficient [94]. It is also worth noting that, whilst riboflavin deficiency levels are under-researched, biochemical deficiency is potentially widespread due to the high prevalence of an inherited restriction of riboflavin absorption/utilisation that affects 10%–15% of the world population [12].
One factor that also continues to exert an upwards pressure on deficiency levels is the paradoxical malnutrition associated with obesity. For instance, thiamine plays an essential role in glucose metabolism, and between 15.5% and 29% of obese patients examined prior to bariatric surgery across a number of studies were found to be deficient. Similarly, thiamine deficiency rates have been reported to vary between 17% and 79% in patients suffering from the gluco-regulatory disease diabetes [9,18]. In a similar vein, both Type II diabetes and increased fasting plasma glucose levels have been found to be associated with lower levels of biotin [18].
Of course, an individual may not be technically deficient in a micronutrient, but may still be in the much more common state of “marginal deficiency” which will still predispose them to an increased risk of a number of more general disease states (e.g., [93,97,98]). The US government, in a recent report on micronutrient levels in the US population [91], gave their first official acknowledgement of the dangers of non-deficient but less than optimal nutritional status when the report stated that, whereas the effects of outright dietary deficiencies are well documented, “In addition, recent findings have determined that less than optimal biochemical concentrations (representing suboptimal status) have been associated with risks of adverse health effects”. Levels of marginal deficiency are, by definition, much higher than levels of outright deficiency for all of the vitamins. As an example, both Smith and Refsum [93] and Tucker et al. [23] noted that the neurological/psychological manifestations of vitamin B12 insufficiency can be evident at much higher serum levels of this vitamin than those marking deficiency. Indeed, Tucker et al. [23] found that whilst 9% of their sample of 3000 adults were frankly deficient in vitamin B12 (<148 pmol/L), over 38% had serum levels (<258 pmol/L) suggesting marginal deficiency. These figures are broadly in line with analyses of US data showing that 17.8% of all adults in the USA were marginally deficient in vitamin B12 using a more stringent cut-off (220 pmol/L) [96], and analyses of more recent population data showing that over 20% of the over 50 years age group in the US were marginally deficient in vitamin B12 between 2001 and 2006 [99]. In terms of other B vitamins, a striking 66% of the UK non-elderly adult population were at least marginally deficient in riboflavin (as assessed by the erythrocyte glutathione reductase activation test (EGRAC)) [89], with a similar figure of 54% derived in another study when a slightly more stringent EGRAC was used [72].
Taken as a whole, these figures suggest that a very sizeable proportion of the populations of developed countries are suffering deficiency or marginal deficiency in one or more B vitamins that may, at the least, dispose them to a variety of chronic diseases. Just as the minimum daily requirement of many micronutrients is simply unknown at present, the optimal level has received no attention at all. As one review paper [100] notes, even the governmental agencies responsible for defining dietary recommendations acknowledge that the benefits of micronutrient consumption may continue on a continuum well above the RDA. Clearly, common sense dictates that the optimal level of consumption of any nutrient will not merely be the level that prevents diseases related to a deficiency, or even marginal deficiency, in that nutrient. In line with this, a wealth of epidemiological evidence suggesting relationships between the increased consumption/biochemical levels of a number of vitamins, and benefits for cardiovascular function, cognitive function and decreased incidence of dementia clearly show that individuals derive additional relevant physiological benefits from consumption of micronutrients well in excess of the RDA, and biochemical levels above those denoting marginal deficiency (see [98,101]). This evidence will be summarised below.
5. How Much Is Enough?
As the B vitamins are water-soluble, any excess is generally excreted in urine. On the one hand, this means they are typically safe at doses much higher than the RDA, but on the other hand, they require a more consistent consumption than the fat soluble vitamins. In terms of safety, only three of the eight B-vitamins have been ascribed any upper limit for daily consumption, with the remainder considered safe at any dose [14,20]. In the case of folic acid, which is ascribed RDAs typically between 200 and 400 µg/day, the upper limit is generally set at 1000 µg/day simply on the basis that increased folate can mask the symptoms of vitamin B12 deficiency, allowing a hidden accumulation of permanent damage related to the latter vitamin [102]. It should also be noted that evidence suggests a potential detrimental effect of consuming high doses of folic acid, and therefore raised levels of un-metabolised folic acid, on normal folate metabolism and immune function. High folate levels may also interfere with the anti-folate medications prescribed for a number of conditions (e.g., rheumatoid arthritis, psoriasis, cancer, bacterial infections, malaria) and exert biphasic effects with regards to cancer; conferring protection at lower concentrations but increasing carcinogenesis at higher concentrations. However, to date there is no consensus as to the blood levels of folates that might cause harm [103]. The upper limit for niacin is set at 35 mg (US/Canada), with this predicated simply on its ability to cause temporary flushing of the skin at doses in excess of 100 mg, although nausea, vomiting, diarrhoea and in very rare cases liver damage have been noted following extended consumption of doses of a gram and more [8]. The final B vitamin with an ascribed upper limit is vitamin B6 which has an upper limit set at 100 mg/day (approximately 75 × RDA) in the US on the basis of case reports of reversible sensory neuropathy following doses in excess of 1000 mg taken for extended periods. However, it is notable that multiple clinical trials entailing consuming up to 750 mg/day of vitamin B6 for a number of years have demonstrated a lack of neuropathic side effects [15].
As noted above, the optimum level of any micronutrient must lie well above the RDA, and the B vitamins can generally be consumed at many times the RDA. This raises the question of how much of these vitamins should we consume? Whilst this issue is poorly understood to date, several strands of evidence suggest that increasing consumption well above the RDA should be a more effective strategy. The first strand of evidence for this comes from dose-ranging studies that have demonstrated increases in bioavailability persisting well above the RDA. For instance, Smithline et al. [104] demonstrated a shallow, linear dose response following single oral doses of thiamine in terms of whole blood and plasma levels up to the maximum administered dose of 1500 mg (corresponding to more than 1000 times the RDA), in healthy subjects. Similarly, one study [105] demonstrated an approximately linear dose-response in serum levels of vitamin B12 among adults which persisted to more than 100 µg/day of supplement use (40 × RDA), but with a plateau in levels at lower doses being evident for middle-aged and older adults. A subsequent meta-analysis [106] of the results of vitamin B12 supplementation studies with doses ranging from approximately 1 RDA up to 400 RDA (i.e., 1000 µg) administered for between four weeks and two years, showed that for every doubling of intake above the RDA, blood levels of Vitamin B12 continued to increase by 11%, while methylmalonic acid levels, an indicator of deficiency, decreased by 7%. This dose response is potentially most relevant to older adults (>50 years), who typically suffer age associated malabsorption of dietary vitamin B12 and therefore high levels of insufficiency. Certainly, in a dose-response study, Eussen [107] found that the most effective dose for normalising vitamin B12 status in marginally deficient older adults was 500 µg/day (200 × RDA). It is also notable that a dose of 1 mg/day folic acid (2.5 × RDA) for 12 months was required to achieve maximal steady state erythrocyte folate concentrations in older adults [108].
In terms of potentially beneficial physiological responses to increased dosage, Eussen et al.’s [107] study was particularly interesting in that it also demonstrated a linear negative dose-response up to the maximum dose of 1000 µg/day Vitamin B12 (i.e., 400 × RDA) with regards to the reductions in plasma levels of homocysteine. A clear dose response was also evident in a meta-analysis of 25 folic acid studies, with 800 µg/day (2–4 × RDA) required to achieve peak reductions in plasma homocysteine of 23%, with the addition of a median dose of 400 µg/day vitamin B12 (i.e., 166 × RDA) associated with a further fall of 7% [109]. Interestingly, single doses of folic acid, and chronic supplementation with vitamin B6, folic acid and their combination, all taken at a minimum of 12 times their RDAs have all been shown to improve endothelial function in patient groups or following laboratory induced endothelial dysfunction. These effects were independent of any effect of these vitamins on homocysteine levels [110,111]. In population studies, intakes of vitamin B6 well in excess of the RDA, along with associated biochemical levels of pyridoxal-5′-phosphate, have also been found to be inversely related to a range of inflammatory biomarkers, with those individuals exhibiting higher levels of inflammatory biomarkers requiring several times the RDA of vitamin B6 merely to avoid deficiency [53,54].
With regards to riboflavin, the highest of two doses (4 mg/day, i.e., 3 × RDA) administered for eight weeks to young females had the greatest effects both on riboflavin status and benefits to haematological parameters [43]. It is also notable that, whilst riboflavin has no demonstrable toxicity, the maximum daily intestinal absorption is approximately 20 times the RDA of 1.3 mg. Doses of this magnitude for up to eight weeks are also required to replenish riboflavin levels and correct enzymatic activity in the 10%–15% of the population who have an inherited restriction in their ability to absorb riboflavin [12].
Evidence (see below) also shows that “mega-doses” of biotin and niacin at between 30 and 500 times the RDA exert beneficial physiological effects, in terms of glycaemic control, insulin sensitivity, and anti-inflammatory properties. As an example, niacin, at pharmacological doses in excess of 1 g/day, has been shown to exert anti-inflammatory properties via niacin receptor interactions [47,112] and improve insulin sensitivity, reduced adipocyte size, and exert anti-atherogenic effects on lipid profiles, whilst increasing the expression of niacin receptors in adipocytes [49].
In general, epidemiological evidence suggests that the benefits of B vitamins extend well beyond the accepted biochemical cut-offs for deficiency or marginal deficiency [101] and that consuming the RDA for some B vitamins would still leave large proportions of the population at risk of insufficiency [16]. Indeed, there would seem to be little evidence for supplementing with the bare minimum requirement (RDA) given the dose-response to B vitamins in terms of bioavailability and physiological benefits.
6. Do B Vitamins Have an Impact on Brain Function?
6.1. Observational Studies
Since Smith’s [64] paper, a number of meta-analyses of data from the more methodologically rigorous, recently published studies have been conducted, although it is notable that these analyses applied differing methodological inclusion criteria, and almost exclusively included studies involving samples of elderly adults. These meta-analyses show a reasonably clear relationship between homocysteine levels and dementia in cross-sectional [113] and prospective studies, with high serum homocysteine at the study outset associated with a 35% increased chance of subsequently developing dementia across eight studies [114] and a 50% greater chance of suffering clinically significant cognitive decline across a further 14 studies [115]. Interestingly, at the other end of the life-span, a single study also demonstrated a positive relationship between dietary folate intake and academic achievement in adolescents [116].
In terms of circulating vitamin status, analysis of the data from 10 cross-sectional studies and one prospective study demonstrated a relationship between low folate and vitamin B12 and depression [117], and analysis of data from 10 cross-sectional and three cohort studies showed that that folate, but not vitamin B12 was associated with cognitive impairment, typically assessed with the Mini Mental State Exam (MMSE) [118].
6.2. Controlled Intervention Trials
6.2.1. Folate, Vitamin B12 and Vitamin B6
Whilst the substantial observational literature in this area suggests a consistent relationship between aspects of brain function and folate/B12 and/or homocysteine, a huge research effort predicated on the hypothesis that supplementation with these vitamins should decrease homocysteine levels and thereby either improve cognitive function or attenuate cognitive decline and the risk of dementia has generated largely equivocal results. Indeed, reviews and meta-analyses published over more than a decade have provided scant evidence to support this hypothesis [122,123,124,125,126,127].
Of course, these demonstrations of a lack of efficacy have elicited a counter-commentary noting that the null findings may be due to a number of methodological factors, including: the study selection; the heterogeneity or insensitivity of the cognitive tests; the good, or bad, cognitive status of the participants at the studies’ outsets; the duration of treatment; and the pooling of data obscuring the positive findings from more methodologically rigorous studies and those in sub-populations that are more likely to see benefits including those with poorer vitamin status [101,129,130,131]. Examples of the latter include positive findings in groups suffering high levels of homocysteine at the outset [132,133]. It has also been noted [129] that more consistent evidence exists for lower vitamin B12 status and higher homocysteine levels being associated with decreased brain volume [134,135] and increased white matter lesions [136] and for supplementation with homocysteine lowering B vitamins attenuating the rate of cerebral atrophy associated with dementia and age related cognitive impairment, particularly in those with higher homocysteine levels at the outset [137,138].
6.2.2. Thiamine, Riboflavin, Biotin, Pantothenic Acid, Niacin
Unfortunately, there is a general dearth of controlled trial research into the effects of the remaining B vitamins on brain function, or indeed any aspect of functioning in humans. Some supportive evidence does exist that shows that several of this group can modulate peripheral cardiovascular and gluco-regulatory function—and it is certainly the case that modulation of these parameters should have an impact on brain function. For instance, administration of 1.6 mg/day of riboflavin attenuated the hypertensive effect of the MTHFR 677TT genotype [140] and up to 4 mg/day led to dose-related increases in the number of circulating red blood cells and the concentration of haemoglobin [43]. Additionally, large doses (60+ × RDA) of biotin, with [141,142] or without additional chromium [143,144] have been shown to improve glycaemic control and/or insulin sensitivity in sufferers from diabetes.
6.2.4. Acute Effects of Multivitamins
Interestingly, the orthodoxy that vitamins have to be administered for an extended period of time in order to elicit any physiological effects is not based on any evidence that vitamins do not exert acute effects. Comparatively few studies have assessed the acute effects of vitamins, but from those studies that have, there is emerging evidence that vitamins have physiological and brain function effects following a single dose. For instance, single doses of a range of single vitamins, including folic acid (as well as vitamins C, E, A), administered at “mega-doses” of between five and 26 times the RDA for that micronutrient, have all been shown to increase vasodilation in groups with disease-related or experimentally induced endothelial dysfunction [149,150,151,152,153]. Acute administration of vitamin B6 has also been shown to elicit increased serotonin synthesis in the primate brain [154], whilst, in a placebo controlled, double blind, cross-over study in humans, the higher of two single doses of vitamin B6 (100 mg, 250 mg) also engendered an increase in dream salience (vividness, bizarreness, emotionality, and color) [155].
7. Summary and Conclusions
The B vitamins represent a group of eight essential dietary micronutrients that work closely in concert at a cellular level and which are absolutely essential for every aspect of brain function. As water soluble nutrients, they are generally safe at levels of consumption well in excess of the recommended minimum consumption levels (possibly with the exception of folic acid, see Section 5). Indeed, bioavailability and functional data suggest that consumption of most B vitamins at levels well above dietary recommendations would be warranted.
Whilst adequate levels of all of the B vitamins should be obtainable from a healthy diet, evidence suggests that large sub-sections of the populations of developed countries are suffering deficiencies or marginal deficiencies in one or more B vitamins that will predispose them to a number of negative health consequences, including less than optimal brain function. Both epidemiological and controlled intervention trial research, driven by the predominant “homocysteine hypothesis”, have overly concentrated on the relationships with brain function, and the effects of supplementation on brain function of a narrow group of three homocysteine lowering B vitamins—folate and vitamin B12 and, to a lesser extent, vitamin B6. The potential roles and effects on brain function of the remaining five inter-related B vitamins have been largely ignored. As a consequence, consistent evidence suggests that biochemical levels of this narrow band of three vitamins, and related levels of the amino-acid homocysteine, correlate positively and negatively with brain function, respectively. However, the evidence that supplementation with one or more of these three homocysteine lowering vitamins in isolation improves brain function is entirely equivocal.
The lack of demonstrable efficacy seen in multiple meta-analyses of supplementation trials involving this small sub-group of homocysteine lowering B vitamins has often prompted a counter commentary that persists with the notion that the underlying homocysteine hypothesis is likely to be correct, suggesting rather that the methodology or focus of the individual studies or meta-analyses are incorrect, and that future research should be directed towards sub-groups of the population more likely to benefit, in trials that employ more sensitive measures (e.g., [131]). This may prove a fruitful approach, but given the inter-related cellular functions of the B vitamins, a more rational approach to research must be to investigate the effects of supplementation with the full range of B vitamins, at doses well in excess of the current governmental RDAs. There is no compelling argument for restricting this research either to a small sub-group of three B vitamins or to the elderly groups of subjects usually employed in these trials. Certainly, the smaller body of research investigating multivitamins, which has largely been undertaken in healthy children and non-elderly adults, suggests significant benefits to brain function following supplementation with multivitamin products containing a full range of B vitamins at levels well in excess of their RDAs.
It is also notable that treatments containing all of the B vitamins will inevitably reduce homocysteine (see [159,165]), and indeed, given the direct contribution of both niacin and riboflavin to the folate/methionine cycles, they should theoretically be more effective than small sub-groups of B vitamins in this regard. It is therefore difficult to conceive of any potential downsides to undertaking research with the full range of B vitamins. Of course, the luxury of being able to attribute any benefits to a single molecule and/or a single mechanism will be lost, but given the equivocal nature of the large body of evidence to date with regards to the homocysteine hypothesis, this loss would appear supportable, if not inevitable.
Naturally, the B vitamins, as a group and individually, also work intricately in concert with other vitamins, minerals and micronutrients. Whilst this topic is outside of the scope of the current review, it is noteworthy that a concerted research effort aimed at elucidating the full range of micronutrient interactions is warranted. For the moment, the foregoing suggests that research should, at a minimum, be redirected towards elucidating the potential benefits for brain function of both the acute and chronic administration of a full range of B vitamins rather than concentrating solely on the chronic effects of a small sub-group of three vitamins.
Referência :
Front Immunol. 2019 Dec 20;10:2904.
