[66] Toole, JF; Malinow, MR; Chambless, LE; Spence, J.D.; Pettigrew, L.C.; Howard, V.J.;

Sides, E.G.; Wang, C.H.; Stampfer, M. Lowering homocysteine in patients with

ischemic stroke to prevent recurrent stroke, myocardial infarction, and death: the

Vitamin Intervention for Stroke Prevention (VISP) randomized controlled trial. JAMA,

2004; 291: 565-575

[67] Spence, J.D.; Bang, H.; Chambless, L.E.; Stampfer, M.J. Vitamin intervention for

stroke prevention trial. An efficacy analysis. Stroke, 2005; 36: 2404-2409.

[68] Bønaa K.H., Njølstad I., Ueland P.M., Schirmer H., Tverdal A., Steigen T., Nordrehaug

J.E., Arnesen E., Rasmussen K., NORVIT Trial Investigators. Homocysteine lowering

and cardiovascular events after acute myocardial infarction. N Engl J Med, 2006; 354:

1578-1588

[69] The Heart Outcomes Prevention Evaluation (HOPE) 2 Investigators. Homocysteine

lowering with folic acid and B vitamins in vascular disease. N Engl J Med, 2006; 354:

1567-1577

[70] Shemin D., Lapane K.L., Bausserman L., Kanaan E., Kahn S., Dworkin L., Bostom

A.G. Plasma total homocysteine and hemodialysis access thrombosis: a prospective

study. J Am Soc Nephrol, 1999; 10: 1095-1099.

[71] Mallamaci F., Bonanno G., Seminara G., Rapisarda F., Fatuzzo P., Candela V., Scudo

P., Spoto B., Testa A., Tripepi G., Tech S., Zoccali C. Hyperhomocysteinemia and

arteriovenous fistola thrombosis in hemodialysis patients. Am J Kidney Dis, 2005; 45:

702-707.

[72] Kim, Y.I. Nutritional epigenetics: impact of folate deficiency on DNA methylation and

colon cancer susceptibility. J Nutr, 2005; 135: 2703-2709.

[73] Kune, G.; Watson, L. Colorectal cancer protective effects and the dietary

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[74] Zoungas S., McGrath B.P., Branley P., Kerr P.G., Muske C., Wolfe R., Atkins R.C.,

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In: Vitamin B: New Research ISBN: 978-1-60021-782-1

Editor: C. M. Elliot, pp. 139-152 © 2008 Nova Science Publishers, Inc.

Chapter VIII

VITAMIN B12, FOLATE DEPLETION AND

HOMOCYSTEINE: WHAT DO THEY MEAN

FOR COGNITION ?

Rita Moretti*

, Paola Torre and Rodolfo M. Antonello

Clinica Neurologica, Dipartimento Medicina Clinica e Neurologia,

Università degli Studi, Trieste

ABSTRACT

Vitamin B12 exerts its physiological effect on two major enzymatic pathways: the

conversion of homocysteine to methionine and the conversion of methylmalonyl

coenzyme A to succinyl coenzyme A. Disruption of either of these pathways due to

vitamin B12 deficiency results in an elevation of both serum homocysteine and

methylmalonic acid. Homocysteine levels are also elevated in the case of folate

deficiency. Serum homocysteine is proposed to be more sensitive for functional

intracellular vitamin B12 deficiency than analysis of vitamin B12 in serum. Hence,

homocysteine, vitamin B12, and folate are closely linked together in the so-called onecarbon cycle. The proposed mechanism relates to the methylation reactions involving

homocysteine metabolism in the nervous system. Vitamin B12 is the necessary coenzyme, adequate for the correct functioning of the methyl donation from 5

Methyltethrahydrofolate in tetrhahydrofolate, necessary for methionine synthetase. On

the other hand, folate is a cofactor in one-carbon metabolism, during which it promotes

the remethylation of homocysteine- a cytotoxic sulfur-containing amino acid that can

induce DNA strand breakage, oxidative stress and apoptosis. What clearly merges from

Literature is the general conviction that vitamin B12 and folate, directly through the

maintenance of two functions, nucleic acid synthesis and the methylation reactions, or

indirectly, due to their lack which cause SAM mediated methylation reactions inhibition

*

 Corresponding Author: Rita Moretti, MD, Clinica Neurologica dell’Università degli Studi di Trieste, Ospedale di

Cattinara, Strada di Fiume, 447, 34149 TRIESTE, Italy; phone:0039-40-3994321; FAX: 0039-40-910861; email: moretti@univ.trieste.it

140 Rita Moretti, Paola Torre and Rodolfo M. Antonello

by its product SAH, and through the related toxic effects of homcystein which cause

direct damage to the vascular endothelium and inhibition of N-methyl-D-Aspartate

receptors, can cause neuropsychiatric disturbances.

INTRODUCTION

It is today well known that vitamin B12 deficiency can be associated with

neuropsychiatric symptoms. Several studies have previously demonstrated that vitamin B12

deficiency is more common in patients with dementia symptoms than in the cognitively nonimpaired. Vitamin B12 deficiency increases with age and is present in 5-40% of the elderly

population. However, the mechanism of neurological damage induced by a quantitative or

functional vitamin B12 deficiency is still unclear.

Vitamin B12 exerts its physiological effect on two major enzymatic pathways: the

conversion of homocysteine to methionine and the conversion of methylmalonyl coenzyme A

to succinyl coenzyme A. Disruption of either of these pathways due to vitamin B12

deficiency results in an elevation of both serum homocysteine and methylmalonic acid.

Homocysteine levels are also elevated in the case of folate deficiency. Serum homocysteine is

proposed to be more sensitive for functional intracellular vitamin B12 deficiency than

analysis of vitamin B12 in serum. Hence, homocysteine, vitamin B12, and folate are closely

linked together in the so-called one-carbon cycle. The proposed mechanism relates to the

methylation reactions involving homocysteine metabolism in the nervous system. It has been

suggested that the brain suffers from a double whammy from hyperhomocysteinaemia:

cerebrovascular damage that triggers or potentiates the effect of Alzheimer pathology

combined with a direct neurotoxic effect of homocysteine [1].

Low levels of vitamin B12 and that of low levels of serum folate still raise debates on

their possible role in cognition. The practice parameter for the diagnosis of dementia

concluded with different recommendations, based on the evidence in the literature. Among

them, screening for depression, B12 deficiency and hypothyroidism should be performed [2,

3,4, 5, 6, 7].

Albeit the theoretical importance of the determination of folate and vitamin B12 blood

levels, there is a general confusion on their possible role in neuropsychiatric alterations.

BYOCHEMISTRY OF FOLATE, VITAMIN B12,

AND HOMCYSTEINE

Congenital B vitamins that participate in one-carbon metabolism (ie folate, vitamin B12,

and vitamin B6) deficiency is associated with severe impairment of brain function [8, 9, 10,

11].

Folate and vitamin B12 are required both in the methylation process. The de novo

synthesis of methionine requires vitamin B12, which is involved directly in the transfer of the

methyl group to homocysteine. In turn, methionine is required in the synthesis of S-

Vitamin B12, Folate Depletion and Homcysteine… 141

adenosylmethionine (SAM) the sole donor in numerous methylation reactions involving

proteins, phospholipids and biogenic amines (figure 1).

Figure 1. Chemical structure of vitamin B12.

The pathway of one-carbon metabolism is characterized by the generation of one-carbon

units, normally from serine, made active through association with tetrahydrofolate (figure 2).

The resulting 5,10-methylentetrahydrofolate is subsequently used for the synthesis of

thymidylate and purines (used for nucleic acid synthesis) and of methionine, which is used

for protein synthesis and biological methylations. The methionine synthesis is preceded by

the irreversible reduction of 5,10 methylentetrahydrofolate to 5-methyltetrahydrofolate in a

142 Rita Moretti, Paola Torre and Rodolfo M. Antonello

reaction that is catalysed by the flavin-containing methylentetrahydrofolate reducatase.

Subsequently, 5-methyltetrahydrofolate serves a substrate to methylate homocysteine in a

reaction that is catalysed by a vitamin B12 containing methyltransferase.

Figure 2. Folate Acid: chemical structure.

Figure 3. Synthesis of SAM for the DNA methylation.

Homocysteine is also methylated by betaine in a reaction not involving vitamin B12. A

considerable proportion of methionine is activated by ATP to form S-adenosylmethionine

(SAM) [12] which serves primarily as a universal methyl donor in a variety of reactions. In

the brain, SAM-dependent methylations are extensive and the products of these reactions

Vitamin B12, Folate Depletion and Homcysteine… 143

include neurotransmitters such as catecholamines and indoleamines, phospholipids and

myelins. Upon transfer of its methyl group, SAM is converted to S-adenosylhomocysteine

(SAH), rapidly and subsequently hydrolysed to homocysteine and adenosine [13] (See figure

3).

This hydrolysis is a reversible reaction that favours SAH synthesis. Thus, in the state of

folate or vitamin B12 deficiency, the sequential inability to methylate homocysteine leads to

SAH intracellular accumulation. If homcysteine is allowed to accumulate, it will be rapidly

metabolised to SAH, which is a strong inhibitor of all methylation reaction, competing with

SAM for the active site on the methyltransferase enzyme protein [14; 15; 16; 17].

It has been hypothesized that a pathway of oxidation of homocystein to homocysteic acid

is the potential explanation of the dangerous effect of homocysteine. In fact, homocysteic

acid is a mixed excitatory agonist preferentially at NMDA receptors [18].

Elevated levels of homocysteine in the blood predispose to arteriosclerosis [19, 20]: as

many as 47% of patients with arterial occlusions manifest modest elevations in plasma

homocysteine [20]. The strength of the association between homocysteine and

cerebrovascular disease appears to be greater than that between homocysteine and coronary

heart disease or peripheral vascular disease. Moreover, homocysteine is also an agonist at the

glutamate site of the NMDA receptor and is therefore a potential excitotoxin. Elevated

glycine levels synergize with homocysteine to overstimulate NMDA receptors and contribute

to neuronal damage. Indeed, the toxicity of cysteine may derive in part from reaction with

bicarbonate and in part from the disulfide cysteine, which is transported into neurons in

exchange for the extracellular transport of glutamate via the anionic cystine glutamate

transporter. In the latter case, the local rise in extracellular excitatory amino acids could then

contribute to neurotoxicity [19].

WHAT HAPPENS IF VITAMIN B12 OR FOLATE

LEVELS ARE UNDER NORMAL RANGE?

Vitamin B12 is the necessary co-enzyme, adequate for the correct functioning of the

methyl donation from 5 Methyltethrahydrofolate in tetrhahydrofolate, necessary for

methionine synthetase. On the other hand, folate is a cofactor in one-carbon metabolism,

during which it promotes the remethylation of homocysteine- a cytotoxic sulfur-containing

amino acid that can induce DNA strand breakage, oxidative stress and apoptosis.

The biochemical basis of the interrelationship between folate and cobalamin is the

maintenance of two functions, nucleic acid synthesis and the methylation reactions. The latter

is particularly important in the brain and relies especially on maintaining the concentration of

S-adenosylmethionine (SAM). SAM mediated methylation reactions are inhibited by its

product S-adneosylhomocysteine (SAH). This occurs when cobalamin is deficient and, as a

result, methionine synthase is inhibited causing a rise of both homcysteine and SAH. Other

potential pathogenic processes related to the toxic effects of homcystein are direct damage to

the vascular endothelium and inhibition of N-methyl-D-Aspartate receptors [21, 22, 23, 24,

25, 26, 27, 28, 29, 30].

144 Rita Moretti, Paola Torre and Rodolfo M. Antonello

CLINICAL IMPLICATIONS

Data obtained from Literature stand that vitamin B12 is somehow bound to cognition and

to the implementation of active strategies to coordinate and well do in active problem

solving.

There are different causes which can produce cobalamin deficiency: an inadequate

intake, malabsorption, drugs, genetic deficiency of transcobalamin II. However, Larner et al.

[31; 32] reported that the effective number of vitamin B12 defect-dementia is extremely

small. Though, elderly individuals with cobalamin deficiency may present with

neuropsychatric or metabolic deficiencies, without frank macrocytic anemia [33, 34].

Psychiatric symptoms attributable to vitamin B12 deficiency have been described for

decades. These symptoms seem to fall into several clinically separate categories: slow

cerebration, confusion, memory changes, delirium with or without hallucinations and or

delusions, depression, acute psychotic states, and more rarely reversible manic and

schizophreniform states. Moreover, acute or subacute changes in a demented patient’s mental

status, specifically a clouding of their consciousness, may be due to a lack of vitamin B12

[35].

A higher prevalence of lower serum vitamin B12 levels have been found in subjects with

AD [36], other dementias [37] and in people with different cognitive impairments [31; 38], as

compared with controls. In contrast, other cross-sectional studies [39; 40] have failed to find

this association. The most recent study [41] on the topic examined the relationship between

vitamin B12 serum levels and cognitive and neuropsyhciatric symptoms in dementia. In AD,

the prevalence of low vitamin B12 serum levels is consistent with that found in community

dwelling elderly persons in general but is associated with greater overall cognitive

impairment.

Furthermore, some intervention studies have shown the effectiveness of vitamin B12

supplementation in improving cognition in demented or cognitively impaired subjects.

Chronic dementia responds poorly but should nevertheless be treated if there is a metabolic

deficiency (as indicated by elevated homocysteine and/or methylmalonic acid levels) [34].

These data have been confirmed by other studies [42; 43; 44 ; 45]. The B12 supplemented

patients who presented with dementia showed no significant improvement, and no less

deterioration, in their neuropsychological function than their matched group. However, a

treatment effect was demonstrated among the patients presenting with cognitive impairment.

These improved significantly compared to matched patients on the verbal fluency test. The

conclusion could be that vitamin B12 treatment may improve frontal lobe and language

function in patients with cognitive impairment, but rarely reverses dementia.

On the contrary, other works have failed to confirm the optimistic results [46, 47].

Cobalamin supplementation was given to al patients and the effect was evaluated after 6

months. When the size and the pattern of individual change scores, and the mean change

scores on all instruments were taken into account, functioning after replacement therapy was

not improved. When change scores of treated patients were compared with those of patients

with AD, vitamin B12 replacement did not result in slowing of the progression of dementia.

Many Different studies have tried to describe a possible consequence of the combined defect

of vitamin B12 and folate. Lower folate and vitamin B12 concentrations were associated with

Vitamin B12, Folate Depletion and Homcysteine… 145

poorer spatial copying skills. In addition, plasma homocysteine concentration, which is

inversely correlated with plasma folate and vitamin B12 concentrations, was a stronger

positive predictor of spatial copying performance than either folate or vitamin B12

concentrations: effective role in a clinical population is at the moment quite controversial [48,

49, 50, 51, 52, 53, 54, 55].

Recent epidemiological and experimental studies have linked folate deficiency and

resultant increased homocystein levels with several neurodegenerative conditions, including

stroke, AD, and Parkinson’s disease [56, 57, 58]. Folate deficiency sensitises mice to

dopaminergic neurodegeneration and motor dysfunction caused by neurotoxin MPTP.

Additional experiments indicate that this effect of folate deficiency may be mediated (again!)

by homocysiteine. These findings suggest that folate deficiency and hyperhomocysteinemia

are risk factors for Parkinsons’s disease.

One of the most recent review on folic acid [56] clearly states its importance in

neuropsychiatric disorders. Depression is commoner in patients with folate deficiency, and

subacute combined degeneration with peripheral neuropathy is more frequent in those with

vitamin B12 deficiency [59, 60, 61, 62, 63]. A close association with dementia and

depression, apathy, withdrawal, and lack of motivation has been noted [64]. One reason far

the apparently high incidence of folate deficiency in elderly people is that folate

concentrations in serum and cerebrospinal fluid fall and plasma homocysteine rises with age,

perhaps contributing to the ageing process [65, 66]. Considering that recent epidemiologic

studies [67; 68; 69; 70; 71] have shown an association between low serum folate levels and

risk of vascular disease, including stroke and various types of vascular cognitive impairment,

this work [66] examined data from the Canadian Study of Health and Ageing. The risk

estimate for an adverse cerebrovascular event associated with the lowest folate quartile

compared with the highest quartile was OR 2.42 (95%CI; 1.04-5.61). Results from stratified

analyses also showed that relatively low serum folate was associated with a significantly

higher risk of an adverse cerebrovascular event among female (OR 4.02, 95%CI; 1.37-11.81)

subjects [72, 73, 74, 75, 76].

Recently, the much larger and longer Framingham community based study confìrmed

that a raised plasma homocysteine (bound to folate level) concentration doubled the risk of

developing Alzheimer's and non-Alzheimer's dementia [77, 78]. In a relatively small sample

[79], serum folate had a strong negative association with the severity of atrophy of the

neocortex, and none of the other nutritional markers examined had significant associations

with atrophy in this study. Folate was related to atophy only among participants with a

significant number of Alzheimer disease lesions in the neocortex. This finding suggests that

folate may exacerbate the likelihood of atrophy only when an atrophying disease process

such as Alzheimer disease is present (with a positive relationship between low level of folate

and an impairment of memory, 80). In other case-control studies in patients with Alzheimer's

disease, cognitive decline was significantly associated with raised plasma homocysteine and

lowered serum folate (and vitamin B12) concentration [81, 82, 83].

146 Rita Moretti, Paola Torre and Rodolfo M. Antonello

HOW LABORATORY HELPS THE CLINICIAN?

Two new markers, plasma homocysteine and serum methylmalonic acid reflect the

functional status of cobalamins and folates in the tissues [84]. Since elevated homocysteine

levels can often be normalized by supplementing the diet with folic acid, pyridoxine

hydrochloride and cyanocobalamin, these observations raise the exciting possibility that this

inexpensive and well-tolerated therapy my be effective in decreasing the incidence of

vascular disease [85]. In addition to its association with cerebrovascular disease,

homocysteine may play a role in neurodegenerative disorders, even if only as a marker of

functional vitamin B12 deficiency.

A recent study [86] showed that B vitamins and homocysteine have been associated with

cognitive variation in old age. Serum total homcysteine levels were significantly higher and

serum folate and vitamin B12 levels were lower in patients with dementia of AD type and

with histologically confirmed AD than in controls [81]. After 3 years of follow-up, there was

significantly greater radiological evidence of disease progression assessed by medial

temporal lobe thickness, among those with total homocysteine levels in the middle and upper

compared with those in the lower tertile, who showed little atrophy [81]. The stability of total

homocysteine levels over time and lack of relationship with duration of symptoms argue

against these findings being a consequence of disease and warrant further studies to assess

the clinical relevance of these associations for AD [81].

Homocysteine has a direct consequence for neurotoxic effects on hippocampal and

cortical neurons [88, 89]. The findings of the study suggest that higher homocysteine levels

may be associated with early Alzheimer pathology.

On the other hand, hyperhomocysteinemia is a strong risk factor for atherosclerotic

vascular disease. In different recent study [90, 91], significantly elevated homocysteine levels

were found in patients with AD as well as in patients with vascular dementia, probably

indicating similar pathophysiological pathways. Prospective double-blind and placebocontrolled intervention studies are not available. If homocysteine-lowering therapy will be in

the running for the prevention and treatment of dementia, the clinician, at the moment, must

be able to diagnose the disease at a preclinical stage (5 to 10 years before the disease

becomes clinically overt for AD).

This might be a key point in the clinical practice in order to define dementia, either the

degenerative either the vascular form, as a complex relationship between oxidative,

inflammatory and degeneration. The last one, probably, might not have the primary role, as it

has, if the first two steps do not occur.

What clearly merges from Literature is the general conviction that vitamin B12 and

folate, directly through the maintenance of two functions, nucleic acid synthesis and the

methylation reactions, or indirectly, due to their lack which cause SAM mediated methylation

reactions inhibition by its product SAH, and through the related toxic effects of homcystein

which cause direct damage to the vascular endothelium and inhibition of N-methyl-DAspartate receptors, can cause neuropsychiatric disturbances.

Vitamin B12, Folate Depletion and Homcysteine… 147

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