The "Cobalaminergic" hypothesis

by Andrew McCaddon, October 2000

Abstract: An association between Alzheimer's Disease (AD) and low CSF and serum vitamin B12 (B12) has recently been described (Van Tiggelen 1983; Cole & Prchal 1984; Karnaze & Carmel 1987). This is apparently independent of nutritional intake (Renvall, Spindler, et al. 1989). It has been suggested that such patients may exhibit an atypical form of cobalamin deficiency (Karnaze & Carmel 1987; Renvall, Spindler, et al. 1989). It is therefore proposed that these deficiencies may be aetiologically important, at least in sub-groups of AD, and a mechanism is described whereby B12 deficiency may result in the characteristic neurotransmitter changes of the disease. The hypothesis generates predictions regarding biochemical evaluation of such patients and suggests associations between the neurochemical disturbances and structural abnormalities of AD.

The metabolic inter-relations of B12 and folate have been extensively documented with regard to the pathogenesis of megaloblastic anaemia. The 'methyl-folate' trap, proposed nearly 30 years ago to explain the biological response to B12 and folate deficiency, is now generally accepted. It is believed that the trap is a physiological response to impending methyl group deficiency due to a low supply of dietary methionine, but results in an inappropriate response to B12 deficiency and to the development of a potentially lethal anaemia. Essentially, the trap revolves around the B12 dependent conversion of homocysteine to methionine by methionine synthetase (MS), with the associated formation of free tetrahydrofolate (THF) from 5-methyl THF (see Fig.1). Free THF is the form from which various polyglutamate folate co-enzymes are subsequently derived via the synthesis of THF-polyglutamate. In the absence of B12, the MS reaction is impaired and a critical lack of free THF results.

This has two important effects:

1. Various methylation reactions are compromised, due to reduced levels of S-adenosyl methionine (SAM), a methionine derivative and important intracellular methyl group donor.
2. Purine (and pyrimidine) synthesis is impaired, due to a lack of formyl polyglutamate forms of folate, which serve as essential one-carbon donors in this pathway . This occurs, not only due to blocked THF synthesis, but also by a release of inhibition of 5-10 methylene THF polyglutamate reductase activity, secondary to falling SAM levels.

In essence, B12 deficiency results in the diversion of folate from nucleotide synthesis towards essential cellular methylation reactions. This response is ineffective, however, due to impaired B12 dependent MS activity and folate becomes trapped in the non-usable 5-methyl form.

It is hypothesised that these two major consequences of B12 deficiency can result in the characteristic cholinergic and monoaminergic deficits of AD.

Monoaminergic deficiency

B12 deficiency and the subsequent sequestration of folate in the 5-methyl form results in deficient supplies of formyl groups necessary for nucleotide biosynthesis . The de novo synthesis of ATP and GTP are both dependent upon the intracellular supply of inosine monophosphate (IMP) . Folate, in the formyl polyglutamate form, is necessary at two stages of IMP synthesis.

Fig.2

B12 deficiency results in reduced intracellular IMP and, hence, ATP and GTP. GTP is an essential precursor of tetrahydrobiopterin (BH4) via dihyroneopterin triphosphate. By way of the methyl-folate trap, B12 deficiency will therefore lead to reduced supplies of BH4, an essential and regulatory co-factor for biosynthesis of the monoamine neurotransmitters, dopamine, noradrenaline, and serotonin . Such an effect on monoamine synthesis has already been reported in B12 deficiency .

Cholinergic deficiency

Choline in cholinergic neurones is derived from three main sources :

Intrasynaptic choline, via degradation of acetylcholine by acetylcholinesterase;

extracellular choline, via a low affinity transport mechanism;

intraneuronal choline, via sequential methylation of membrane phosphatidylethanolamine (PE) or ethanolamine plasmalogens.

It is hypothesised that B12 deficiency may reduce both extracellular choline supplies and intraneuronal synthesis and thereby result in the characteristic cholinergic deficit of AD.

As discussed previously, one effect of B12 deficiency is impairment of essential methylation reactions, due to reduced formation of SAM, and important source of intracellular methyl-groups . Nitrous oxide induced cobalamin inactivation in the rat results in lowered levels of hepatic SAM and methylation reactions are compromised . These animals remain well, however, and it has been shown that an alternative B12 independent pathway for methylation of homocysteine to methionine is induced . This is the betaine homocysteine methyltransferase pathway, betaine supplying the methyl group instead of methyl-folate. Betaine is derived from the oxidation of choline via betaine aldehyde. Hence, B12 deficiency results in the diversion of endogenous and dietary choline to overcome the MS block, with consequent reduction in plasma and extraneuronal supplies.

It is also proposed that intraneuronal choline synthesis is compromised in B12 deficiency. Choline may be released from phosphatidylcholine (PC) by base exchange of phospho-lipase mediated hydrolyses . PC is formed either by the incorporation of existing choline, or by de novo synthesis. The latter process involves three sequential methylations of PC by SAM . B12 deficiency should therefore result in a decreased supply of neuronal SAM. Furthermore, betaine homocysteine methyltransferase is absent in the brain , which will further exacerbate this process. Sequential methylation of PE to PC will therefore be inhibited in B12 deficiency, resulting in impaired de novo synthesis of intraneuronal choline. There will also be an inversion of the SAM/SAH ratio, thereby inhibiting all transmethylation reactions .

This hypothesis outlines established biochemical pathways by which B12 deficiency may result in the cholinergic and monoaminergic deficits observed in AD. The hypothesis lends itself to certain neurochemical predictions and suggests possible mechanisms of structural change in AD which are now discussed more fully.

Neurochemical predictions

The B12 dependent MS block should result in an elevation of serum homocysteine, a potentially useful indicator of subtle and atypical B12 deficient states .

Impaired monoaminergic synthesis should be apparent by increased urinary excretion of amino-imidazole carboxamide, due to blocked IMP synthesis . Furthermore, the concentrations of total biopterin in brains of patients with AD should be reduced. This has already been demonstrated in one recent study .

Three further neurochemical implications have already been established. Firstly, as nucleotide synthesis is of prime importance for the formation of nucleic acid and protein, some derangement of this process would be expected in AD if B12 or folate deficiencies are indeed pathogenic. In fact, a 30% reduction in RNA synthesis has been described and protein formation is known to be similary deranged . In addition, the amount of RNA depletion exceeds that predicted solely on the basis of neuronal loss or neurofibrillary tangle formation . Secondly, and more specifically, reductions in adenine nucleotide content of neocortical samples in AD have been shown . Thirdly, as predicted, levels of CSF SAM are reduced in AD patients suggesting a disturbance of methylation reactions in the disease process .

Neuropathological implications

The hypothesis that B12 deficiency is of primary aetiological importance for sub-groups of AD presupposes that neuropathological changes of the disease occur secondary to metabolic derangement. The hypothesis raises interesting possibilities regarding this.

Reduced supplies of free choline, precipitated by B12 deficiency, could be the trigger factor for membrane disruption or "autocannabilism" which may occur when neuronal tissue resorts to this alternative choline supply . This membrane disruption could result in increased permeability, with subsequent leakage of protein and enzymes. There are two major consequences of such a process, which may correlate with recent findings. Membrane disruption may facilitate proteolytic cleavage of the putative A4 precursor (A4CT) into amyloid A4 protein, which would then aggregate into pathological fibrils, fibril bundles and amyloid . Secondly, altered membrane permeability, with protein and enzyme leakage, may account for cholinergic auto-antibody formation observed in AD, which may then further exacerbate this process .

Any hypothesis of the pathogenesis of AD must also account for the selective destruction of central cholinergic neurones. Interestingly, homocysteic acid is an endogenous agonist of the NMDA receptor which has an anatomical distribution correlating with the distribution of neurofibrillary tangles (NFT) and senile plaques (SP) seen in AD . It has been suggested that hyper-activation of this receptor may result in neuronal death . Elevated homocysteine, as predicted by this cobalaminergic hypothesis, could therefore account for the characteristic NFT and SP distribution of AD.

Although the effects of B12 deficiency have been discussed throughout it is interesting to note the predicted effects of a pure folate deficiency with regard to this hypothesis. Folate deficiency will result in reduced levels of SAM and the diversion of folate from purine synthesis to methylation reactions. B12 dependent MS activity remains intact and the strategy is successful with regard to methylation. Purine synthesis declines, however, with subsequent monoaminergic deficit. An affective disorder should therefore predominate which is, in fact, the commonest neuropsychiatric feature of this deficiency . Furthermore, the efficacy of SAM in the treatment of depression may be explained by its inhibition of 5-10 methylene THF reductase, resulting in an increased availability of folate co-enzymes necessary for biosynthesis of purines and, hence, monoamines.

It would be interesting to observe the effects of folinic acid supplementation for folate deficiency depression (and indeed for the emotional lability of AD) as this is a more direct substrate for formyl-folate co-enzyme synthesis .

This hypothesis delineates several mechanisms whereby B12 deficiency could result in some of the characteristic neurotransmitter deficits and structural abnormalities of AD (see Fig.3) Although it is not proposed that B12 deficiency is the primary cause of neurotransmitter and structural changes in all patients with AD, evidence is accumulating that a significant sub-group exists in which such deficits may be of primary aetiological importance . Furthemore, the 30% prevalence of sub-normal B12 levels observed in these studies may not reflect the true prevalence of low B12 in AD. A recent model of B12 deficiency suggests that subtle B12 deficient states could exist in the early stages of B12 malabsorption, before a measurable decline in serum B12 levels . It is also possible that B12 may be functionally inactive (although present in normal serum concentration), as a result of increased oxidative damage known to occur in AD . Nitrous oxide is known to inactivate B12 by the oxidation of Cob(I)alamin to Cob(III)alamin . The age-related increase in free radical formation could exert a similar oxidative effect on B12 resulting in the development of a "cryptic" cerebral B12 deficient state, not unlike that observed after prolonged N2O exposure . Indeed, such a mechanism may account for the well-established association between Down's syndrome and AD, as the gene dosage effect of superoxide dismutase (SOD I) located on chromosome 21 could also result in oxidative inactivation of B12 . Furthermore, the effects of aluminium on cellular redox potential and subsequent inactivation of cerebral B12 may explain the link between aluminium and AD . In these situations, serum homocysteine concentration may prove to be a more accurate indicator of B12 tissue status .

The effects of B12 supplementation on restoration of cognitive function in B12 deficient subjects with AD remains to be seen. By virtue of the pivotal status of B12/folate inter-relationships, early supplementation may well lead to a restoration of balanced neurotransmission, but correction of subsequent structural abnormalities, namely senile plaques and neurofibrillary tangles, would seem unlikely.

Finally, if, as is suspected, B12 deficiency is found to be of primary importance, the possibility of a simple screening procedure may be realised. Ideally, estimation of serum homocysteine or transcobalamin II saturation may provide the earliest evidence of a deficiency state. Appropriate intervention could then be provided for such patients before the onset of deteriorating cognitive function so characteristic of this devastating disease.

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Reprinted from Medical Hypotheses, 37,(3), McCaddon A, and Kelly C, "Alzheimer's Disease: A "cobalaminergic" hypothesis, 161-165, 1992, by permission of the publisher Churchill Livingstone. www.harcourt-international.com/journals/mehy