Vitamin B-12

Vitamin B-12, known also as vitamin B₁₂ (or commonly B₁₂ or B-12 for short), is a collection of cobalt and corrin-ring molecules which are defined by their particular vitamin function in the body. All of the substrate cobalt-corrin molecules from which B-12 is made must be synthesized by bacteria. However, after this synthesis is complete, the body has a limited power to convert any form of B-12 to another, by means of enzymatically removing certain prosthetic chemical groups from the cobalt atom.

Cyanocobalamin is one such compound that is a vitamin in this B complex, because it can be metabolized in the body to an active co-enzyme form. However, the cyanocobalamin form of B-12 does not occur in nature normally, but is a byproduct of the fact that other forms of B-12 are avid binders of cyanide (-CN) which they pick up in the process of activated charcoal purification of the vitamin after it is made by bacteria in the commercial process. Since the cyanocobalamin form of B-12 is deeply red colored, easy to crystallize, and is not sensitive to air-oxidation, it is typically used as a form of B-12 for food additives and in many common multivitamins. However, this form is not perfectly synonymous with B-12, inasmuch as many subtances have B-12 vitamin activity and can properly be labeled vitamin B-12, but cyanocobalamin is just one of them. (Thus, all cyanocobalamin is vitamin B-12, but not all vitamin B-12 is cyanocobalamin).

Vitamin B-12 is important for the normal functioning of the brain and nervous system and for the formation of blood. It is normally involved in the metabolism of every cell of the body, especially affecting the DNA synthesis and regulation but also fatty acid synthesis and energy production. However, many (though not all) of the effects of functions of B-12 can be replaced by sufficient quantities of folic acid (another B vitamin), since B-12 is used to regenerate folate in the body. Most “B-12 deficient symptoms” are actually folate deficient symptoms, since they include all the effects of pernicious anemia and megaloblastosis, which are due to poor synthesis of DNA when the body does not have a proper supply of folic acid for the production of thymine. When sufficient folic acid is available, all known B-12 functions normalize, save those connected with the enzyme Methylmalonyl Coenzyme A mutase (MUT), and its products (S-adenosylmethionine, SAMe) and substrates (methylmalonic acid, MMA).

Terminology

The name vitamin B-12 generally refers to all forms of the vitamin. Some medical practitioners have suggested that its use be split into two different categories, however.

• In a broad sense B-12 still refers to a group of cobalt-containing vitamer compounds known as cobalamins: these include cyanocobalamin (an artifact formed as a result of the use of cyanide in the purification procedures), hydroxocobalamin (another medicinal form), and finally, the two naturally occurring cofactor forms of B-12: methylcobalamin (MeB-12), the cofactor of Methylmalonyl Coenzyme A mutase (MUT), and 5-deoxyadenosylcobalamin (adenosylcobalamin—AdoB-12), the cofactor of 5-methyltetrahydrofolate-homocysteine methyltransferase (MTR).

• The term B-12 may be properly used to refer to cyanocobalamin, the principal B-12 form used for foods and in nutritional supplements. This ordinarily creates no problem, except perhaps in rare cases of eye nerve damage, where the body is only marginally able to use this form due to high cyanide levels in the blood due to cigarette smoking, and thus requires cessation of smoking, or else B-12 given in another form, for the optic symptoms to abate. However, tobacco amblyopia is a rare enough condition that debate continues about whether or not it represents a peculiar B-12 deficiency which is resistant to treatment with cyanocobalamin.

Finally, so-called Pseudo-B-12 refers to B-12-like substances which are found in certain organisms, including spirulina (a cyanobacterium) and some algae. These substances are active in tests of B-12 activity by highly sensitive antibody-binding serum assay tests, which measure levels of B-12 and B-12 like compounds in blood. However, these substances do not have B-12 biological activity for humans, a fact which may pose a theoretical danger to vegans and others on limited diets who do not ingest B-12 producing bacteria, but who nevertheless may show normal “B-12″ levels in the standard immunoassay which has become the normal medical method for testing for B-12 deficiency.

Structure

B-12 is the most chemically complex of all the vitamins. The structure of B-12 is based on a corrin ring, which is similar to the porphyrin ring found in heme, chlorophyll, and cytochrome. The central metal ion is Co (cobalt). Four of the six coordination sites are provided by the corrin ring, and a fifth by a dimethylbenzimidazole group. The sixth coordination site, the center of reactivity, is variable, being a cyano group (-CN), a hydroxyl group (-OH), a methyl group (-CH₃) or a 5′-deoxyadenosyl group (here the C5′ atom of the deoxyribose forms the covalent bond with Co), respectively, to yield the four B-12 forms mentioned above. The covalent C-Co bond is one of first examples of carbon-metal bonds in biology. The hydrogenases and, by necessity, enzymes associated with cobalt utilization, involve metal-carbon bonds.

Synthesis

Vitamin B-12 cannot be made by plants or animals, as the only type of organism that have the enzymes required for the synthesis of B-12 is bacteria. The total synthesis of B-12 was reported by Robert Burns Woodward and Albert Eschenmoser, and remains one of the classic feats of total synthesis.

Species from the following genera are known to synthesize B-12: Aerobacter, Agrobacterium, Alcaligenes, Azotobacter, Bacillus, Clostridium, Corynebacterium, Flavobacterium, Micromonospora, Mycobacterium, Nocardia, Propionibacterium, Protaminobacter, Proteus, Pseudomonas, Rhizobium, Salmonella, Serratia, Streptomyces, Streptococcus and Xanthomonas. Industrial production of B-12 is through fermentation of selected microorganisms. The most used species are Pseudomonas denitrificans and Propionibacterium shermanii, often genetically engineered and grown under special conditions to enhance yield.

Functions

Coenzyme B-12’s reactive C-Co bond participates in two types of enzyme-catalyzed reactions.

1. Rearrangements in which a hydrogen atom is directly transferred between two adjacent atoms with concomitant exchange of the second substituent, X, which may be a carbon atom with substituents, an oxygen atom of an alcohol, or an amine.
2. Methyl (-CH₃) group transfers between two molecules.

In humans, only two coenzyme B-12-dependent enzymes are known:

1. Methylmalonyl Coenzyme A mutase (MUT) which uses the AdoB-12 form and reaction type 1 to catalyze a carbon skeleton rearrangement (the X group is -COSCoA). MUT’s reaction converts MMl-CoA to Su-CoA, an important step in the extraction of energy from proteins and fats (for more see MUT’s reaction mechanism). This functionality is always lost in vitamin B-12 deficiency, and can be measured clinically as an increased methylmalonic acid (MMA) level. Unfortunately, an elevated MMA, though sensitive to B-12 deficiency, is probably overly sensitive, and not all who have it actually have B-12 deficiency. For example, MMA is elevated in 90-98% of patients with B-12 deficiency; however 25-20% of patients over the age of 70 have elevated levels of MMA, yet 25-33% of them do not have B-12 deficiency. For this reason, MMA is not routinely recommended in the elderly. The “gold standard” test for B-12 deficiency continues to be low blood levels of the vitamin.The MUT function, however, remains the one B-12 function which cannot be restored with folate supplementation, and which is necessary for myelin synthesis (see mechanism below) and certain other functions of the central nervous system. Other functions of B-12 related to DNA, as well as elevated homocysteine levels (see below) can often be corrected with supplementation with the vitamin folic acid.In a vitamin B12-dependent MUT reaction, methionine is subsequently converted to S-adenosyl-methionine. S-adenosyl-methionine is necessary for methylation of myelin sheath phospholipids. In a second reaction dependent on MUT, B-12 is used to convert methylmalonyl coenzyme A into succinyl coenzyme A. Failure of this second reaction to occur results in elevated levels of methylmalonic acid. Excessive methylmalonic acid will prevent normal fatty acid synthesis, or it will be incorporated into fatty acid itself rather than normal malonic acid. If this abnormal fatty acid subsequently is incorporated into myelin or if the methylation of the myelin sheath phospholipids fails to occur, the resulting myelin will be too fragile, and demyelination will occur. The result is subacute combinded degeneration of central nervous system and spinal cord.
2. 5-methyltetrahydrofolate-homocysteine methyltransferase (MTR), a methyl transfer enzyme, which uses the MeB-12 and reaction type 2 to catalyze the conversion of the amino acid Hcy into Met (for more see MTR’s reaction mechanism). This functionality is lost in vitamin B-12 deficiency, and can be measured clinically as an increased homocysteine level in vitro. Increased homocysteine can also be caused by a folic acid deficiency, since B-12 helps to regenerate folic acid, and thus decrease the need for it in the diet. There is some controversy over whether it is the reduced availability of methionine, or the reduced availability of THF (produced in the conversion of homocysteine to methionine) that is responsible for the reduced availability of 5,10-methylene-THF. In any case, 5,10-methylene-THF is involved in the synthesis of thymine, and hence reduced availability of 5,10-methylene-THF results in problems with DNA synthesis, and ultimately in ineffective production of blood cells, and also in intestinal wall cells which are responsible for absorption, in the once-dreaded and fatal disease, pernicious anemia. All of these effects, including the anemia of pernicious anemia, resolve if sufficient folate is at hand, making this best known function of B-12 (the one involved with DNA synthesis and restoration of cell-division and anemia) actually a facultative function which is mediated by folate.

On the other hand, the absolutely B-12 dependent MUT reaction (mentioned first) is now the one with the most important secondary effects, now that folic acid is being added to fortify flour in many countries (so that folate deficiency is now more rare), and now that folate-sensitive tests for anemia and erythrocyte size are routinely done in even simple medical test clinics (so these biochemical effects are more often directly detected). In the MUT reaction, the transmethylating agent S-adenosylmethionine (SAMe) is produced, which is involved in the synthesis of myelin, necessary for normal nerve function. Methylmalonic acid (MMA), produced when B-12 is deficient and MUT is inactive, is also a myelin destabalizer, as noted. These reasons explain why B-12 deficiency causes neuropathies, even if folic acid is present in good supply and anemia is not present. In addition, SAMe is involved in the manufacture of certain neurotransmitters, catecholamines and in brain metabolism. These neurotransmitters are important for maintaining mood, possibly explaining why depression is associated with B-12 deficiency.

Human absorption and distribution

The human physiology of vitamin B-12 is complex, and therefore is prone to mishaps leading to vitamin B-12 deficiency. The vitamin as it occurs in foods enters the digestive tract bound to proteins, known as salivary R-binders. Stomach proteolysis of these proteins requires an acid pH, and also requires proper pancreatic release of proteolytic enzymes. (Even small amounts of B-12 taken in supplements bypasses these steps and thus any need for gastric acid, which may be blocked by antacid drugs).

The free B-12 then attaches to gastric intrinsic factor, which is generated by the gastric parietal cells. If this step fails due to gastric parietal cell atrophy (the problem in pernicious anemia), sufficient B-12 is not absorbed later on, unless administered orally in relatively massive doses (500 to 1000 mcg/day).

The conjugated vitamin B-12-intrinsic factor complex (IF/B-12) is then normally absorbed by the terminal ileum of the small bowel. Absorption of food vitamin B-12 therefore requires an intact and functioning stomach, exocrine pancreas, intrinsic factor, and small bowel. Problems with any one of these organs makes a vitamin B-12 deficiency possible.

Once the IF/B-12 complex is recognized by specialized ileal receptors, it is transported into the portal circulation. The vitamin is then transferred to transcobalamin II (TC-II/B12), which serves as the plasma transporter of the vitamin. Genetic deficiencies of this protein are known, also leading to functional B-12 deficiency.

For the vitamin to serve inside cells, the TC-II/B-12 complex must bind to a cell receptor, and be endocytosed. The transcobalamin-II is degraded within a lysozyme, and the B-12 is finally released into the cytoplasm, where it may be transformed into the proper coenzyme, by certain cellular enzymes (see above).

Hereditary defects in production of the transcobalamins and their receptors may produce functional deficiencies in B-12 and infantile megaloblastic anemia, and abnormal B-12 related biochemistry, even in some cases with normal blood B-12 levels.

History as a treatment for anemi

B-12 deficiency is the cause of pernicious anemia, a usually-fatal disease of unknown etiology when it was first described in medicine. The cure was discovered by accident. William Murphy had been inducing anemia in dogs by bleeding them, and then conducting experiments in which he fed them various foods to observe which diets allowed them fastest recovery from the anemia. In the process, he discovered that ingesting large amounts of liver seemed to most-rapidly cure the anemia of traumatic blood loss, and suggested that therefore liver extracts be tried for pernicious anemia, an anemic disease of the time with no known cause or cure. The treatment of pernicious anemia with raw liver juice proved a success, but when George Minot and George Whipple set about to chemically isolate the curative substance in liver which cured anemia in dogs, they found it was iron. They found further that the partly isolated water-soluble liver-substance which cured pernicious anemia in humans, was something else entirely– and which had no effect at all on dogs under the conditions used. The cure for pernicious anemia had been found by coincidence. Minot and Whipple ultimately were able to isolate previously unsuspected vitamin B-12 from the extract of the liver which cured pernicious anemia. For this, the three men shared the 1934 Nobel Prize in Medicine.

The chemical structure of the molecule was determined by Dorothy Crowfoot Hodgkin and her team in 1956, based on crystallographic data.

Symptoms and damage from deficiency

Vitamin B-12 deficiency can potentially cause severe and irreversible damage, especially to the brain and nervous system. At levels only slightly lower than normal, a range of symptoms such as fatigue, depression, and poor memory may be felt. However, these symptoms by themselves are too nonspecific to diagnose deficiency of the vitamin.

B-12 can be supplemented in healthy subjects by oral pill; sublingual pill, liquid, or strip; by injection; or by nasal spray. B-12 is available singly or in combination with other supplements.

The Dietary Reference Intake for an adult ranges from 2 to 3 µg (micrograms). The recommended optimal daily intake (ODI) is 10 to 15 µg.

Sources

Vitamin B-12 is naturally found in foods of animal origin including meat (especially liver and shellfish) and milk products. Eggs are often mentioned as a good source, but they also contain a factor that blocks absorption. Interestingly, certain insects such as termites have been found to contain B-12. An NIH Fact Sheet lists a variety of food sources of vitamin B-12.

Plants only supply B-12 when the soil containing B-12 producing microorganisms has not been washed from them. However, it is rare to eat unwashed vegetables in the West, so vegans need to take special care to supplement their diets accordingly. According to the U.K. [Vegan Society], the only reliable vegans sources of B-12 are foods fortified with B-12 (including some plant milks, some soy products and some breakfast cereals) and B-12 supplements. Fortified breakfast cereals are a particularly valuable source of vitamin B-12 for vegetarians and vegans.

While lacto-ovo vegetarians usually get enough B-12 through dairy products, it may be found lacking in those practicing vegan diets who do not use multivitamin supplements or eat B-12 fortified foods, such as fortified breakfast cereals, fortified soy-based products, and fortified energy bars. Claimed sources of B-12 that have been shown through direct studies of vegans to be inadequate or unreliable include, nori (a seaweed), barley grass, and human gut bacteria. People on a vegan raw food diet are also susceptible to B-12 deficiency if no supplementation is used. However, the more alkaline intestines of vegans are better able to metabolize hydroxocobalamin, a more efficient cobalamin than cyanocobalamin.

The Vegan Society, the Vegetarian Resource Group and the Physicians Committee for Responsible Medicine, among others, recommend that vegans either consistently eat foods fortified with B-12 or take a daily or weekly B-12 supplement.

Cyanocobalamin is converted to its active forms, first hydroxocobalamin and then methylcobalamin and adenosylcobalamin in the liver. The sublingual route, in which B-12 is supposed to be absorbed more directly under the tongue, has not proven to be necessary or helpful. A 2003 study found no significant difference in absorption for serum levels from oral vs. sublingual delivery of 500 micrograms of cobalamin.

Injection is sometimes used in cases where digestive absorption is impaired, but there is some evidence that it may not necessary with modern high potency oral supplements (such as 500 to 1000 mcg or more). These supplements carry such large doses of the vitamin that the many different components of the B-12 absorption system are not required, and enough of the vitamin (only a few mcg a day) is obtained simply by mass-action transport across the gut. Even pernicious anemia can be treated entirely by the oral route.

For the much lower amounts of B-12 found in food sources, however, oral absorption is complex and requires stomach acid and also specific intestinal transport proteins (intrinsic factor) produced in the stomach. Lack of function in these systems is the causes of much of the increased risk in many elderly persons who develop B-12 deficiency later in life. However, it can be treated with a simple high dose oral B-12 supplement. Cyanocobalamin is also sometimes added to beverages including Diet Coke Plus and many energy drinks, in some cases with over 80 times the recommended intake. It is not known if these amounts are enough to correct malabsorbtion of B-12 due to lack of gastric acid or intrinsic factor.