British Journal of Nutrition (2001), 85, Suppl. 2, S109±S113 DOI: 10.

1049/BJN2000302
q The Author 2001

Folate, vitamin B12 and homocysteine in relation

Helga Refsum
Department of Pharmacology, University of Bergen, Armauer Hansens Hus, 5021 Bergen, Norway

Increased folate intake reduces the risk of neural tube defects, other malformations and also
possibly, pregnancy complications. Increasing evidence suggests that the beneficial effect of
folate may be related to improved function of methionine synthase, a vitamin B12-dependent
enzyme that converts homocysteine to methionine. In India, the majority of the population
adheres to a vegetarian diet known to be deficient in vitamin B12. In such a population,
increased folate intake may offer minimal protection against birth defects, whereas vitamin B12
administration should be considered. In this review, is described the metabolism of and
interrelations between folate, vitamin B12 and homocysteine. This is followed by a brief
discussion of some of the proposed mechanisms for their biological effects in relation to birth
defects and pregnancy outcome.

Folate: Neural tube defects: Pregnancy complications

Periconceptional intake of folic acid reduces the incidence so formed is either directed to the transsulfuration pathway
of neural tube defects (NTD), one of the most common which irreversibly converts Hcy to cysteine. Alternatively, it
birth defects, by more than 50% (MRC-Vitamin-Study- is remethylated to methionine, a reaction which in most
Research-Group, 1991; Czeizel & Dudas, 1992; Berry et al. tissues is catalysed by methionine synthase. This enzyme
1999; Botto et al. 1999). Folic acid may have a beneficial uses vitamin B12 as a cofactor and methyltetrahydrofolate
effect on other malformations (Czeizel 1993; Allen, 1996) (methyl-THF) as a substrate (Finkelstein, 1990), and this
and pregnancy complications as well (Scholl et al. 1996; explains the close relation between folate, vitamin B12 and
Neggers et al. 1997; Leeda et al. 1998). Recent studies Hcy. Deficiency of either vitamin leads to elevated total Hcy
suggest that impaired vitamin B12 status (Adams et al. (tHcy) level in plasma or serum (Allen et al. 1994), referred
1995; Rowland et al. 1995; Wilson et al. 1999), and to as hyperhomocysteinemia.
elevated blood homocysteine (Hcy) (Burke et al. 1992; The provision of methyl-THF for the methionine
Steegers-Theunissen et al. 1992; de Vries et al. 1997; Ray synthase reaction is from two sources. The major fraction
& Laskin, 1999; Vollset et al. 2000) may also be associated derives from the common cellular folate pool and is
with birth defects and common pregnancy complications provided by the methylenetetrahydrofolate reductase
such as spontaneous abortions, placental abruption, pre- (MTHFR) reaction (Fig. 1) (Rozen, 1996). A second
eclampsia and low birth weight. It is now recommended source, particularly in dividing cells, is through uptake of
that all women in their reproductive years should increase the circulating methyl-THF monoglutamate (ˆserum
their folate intake to at least 400 mg per day, whereas the folate). The initial demethylation of this newly absorbed
possible significance of vitamin B12 status has received folate through the methionine synthase reaction is critical
less attention. for providing the cells with the folates used in DNA
In this presentation, the focus will be on the metabolism synthesis reaction. Moreover, the THF formed becomes
of and interrelations between folate, vitamin B12 and Hcy, polyglutamated, a process which ensures that the cellular
and then some of the proposed mechanisms for their folates are retained (Shane & Stokstad, 1985). Interestingly,
biological effects will be discussed. both Hcy elimination and methyl-THF formation are under
strict regulation of AdoMet and therefore the methionine
level (Finkelstein, 1990). When AdoMet is in excess, the
Interrelation between folate, vitamin B12 and
transsulfuration pathway is activated, leading to elimina-
homocysteine
tion of methionine. On the other hand, when AdoMet is
Homocysteine is formed from methionine as a product low, MTHFR is stimulated, and this directs the folates from
of numerous S-adenosylmethionine (AdoMet)-dependent DNA and RNA synthesis to methionine conservation (Scott
transmethylation reactions (Fig. 1) (Mudd et al. 1995). Hcy & Weir, 1981).

Corresponding author: Prof Helga Refsum, tel 147 55974680, fax 147 55974605, email helga.refsum@farm.uib.no

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https://doi.org/10.1049/BJN2000302
 S110 H. Refsum

Fig. 1. Interrelation between folate, vitamin B12 and homocysteine metabolism. AdoHcy, adenosylhomocysteine; AdoMet, adenosylmethionine;
DHF, dihydrofolate; DMG, dimethylglycine; MS, methionine synthase; MTHFR, methylenetetrahydrofolate reductase; THF, tetrahydrofolate.

Both folate and vitamin B12 deficiency will lead to congenital malformations are related to inhibited folate
reduced methionine formation, and if provision of dependent dTMP formation are the observations related to
methionine is limited, the MTHFR activity will be the common C677T polymorphism in the MTHFR gene.
stimulated, thereby providing more methyl-THF for Homozygosity for this polymorphism, the TT genotype, is
methionine synthase. During vitamin B12 deficiency, associated with NTD (Van der Put et al. 1995; Christensen
however, the enzyme function remains impaired, and a et al. 1999; Shields et al. 1999). This enzyme variant has
situation referred to as the methylfolate trap develops low activity, and the afflicted subjects frequently have
(Herbert & Zalusky, 1962; Scott & Weir, 1981): The hyperhomocysteinemia. Notably, TT subjects usually have
methyl-THF accumulates at the expense of the other a normal or even high folate content, but their methyl-THF
cellular folates, and the uptake of folate from serum is level is low (Bagley & Selhub, 1998). These data point in
prevented. Thus, the biochemical effects of both folate the direction of impaired methionine synthase function or a
and vitamin B12 deficiency are quite similar and include disturbed ratio between methionine and Hcy as the cause of
a functional folate deficiency, hyperhomocysteinemia, major malformations (Lucock et al. 1997). In further
and a methionine level that is too low relative to the support of this, is a recent finding that the common A66G
Hcy level. The possible consequences of these three polymorphism in methionine synthase reductase (MTRR),
biochemical conditions will be now discussed. the enzyme that activates cobalamin-dependent methionine
synthase, increases NTD risk when cobalamin status is low
(Wilson et al. 1999).
Cellular folate deficiency
In addition to being a substrate in the methionine synthase
Imbalance between methionine and Hcy levels
reaction, folate is required for synthesis of both DNA and
RNA. In severe folate deficiency, cell division is impaired, Methionine is required for protein synthesis and it is the
and a characteristic morphologic picture arises, i.e. the precursor of AdoMet which is required in polyamine
megaloblastic changes (Wickramasinghe, 1999). Presum- synthesis and in the numerous transmethylation reactions
ably, severe deficiency is incompatible with normal fetal (Finkelstein, 1990). In relation to birth defects, it is
growth and development. Originally, it was believed that particularly AdoMet and its role in the transmethylation
the cause of megaloblastosis was explained by reduced reactions that has received attention (Fig. 1). AdoMet is
folate dependent formation of dTMP from dUMP used in the methylation of phospholipids, proteins, DNA,
(Fig. 1) and possibly also inhibited purine synthesis RNA, amino acids, neurotransmitters and a number of other
(Wickramasinghe, 1999). However, more recent data small molecules. Methylation and demethylation of critical
suggest that under conditions of low folate, uracil will CpG loci in DNA may cause gene silencing and gene
frequently be incorporated into DNA instead of thymine, activation, respectively, thereby regulating mammalian
and the normal repair processes to remove the misincorpo- gene expression and cellular differentiation. In addition,
rated uracil often fails. This will ultimately lead to double methylation of ribosomal RNAs plays an important role in
strand break and chromosome instability which again mRNA function and integrity (Chiang et al. 1996). Thus, a
promotes apoptosis (Blount et al. 1997; Koury et al. disturbed methylation activity may interfere with normal
1997). It appears that chromosomal damage also occurs in fetal growth and development in a number of different
subjects without clinical symptoms but with low folate or ways.
vitamin B12 status or increased tHcy (Blount et al. 1997; Reduced methylation activity may occur when the
Fenech et al. 1998). AdoMet level is very low. Indeed, a recent study observed
Perhaps the strongest evidence against the theory that that women with low methionine intake had an increased

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https://doi.org/10.1049/BJN2000302
 Folate, vitamin B12 and homocysteine S111

risk of having a NTD child (Shaw et al. 1997). However, a so, the relation between hyperhomocysteinemia and the
more common cause may be an unfavourable (low) ratio pregnancy complications involving placental ischemia can
between AdoMet and adenosylhomocysteine (AdoHcy). be explained.
This latter compound is the product formed after demethy- In relation to congenital abnormalities, an interesting
lation of AdoMet, and it is the immediate precursor of Hcy observation is that Hcy interacts with the N-methyl-D-
(Fig. 1). A high AdoHcy exerts a negative feed back on the asparate (NMDA) receptor system which is involved in
transmethylation reactions. A high Hcy by increasing neuronal development and migration. A recent study
AdoHcy will have a similar effect. Notably, a deficiency showed that agents interfering with the NMDA receptor,
of either vitamin B12 or folate will be particularly are potent teratogens in animal embryo models (Andaloro
deleterious since both Hcy (AdoHcy) and methionine et al. 1998), and it has been suggested that hyperhomo-
(AdoMet) change in an unfavourable direction. A high cysteinemia may cause NTD through such mechanisms
intake of methionine will circumvent the effect of a vitamin (Rosenquist et al. 1997).
deficiency because the AdoMet: AdoHcy ratio is restored.
Moreover, AdoMet has a folate sparing effect through its
Conclusions
effect on MTHFR (Shane & Stokstad, 1985).
In this respect, a dietary vitamin B12 deficiency should Worldwide, a unified strategy has been adopted to reduce
be briefly mentioned. Vitamin B12 is only provided in the incidence of NTD, i.e. that all women in their
animal derived proteins, and a vegetarian diet therefore reproductive years should substantially increase their folate
contains little vitamin B12. Unfortunately, such a diet is intake (Locksmith & Duff, 1998), preferably by taking folic
also a poor source of methionine, thus, vegetarians may be acid supplements (Cuskelly et al. 1996). However, in
at particularly high risk of developing conditions associated addition to folate status, low vitamin B12 level and
with reduced methylation activity. Indeed, low dietary elevated levels of tHcy have also been associated with
intake or malabsorption of vitamin B12 may be the reason birth defects and pregnancy complications. The biochem-
for the high risk of NTD in countries such as India (Sharma ical basis for their effect on the fetus and the pregnant
et al. 1994) and Mexico (Allen et al. 1995; Botto et al. woman remains uncertain, but increasing evidence points in
1999) where the reported incidence in some regions is the direction of altered AdoMet dependent transmethylase
nearly ten times higher than that observed in the United activity, impaired methionine synthase function and
States (Lary & Edmonds, 1996; Botto et al. 1999). Notably, hyperhomocysteinemia. From a practical point of view,
the harmful effect of vitamin B12 deficiency is not the identification of the exact mechanisms may seem only
confined to birth defects and pregnancy complications. of academic interest. However, an appropriate strategy for
The babies of vitamin B12 deficient mothers have low the prevention of these common conditions may critically
stores of vitamin B12, and breast feeding may further depend on the underlying biochemical defect. For instance,
aggravate the condition since maternal milk is low in B12 increased folic acid intake may be appropriate for the
(Specker et al. 1990; Allen et al. 1995). Even a mild Western society where impaired folate status and hyper-
vitamin B12 deficiency in the mother may later in infancy homocysteinemia usually are related to the C677T poly-
cause growth retardation, delayed psychomotor develop- morphism, poor diet or unhealthy life style (Guttormsen
ment and in some instances, permanent effects on the et al. 1996; NygaÊrd et al. 1998). However, intervention
developing brain (Schneede et al. 1994). with folic acid alone may not only be inefficient, but may
even cause harm to women living in regions where vitamin
B12 deficiency is endemic. The scientists, clinicians and
Homocysteine accumulation
policy-makers in different countries should carefully
Hcy accumulation may have multiple biological effects. It investigate and evaluate the relevance of the present
may impair the methylation activity as described above. In guidelines in their own populations.
addition, a high plasma tHcy level is believed to be
thrombogenic and atherogenic (Refsum et al. 1998). The
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