Chapter 15

The Role of B Group Vitamins and Choline in Cognition and Brain Aging

Francesco Bonetti, Gloria Brombo and Giovanni Zuliani,    University of Ferrara, Ferrara, Italy


Brain aging is a balance between homeostatic processes and harmful interactions with the environment. Each individual has a unique aging path. Healthy neurological aging requires maintenance of normal brain functioning and prevention of neuronal damage. Diet is a cornerstone of disease prevention and health promotion. B group vitamins (BGVs) and choline are micronutrients that share a key role in multiple metabolic pathways related to the nervous system’s structural and functional integrity. Almost all BGVs show clinically relevant neurological manifestations in case of insufficient dietary intake. BGVs and choline seem to have a role in optimizing brain energy metabolism, containing oxidative stress and inflammation, maintaining deoxyribonucleic acid stability and regulation of gene expression, modulating neurotransmission, and preventing vascular damage. A possible role in neurodegenerative disease prevention has been hypothesized. BGV deficiency-related disturbances in homocysteine metabolism result in hyperhomocysteinemia, a plausible contributor in many of these processes.


B group vitamins; choline; homocysteine; hyperhomocysteinemia; aging; diet; prevention; neurodegenerative


Aging is an inescapable process of our body, the cumulative modifications of our organs and systems usually result in a progressive loss of function due to the unbalanced relationship between trophic and homeostatic stimuli and harmful interactions with the environment. Brain aging is no exception. How we age, however, is unique for each individual. Pathological and instrumental exploration of the brain in both demented and nondemented older subjects often shows a mixed pattern of lesions (amyloid plaques, tau protein fibrillary tangles, synucleinopathies, vascular damage, and white matter rarefaction) possibly but not inevitably resulting in cognitive impairment (Fernando and Ince, 2004; Schneider et al., 2007; Kang et al., 2012; Nelson et al., 2012; Seo et al., 2013; Xekardaki et al., 2015; Yu et al., 2015). The finding that cerebral lesions as confirmed by pathology do not always reflect correctly clinical diagnoses and functional status of individuals (Schneider et al., 2007; Jellinger and Attems, 2013; Xekardaki et al., 2015) opens a new window of insight into brain aging, cognitive decline, and dementia. It is imperative to shift from morphological analysis alone to integrated functional evaluations, considering the pathological damage in the context of a system that relies primarily on networks, where on the one hand connections and integrations could somehow survive (even if slightly impaired) in a structurally damaged organ and on the other hand a relatively conserved cellular mass is not surely sufficient to supply loss of interconnections and functional disturbances (Reijmer et al., 2013; Lawrence et al., 2014). Even if individual resilience to pathological brain damage is hardly predictable, possibly due to differences in cognitive reserve (Xekardaki et al., 2015), the global burden of brain lesions correlates with cognitive outcomes for a large number of subjects (Yu et al., 2015; White et al., 2016). Neuroplasticity, in terms of neurogenesis and rearrangement of neural networks, has emerged as one possible target to be enhanced in order to counterbalance the age-related degeneration of the central nervous system (CNS). Sadly, this ability has the tendency to wane even in subjects defined to have “successful aging” (Jellinger and Attems, 2013). Because it is now still impossible to fully revert or compensate neuronal damage when an extensive loss of neurons occurs, to preserve our functional status we need to sustain a tireless and expensive tendency to homeostasis, preventing avoidable brain damage and providing our organism with the essential elements required for normal functioning. One field of growing interest that covers both health promotion and disease prevention with a safe high-impact intervention is nutrition.

Dietary patterns have shown a noteworthy relevance in preventing cognitive decline (Cheung et al., 2014), and it has been known for decades that poor nutritional status, especially in terms of micronutrients deficiency, has a role in the development of neuropsychiatric conditions and neurodegenerative diseases (Bourre, 2006; Del Parigi et al., 2006; Waserman et al., 2015). To better understand the role of micronutrients in neuroprotection and healthy brain aging, it is useful to establish the relationship between a micronutrient intake level and modulation of the physiological events that are regulated, promoted, or contained because of the micronutrient availability. Almost all nutrients have an inverted U-shaped relationship between their concentrations and their physiological functions: the optimal effect occurs over a variable range of intake levels, while both deficiency and excess of the substance could result in detrimental and even life-threatening effects (Morris, 2011; Morris, 2015). Moreover, the dietary requirements and the maximum tolerable intake levels can vary among individuals on the basis of constitutional, clinical, and physiological characteristics. These basic rules are determinant in understanding why there is no “standardized approach” that perfectly fits the requirements of an entire population. However, an estimation of the population needs based on epidemiological data united with a documented knowledge of the risk-benefit ratio could allow planning for a fairly safe and potentially beneficial intervention at the population level. Clear examples are dietary recommendations implemented for health-promotion and food-fortification programs. To accurately analyze the amount of substances needed to maintain a normal metabolic function, it is necessary to introduce the concepts of estimated average requirement (EAR), or intake with 50% risk of inadequacy; recommended dietary allowance (RDA), or intake with about 3% risk of inadequacy; and tolerable upper intake level (UL), or intake with a clinically relevant risk of toxicity. The adequate intake (AI) represents the span of intake of a nutrient that almost certainly satisfies the physiological requirements of an individual with a low risk of developing toxic effects; theoretically, the AI is higher than RDA (almost certainly sufficient) and lower than UL (possibly toxic). AI is the reference value if sufficient scientific evidence is not available to establish an EAR on which to base an RDA.

Many micronutrients have a role in modulating brain activity or in neurodegeneration, so we will focus here on B group vitamins (BGVs), a group that is essential for normal cognitive development and is supposed to be helpful in preventing cognitive decline, as well as on choline, a precursor of membrane lipids that share BGV metabolic pathways.

Role of B Group Vitamins and Choline in Normal Brain Functioning and Neuroprotection

BGVs are a heterogeneous group of hydrosoluble vitamins whose chemical and functional diversity is associated with an equally varied pool of food sources. Almost all deficiency syndromes related to the compounds that belong to BGVs have neurological manifestations, either as direct effects of deficiency or as consequences of the accumulation of neurotoxic metabolites (Lanska, 2010; Abdou and Hazell, 2015; Nardone et al., 2013; Bowman et al., 2012; Powers, 2003; Ghavanini and Kimpinski, 2014; Gerlach et al., 2011; Said, 2012). Before detailing the possible mechanisms underlying BGV-related neuronal damage and subsequent protective activities, we remind readers of the physiological role of each vitamin for a better understanding of their contribution in complex metabolic processes (often the same detrimental condition involves more than one micronutrient metabolism, which makes interactions between micronutrients essential for broadly seeing the phenomenon). Piridoxyne (vB6), folate (vB9), cobalamin (vB12), and choline will be discussed together because they share biochemical pathways that are highly relevant to normal brain functioning and common neurodegenerative processes. Dietary recommendations for each micronutrient are summarized in Table 15.1.

Table 15.1

Summary of Multiple-Source Dietary Recommendations for BGVs and Choline

Micronutrient Nonfortified Food Sourcea RDA: M/F (Elderlyd) AI: M/F UL UL/RDAb: M/F (Elderlyd)
Vitamin B1 Lean pork, legumes, and cereal grains (germ fraction) 1.2/1.1 mg/day IOMf NA NA; presumably very high
Vitamin B2 Especially yeast and liver but also milk, egg white, fish roe, kidney, and leafy vegetables 1.3/1.1 mg/day IOMf NA NA; presumably very high
Vitamin B3 Meat, fish, or poultry, roasted coffee 16/14 mg/day IOMf 35 mg/day IOMf 2.2/2.5
Vitamin B5 Chicken, beef, potatoes, oat cereals, tomato products, liver, kidney, yeast, egg yolk, broccoli, and whole grains NA 5 mg/day,e IOMf NA NA; presumably very high
Vitamin B6 Fish and meat, seeds, noncitrus fruits (bananas, watermelons) 1.3/1.4 mg/day IOMf (1.5/1.7) IOMf 25 mg/day EFSAg 19.2/17.8 (16.6/14.7)
Vitamin B7 Liver, kidney, egg yolk, soybeans, nuts, spinach, mushrooms, lentils NA 30 μg/day,e IOMf NA NA; presumably very high
Vitamin B9 Fruits and green leafy vegetables, yeast, liver 400 μg/day,e IOMf 1 mg/day IOMf 2.2e
Vitamin B12 Meat, fish, liver, dairy products 2.4 μg/day,e IOMf 4 μg/day,e EFSAg NA NA; presumably extremely high
Choline Milk, liver, eggs, peanuts NA 550/425 mg/day IOMf 3500 g/day IOMf 6.4/8.2c


This table is derived from data obtained by NDA panels (EFSA, 2006) and Institute of Medicine (1998) statements on dietary references and tolerable upper limits of the listed micronutrients. If the two considered statements presented different recommendations, we chose on the basis of our clinical and scientific knowledge on the possible clinical efficacy of neuroprotection and benefit–risk considerations. RDA, recommended daily allowance; AI, adequate intake; UL, tolerable upper intake level; NA, not available; M, male; F, female.

aThese lists of food sources are not complete but represent examples of foods with relatively high contents of each micronutrient.

bWhen possible, we indicated the ratio between UL and RDA (or AI if RDA was not available) to quantify the safety span of a possible integration into the diet.

cAI was used instead of RDA (lack of sufficient quality evidences).

dReference values for elderly individuals when available.

eNo gender differences.

fIOM derived value (Institute of Medicine, 1998).

gEFSA value derived by European NDA panel statement (EFSA, 2006).


Thiamin (vB1), the first BGV identified, is an essential nutrient involved in carbohydrate and amino acid metabolism and in energy production (El-Sohemy et al., 2013). The vB1 active form, thiamine diphosphate, acts as a cofactor of three step-limiting enzymes of carbohydrate metabolism (transketolase in the pentose phosphate pathway and pyruvate dehydrogenase and α-ketoglutarate dehydrogenase in the Krebs cycle) (Zhao et al., 2009). The RDA for vB1 is set at 1.2 mg/day for adult men and 1.1 mg/day for adult women. These values do not vary for people aged 50 and older but can be affected by energy-demanding situations such as physical activity or diseases with increased energy metabolism (e.g., hyperthyroidism) (Institute of Medicine, 1998; El-Sohemy et al., 2013). It is absorbed in the jejunum through active carriers at low doses (with about 90% of absorption at administration up to 5 mg) (El-Sohemy et al., 2013) and via passive diffusion with lower rates of absorption at higher doses (Institute of Medicine, 1998). Since the intake of several hundred mg of vB1 gave no measurable adverse effects, no UL for this nutrient has been established (Institute of Medicine, 1998; El-Sohemy et al., 2013). The European Food Safety Authority (EFSA) states that even if vB1 is an essential contributor to normal cognitive functioning, a balanced diet should easily meet the daily requirements of the nutrient (in the absence of pathological conditions) (EFSA, 2010a). Historically, a vB1 deficiency was identified with beriberi, a disease known for centuries: deficiency-related symptoms are anorexia, weight loss, apathy, short-term memory loss, confusion, irritability, muscle weakness (El-Sohemy et al., 2013), and cardiovascular effects such as an enlarged heart and heart failure (Azizi-Namini et al., 2012). Today vB1 deficiency is mainly associated with alcoholism and malnutrition (such as in obese patients, both during weight loss in preparation for bariatric surgery and after surgery if not adequately treated, and in elderly people in different countries) (Kerns et al., 2015; de Carvalho et al., 1996; Yang et al., 2005). Wernike encephalopathy (vision and muscle coordination impairment) and Korsakoff syndrome (memory loss, confabulation, hallucination) are vB1-related neurological manifestations that occur more frequently in alcoholic patients (Morris et al., 2006). The neuropsychological symptoms of Wernike–Korsakoff syndrome (confusion, episodic memory deficit with relatively spared working memory, confabulation, impairment in verbal fluency and flexibility, perseverative responding with strong frontal signs) seem caused by a severe impairment in the cholinergic networks, primarily due to neuronal cell loss (Nardone et al., 2013). These neurological manifestations are supported by the fact that vB1 deficiency has been proposed to lead to mitochondrial dysfunction, impaired cellular metabolism, glutamate excitotoxicity, and oxidative stress (OS) in deep brain structures (thalamus, mammillary bodies, and other diencephalic sites) (Nardone et al., 2013). Concern is rising over the possibility that vB1 could contribute to other chronic diseases even in general population subjects. In particular, some authors suggest that vB1 deficiency could impair neuroplasticity via inhibition of hippocampal neurogenesis (Zhao et al., 2009); this finding places vB1 deficiency in the pool of harmful conditions that theoretically could hasten or worsen Alzheimer’s disease (AD) symptoms in predisposed individuals. The report of reduced thiamin plasma levels in AD patients supports the latter theory (Gold et al., 1995, 1998). Moreover, abnormal vB1-related enzyme activity has been observed in both brain and peripheral tissues in AD patients (Gibson et al., 1988; Butterworth and Besnard, 1990; Héroux et al., 1996), and vB1 deficiency in preclinical models seems to be able to promote β-amyloid (Aβ) deposition and tau protein hyperphosphorylation in the brain (Zhao et al., 2011), possibly via increased β-secretase activity (Zhang et al., 2011) and OS promotion (Karuppagounder et al., 2009).


Riboflavin (vB2) is a yellow fluorescent compound whose biological role is as an integral component of two coenzymes—flavin mononucleotide (FMN) and flavin-adenine dinucleotide (FAD)—that are involved in several redox reactions including important steps in energy production. In addition, these flavocoenzymes regulate crucial intersections between other vitamins pathways, the most relevant being their impact on neuronal damage through the FMN-mediated generation of pyridoxal 5ʹ-phosphate (a vitamin B6–derived coenzyme) and the FAD-dependent reduction of 5,10-methylene-tetrahydrofolate to 5ʹ-methyl-tetrahydrofolate, which reacts with vB12 in homocysteine (Hcy) remethylation to metionine (Met) (Institute of Medicine, 1998). RDA for men and women between ages 30 and 70 is set at 1.3 and 1.1 mg/day, respectively; on the basis of few studies, the same amount seems to be reasonably sufficient even for individuals older than 70 (Institute of Medicine, 1998). No UL has been set for vB2. The majority of vB2 of food origin is from flavocoenzymes complexed with proteins that require a normal gastric acid environment to be separated; riboflavin is then released from coenzymes via nonspecific enzymatic activity in the upper gut (Institute of Medicine, 1998). vB2 is absorbed primarily in the proximal small intestine via a saturable transport system while a small amount is subject to enterohepatic circulation or is absorbed in the large intestine (Institute of Medicine, 1998). The absorption is proportional to intake and is facilitated by concomitant ingestion of other food (Institute of Medicine, 1998). The main signs of vB2 deficiency are dermatological alterations, edema, and mucosal alterations of the larynx and oral cavity and possible interference with iron handling and hemopoiesis. However, as previously stated, vB2 deficiency may exert some of its effects by reducing the metabolism of other BGVs (folate and vB6), raising concerns about possible Hcy metabolism perturbation (Powers, 2003) (see the later section “The Homocysteine Cycle: Biochemistry and Clinical Implications”). Moreover, laboratory data from animal experiments suggest a possible neurological involvement in severe deficiencies, but little evidence is available for humans (Powers, 2003). On these bases, EFSA declared vB2 as essential for maintenance of normal nervous system function (EFSA, 2010b).


Niacin (vB3) is defined as a group of compounds with biological activities similar to nicotinamide (nicotinic acid amide itself, nicotinic acid, and other molecules with similar structures and functions) (Institute of Medicine, 1998). Nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) are niacin-derived coenzymes fundamental for a wide range of redox reactions. NAD is also involved in reactions crucial to DNA repair and calcium signaling (Institute of Medicine, 1998). RDA for vB3 is set at 16 mg/day for men and 14 mg/day for women; these values are the same for both adult and elderly individuals. Tryptophan can be transformed into niacin, so its intake could reduce the requirements of vB3, but because vB2, vB6, and iron are needed in the tryptophan to niacin conversion pathway, a multiple deficiency of these nutrients could impair this alternative endogenous supply route (Institute of Medicine, 1998). The UL for adult individuals is set at 35 mg/day of niacin on the basis of the onset of flushing (vasodilation of face and extremities that produces red skin changes along with tingling, burning, or itching), which is the first adverse effect presented at low doses (recently other authors suggested a safer threshold at 10 mg/day) (Scientific Committee on Food, 2002). Higher doses could induce ocular and gastrointestinal disturbances and, at dosages of 3 g/day of nicotinamide (1.5 g/day of nicotinic acid), severe and possibly life-threatening hepatotoxicity (Institute of Medicine, 1998). In addition, individual susceptibility to niacin and the assumed form of the vitamin could greatly influence the genesis of adverse effects; in fact, severe forms of hepatitis have been observed for dosages as low as 500 mg/day (Eeuwijk et al., 2012).

Vitamin B3 is absorbed readily by the stomach and small intestine, with an active transport prevalent at low doses overruled by passive diffusion at higher doses (Institute of Medicine, 1998). The main manifestation of deficiency is pellagra, which consists of skin pigmentation, gastrointestinal symptoms, and neurasthenia followed by psychosis, disorientation, memory loss, and confusion. Nowadays pellagra has almost disappeared in industrialized countries, but niacin deficiency is still a possible problem in long-term alcoholism or malabsorption conditions and severe malnutrition (Hegyi et al., 2004), which is more likely to occur in older patients. Niacin acts as a lipid-lowering drug and is able to reduce cardiovascular risk in hypercholesterolemic patients (Lavigne and Karas, 2013), but it is less effective than statins and not the therapy of choice for dyslipidemia, being relegated as an option for patients with multiple drug intolerances (Sando and Knight, 2015). That said, on the basis of preclinical data, some authors have suggested that vB3 activity in preventing the progression of atherosclerosis does not rely only on reduction of cholesterol (Lavigne and Karas, 2013). An antiinflammatory activity observed on vasal walls cells (Su et al., 2015), adipocytes (Digby et al., 2010), and macrophages (Digby, 2012) could be partially responsible for vB3 effects, opening a frame of possible multilevel interactions between vB3 and CNS damage, not only contributing to cerebrovascular prevention (known also to affect AD progression) (Saito and Ihara, 2016) but also reducing the systemic inflammatory burden, which is known to be related to cognitive decline and dementia (Zuliani et al., 2007; Marsland et al., 2015).

The antiinflammatory effects of niacin are probably mediated by the GPR109a nicotinic acid receptor; its activation in macrophages results in reduced production of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and monocyte chemoattractant protein-1 along with inhibition of chemotaxis and adhesion in experimental conditions (Digby, 2012). Recent findings suggest a role for niacin and the nicotinic acid GPR109a receptor in Parkinson’s disease (PD): vB3 levels were lower in PD patients compared to age-matched controls, and they correlated with a rise in inflammation markers and clinical reports of higher body pain and more pronounced sleep disturbances (Wakade et al., 2014).

Other authors have explored the possible impact of 1-g extended-release niacin on cholesterol metabolism in the CNS of AD patients (Vega et al., 2003). This suggests that an investigation might be worth conducting into the possible mechanisms underlying the debated reduced incidence of AD in previously healthy subjects treated with lipid-lowering drugs (reported in an observational study but at the moment not confirmed by good quality evidences) (McGuinness et al., 2016). Anyway, Vega and colleagues demonstrated a reduction in plasma 24S-hydroxycholesterol concentrations in niacin-treated AD patients (Vega et al., 2003). As a marker of neurodegeneration with hypothesized neurotoxic properties, 24S-hydroxycholesterol (Bogdanovic et al., 2001; Lütjohann et al., 2002) has been found to be higher in the plasma and cerebrospinal fluid of AD patients compared to the general population (Zuliani et al., 2011; Lütjohann and von Bergmann, 2003) and is possibly implicated in Aβ pathology (Lütjohann and von Bergmann, 2003). Even if there are rational bases to hypothesize a possible role for vB3 in modulating neuronal damage, the literature data are scant and come from studies with low qualities of evidence. Lower niacin plasma levels have been observed in demented patients in respect to controls (along with multiple deficiencies of other nutrients in a small sample) (Thomas et al., 1986), and a possible role for niacin dietary intake levels in AD prevention has been postulated on the basis of observational data (findings with a high risk of bias due to the many possible confounders) (Morris et al., 2004). In our opinion, better-designed studies are needed to clarify this subject. Alas, a main bias of nutritional investigations in demented patients is that dementia is often a cause of malnutrition itself, so even longitudinal data should be critically considered.

Pantothenic Acid

Pantothenic acid (vB5) is involved in the synthesis of coenzyme A (CoA) and acyl carrier proteins. CoA in its different forms is an essential cofactor in a wide range of biological reactions, including the regulation of lipid metabolism, the production of molecules fundamental for the structure of the body machinery (such as amino acids, cholesterol, and membrane phospholipids), and the synthesis of substances with high-impact functional roles such as steroid hormones, vitamin D, and neurotransmitters (El-Sohemy et al., 2013). Acetyl-CoA and succinyl-CoA have a key role in the tricarboxylic acid cycle. Pantothenate kinase is the enzyme that transforms pantothenate in CoA, and it is regulated by its end product (CoA), so CoA concentrations do not exactly reflect the pantothenate availability for the body (Institute of Medicine, 1998).

The RDA for vB5 has not been established due to the lack of scientific data to support it, but an AI of 5 mg/day for both adult and elderly men and women has been considered sufficient on the basis of the balance between intake and excretion (Institute of Medicine, 1998; El-Sohemy et al., 2013). No adverse effects have been associated with high intakes of pantothenic acid, so no UL has been set (Institute of Medicine, 1998). Even if vB5 is not synthesized in humans, deficiency is unlikely to occur because vB5 itself and its derivatives are widely available in food; moreover, it is probable that resident microbiota of the intestine contribute to the pantothenic acid supply (an active synthesis has been observed in mice, but it is not clear whether the phenomenon is relevant to humans) (Institute of Medicine, 1998). Pantothenic acid absorption is mediated by a saturable active transport system at low concentrations and with greater contribution of passive diffusion at higher levels of intake (Institute of Medicine, 1998).

Data on deficiency-related clinical manifestations are deduced by historical reports of plausible peripheral neuropathy (“burning feet”) reversible with vB5 administration in war prisoners in Asia during World War II (Glusman, 1947). Moreover, it has been observed that patients fed a diet poor in vB5 or treated with a pantothenate antagonist (Hodges et al., 1958, 1959; Fry et al., 1976) exhibited irritability, restlessness, fatigue, apathy, sleep disturbances, gastrointestinal manifestations (nausea, vomiting, abdominal pain), and neurological symptoms involving the peripheral nervous system (numbness, paresthesias, muscle cramps, and gait disturbances). Preclinical data suggest that pantothenate could have a role in cellular protection from apoptosis at a subsequent OS exposure (Wojtczak and Slyshenkov, 2003), plausibly via mitochondrial activity optimization and increased glutathione synthesis (Slyshenkov et al., 2004). A striking example of a possible neurological repercussion with severe impairment in pantothenic acid metabolism on CNS is pantothenate kinase–associated neurodegeneration (PKAN), the most frequent of a group of neurodegenerative disorders characterized by high iron accumulation in the brain, which has as major clinical expression in extrapyramidal symptoms and cognitive impairment. PKAN is secondary to rare autosomal recessive mutations (about 1:500 carriers in the general population) of the gene pantothenate kinase type 2, which is a mitochondrial protein necessary to CoA biosynthesis (Brunetti et al., 2012). The depletion in CoA availability due to impaired vB5 metabolism (and subsequent disturbances in mitochondrial bioenergetics and cellular metabolic reactions) has been proposed as a possible explanation of this disease (Shumar et al., 2015).

PKAN has a marginal role in adult onset neurodegenerative diseases, considering that it is a rare syndrome with usual onset during childhood and rapid progression, but adult variants with slower progression have been documented (Doi et al., 2010). Pantethine, a vB5-related molecule composed of two molecules of pantothenic acid linked by cysteamine bridging groups, seems able to bypass pantothenate kinase impairment in animal models of PKAN (Rana et al., 2010; Brunetti et al., 2014). These considerations open a possible future perspective in investigating the eventual role of dietary pantothenic acid or vB5 derivative supplements in brain containment of OS and optimization of energy metabolism, two key points in normal brain function maintenance often suggested to be contributors in age-related neurodegenerative diseases.


Biotin (vB7) is a structural part of enzymes involved in bicarbonate-dependent carboxylation reactions, some of which are of exclusive mitochondrial pertinence (namely, pyruvate carboxylase, methylcrotonyl-CoA carboxylase, and propionyl-CoA carboxylase), while others are found also in the cytosol (acetyl-CoA carboxylase 1 and 2) (Institute of Medicine, 1998; Zempleni et al., 2012). Holocarboxylase synthetase (HLCS), a chromatin protein, catalyzes the covalent binding of biotin to carboxylases, while biotinidase releases biotin, which disrupts the covalent bond in enzymes to render it available for recycling (Zempleni et al., 2012). These enzymatic reactions are linked to tricarboxylic acid cycle, gluconeogenesis, fatty acid elongation, and branch-chained amino-acids (leucine) degradation (Institute of Medicine, 1998). Moreover, biotin seems to have a role in DNA stability maintenance and gene expression (Zempleni et al., 2011, 2012; Liu and Zempleni, 2014). HLCS is able to covalently bind biotin to histones; biotinylated histones play a role in transcriptional repression of genes and are particularly represented in long terminal repeats, although in humans <0.001% of histones are biotinylated (Li et al., 2013). Derepression of long terminal repeats in biotin-depleted cell cultures is associated with genome instability (Zempleni et al., 2011). Some authors suggest that an epigenomic synergy between biotinylation and methylation reactions on histones could significantly amplify the relative selectivity of histone biotinylation and render this phenomenon more relevant in gene expression (Li et al., 2013). Furthermore, it seems that biotin and folate can act in concert to promote the suppression of long terminal repeats via interactions between HLCS and other chromatin proteins (Xue and Zempleni, 2013).

Due to the lack of data on biotin requirements, a RDA cannot be deduced, although an AI has been set at 30  μ g/day for both men and women of all ages as a weight-adjusted estimation based on inferred infant requirements: biotin intake from exclusive human milk feeding. For similar reasons (scant data on possible toxic effects), it is not possible to establish a UL (Institute of Medicine, 1998). Hemodialysis, inborn errors of enzymes involved in vB7 absorption or metabolism, dietary habits (heavy raw egg white consumption), and lactation could represent conditions with higher requirements for biotin (Institute of Medicine, 1998). Doses up to 200 mg orally and 20 mg intravenously were administered to treat human patients with biotin-responsive inborn errors of metabolism or acquired biotin deficiency without reports of toxicity (Mock, 1996). Preclinical data on pregnant rats reported a possible risk of implant prevention or miscarriage at very high doses of vB7 (about 100 mg/kg). The data could be severely biased because this dose is more than 108 times the estimated AI and vB7 was coadministered along with toxic substances via an unusual route (subcutaneous); a 10-mg daily administration in a woman at the ninth month of pregnancy did not measurably affect either mother or infant (Institute of Medicine, 1998).

Vitamin B7 is absorbed in its free form in the small intestine and possibly in the proximal colon with both active and passive mechanisms (Institute of Medicine, 1998). Vitamin B7 is partially produced by colonic microflora, but its real role in supplying vitamins in humans is not clear. In food, vB7 is found as both free biotin and protein bound; biotinidase is essential to release biotin from proteins to get it ready to absorption (and to recycle endogenous biotin bound to proteins). Patients deficient in this enzyme usually require a vB7 supplement in its unbound form to meet daily requirements. Other conditions observed to be at risk of deficiency are short gut syndrome, inadequate total parenteral nutrition, and diets characterized by high prolonged consumption of raw egg white (Baugh et al., 1968), which contains avidin, a protein able to sequester biotin in the small intestine and prevent its absorption (Institute of Medicine, 1998). Low biotin plasma levels have also been reported in patients with glucose metabolism dysfunctions such as type 2 diabetes, and an inverse correlation between biotin and fasting plasma glucose has been observed (Kennedy, 2016). The clinical findings of biotin deficiency include dermatitis (periorificial red scaly skin rash), conjunctivitis, alopecia, and neurological abnormalities (depression, lethargy, hallucinations, and paresthesia of the extremities) (Institute of Medicine, 1998).

In an animal model of biotinidase deficiency, the neurological symptoms were associated with demyelination and axonal degeneration in the CNS. Confirmation that these pathological manifestations are subsequent to biotin deficiency itself and not to the accumulation of biotinylated products was supported by rapid regression of both neurological symptoms and pathology after free biotin supplementation (Pindolia et al., 2012). It is not completely understood if the neurological manifestations of vB7 deficiency are consequent to epigenetic modifications or impaired glucose metabolism and energy production or to other unknown causes; neither is it clear if a marginal deficiency of this vitamin could have a clinically relevant impact on aging adults nor if exceeding the normal daily requirements with supplements could ameliorate energy metabolism in healthy individuals. Considering that vB7 seems safe even when administered at high dosages and that prolonged deficiency of this vitamin has sure neurological consequences, it would be recommended to treat promptly in case of suspected deficiency.

Pyridoxine (and Related Compounds), Folate, Cobalamin, and Choline

The link between BGVs and choline is the Met metabolism and Hcy cycle. Specifically, pyridoxine, folate, and cobalamin (with an indirect contribution of riboflavin) are the BGV that intersect one of the numerous metabolic pathways of choline metabolism. After a brief summary for each micronutrient and its function, we will discuss such interactions (see “The Homocysteine Cycle: Biochemistry and Clinical Implications” later in this chapter).

Pyridoxine and Related Compounds

Pyridoxine, pyridoxal, pyridoxamine, and their 5ʹ-phosphate forms are all grouped under the definition of vitamin B6 (vB6) because interconversion between forms is possible (Bender, 1989), and they can be transformed to pyridoxal-phosphate, the main active form of vB6 in the human body (Institute of Medicine, 1998). Vitamin B6 is a coenzyme in reactions involved in the metabolism of amino acids, glycogen, and sphingoid bases. Relevant biochemical reactions in which vB6 plays a fundamental role are heme biosynthesis and transsulfuration reactions that transform Hcy to cysteine (Cys) via cystathionine β-synthase and cystathionine γ-lyase activity (Institute of Medicine, 1998). Moreover, pyridoxine is an essential cofactor in monoamine synthesis, mediating a step in the conversion of tryptophan and tyrosine in serotonin and dopamine, respectively (Rotstein and Kang, 2009). The RDA for vB6 is set at 1.3 and 1.4 mg/day for adult men and women, rising to 1.5 and 1.7 mg/day in elderly men and women (Institute of Medicine, 1998). The EFSA reported a possible adjustment for daily protein intake because it has been observed that vB6 plasma values tend to decrease more rapidly in individuals with higher protein intake. The suggested RDA in relation to dietary protein load would be 15  μ g of vB6 per gram of protein, reaching an average RDA in adults of 2–3 g/day (EFSA, 2006). Absorption in the gut is mediated by a not saturable passive diffusion system for nonphosphorylated forms of vB6 (a phosphatase hydrolyze 5 ′ -phosphate forms of vB6, no saturation effect has been seen even for extremely high doses) (Institute of Medicine, 1998). Bioavailability varies among different vitamers of vB6 group; in a mixed diet, the mean absorption of the vitamers has been estimated to reach values of 75% of the ingested dose. The absorbed part of dietary source is phosphorylated in the liver and stored bound to proteins (Institute of Medicine, 1998). Vitamin B6 is the BGV with the most established and relevant adverse effect; the UL has been set at 100 mg/day due to onset of neurotoxicity, consisting mainly in a symptomatic peripheral neuropathy (Institute of Medicine, 1998). Concerns of possible neurotoxicity associated to prolonged intake of lower daily doses impose a critical approach to supplementation, and some authors suggest caution in choosing dosages (Ghavanini and Kimpinski, 2014). Because this side effect is dependent on individual susceptibility, dose, and time of exposure, the EFSA suggested a safer UL: 25 mg/day (EFSA, 2006). Clinical signs of deficiency are retarded growth, acrodynia, hair loss and thinning, bone abnormalities, and microcytic anemia (Institute of Medicine, 1998; EFSA, 2006). On the neurological side, the perturbation of neurotransmission (depletion of dopamine, serotonin, norepinephrine, tryptamine, tyramine, histamine, gamma-aminobutyric acid, and taurine) probably contributes to psychological and cognitive alterations and seizures (EFSA, 2006). The link between vB6 deficiency and high Hcy plasma levels will be discussed separately in the section “The Homocysteine Cycle: Biochemistry and Clinical Implications”).


Folate (vB9) is a generic name for a group of vitamers (compounds with similar vitamin biological activities). Naturally occurring folates have a chain of molecules of glutamate joined to a γ-carboxyl site in peptide linkage; variable in their numbers of molecules, these vitamers are defined pteroylpolyglutamates (Institute of Medicine, 1998). Not all the vitamers have the same bioavailability, but all can be converted to tetrahydrofolate (the main biologically active form). Folic acid (pteroylmonoglutamic acid) is the vitamer most used in supplements and fortified foods due to its stability, but it is scarcely represented in foods of natural origin; it has nearly 1.7 times the availability of natural occurring folates (Gregory, 2012). Folate participates in DNA synthesis and one-carbon metabolisms (methylation reactions); folate-dependent enzymatic steps are also described in amino acid interconversion reactions (histidine to glutamic acid, serine to glycine, and Met to Hcy) (Institute of Medicine, 1998).

The RDA for folates has been set at 400 μg/day for both adult men and women; it has been decided to maintain this threshold even for elderly individuals (Institute of Medicine, 1998). More recently, a lower RDA has been proposed by the EFSA (250 μg/day) (EFSA, 2014), but in our opinion, thinking at the good therapeutic index of folates, probably 400 μg/day is a more suitable RDA. Absorption in the small intestines is favored by a proximal active saturable transport system that is overcome by distal passive diffusion at higher dosages (EFSA, 2014). To be internalized by the gut mucosa, polyglutamic forms must be hydrolyzed to monoglutamates by γ-glutamyl carboxypeptidase (an enzyme of the intestinal brush border membrane), then it is mainly transported in this form bound to plasma proteins and finally retransformed in a functional form in target organs (EFSA, 2014). Several situations could increase the requirements of folates. Probably one of the most clinically relevant is a mutation of the methylenetetrahydrofolate reductase (MTHFR) gene. MTHFR mutation reduces the enzyme activity inhibiting the normal folate metabolism (with consequent raise Hcy concentration and reduction of methyl donors). The most common variant reduces enzymatic activity up to 45% of the wild type (Leclerc et al., 2000). It consists of an amino acid substitution of an alanine with a valine (a cytosine in position 677 into thymine—MTHFR C677T). The frequency of the mutated gene varies greatly geographically: about 30% in Europe and Japan and only about 11% in African Americans (Clarke and Armitage, 2000). In the homozygote condition, it is associated with a mild to moderate rise in Hcy plasma levels (Carmel et al., 2003) and low red blood cell folates (Molloy et al., 1997). In Asian populations, MTHFR C677T polymorphism has been linked by epidemiological data to vascular dementia (Liu et al., 2010), AD (Hua et al., 2011), and depression (Jiang et al., 2015). In individuals carriers of MTHFR C677T homozygous mutation, a higher daily dose of folic acid supplement may be useful to force the enzymatic pathway and reduce the detrimental effects of this condition (Jacques et al., 1999; Ashfield-Watt et al., 2002; de Bree et al., 2003). A second polymorphism of MTHFR at base pair (bp) 1298, resulting in a substitution of a glutamate with an alanine (MTHFR A1298C), has lower clinical significance. The enzyme mutated at bp 1298 shows a minor reduction in its activity (68% of wild type vs, 45% of the one mutated in bp 677), and homozygous individuals (about 8% in European populations) are reported to have near normal Hcy levels. Individuals who are compound heterozygotes for the MTHFR A1298C and MTHFR C677T alleles tend to have a biochemical profile closer to that seen among MTHFR C677T homozygotes (Leclerc et al., 2000). Moreover, some authors suggest that the cis configuration of mutation A1298C and C677T is rarely observed; even in this case, it seems that the colocalization of both alleles on the same chromosome would not significantly affect enzyme activity compared to MTHFR C677T alone (Leclerc et al., 2000).

High intake of folates of food origin do not seem to cause severe adverse effects, while folic acid supplements gave rise to concerns about possible masking of vB12 deficiency and induction of neurotoxicity and carcinogenesis. The so-called folate trap is a major concern, especially in nations in which isolated folate fortification of foods has been implemented. It consists of masking the hematological consequences of cobalamin deficiency (macrocytic anemia) without reversing the neurological damage (or enhancing it) (EFSA, 2014). The subsequent delay in diagnosis due to the initially paucisymptomatic onset of cognitive, psychological, and sensory impairment (subacute combined degeneration) secondary to cobalamin deficiency can impede a full recovery (Martin, 1992; Abyad, 2002). Some authors also reported concerns of possible direct neurotoxic effect of high dosage supplements with folic acid (>400 μ g/day) (Morris et al., 2005). These concerns are apparently open to discussion since the same authors later identified the signs of vB12 deficiency as the strongest risk factors for cognitive decline in a subgroup of the same population (exposed to folic acid fortification and consequently to a high risk of undetected progression of vB12 deficiency-related neurological complications) (Tangney et al., 2009). Other authors have found similar results in a different cohort of individuals subjected to folate fortification, in which low vB12 in presence of normal folate was associated to cognitive decline, while high folic acid intake in individuals with normal vB12 status emerged as a protective factor (Morris et al., 2007). Adequate folate intake is reported to reduce the risk of colon and breast cancer (EFSA, 2014), but some contrasting evidences seem to correlate folate intake and cancer development or recurrence (Cole et al., 2007; Figueiredo et al., 2009). This has been sustained by the hypothesis that folate can be protective for normal tissue but has, on the other hand, the ability to fuel ongoing neoplastic foci (EFSA, 2014; Ulrich, 2006). A recent meta-analysis confuted the hypothesized relationship between folate intake and cancer development (Mackerras et al., 2014), but the topic is still widely debated and caution in general is suggested (Choi et al., 2014).

In conclusion, there is no sufficient strength of evidence to express a solid statement on this matter (EFSA, 2014). The UL has been set at 1 mg/day from fortified foods and supplements because of the described concerns about possible carcinogenic properties and most of all disease masking and neurotoxic effects in the case of vB12 deficiency (Institute of Medicine, 1998; EFSA, 2014). It should be a fairly safe dosage even in the latter case (EFSA, 2014). Folate deficiency syndrome shares multiple features with cobalamin deficiency: macrocytic anemia and neuronal symptoms (psychiatric and cognitive disorders and less commonly peripheral neuropathy) are the main manifestations (Young and Ghadirian, 1989; Reynolds, 2014). The observation of higher prevalence of folate deficiency in psychiatric patients and the (partial) reversibility of psychiatric symptoms in deficient patients confirms that folate deficiency alone could induce neurological symptoms (Young and Ghadirian, 1989) without the contribution of a concomitant vB12 deficiency (in this case, a folate supplement would have had no effect or could even have a detrimental contribution). A partial explanation of the neurological impact of folate deficiency probably resides in an elevation of Hcy plasma levels (see “The Homocysteine Cycle: Biochemistry and Clinical Implications” later in this chapter).


Cobalamin (vB12) is a generic description of compounds characterized by a corrinic ring structure (a cobalt atom bound to six ligands) with similar biological activity. The upper (or β-axial) ligand varies and defines the vitamer of vB12 (cyano, hydroxo, aquo, methyl, sulfito, nitrite, glutathionyl, or adenosyl group). The bioactive forms of the vitamin are methylcobalamin and 5′-deoxyadenosylcobalamin. Cyanocobalamin is a stable synthetic compound usually found in supplements and drugs. Cobalamin is known to participate as a coenzyme in two biochemical reactions in humans: (1) remethylation of Hcy to Met via methionine synthase in the cytosol and (2) rearrangement of methylmalonyl-CoA to succinyl-CoA in mitochondria via methylmalonyl-CoA mutase (EFSA, 2015). Globally, vB12 contributes to DNA regulation via epigenetic modifications and to amino acids and fatty acid metabolism. Succinyl-CoA is directly involved in the tricarboxylic acid cycle, while remethylation of Hcy is fundamental for recycling this neurotoxic sulfur-containing nonessential amino acid (see “The Homocysteine Cycle: Biochemistry and Clinical Implications” later in the chapter) and for Met pool maintenance and subsequent S-adenosyl-methionine (SAM) synthesis. As better described later, SAM is a methyl donor involved in genetic regulation (see the later section “Epigenetic Theory of BGV Deficiency-Related Neurodegeneration”) and neurotransmitter synthesis (see “Impact of BGV and Choline on Neurotransmission”) along with many other methylation reactions (EFSA, 2015). In 1998, the US Institute of Medicine set the RDA for vB12 at 2.4 μg/day for both men and women older than 19 (Institute of Medicine, 1998). Recently, the EFSA, given a reported lack of information necessary to calculate the average requirement for vB12, only set an AI at 4 μg/day for both adults and the elderly without gender differences (EFSA, 2015).

The absorption process of vB12 is quite different from other BGVs; indeed, being a complex molecule, its transport into the blood stream from the intestinal lumen necessitates an active system at normal dietary intake levels. Effective passive diffusion occurs only with very high dosages of the micronutrient, up to 1–2 g, but can be a viable alternative to parenteral administration for supplements in patients with vB12 malabsorption due to inactivity of the normal facilitated transport (Vidal-Alaball et al., 2005; Stabler, 2013). The absorption process initiates in the proximal digestive tract with haptocorrin (or transcobalamin 1) production. Haptocorrin is a glycoprotein secreted by the salivary glands that binds vB12 in the stomach after it has been released from food proteins by acid and enzymatic processes. The vB12–haptocorrin complex is able to resist the gastric acid environment and then in the duodenum, haptocorrin is digested by pancreatic enzymes and substituted by intrinsic factor (a transport glycoprotein produced by gastric parietal cells) to form the complex that is able to be absorbed in the ileum by a specific transport system. This complex system is particularly prone to inefficiency due to its multiple step-limiting passages. Different conditions (especially prevalent in elderly individuals) can lead to gastric pH modifications and lower intrinsic factor production that affects vB12 availability (antacid medications for increments in pH, autoimmune processes such as pernicious anemia for intrinsic factor-limited production, and chronic gastritis for both mechanisms) (Andrès et al., 2008). Moreover, integrity of the distal ileum is essential to reach a good absorption rate at intake levels normally reached with food (Institute of Medicine, 1998). Only about 1.2% of vB12 is absorbed via passive diffusion (Vidal-Alaball et al., 2005). No adverse effects have ever been related to cobalamin administration, even at high dosages, so it is impossible at the moment to establish an UL.

A vB12 deficiency develops after a prolonged inadequate cobalamin intake, since usually the biological stores of the vitamin in a healthy subject without prior deficient intake are sufficient for several months (Loew et al., 1999). The main clinical manifestations of deficiency are a macrocytic anemia that is very similar to the one observable in folate deficiency and which evolves to pancytopenia at later stages (leukopenia associated with typical hypersegmented neutrophils and thrombocytopenia; elevated lactic dehydrogenase may be found due to ineffective erythropoiesis), neurological abnormalities (spinal cord degeneration, depression, and cognitive impairment), and gastrointestinal symptoms (even if the latter are debated because the cause of vB12 deficiency itself often can be responsible for the symptoms) (Institute of Medicine, 1998).

The neurological consequences of vB12 deficiency can be the only clinical manifestations of the disease (Stabler, 2013). In some cohorts of patients with neuropsychiatric disorders secondary to vB12 deficiency, macrocytosis, and anemia were absent in about one-third of subjects (Lindenbaum et al., 1988). At early stages, neurological symptoms can be subtle and difficult to diagnose because of the possible polymorphic presentation symptoms ranging from sensory alterations (paresthesias, dysesthesias, hypoesthesias, hypopallesthesia, and altered proprioception) to mild cognitive impairment and mood changes. In advanced stages, patients could present ataxia, double incontinence, impotence, optic-nerve atrophy, severe cognitive impairment or frank dementia, and various psychiatric disorders (depression, psychosis, and abnormal behavior) (Stabler et al., 1990). The spongy degeneration (swelling of myelin sheets that subsequently evolves to full demyelination) historically observed in the dorsolateral columns of the spinal cord is now known to occur systemically (subacute combined degeneration). Magnetic resonance imaging (MRI) scans can observe signs of spinal cord degeneration early in the disease (Senol et al., 2008), but leukoencephalopathy and peripheral nerves demyelination has also been described (Pacheco et al., 2015). A possible sequential involvement due to regional differences in myelin composition and resilience to vB12 deficiency in central and peripheral nervous system has been postulated (Minn et al., 2012). Part of the neurotoxic effect of vB12 deficiency is probably due to a rise in plasma Hcy with subsequent associated damage (see the later section “The Homocysteine Cycle: Biochemistry and Clinical Implications”). It has been observed that symptoms related to cobalamin deficiency can be found also in individuals with near-normal vB12 plasmatic levels; this could be a sign of scarce utilization of the micronutrient. Surrogate markers have been proposed for confirmation of suspected deficiency even in the presence of normal vitamin levels; one of these is Hcy, even if it has low specificity due to its multifactorial biochemistry (vB9 and vB6 deficits, glomerular filtration-rate reduction, many drugs and illnesses can cause a rise in Hcy plasma levels). Methylmalonic acid instead seems to be more reliable: some authors suggest that subjects with normal vB12 and raised levels of Hcy and methylmalonic acid should be considered functionally deficient and that surrogate markers of deficiency have a higher sensibility and specificity than vB12 plasma levels themselves (Carmel, 2000; Carmel et al., 2003; Ulrich et al., 2015).


Choline pool is balanced by both endogenous synthesis and exogenous supply. Usually the normal choline biosynthesis is not sufficient to support the numerous physiological functions for which it is required (Institute of Medicine, 1998), so generally dietary intake assumes a notable importance even if the micronutrient is not formally considered a vitamin. Phosphatidylcholine (PC) accounts for about 95% of the body storage of choline in mammalians; the remaining pool is divided by free choline, phosphocholine, glycerophosphocholine, cytidine 5-diphosphocholine, and acetylcholine (Ach) (Ueland, 2011). Choline is present in food as free choline and in its esterified forms—mainly as lecithin, Ach, and citicoline (El-Sohemy et al., 2013). Lecithin, in which PC is usually the most represented phospholipid, is a common supplement also used for its amphipathic properties as an emulsifier in foods (Institute of Medicine, 1998).

To understand the wide metabolic implications of choline, it is to consider that it is involved in membrane structure maintenance, lipid metabolism, neurotransmission, methylation reactions, and Hcy balance (Institute of Medicine, 1998; Ueland, 2011). Choline intake accelerates the synthesis and release of Ach, thus affecting global brain functions (with special reference to memory storage and recall), autonomic nervous system regulation, and muscle activity (Institute of Medicine, 1998). Betaine (Bet), a choline metabolite, is an important methyl donor involved in the Hcy cycle (the remethylation of Hcy to Met via betaine-homocysteine S-methyltransferase, a non-BGV–dependent shunt for BGV-dependent reactions in the Hcy cycle) with osmotic properties essential for normal renal physiology (Institute of Medicine, 1998). De novo biosynthesis of PC (the most used form of choline in the body and obtained by the methylation of phosphoethanolamide) is strictly bound to SAM availability, so indirectly Met, vB9, and vB12 intake and metabolism regulate the requirements of exogenous choline provision (Institute of Medicine, 1998). PC synthesis is probably the most demanding SAM-dependent methylation reaction in human body, so this reaction is a candidate to be the principal source of Hcy in human metabolism once deprived of the methyl group SAM, which becomes S-adenosyl-homocysteine (SAH) and then Hcy (Stead et al., 2006).

The AI for choline has been set at 550 mg/day for men and 425 mg/day for women of all ages (19 and older) (Institute of Medicine, 1998). Folate restriction or MTHFR polymorphisms with reduced enzymatic activity along with intense physical activity can increase the daily choline requirements (Institute of Medicine, 1998; El-Sohemy et al., 2013). The UL has been set at 3.5 g/day for choline. The main manifestation of excessive intake are hypotension at high dosages (observed for 7.5 g/day supplements in AD patients), gastrointestinal symptoms, and sweating with a fishy odor (probably due to increased vagal tone and trimethylamine production) (Institute of Medicine, 1998). Choline deficiency, although rare in subjects with a varied diet, has been associated with liver damage, including elevated alanine aminotransferase and the development of fatty liver. Moreover, insufficient choline intake can result in increased Hcy plasma levels, especially in folate poor diets (Ueland, 2011) (see “The Homocysteine Cycle: Biochemistry and Clinical Implications” section). This may be attributable to Bet promoting remethylation of homocysteine to methionine in the liver (El-Sohemy et al., 2013).

The Homocysteine Cycle: Biochemistry and Clinical Implications

In the last few decades, clinical research in the fields of brain aging and dementia treatment and prevention has focused mostly on two BGVs—namely, vB9 and vB12 (Morris, 2006)—mainly because epidemiological data reported a possible association with cognition and dementia development (Hinterberger and Fischer, 2013; O’Leary et al., 2012). These results are still controversial, and some interventional clinical trials did not show any benefit from vB9 or vB12 supplementation in terms of cognition (Malouf and Areosa Sastre, 2003; Malouf and Grimley Evans, 2008; Ford and Almeida, 2012; Clarke et al., 2014). A hypothesized path of neuronal damage involved in BGV-related defects in cognition is related to an intermediate product of Met metabolism: Hcy. Hcy is a sulfur amino acid with several interactions with the vascular and neuronal systems (Selhub et al., 1996; Kalmijn et al., 1999; Seshadri et al., 2002; Hassan et al., 2004; Martí-Carvajal et al., 2009; Ford and Flicker, 2012) that accumulates in conditions of vB9 and vB12 deficiency. Another key micronutrient in Hcy metabolism is vB6, and for this reason it has been proposed as having a possible role in determining Hcy-related neuronal damage. As for the other two just cited BGVs, studies about vB6 supplementation still have not shown any benefit in terms of cognition (Malouf and Grimley Evans, 2003). As a precursor of Bet, choline seems to be useful in partially compensating for the Hcy rise (Ueland, 2011). To better understand the possible role of vB6, vB9, vB12, and choline in Hcy-mediated neuronal damage, we will discuss the metabolic interconnections among these molecules and the supposed mechanisms that underlie their detrimental effects on the CNS. Met metabolism has a crucial role in methylation processes, via S-adenosyl-methionine synthase it generates SAM, one of the main methyl donors in the human body. Methylation processes regulate DNA synthesis and are essential in a large number of enzymatic reactions. Losing a methyl group, SAM becomes SAH, which is then hydrolyzed in Hcy. Hcy represents a juncture between remethylation reactions that restore the Met pool and transsulfuration reactions that transform Hcy in Cys. The remethylation pathway requires vB9 and vB12 (Carmel et al., 2003). In conditions where one of the aforementioned vitamins is deficient, it can only be partially shunted via betaine–homocysteine–methyltransferase, which requires Bet as a cofactor (Ueland, 2011). The transsulfuration enzymatic pathway, on the other hand, requires vB6 as a cofactor. Moreover, as previously described (see the riboflavin in the section titled “Riboflavin”), vB2 is necessary for both vB9 and vB6 metabolism.

It is simple to deduce that a deficit of vB6, vB9, vB12 (and vB2 indirectly), or choline results in hyperhomocysteinemia (HHcy), which is defined as a plasma concentration higher than 15 μmol/L. In animal models, HHcy is clearly related to a rapid and massive development of cognitive impairment and pathological findings of microvascular damage and tissue phlogosis in the brain (Sudduth et al., 2013). Even if the HHcy-induced damage seems to be incontrovertible in preclinical models (Sudduth et al., 2013), the experimental conditions used to simulate a HHcy exposure are far from the prevalent forms of human clinical expression of HHcy, which is usually a mild or moderate increase of the metabolite. Currò and colleagues demonstrated that also prolonged exposure to mildly elevated Hcy levels can be toxic in neuronal cell cultures promoting genotoxicity and OS (Currò et al., 2014). HHcy can reach a severe degree (values of 100 μmol/L or more) in rare cases of genetic defects (homocystinuria), long standing multiple BGV deficiency and/or severe renal disease (Carmel et al., 2003). In humans it is reported a higher rate of microvascular brain damage, on CT and MRI scans, in hyperhomocysteinemic patients (Vermeer et al., 2002; Tangney et al., 2011), the instrumental findings of leukoaraiosis and small white matter infarcts, though, did not seem to fully explain the decline in cognitive performance observed in HHcy (Nilsson et al., 2012; Dufouil et al., 2003). Numerous epidemiological studies also associate HHcy with neurodegenerative diseases and neuropsychiatric manifestations in elderly patients (Lehmann et al., 1999; Seshadri et al., 2002; Quadri et al., 2004). The possible relation between HHcy and AD (the neurodegenerative dementia with the highest prevalence in elderly people) or other types of dementia is supported by epidemiological correlation (Seshadri et al., 2002; Ford and Flicker, 2012). Even if AD symptoms could be anticipated and worsened by brain vascular damage (Snowden et al., 1997), recent evidence has shown that HHcy also contributes to AD development with other mechanisms (Fuso et al., 2012). Since HHcy is a consequence of low BGV intake or activity, it is difficult to separate the neurological effects of raised Hcy plasma levels and BGV deficiencies, but it has been reported that HHcy is correlated to cognitive decline and dementia independently of BGV status (Bonetti et al., 2015). Preclinical studies linked HHcy to many detrimental effects with possible neurological repercussions that deserve a detailed description.

Excitotoxicity, Endoplasmatic Reticulum Stress, and Reactive Oxygen Species Production

In both acute ischemic brain damage and chronic neurodegenerative disorders (including AD and PD), a common activation of glutamatergic receptors induces or worsens cellular dysfunction, which leads ultimately to cell death (Prentice et al., 2015). Hcy is a potent agonist of N-methyl-D-aspartate (NMDA) glutamatergic receptors (McCully, 2009). Activation of NMDA receptors results in intracellular calcium overload and mitochondrial dysfunction (Prentice et al., 2015). A heavy activation of NMDA receptors brings intracellular calcium concentrations at levels scarcely compatible with cell survival (oxidative phosphorylation inhibition, phosphate depletion, and even deposition of insoluble salts of calcium and phosphate). The degree and the time of exposure to this dysfunctional process influence the type of damage: tissutal necrosis when it is intense and acute or programmed cellular death if it does not completely overcome the essential cellular reactions (McCully, 2009; Prentice et al., 2015). Impaired mitochondrial activity determines a reduction in energy production with subsequent dysfunction of cellular machinery due to depletion of adenosine triphosphate and augmented production of reactive oxygen species. Furthermore, NMDA activation induces neuronal nitric oxide synthase, which accelerates nitric oxide production, and OS is increased by interaction of nitric oxide with superoxide with the generation of reactive nitrogen species (Prentice et al., 2015). Moreover, the altered cytosolic ambient and the strong OS insult result in protein misfolding and activation of endoplasmatic reticulum stress reactions defined as an unfolded protein response (UPR) (Doyle et al., 2011; Prentice et al., 2015). If prolonged over time, UPR contributes to the induction of apoptotic pathways and has been postulated as an etiological contributor to neurodegenerative diseases (Zhang et al., 2001; Doyle et al., 2011; Penke et al., 2016). HHcy has been linked to apoptosis and prolonged UPR activation, which seems to be one of the mechanisms that underlie this correlation (Zhang et al., 2001; Perla-Kajan et al., 2007).

Protein Homocysteinylation

Circulating Hcy is mainly found bound to proteins. One particularly relevant form of covalent modification of circulating proteins occurs as a consequence of the high reactivity of a Hcy derivative: homocysteine thiolactone (Htl) (Sharma et al., 2015). Htl is a cyclic thioester that results from an error-editing reaction by methionyl–tRNA synthetase (Jakubowski and Goldman, 1993; Jakubowski et al., 2000). One possible mechanism of Htl toxicity is the nonenzymatic formation of amide bonds with ε-amino groups of protein lysine residues; this reaction is a defined protein N-homocysteinylation (Sharma et al., 2014). Htl production and the subsequent protein homocysteinylation rate depend on Hcy concentration and result in enzyme inactivation, protein denaturation, aggregation, and precipitation (Sharma et al., 2015). On the basis of these observations, it has been postulated a role of Htl-mediated homocysteinylation in AD progression via promotion of amyloid deposition in the brain and tau protein aggregation (Sharma et al., 2015) and enhancement of Aβ peptide neurotoxicity via stabilization of its oligomeric form (the most reactive and dangerous) (Khodadadi et al., 2012). Moreover, homocysteinylation produces new epitopes on proteins usually recognized as self (Undas et al., 2004), and it has been postulated that autoantibodies versus homocysteinylated proteins could contribute to immunomediated damage to vessels walls and the promotion of atherosclerosis (Sharma et al., 2015). Furthermore, homocysteinylation of low-density lipoproteins (LDLs) renders them more prone to oxidation (augmented also due to Hcy-induced OS) and to uptake by macrophages to form foam cells and promote atherosclerotic plaque progression (Bełtowski, 2005; McCully, 2009).

Endothelial Damage and Atherothrombosis

Early atherosclerosis and thrombotic events are the hallmarks of vascular damage in homocystinuria, an inherited disorder in Met metabolism due to a cystathionine beta synthase deficiency that results in markedly high Hcy. Endothelial dysfunction is initially represented by impaired vasodilation capability, probably due to reduction in nitric oxide availability (McCully, 2009). Later stages exhibit various pathological manifestations such as vacuolization, cytoplasmic swelling, and hyperplasia of endothelial cells (McCully, 2009), vascular smooth cells growth, and enhanced collagen deposition in arterial intima and media (Tsai et al., 1994; Majors et al., 1997). So atherogenesis is promoted and sustained by endothelial dysfunction, OS and protein homocysteinylation consequences (especially oxidized homocysteinylated LDLs), and phlogistic or immunologic processes (activation of macrophages and production of IL-1, IL-6, and TNF-α) (McCully, 2009). A pronounced intimal damage in the absence of significant lipid deposition in vessel walls of young homocystinuric patients suggests that inflammatory modifications are the primary movers of Hcy-induced arterial alterations (McCully, 1969). Another Hcy effect able to further mine the stability of these severely phlogistic plaques is the promotion of platelet aggregation (McCully, 1969; Harker et al., 1974) and thrombosis (Genoud, 2014). Thus, vascular brain damage subsequent to prolonged exposure to HHcy can lead to stroke and vascular cognitive impairment or favor the progression of other neurodegenerative diseases (e.g., AD and PD).

The Epigenetic Theory of BGV Deficiency-Related Neurodegeneration

In the last decades, epigenetics gained noteworthy attention in the field of brain health maintenance and neurodegeneration (Fuso, 2013). DNA methylation is essential to the regulation of gene expression and genome stability, and recently growing evidence seems to suggest a relationship with neurodegenerative diseases such as AD and PD (Kwok, 2010). Methylated sequences are silenced, but the silencing could interfere with both inhibitory and promoting genes, resulting in a complex and varied, although specific, modified DNA expression. Numerous environmental factors could affect DNA expression; nutritional factors in particular have a potentially critical role (Fuso, 2013). SAM is the main substrate of DNA-methyl-transferases (DNMT), a class of enzymes responsible for the transposition of methyl groups from SAM to DNA cytosine, leaving SAH as a by-product of the reaction, which is then transformed to Hcy (Fuso et al., 2011). DNA-demethylase (DDM) conversely demethylates DNA, delineating a finely regulated equilibrium that depends on DNMTs and DDM activity and the availability of their substrates (Fuso et al., 2011). The SAM-to-SAH ratio defines the methylation potential (MP) of the body. Reduced SAM synthesis (due to Met or BGV deficiency) or SAH clearance (e.g., the inhibited transformation of SAH in Hcy even simply due to excess product) determines a reduction of the MP, resulting in DNA hypomethylation and aberrant gene expression (Fuso et al., 2011). SAM synthesis requires Met, and Hcy remethylation promotes an increment in MP in two ways: (1) by increasing Met availability (and as a consequence SAM availability) and (2) by scavenging Hcy (that, as already said, favors SAH degradation). Hcy remethylation is a key point in methyl donors synthesis, so it is clear that both the enzymatic pathways mediated by betaine–homocysteine–methyltransferase (which requires Bet, a metabolite of choline) and methionine synthase (which uses 5ʹ-methylene-tetrahydrofolate as a substrate and cobalamin as cofactor) have repercussions for DNA methylation processes. Moreover, as previously described, vB2 could also be considered to have an impact in SAM synthesis by being a cofactor for MTHFR in the transformation of 5,10-methylene-tetrahydrofolate to 5ʹ-methyl-tetrahydrofolate. Finally, though via mechanisms not completely understood, vB7-mediated histone biotinylation could also interfere with DNA methylation and further modify DNA expression.

Fuso and colleagues demonstrated with preclinical models of HHcy, obtained with BGV deficiency, that SAM depletion and SAH accumulation inhibit DNMTs and promote DDM activity. That results in DNA hypomethylation of the presenilin 1 gene promoter and increased γ-secretase synthesis (Fuso et al., 2011). Increased amyloidogenesis via γ-secretase cleavage of amyloid precursor protein into Aβ delineates the final steps of the complex relationship between BGV deficiency epigenetic consequences and AD development. Even if not studied in relation to BGV deficiency specific hypomethylation pattern, some authors have hypothesized that similar epigenetic mechanisms (in particular, DNA methylation pattern modifications) could be the expression of both “normal” age-related brain changes and neurodegeneration with variations in the entity and topography of DNA alterations (Keleshian et al., 2013). Other authors report that similar hypomethylation patterns have been observed in AD and AD-like syndromes such as in PD and Lewy body dementia (Sanchez-Mut et al., 2016). In conclusion, BGV deficiency seems to have epigenetic repercussions that could interfere with normal brain aging by promoting AD-like pathology and possibly other neurodegenerative pathways. More research is needed to confirm and eventually clarify these relationships.

An observation that could have an enormous impact on BGV fortification policies is that recent evidence from animal models supports the transmission of a hypomethylation pattern induced by BGV deficiency in pregnant rats to the brain cells of their offspring with permanent consequences until adult age (Sable, 2015). If such findings are observed and confirmed in humans, then new perspectives will open in the field of nutritional programs for disease prevention and health promotion on a generational level. An adequate fortification of foods with all of the BGV involved in one-carbon metabolism would reduce drastically the consequences of the detrimental genetic processes just described, possibly modifying the clinical landscape of neurodegenerative diseases as we know it.

Impact of B Group Vitamins and Choline on Neurotransmission

A balanced activity of neural networks sustained by different neurotransmitters is essential for normal brain functioning. Disturbances in the availability of neurotransmitters have been recognized as etiological in many neuropsychiatric and neurological disorders. Nowadays, therapies that directly or indirectly substitute or enhance the activity of neurotransmitters remain cornerstones for the treatment of depression, PD, AD, and other neurological conditions. The synthesis of monoamines (dopamine, serotonin, noradrenaline) depends on vB6 availability, and SAM is a cofactor in their metabolism (Bottiglieri, 1996; Rotstein and Kang, 2009). SAM administration or promotion of its synthesis via BGV has been reported as effective in increasing CNS monoaminergic systems and in treating depressive disorders (Bottiglieri, 1996, 1997). Moreover, the observation that SAM administration also enhances cholinergic neurotransmission (increased brain concentrations of acetylcholine and the expression of muscarinic receptors) led to the finding that it could be beneficial in treating the cognitive symptoms of major depression (Levkovitz et al., 2012) and to the hypothesis that it, along with the BGV implicated in its maintenance levels, could also have a role in treating demented patients (Bottiglieri, 2013). Being the cholinergic system strongly related to attention, learning, memory, and motivation (Luchicchi et al., 2014), it has been selected as main target for nootropic drugs, and its augmentation remains the main therapeutic strategy in AD patients (Allgaier and Allgaier, 2014). Both dietary choline (Hollenbeck, 2012) and its pharmacological derivatives (Gareri et al., 2015) promote acetylcholine synthesis. Moreover, in combination with other micronutrients (uridine monophosphate and omega-3 fatty acids), it has been proposed to modulate synaptic plasticity and consequently neurotransmission efficacy (Engelborghs et al., 2014; Wurtman, 2014).


BGV and choline are certainly essential nutrients for adequate brain development, function, and protection. A balanced diet rich in fruits and vegetables and minimal intake of foods of animal origin, in the absence of malabsorption and severe comorbidities, should ensure a sufficient provision of the necessary micronutrients for the maintenance of normal neurological functions. Elderly individuals often require a higher intake of BGVs, probably because of an expected reduced absorption capability and the onset of concomitant age-related conditions (e.g., increased OS, burden of acquired organ damage, and impaired buffer mechanisms due to abnormal metabolic activity). Most BGVs (with vB3, vB6, and perhaps vB9 as exceptions) are almost surely safe even at intake levels reached with food fortification or supplements. In the latter case, considering the complex interaction between all BGVs and choline, a complete supplementation of all of these micronutrients is safer and surely more effective than the administration of a single vitamin. In the presence of initial cognitive decline or, even worse, frank dementia, supplementation is probably useful but with limited rate of efficacy in the absence of a severe deficiency of the micronutrients supplemented. A neuroprotective dietary pattern should be adopted as soon as possible (even in early adulthood) to obtain the maximum result when all of the systems are still functional. Moreover, given the repercussion of systemic state on brain aging, a global approach that also contemplates regular physical activity and the adoption of a stimulating and challenging but healthy lifestyle is recommended.


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