Vitamin B12

Vitamin B12

Role of vitamin B12 in human metabolic processes

lthough the nutritional literature still uses the term vitamin B12, a more specific name

for vitamin B12 is cobalamin. Vitamin B12 is the largest of the B complex vitamins,

with a molecular weight of over 1000. It consists of a corrin ring made up of four

pyrroles with cobalt at the center of the ring (1, 2).

There are several vitamin B12–dependent enzymes in bacteria and algae, but no species

of plants have the enzymes necessary for vitamin B12 synthesis. This fact has significant

implications for the dietary sources and availability of vitamin B12. In mammalian cells there

are only two vitamin B12–dependent enzymes (3). One of these enzymes, methionine

synthase, uses the chemical form of the vitamin which has a methyl group attached to the

cobalt and is called methylcobalamin (see Figure 7 in Chapter 4.). The other enzyme,

methylmalonyl CoA mutase, uses vitamin B12 with a 5’-adeoxyadenosyl moiety attached to

the cobalt and is called 5’-deoxyaldenosylcobalamin, or coenzyme B12. In nature there are two

other forms of vitamin B12: hydroxycobalamin and aquacobalamin, where hydroxyl and water

groups, respectively, are attached to the cobalt. The synthetic form of vitamin B12 found in

supplements and fortified foods is cyanocobalamin, which has cyanide attached to the cobalt.

These three forms of B12 are enzymatically activated to the methyl- or

deoxyadenosylcobalamins in all mammalian cells.

Dietary sources and availability

Most microorganisms, including bacteria and algae, synthesise vitamin B12, and they

constitute the only source of the vitamin (4). The vitamin B12 synthesised in microorganisms

enters the human food chain through incorporation into food of animal origin. In many

animals gastrointestinal fermentation supports the growth of these vitamin B12–synthesising

microorganisms, and subsequently the vitamin is absorbed and incorporated into the animal

tissues. This is particularly true for the liver, where vitamin B12 is stored in large

concentrations. Products from these herbivorous animals, such as milk, meat, and eggs,

constitute important dietary sources of the vitamin unless the animal is subsisting in one of

the many regions known to be geochemically deficient in cobalt (5). Milk from cows and

humans contains binders with very high affinity for vitamin B12, whether they hinder or

promote intestinal absorption is not entirely clear. Omnivores and carnivores, including

humans, derive dietary vitamin B12 from animal tissues or products (i.e., milk, butter, cheese,

eggs, meat, poultry, etc.). It appears that no significant amount of the required vitamin B12 by

humans is derived from microflora, although vegetable fermentation preparations have also

been reported as being possible sources of vitamin B12 (6).

Absorption

The absorption of vitamin B12 in humans is complex (1, 2). Vitamin B12 in food is bound to

proteins and is released from the proteins by the action of a high concentration of

hydrochloric acid present in the stomach. This process results in the free form of the vitamin,

which is immediately bound to a mixture of glycoproteins secreted by the stomach and

salivary glands. These glycoproteins, called R-binders (or haptocorrins), protect vitamin B12

from chemical denaturation in the stomach. The stomach’s parietal cells, which secrete

hydrochloric acid, also secrete a glycoprotein called intrinsic factor. Intrinsic factor binds

vitamin B12 and ultimately enables its active absorption. Although the formation of the

vitamin B12 – intrinsic factor complex was initially thought to happen in the stomach, it is now

clear that this is not the case. At an acidic pH the affinity of the intrinsic factor for vitamin B12

is low whereas its affinity for the R-binders is high. When the contents of the stomach enter

the duodenum, the R-binders become partly digested by the pancreatic proteases, which

causes them to release their vitamin B12. Because the pH in the duodenum is more neutral

than that in the stomach, the intrinsic factor has a high binding affinity to vitamin B12, and it

quickly binds the vitamin as it is released from the R-binders. The vitamin B12–intrinsic factor

complex then proceeds to the lower end of the small intestine, where it is absorbed by

phagocytosis by specific ileal receptors (1, 2).

Populations at risk for and consequences of vitamin B12 deficiency

Vegetarians

Because plants do not synthesise vitamin B12, individuals who consume diets completely free

of animal products (vegan diets) are at risk of vitamin B12 deficiency. This is not true of lactoovo-

vegetarians, who consume the vitamin in eggs, milk, and other dairy products.

Pernicious anaemia

Malabsorption of vitamin B12 can occur at several points during digestion (1, 4). By far the

most important condition resulting in vitamin B12 malabsorption is the auto-immune disease

called pernicious anaemia (PA). In most cases of PA, antibodies are produced against the

parietal cells causing them to atrophy, lose their ability to produce intrinsic factor, and secrete

hydrochloric acid. In some forms of PA the parietal cells remain intact but auto-antiobodies

are produced against the intrinsic factor itself and attach to it, thus preventing it from binding

vitamin B12. In another less common form of PA, the antibodies allow vitamin B12 to bind to

the intrinsic factor but prevent the absorption of the intrinsic factor–vitamin B12 complex by

the ileal receptors. As is the case with most auto-immune diseases, the incidence of PA

increases markedly with age. In most ethnic groups it is virtually unknown to occur before the

age of 50, with a progressive rise in incidence thereafter (4). However, African American

populations are known to have an earlier age of presentation (4). In addition to causing

malabsorption of dietary vitamin B12, PA also results in an inability to reabsorb the vitamin

B12 which is secreted in the bile. Biliary secretion of vitamin B12 is estimated to be between

0.3 and 0.5 μg/day. Interruption of this so-called enterohepatic circulation of vitamin B12

causes the body to go into a significant negative balance for the vitamin. Although the body

typically has sufficient vitamin B12 stores to last 3–5 years, once PA has been established the

lack of absorption of new vitamin B12 is compounded by the loss of the vitamin because of

negative balance. When the stores have been depleted, the final stages of deficiency are often

quite rapid, resulting in death in a period of months if left untreated.

Atrophic gastritis

Historically, PA was considered to be the major cause of vitamin B12 deficiency, but it was a

fairly rare condition, perhaps affecting 1 percent to a few percent of elderly populations. More

recently it has been suggested that a far more common problem is that of hypochlorhydria

associated with atrophic gastritis, where there is a progressive reduction with age of the ability

of the parietal cells to secrete hydrochloric acid (7). It is claimed that perhaps up to onequarter

of elderly subjects could have various degrees of hypochlorhydria as a result of

atrophic gastritis. It has also been suggested that bacterial overgrowth in the stomach and

intestine in individuals suffering from atrophic gastritis may also reduce vitamin B12

absorption. This absence of acid is postulated to prevent the release of protein-bound vitamin

B12 contained in food but not to interfere with the absorption of the free vitamin B12 found in

fortified foods or supplements. Atrophic gastritis does not prevent the reabsorption of bilary

vitamin B12 and therefore does not result in the negative balance seen in individuals with PA.

However, it is agreed that with time, a reduction in the amount of vitamin B12 absorbed from

the diet will eventually deplete even the usually adequate vitamin B12 stores, resulting in overt

deficiency.

When considering recommended nutrient intakes (RNIs) for vitamin B12 for the

elderly, it is important to take into account the absorption of vitamin B12 from sources such as

fortified foods or supplements as compared with dietary vitamin B12. In the latter instances, it

is clear that absorption of intakes of less than 1.5–2.0 μg/day is complete – that is, for intakes

of less than 1.5–2.0 μg of free vitamin B12, the intrinsic factor – mediated system absorbs all

of that amount. It is probable that this is also true of vitamin B12 in fortified foods, although

this has not specifically been examined. However, absorption of food-bound vitamin B12 has

been reported to vary from 9 percent to 60 percent depending on the study and the source of

the vitamin, which is perhaps related to its incomplete release from food (8). This has led

many to estimate absorption as being up to 50 percent to correct for bio-availability of

absorption from food.

Vitamin B12 interaction with folate or folic acid

One of the vitamin B12 – dependent enzymes, methionine synthase, functions in one of the

two folate cycles   – the methylation cycle. This cycle is necessary to maintain

availability of the methyl donor S-adenosylmethionine; interruption reduces the wide range of

methylated products. One such important methylation is that of myelin basic protein.

Reductions in the level of S-adenosylmethionine seen in PA and other causes of vitamin B12

deficiency produce demyelination of the peripheral nerves and the spinal column, called subacute

combined degeneration (1, 2). This neuropathy is one of the main presenting conditions

in PA. The other principal presenting condition in PA is a megaloblastic anaemia

morphologically identical to that seen in folate deficiency. Disruption of the methylation cycle

should cause a lack of DNA biosynthesis and anaemia.

The methyl trap hypothesis is based on the fact that once the cofactor 5,10-

methylenetetrahydrofolate is reduced by its reductase to form 5-methyltetrahydrofolate, the

reverse reaction cannot occur. This suggests that the only way for the methyltetrahydrofolate

to be recycled to tetrahydrofolate, and thus to participate in DNA biosynthesis and cell

division, is through the vitamin B12 – dependent enzyme methionine synthase. When the

activity of this synthase is compromised, as it would be in PA, the cellular folate will become

progressively trapped as 5-methyltetrahydrofolate. This will result in a cellular pseudo folate

deficiency where despite adequate amounts of folate an anaemia will develop that is identical

to that seen in true folate deficiency. Clinical symptoms of PA, therefore, include neuropathy,

anaemia, or both. Treatment with vitamin B12, if given intramuscularly, will reactivate

methionine synthase, allowing myelination to restart. The trapped folate will be released and

DNA synthesis and generation of red cells will cure the anaemia. Treatment with high

concentrations of folic acid will treat the anaemia but not the neuropathy of PA. It should be

stressed that the so-called masking of the anaemia of PA is generally agreed not to occur at

concentrations of folate found in food or at intakes of the synthetic form of folic acid found at

usual RNI levels of 200 or 400 μg/day (1). However, there is some evidence that amounts less

than 400 μg may cause a haematologic response and thus potentially treat the anaemia (9).

The masking of the anaemia definitely occurs at high concentrations of folic acid (>1000

μg/day). This becomes a concern when considering fortification with synthetic folic acid of a

dietary staple such as flour.

 Vitamin B12

In humans the vitamin B12 – dependent enzyme methylmalonyl coenzyme A (CoA)

mutase functions in the metabolism of propionate and certain of the amino acids, converting

them into succinyl CoA, and in their subsequent metabolism via the citric acid cycle. It is

clear that in vitamin B12 deficiency the activity of the mutase is compromised, resulting in

high plasma or urine concentrations of methylmalonic acid (MMA), a degradation product of

methylmalonyl CoA. In adults this mutase does not appear to have any vital function, but it

clearly has an important role during embryonic life and in early development. Children

deficient in this enzyme, through rare genetic mutations, suffer from mental retardation and

other developmental defects.

Assessment of vitamin B12 status

Traditionally it was thought that low vitamin B12 status was accompanied by a low serum or

plasma vitamin B12 level (4). Recently this has been challenged by Lindenbaum et al.(10),

who suggested that a proportion of people with normal vitamin B12 levels are in fact vitamin

B12 deficient. They also suggested that elevation of plasma homo-cysteine and plasma MMA

are more sensitive indicators of vitamin B12 status. Although plasma homo-cysteine may also

be elevated because of folate or vitamin B6 deficiency, elevation of MMA apparently always

occurs with poor vitamin B12 status. There may be other reasons why MMA is elevated, such

as renal insufficiency, so the elevation of itself is not diagnostic. Many would feel that low or

decreased plasma vitamin B12 levels should be the first indication of poor status and that this

could be confirmed by an elevated MMA if this assay was available.

Evidence on which to base a recommended intake

Recommendations for nutrient intake

The Food and Nutrition Board of the National Academy of Sciences (NAS) Institute of

Medicine (8) has recently exhaustively reviewed the evidence of intake, status, and health for

all age groups and during pregnancy and lactation. This review has lead to calculations of

what they have called an estimated average requirement (EAR). The EAR is defined by NAS

as “the daily intake value that is estimated to meet the requirement, as defined by the specific

indicator of adequacy, in half of the individuals in a life-stage or gender group” (8). They

have estimated the recommended dietary allowances to be this figure plus 2 standard

deviations (SDs). Some members of the Food and Agriculture Organization of the United

Nations and World Health Organization (FAO/WHO) Expert Consultation were involved in

the preparation and review of the NAS recommendations and judge them to have been the

best estimates based on available scientific literature. The FAO/WHO expert group felt it

appropriate to adopt the same approach used by the NAS in deriving the RNIs. Therefore the

RNIs suggested in Table 14 are based on the NAS EARs plus 2 SDs.

Adults

Several lines of evidence point to an adult average requirement of about 2.0 μg/day. The

amount of intramuscular vitamin B12 needed to maintain remission in people with PA

suggests a requirement of about 1.5 μg/day (10), but they would also be losing 0.3–0.5μg/day

through interruption of their enterohepatic circulation, which is not typical. This might

suggest a requirement of 0.7–1.0 μg/day. Because vitamin B12 is not completely absorbed

from food, an adjustment of 50 percent has to be added giving a range of 1.4–2.0 μg/day (4).

Therapeutic response to ingested food vitamin B12 suggests a minimum requirement of

something less than 1.0 μg/day (8). Diets containing 1.8 μg/day seemed to maintain adequate

status but lower intakes showed some signs of deficiency. (8). Dietary intakes of less than 1.5