Vitamin B₁₂, also known as cobalamin, is a water-soluble vitamin involved in metabolism. It is one of eight B vitamins. It is required by animals, which use it as a cofactor in DNA synthesis, and in both fatty acid and amino acid metabolism. It is important in the normal functioning of the nervous system via its role in the synthesis of myelin, and in the circulatory system in the maturation of red blood cells in the bone marrow. Plants do not need cobalamin and carry out the reactions with enzymes that are not dependent on it.

Vitamin B₁₂ is the most chemically complex of all vitamins, and for humans the only vitamin that must be sourced from animal-derived foods or supplements. Only some archaea and bacteria can synthesize vitamin B₁₂. Vitamin B₁₂ deficiency is a widespread condition that is particularly prevalent in populations with low consumption of animal foods. This can be due to a variety of reasons, such as low socioeconomic status, ethical considerations, or lifestyle choices such as veganism.

Foods containing vitamin B₁₂ include meat, shellfish, liver, fish, poultry, eggs, and dairy products. Many breakfast cereals are fortified with the vitamin. Supplements and medications are available to treat and prevent vitamin B₁₂ deficiency. They are usually taken by mouth, but for the treatment of deficiency may also be given as an intramuscular injection.

Vitamin B₁₂ deficiencies have a greater effect on young children, pregnant and elderly people, and are more common in middle and lower developed countries due to malnutrition. The most common cause of vitamin B₁₂ deficiency in developed countries is impaired absorption due to a loss of gastric intrinsic factor (IF) which must be bound to a food-source of B₁₂ in order for absorption to occur. A second major cause is an age-related decline in stomach acid production (achlorhydria), because acid exposure frees protein-bound vitamin. For the same reason, people on long-term antacid therapy, using proton-pump inhibitors, H₂ blockers or other antacids are at increased risk.

The diets of vegetarians and vegans may not provide sufficient B₁₂ unless a dietary supplement is taken. A deficiency may be characterized by limb neuropathy or a blood disorder called pernicious anemia, a type of anemia in which red blood cells become abnormally large. This can result in fatigue, decreased ability to think, lightheadedness, shortness of breath, frequent infections, poor appetite, numbness in the hands and feet, depression, memory loss, confusion, difficulty walking, blurred vision, irreversible nerve damage, and many others. If left untreated in infants, deficiency may lead to neurological damage and anemia. Folate levels in the individual may affect the course of pathological changes and symptomatology of vitamin B₁₂ deficiency. Vitamin B₁₂ deficiency in pregnant women is strongly associated with an increased risk of spontaneous abortion, congenital malformations such as neural tube defects, problems with brain development growth in the unborn child.

Vitamin B₁₂ was discovered as a result of pernicious anemia, an autoimmune disorder in which the blood has a lower than normal number of red blood cells, due to a deficiency of vitamin B₁₂. The ability to absorb the vitamin declines with age, especially in people over 60.

Definition

Vitamin B₁₂ is a coordination complex of cobalt, which occupies the center of a corrin ligand and is further bound to a benzimidazole ligand and adenosyl group. A number of related species are known and these behave similarly, in particular all function as vitamins. This collection of compounds is sometimes referred to as "cobalamins". These chemical compounds have a similar molecular structure, each of which shows vitamin activity in a vitamin-deficient biological system, they are referred to as vitamers. The vitamin activity is as a coenzyme, meaning that its presence is required for some enzyme-catalyzed reactions.

• adenosylcobalamin
• cyanocobalamin, the adenosyl ligand in vitamin B₁₂ is replaced by cyanide.
• hydroxocobalamin, the adenosyl ligand in vitamin B₁₂ is replaced by hydroxide.
• methylcobalamin, the adenosyl ligand in vitamin B₁₂ is replaced by methyl.

Cyanocobalamin is a manufactured form of B₁₂. Bacterial fermentation creates AdoB₁₂ and MeB₁₂, which are converted to cyanocobalamin by the addition of potassium cyanide in the presence of sodium nitrite and heat. Once consumed, cyanocobalamin is converted to the biologically active AdoB₁₂ and MeB₁₂. The two bioactive forms of vitamin B
₁₂ are methylcobalamin in cytosol and adenosylcobalamin in mitochondria.

Cyanocobalamin is the most common form used in dietary supplements and food fortification because cyanide stabilizes the molecule against degradation. Methylcobalamin is also offered as a dietary supplement. There is no advantage to the use of adenosylcobalamin or methylcobalamin forms for the treatment of vitamin B₁₂ deficiency.

Hydroxocobalamin can be injected intramuscularly to treat vitamin B₁₂ deficiency. It can also be injected intravenously for the purpose of treating cyanide poisoning, as the hydroxyl group is displaced by cyanide, creating a non-toxic cyanocobalamin that is excreted in urine.

"Pseudovitamin B₁₂" refers to compounds that are corrinoids with a structure similar to the vitamin but without vitamin activity. Pseudovitamin B₁₂ is the majority corrinoid in spirulina, an algal health food sometimes erroneously claimed as having this vitamin activity.

Deficiency

Vitamin B₁₂ deficiency can potentially cause severe and irreversible damage, especially to the brain and nervous system. Deficiency at levels only slightly lower than normal can cause a range of symptoms such as fatigue, feeling weak, lightheadedness, dizziness, breathlessness, headaches, mouth ulcers, upset stomach, decreased appetite, difficulty walking (staggering balance problems), muscle weakness, depression, poor memory, poor reflexes, confusion, and pale skin, feeling abnormal sensations, among others, especially in people over age 60. Vitamin B₁₂ deficiency can also cause symptoms of mania and psychosis. Among other problems, weakened immunity, reduced fertility and interruption of blood circulation in women may occur.

The main type of vitamin B12 deficiency anemia is pernicious anemia, characterized by a triad of symptoms:

1. Anemia with bone marrow promegaloblastosis (megaloblastic anemia). This is due to the inhibition of DNA synthesis (specifically purines and thymidine).
2. Gastrointestinal symptoms: alteration in bowel motility, such as mild diarrhea or constipation, and loss of bladder or bowel control. These are thought to be due to defective DNA synthesis inhibiting replication in tissue sites with a high turnover of cells. This may also be due to the autoimmune attack on the parietal cells of the stomach in pernicious anemia. There is an association with gastric antral vascular ectasia (which can be referred to as watermelon stomach), and pernicious anemia.
3. Neurological symptoms: sensory or motor deficiencies (absent reflexes, diminished vibration or soft touch sensation) and subacute combined degeneration of the spinal cord. Deficiency symptoms in children include developmental delay, regression, irritability, involuntary movements and hypotonia.

Vitamin B₁₂ deficiency is most commonly caused by malabsorption, but can also result from low intake, immune gastritis, low presence of binding proteins, or use of certain medications. Vegans—people who choose to not consume any animal-sourced foods—are at risk because plant-sourced foods do not contain the vitamin in sufficient amounts to prevent vitamin deficiency. Vegetarians—people who consume animal byproducts such as dairy products and eggs, but not the flesh of any animal—are also at risk. Vitamin B₁₂ deficiency has been observed in between 40% and 80% of the vegetarian population who do not also take a vitamin B₁₂ supplement or consume vitamin-fortified food. In Hong Kong and India, vitamin B₁₂ deficiency has been found in roughly 80% of the vegan population. As with vegetarians, vegans can avoid this by consuming a dietary supplement or eating B₁₂ fortified food such as cereal, plant-based milks, and nutritional yeast as a regular part of their diet. The elderly are at increased risk because they tend to produce less stomach acid as they age, a condition known as achlorhydria, thereby increasing their probability of B₁₂ deficiency due to reduced absorption.

Nitrous oxide overdose or overuse converts the active monovalent form of vitamin B12 to the inactive bivalent form.

Pregnancy, lactation and early childhood

The U.S. Recommended Dietary Allowance (RDA) for pregnancy is 2.6 μg/d, for lactation 2.8 μg/d. Determination of these values was based on an RDA of 2.4 μg/d for non-pregnant women, plus what will be transferred to the fetus during pregnancy and what will be delivered in breast milk. However, looking at the same scientific evidence, the European Food Safety Authority (EFSA) sets adequate intake (AI) at 4.5 μg/d for pregnancy and 5.0 μg/d for lactation. Low maternal vitamin B₁₂, defined as serum concentration less than 148 pmol/L, increases the risk of miscarriage, preterm birth and newborn low birth weight. During pregnancy the placenta concentrates B₁₂, so that newborn infants have a higher serum concentration than their mothers. As it is recently absorbed vitamin content that more effectively reaches the placenta, the vitamin consumed by the mother-to-be is more important than that contained in her liver tissue.

Women who consume little animal-sourced food, or who are vegetarian or vegan, are at higher risk of becoming vitamin depleted during pregnancy than those who consume more animal products. This depletion can lead to anemia, and also an increased risk that their breastfed infants become vitamin deficient. Vitamin B₁₂ is not one of the supplements recommended by the World Health Organization for healthy women who are pregnant, however vitamin B₁₂ is often suggested during pregnancy in a multivitamin along with folic acid especially for pregnant mothers who follow a vegetarian or vegan diet.

Low vitamin concentrations in human milk occur in families with low socioeconomic status or low consumption of animal products. Only a few countries, primarily in Africa, have mandatory food fortification programs for either wheat flour or maize flour; India has a voluntary fortification program. What the nursing mother consumes is more important than her liver tissue content, as it is recently absorbed vitamin that more effectively reaches breast milk. Breast milk B₁₂ decreases over months of nursing in both well-nourished and vitamin-deficient mothers. Exclusive or near-exclusive breastfeeding beyond six months is a strong indicator of low serum vitamin status in nursing infants. This is especially true when the vitamin status was poor during the pregnancy and if the early-introduced foods fed to the still breastfeeding infant are vegan.

Risk of deficiency persists if the post-weaning diet is low in animal products. Signs of low vitamin levels in infants and young children can include anemia, poor physical growth and neurodevelopmental delays. Children diagnosed with low serum B₁₂ can be treated with intramuscular injections, then transitioned to an oral dietary supplement.

Gastric bypass surgery

Various methods of gastric bypass or gastric restriction surgery are used to treat morbid obesity. Roux-en-Y gastric bypass surgery (RYGB) but not sleeve gastric bypass surgery or gastric banding, increases the risk of vitamin B₁₂ deficiency and requires preventive post-operative treatment with either injected or high-dose oral supplementation. For post-operative oral supplementation, 1000 μg/d may be needed to prevent vitamin deficiency.

Diagnosis

According to one review: "At present, no 'gold standard' test exists for the diagnosis of vitamin B₁₂ deficiency and as a consequence the diagnosis requires consideration of both the clinical state of the patient and the results of investigations." The vitamin deficiency is typically suspected when a routine complete blood count shows anemia with an elevated mean corpuscular volume (MCV). In addition, on the peripheral blood smear, macrocytes and hypersegmented polymorphonuclear leukocytes may be seen. Diagnosis is supported based on vitamin B₁₂ blood levels below 150–180 pmol/L (200–250 pg/mL) in adults. However, serum values can be maintained while tissue B₁₂ stores are becoming depleted. Therefore, serum B₁₂ values above the cut-off point of deficiency do not necessarily confirm adequate B₁₂ status. For this reason, elevated serum homocysteine over 15 micromol/L and methylmalonic acid (MMA) over 0.271 micromol/L are considered better indicators of B₁₂ deficiency, rather than relying only on the concentration of B₁₂ in blood. However, elevated MMA is not conclusive, as it is seen in people with B₁₂ deficiency, but also in elderly people who have renal insufficiency, and elevated homocysteine is not conclusive, as it is also seen in people with folate deficiency. In addition, elevated methylmalonic acid levels may also be related to metabolic disorders such as methylmalonic acidemia. If nervous system damage is present and blood testing is inconclusive, a lumbar puncture may be carried out to measure cerebrospinal fluid B₁₂ levels.

Serum haptocorrin binds 80-90% of circulating B₁₂, rendering it unavailable for cellular delivery by transcobalamin II. This is conjectured to be a circulating storage function. Several serious, even life-threatening diseases cause elevated serum HC, measured as abnormally high serum vitamin B₁₂, while at the same time potentially manifesting as a symptomatic vitamin deficiency because of insufficient vitamin bound to transcobalamin II which transfers the vitamin to cells.

Medical uses

Treatment of deficiency

Severe vitamin B₁₂ deficiency is initially corrected with daily intramuscular injections of 1000 μg of the vitamin, followed by maintenance via monthly injections of the same amount or daily oral dosing of 1000 μg. The daily dose is far in excess of the vitamin requirement because the normal transporter protein mediated absorption is absent, leaving only very inefficient intestinal passive absorption. Injection side effects include skin rash, itching, chills, fever, hot flushes, nausea and dizziness. Oral maintenance treatment avoids this problem and significantly reduces cost of treatment.

Cyanide poisoning

For cyanide poisoning, a large amount of hydroxocobalamin may be given intravenously and sometimes in combination with sodium thiosulfate. The mechanism of action is straightforward: the hydroxycobalamin hydroxide ligand is displaced by the toxic cyanide ion, and the resulting non-toxic cyanocobalamin is excreted in urine.

Dietary recommendations

Some research shows that most people in the United States and the United Kingdom consume sufficient vitamin B₁₂. However, other research suggests that the proportion of people with low or marginal levels of vitamin B₁₂ is up to 40% in the Western world. Grain-based foods can be fortified by having the vitamin added to them. Vitamin B₁₂ supplements are available as single or multivitamin tablets. Pharmaceutical preparations of vitamin B₁₂ may be given by intramuscular injection. Since there are few non-animal sources of the vitamin, vegans are advised to consume a dietary supplement or fortified foods for B₁₂ intake, or risk serious health consequences. Children in some regions of developing countries are at particular risk due to increased requirements during growth coupled with diets low in animal-sourced foods.

The US National Academy of Medicine updated estimated average requirements (EARs) and recommended dietary allowances (RDAs) for vitamin B₁₂ in 1998. The EAR for vitamin B₁₂ for women and men ages 14 and up is 2.0 μg/day; the RDA is 2.4 μg/d. RDA is higher than EAR so as to identify amounts that will cover people with higher than average requirements. RDA for pregnancy equals 2.6 μg/day. RDA for lactation equals 2.8 μg/d. For infants up to 12 months the adequate intake (AI) is 0.4–0.5 μg/day. (AIs are established when there is insufficient information to determine EARs and RDAs.) For children ages 1–13 years the RDA increases with age from 0.9 to 1.8 μg/day. Because 10 to 30 percent of older people may be unable to effectively absorb vitamin B₁₂ naturally occurring in foods, it is advisable for those older than 50 years to meet their RDA mainly by consuming foods fortified with vitamin B₁₂ or a supplement containing vitamin B₁₂. As for safety, tolerable upper intake levels (known as ULs) are set for vitamins and minerals when evidence is sufficient. In the case of vitamin B₁₂ there is no UL, as there is no human data for adverse effects from high doses. Collectively the EARs, RDAs, AIs and ULs are referred to as dietary reference intakes (DRIs).

The European Food Safety Authority (EFSA) refers to the collective set of information as "dietary reference values", with population reference intake (PRI) instead of RDA, and average requirement instead of EAR. AI and UL are defined by EFSA the same as in the United States. For women and men over age 18 the adequate intake (AI) is set at 4.0 μg/day. AI for pregnancy is 4.5 μg/day, for lactation 5.0 μg/day. For children aged 1–14 years the AIs increase with age from 1.5 to 3.5 μg/day. These AIs are higher than the U.S. RDAs. The EFSA also reviewed the safety question and reached the same conclusion as in the United States—that there was not sufficient evidence to set a UL for vitamin B₁₂.

The Japan National Institute of Health and Nutrition set the RDA for people ages 12 and older at 2.4 μg/day. The World Health Organization also uses 2.4 μg/day as the adult recommended nutrient intake for this vitamin.

For U.S. food and dietary supplement labeling purposes, the amount in a serving is expressed as a "percent of daily value" (%DV). For vitamin B₁₂ labeling purposes, 100% of the daily value was 6.0 μg, but on May 27, 2016, it was revised downward to 2.4 μg. Compliance with the updated labeling regulations was required by 1 January 2020 for manufacturers with US$10 million or more in annual food sales, and by 1 January 2021 for manufacturers with lower volume food sales. A table of the old and new adult daily values is provided at Reference Daily Intake.

Sources

Bacteria and archaea

Vitamin B₁₂ is produced in nature by certain bacteria, and archaea. It is synthesized by some bacteria in the gut microbiota in humans and other animals, but it has long been thought that humans cannot absorb this as it is made in the colon, downstream from the small intestine, where the absorption of most nutrients occurs. Ruminants, such as cows and sheep, are foregut fermenters, meaning that plant food undergoes microbial fermentation in the rumen before entering the true stomach (abomasum), and thus they are absorbing vitamin B₁₂ produced by bacteria.

Other mammalian species (examples: rabbits, pikas, beaver, guinea pigs) consume high-fibre plants which pass through the gastrointestinal tract and undergo bacterial fermentation in the cecum and large intestine. In this hindgut fermentation, the material from the cecum is expelled as "cecotropes" and are re-ingested, a practice referred to as cecotrophy. Re-ingestion allows for absorption of nutrients made available by bacterial fermentation, and also of vitamins and other nutrients synthesized by the gut bacteria, including vitamin B₁₂.

Non-ruminant, non-hindgut herbivores may have an enlarged forestomach and/or small intestine to provide a place for bacterial fermentation and B-vitamin production, including B₁₂. For gut bacteria to produce vitamin B₁₂, the animal must consume sufficient amounts of cobalt. Soil that is deficient in cobalt may result in B₁₂ deficiency, and B₁₂ injections or cobalt supplementation may be required for livestock.

Animal-derived foods

Animals store vitamin B₁₂ from their diets in their livers and muscles and some pass the vitamin into their eggs and milk. Meat, liver, eggs and milk are therefore sources of the vitamin for other animals, including humans. For humans, the bioavailability from eggs is less than 9%, compared to 40% to 60% from fish, fowl and meat. Insects are a source of B₁₂ for animals (including other insects and humans). Animal-derived food sources with a high concentration of vitamin B₁₂ include liver and other organ meats from lamb, veal, beef, and turkey; also shellfish and crab meat.

Plants and algae

There is some evidence that bacterial fermentation of plant foods and symbiotic relationships between algae and bacteria can provide vitamin B₁₂. However, the Academy of Nutrition and Dietetics considers plant and algae sources "unreliable", stating that vegans should turn to fortified foods and supplements instead.

Natural plant and algae sources of vitamin B₁₂ include fermented plant foods such as tempeh and seaweed-derived foods such as nori and laverbread. Methylcobalamin has been identified in Chlorella vulgaris. Since only bacteria and some archea possess the genes and enzymes necessary to synthesize vitamin B₁₂, plant and algae sources all obtain the vitamin secondarily from symbiosis with various species of bacteria, or in the case of fermented plant foods, from bacterial fermentation.

Fortified foods

Foods for which vitamin B₁₂-fortified versions are available include breakfast cereals, plant-derived milk substitutes such as soy milk and oat milk, energy bars, and nutritional yeast. The fortification ingredient is cyanocobalamin. Microbial fermentation yields adenosylcobalamin, which is then converted to cyanocobalamin by addition of potassium cyanide or thiocyanate in the presence of sodium nitrite and heat.

As of 2019, nineteen countries require food fortification of wheat flour, maize flour or rice with vitamin B₁₂. Most of these are in southeast Africa or Central America.

Vegan advocacy organizations, among others, recommend that every vegan consume B₁₂ from either fortified foods or supplements.

Supplements

Vitamin B₁₂ is included in multivitamin pills; in some countries grain-based foods such as bread and pasta are fortified with B₁₂. In the US, non-prescription products can be purchased providing up to 5,000 μg each, and it is a common ingredient in energy drinks and energy shots, usually at many times the recommended dietary allowance of B₁₂. The vitamin can also be supplied on prescription and delivered via injection or other means.

Sublingual methylcobalamin, which contains no cyanide, is available in 5 mg tablets. The metabolic fate and biological distribution of methylcobalamin are expected to be similar to that of other sources of vitamin B₁₂ in the diet. The amount of cyanide in cyanocobalamin is generally not a concern, even in the 1,000 μg dose, since the amount of cyanide there (20 μg in a 1,000 μg cyanocobalamin tablet) is less than the daily consumption of cyanide from food, and therefore cyanocobalamin is not considered a health risk.

Intramuscular or intravenous injection

Injection of hydroxycobalamin is often used if digestive absorption is impaired, but this course of action may not be necessary with high-dose oral supplements (such as 0.5–1.0 mg or more), because with large quantities of the vitamin taken orally, even the 1% to 5% of free crystalline B₁₂ that is absorbed along the entire intestine by passive diffusion may be sufficient to provide a necessary amount.

A person with cobalamin C disease (which results in combined methylmalonic aciduria and homocystinuria) may require treatment with intravenous or intramuscular hydroxocobalamin or transdermal B₁₂, because oral cyanocobalamin is inadequate in the treatment of cobalamin C disease.

Nanotechnologies used in vitamin B₁₂ supplementation

Conventional administration does not ensure specific distribution and controlled release of vitamin B₁₂. Moreover, therapeutic protocols involving injection require health care people and commuting of patients to the hospital thus increasing the cost of the treatment and impairing the lifestyle of patients. Targeted delivery of vitamin B₁₂ is a major focus of modern prescriptions. For example, conveying the vitamin to the bone marrow and nerve cells would help myelin recovery. Currently, several nanocarriers strategies are being developed for improving vitamin B₁₂ delivery with the aim to simplify administration, reduce costs, improve pharmacokinetics, and ameliorate the quality of patients' lives.

Pseudovitamin-B₁₂

Pseudovitamin-B₁₂ refers to B₁₂-like analogues that are biologically inactive in humans. Most cyanobacteria, including Spirulina, and some algae, such as Porphyra tenera (used to make a dried seaweed food called nori in Japan), have been found to contain mostly pseudovitamin-B₁₂ instead of biologically active B₁₂. These pseudo-vitamin compounds can be found in some types of shellfish, in edible insects, and at times as metabolic breakdown products of cyanocobalamin added to dietary supplements and fortified foods.

Pseudovitamin-B₁₂ can show up as biologically active vitamin B₁₂ when a microbiological assay with Lactobacillus delbrueckii subsp. lactis is used, as the bacteria can utilize the pseudovitamin despite it being unavailable to humans. To get a reliable reading of B₁₂ content, more advanced techniques are available. One such technique involves pre-separation by silica gel and then assessment with B₁₂-dependent E. coli bacteria.

A related concept is antivitamin B₁₂, compounds (often synthetic B₁₂ analogues) that not only have no vitamin action, but also actively interfere with the activity of true vitamin B₁₂. The design of these compounds mainly involve replacement of the metal ion with rhodium, nickel, or zinc; or the attachment of an inactive ligand such as 4-ethylphenyl. These compounds have the potential to be used for analyzing B₁₂ utilization pathways or even attacking B₁₂-dependent pathogens.

Drug interactions

H₂-receptor antagonists and proton-pump inhibitors

Gastric acid is needed to release vitamin B₁₂ from protein for absorption. Reduced secretion of gastric acid and pepsin, from the use of H₂ blocker or proton-pump inhibitor (PPI) drugs, can reduce absorption of protein-bound (dietary) vitamin B₁₂, although not of supplemental vitamin B₁₂. H₂-receptor antagonist examples include cimetidine, famotidine, nizatidine, and ranitidine. PPIs examples include omeprazole, lansoprazole, rabeprazole, pantoprazole, and esomeprazole. Clinically significant vitamin B₁₂ deficiency and megaloblastic anemia are unlikely, unless these drug therapies are prolonged for two or more years, or if in addition the person's dietary intake is below recommended levels. Symptomatic vitamin deficiency is more likely if the person is rendered achlorhydric (a complete absence of gastric acid secretion), which occurs more frequently with proton pump inhibitors than H₂ blockers.

Metformin

Reduced serum levels of vitamin B₁₂ occur in up to 30% of people taking long-term anti-diabetic metformin. Deficiency does not develop if dietary intake of vitamin B₁₂ is adequate or prophylactic B₁₂ supplementation is given. If the deficiency is detected, metformin can be continued while the deficiency is corrected with B₁₂ supplements.

Other drugs

Certain medications can decrease the absorption of orally consumed vitamin B₁₂, including colchicine, extended-release potassium products, and antibiotics such as gentamicin, neomycin and tobramycin. Anti-seizure medications phenobarbital, pregabalin, primidone and topiramate are associated with lower than normal serum vitamin concentration. However, serum levels were higher in people prescribed valproate. In addition, certain drugs may interfere with laboratory tests for the vitamin, such as amoxicillin, erythromycin, methotrexate and pyrimethamine.

Chemistry

Vitamin B₁₂ is the most chemically complex of all the vitamins. The structure of B₁₂ is based on a corrin ring, which is similar to the porphyrin ring found in heme. The central metal ion is cobalt. As isolated as an air-stable solid and available commercially, cobalt in vitamin B₁₂ (cyanocobalamin and other vitamers) is present in its +3 oxidation state. Biochemically, the cobalt center can take part in both two-electron and one-electron reductive processes to access the "reduced" (B_12r, +2 oxidation state) and "super-reduced" (B_12s, +1 oxidation state) forms. The ability to shuttle between the +1, +2, and +3 oxidation states is responsible for the versatile chemistry of vitamin B₁₂, allowing it to serve as a donor of deoxyadenosyl radical (radical alkyl source) and as a methyl cation equivalent (electrophilic alkyl source).

Four of the six coordination sites are provided by the corrin ring, and a fifth by a dimethylbenzimidazole group. The sixth coordination site, the reactive center, is variable, being a cyano group (–CN), a hydroxyl group (–OH), a methyl group (–CH₃) or a 5′-deoxyadenosyl group. Historically, the covalent carbon–cobalt bond is one of the first examples of carbon–metal bonds to be discovered in biology. The hydrogenases and, by necessity, enzymes associated with cobalt utilization, involve metal–carbon bonds. Animals have the ability to convert cyanocobalamin and hydroxocobalamin to the bioactive forms adenosylcobalamin and methylcobalamin by means of enzymatically replacing the cyano or hydroxyl groups.

Methods for the analysis of vitamin B₁₂ in food

Several methods have been used to determine the vitamin B₁₂ content in foods including microbiological assays, chemiluminescence assays, polarographic, spectrophotometric and high-performance liquid chromatography processes. The microbiological assay has been the most commonly used assay technique for foods, utilizing certain vitamin B₁₂-requiring microorganisms, such as Lactobacillus delbrueckii subsp. lactis ATCC7830. However, it is no longer the reference method due to the high measurement uncertainty of vitamin B₁₂.

Furthermore, this assay requires overnight incubation and may give false results if any inactive vitamin B₁₂ analogues are present in the foods. Currently, radioisotope dilution assay (RIDA) with labelled vitamin B₁₂ and hog IF (pigs) have been used to determine vitamin B₁₂ content in food. Previous reports have suggested that the RIDA method is able to detect higher concentrations of vitamin B₁₂ in foods compared to the microbiological assay method.

Biochemistry

Coenzyme function

Vitamin B₁₂ functions as a coenzyme, meaning that its presence is required in some enzyme-catalyzed reactions. Listed here are the three classes of enzymes that sometimes require B₁₂ to function (in animals):

1. Isomerases

   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. These use the adoB₁₂ (adenosylcobalamin) form of the vitamin.

2. Methyltransferases

   Methyl (–CH₃) group transfers between two molecules. These use the MeB₁₂ (methylcobalamin) form of the vitamin.

3. Dehalogenases

   Some species of anaerobic bacteria synthesize B₁₂-dependent dehalogenases, which have potential commercial applications for degrading chlorinated pollutants. The microorganisms may either be capable of de novo corrinoid biosynthesis or are dependent on exogenous vitamin B₁₂.

In humans, two major coenzyme B₁₂-dependent enzyme families corresponding to the first two reaction types, are known. These are typified by the following two enzymes:

Methylmalonyl coenzyme A mutase (MUT) is an isomerase enzyme which uses the AdoB₁₂ form and reaction type 1 to convert L-methylmalonyl-CoA to succinyl-CoA, an important step in the catabolic breakdown of some amino acids into succinyl-CoA, which then enters energy production via the citric acid cycle. This functionality is lost in vitamin B₁₂ deficiency, and can be measured clinically as an increased serum methylmalonic acid (MMA) concentration. The MUT function is necessary for proper myelin synthesis. Based on animal research, it is thought that the increased methylmalonyl-CoA hydrolyzes to form methylmalonate (methylmalonic acid), a neurotoxic dicarboxylic acid, causing neurological deterioration.

Methionine synthase, coded by MTR gene, is a methyltransferase enzyme which uses the MeB₁₂ and reaction type 2 to transfer a methyl group from 5-methyltetrahydrofolate to homocysteine, thereby generating tetrahydrofolate (THF) and methionine. This functionality is lost in vitamin B₁₂ deficiency, resulting in an increased homocysteine level and the trapping of folate as 5-methyl-tetrahydrofolate, from which THF (the active form of folate) cannot be recovered. THF plays an important role in DNA synthesis, so reduced availability of THF results in ineffective production of cells with rapid turnover, in particular red blood cells, and also intestinal wall cells which are responsible for absorption. THF may be regenerated via MTR or may be obtained from fresh folate in the diet. Thus all of the DNA synthetic effects of B₁₂ deficiency, including the megaloblastic anemia of pernicious anemia, resolve if sufficient dietary folate is present. Thus the best-known "function" of B₁₂ (that which is involved with DNA synthesis, cell-division, and anemia) is actually a facultative function which is mediated by B₁₂-conservation of an active form of folate which is needed for efficient DNA production. Other cobalamin-requiring methyltransferase enzymes are also known in bacteria, such as Me-H₄-MPT, coenzyme M methyltransferase.

Physiology

Absorption

Vitamin B₁₂ is absorbed by a B₁₂-specific transport proteins or via passive diffusion. Transport-mediated absorption and tissue delivery is a complex process involving three transport proteins: haptocorrin (HC), intrinsic factor (IF) and transcobalamin II (TC2), and respective membrane receptor proteins. HC is present in saliva. As vitamin-containing food is digested by hydrochloric acid and pepsin secreted into the stomach, HC binds the vitamin and protected it from acidic degradation. Upon leaving the stomach the hydrochloric acid of the chyme is neutralized in the duodenum by bicarbonate, and pancreatic proteases release the vitamin from HC, making it available to be bound by IF, which is a protein secreted by gastric parietal cells in response to the presence of food in the stomach. IF delivers the vitamin to receptor proteins cubilin and amnionless, which together form the cubam receptor in the distal ileum. The receptor is specific to the IF-B₁₂ complex, and so will not bind to any vitamin content that is not bound to IF.

Investigations into the intestinal absorption of B₁₂ confirm that the upper limit of absorption per single oral dose is about 1.5 μg, with 50% efficiency. In contrast, the passive diffusion process of B₁₂ absorption — normally a very small portion of total absorption of the vitamin from food consumption — may exceed the haptocorrin- and IF-mediated absorption when oral doses of B₁₂ are very large, with roughly 1% efficiency. Thus, dietary supplement B₁₂ supplementation at 500 to 1000 μg per day allows pernicious anemia and certain other defects in B₁₂ absorption to be treated with daily oral megadoses of B₁₂ without any correction of the underlying absorption defects.

After the IF/B₁₂ complex binds to cubam the complex is disassociated and the free vitamin is transported into the portal circulation. The vitamin is then transferred to TC2, which serves as the circulating plasma transporter, Hereditary defects in production of TC2 and its receptor may produce functional deficiencies in B₁₂ and infantile megaloblastic anemia, and abnormal B₁₂ related biochemistry, even in some cases with normal blood B₁₂ levels. For the vitamin to serve inside cells, the TC2-B₁₂ complex must bind to a cell receptor protein and be endocytosed. TC2 is degraded within a lysosome, and free B₁₂ is released into the cytoplasm, where it is transformed into the bioactive coenzyme by cellular enzymes.

Malabsorption

Antacid drugs that neutralize stomach acid and drugs that block acid production (such as proton-pump inhibitors) will inhibit absorption of B₁₂ by preventing release from food in the stomach. Other causes of B12 malabsorption include intrinsic factor deficiency, pernicious anemia, bariatric surgery pancreatic insufficiency, obstructive jaundice, tropical sprue and celiac disease, and radiation enteritis of the distal ileum. Age can be a factor. Elderly people are often achlorhydric due to reduced stomach parietal cell function, and thus have an increased risk of B₁₂ deficiency.

Storage and excretion

How fast B₁₂ levels change depends on the balance between how much B₁₂ is obtained from the diet, how much is secreted and how much is absorbed. The total amount of vitamin B₁₂ stored in the body is about 2–5 mg in adults. Around 50% of this is stored in the liver. Approximately 0.1% of this is lost per day by secretions into the gut, as not all these secretions are reabsorbed. Bile is the main form of B₁₂ excretion; most of the B₁₂ secreted in the bile is recycled via enterohepatic circulation. Excess B₁₂ beyond the blood's binding capacity is typically excreted in urine. Owing to the extremely efficient enterohepatic circulation of B₁₂, the liver can store 3 to 5 years' worth of vitamin B₁₂; therefore, nutritional deficiency of this vitamin is rare in adults in the absence of malabsorption disorders. In the absence of intrinsic factor or distal ileum receptors, only months to a year of vitamin B₁₂ are stored.

Cellular reprogramming

Vitamin B₁₂ through its involvement in one-carbon metabolism plays a key role in cellular reprogramming and tissue regeneration and epigenetic regulation. Cellular reprogramming is the process by which somatic cells can be converted to a pluripotent state. Vitamin B₁₂ levels affect the histone modification H3K36me3, which suppresses illegitimate transcription outside of gene promoters. Mice undergoing in vivo reprogramming were found to become depleted in B₁₂ and show signs of methionine starvation while supplementing reprogramming mice and cells with B₁₂ increased reprogramming efficiency, indicating a cell-intrinsic effect.

Synthesis

Biosynthesis

Vitamin B₁₂ is derived from a tetrapyrrolic structural framework created by the enzymes deaminase and cosynthetase which transform aminolevulinic acid via porphobilinogen and hydroxymethylbilane to uroporphyrinogen III. The latter is the first macrocyclic intermediate common to heme, chlorophyll, siroheme and B₁₂ itself. Later steps, especially the incorporation of the additional methyl groups of its structure, were investigated using C methyl-labelled S-adenosyl methionine. It was not until a genetically engineered strain of Pseudomonas denitrificans was used, in which eight of the genes involved in the biosynthesis of the vitamin had been overexpressed, that the complete sequence of methylation and other steps could be determined, thus fully establishing all the intermediates in the pathway.

Species from the following genera and the following individual species are known to synthesize B₁₂: Propionibacterium shermanii, Pseudomonas denitrificans, Streptomyces griseus, Acetobacterium, Aerobacter, Agrobacterium, Alcaligenes, Azotobacter, Bacillus, Clostridium, Corynebacterium, Flavobacterium, Lactobacillus, Micromonospora, Mycobacterium, Nocardia, Proteus, Rhizobium, Salmonella, Serratia, Streptococcus and Xanthomonas.

Industrial

Industrial production of B₁₂ is achieved through fermentation of selected microorganisms. Streptomyces griseus, a bacterium once thought to be a fungus, was the commercial source of vitamin B₁₂ for many years. The species Pseudomonas denitrificans and Propionibacterium freudenreichii subsp. shermanii are more commonly used today. These are grown under special conditions to enhance yield. Rhone-Poulenc improved yield via genetic engineering P. denitrificans. Propionibacterium, the other commonly used bacteria, produce no exotoxins or endotoxins and are generally recognized as safe (have been granted GRAS status) by the Food and Drug Administration of the United States.

The total world production of vitamin B₁₂ in 2008 was 35,000 kg (77,175 lb).

Laboratory

The complete laboratory synthesis of B₁₂ was achieved by Robert Burns Woodward and Albert Eschenmoser in 1972. The work required the effort of 91 postdoctoral fellows (mostly at Harvard) and 12 PhD students (at ETH Zurich) from 19 nations. The synthesis constitutes a formal total synthesis, since the research groups only prepared the known intermediate cobyric acid, whose chemical conversion to vitamin B₁₂ was previously reported. This synthesis of vitamin B₁₂ is of no practical consequence due to its length, taking 72 chemical steps and giving an overall chemical yield well under 0.01%. Although there have been sporadic synthetic efforts since 1972, the Eschenmoser–Woodward synthesis remains the only completed (formal) total synthesis.

History

Descriptions of deficiency effects

Between 1849 and 1887, Thomas Addison described a case of pernicious anemia, William Osler and William Gardner first described a case of neuropathy, Hayem described large red cells in the peripheral blood in this condition, which he called "giant blood corpuscles" (now called macrocytes), Paul Ehrlich identified megaloblasts in the bone marrow, and Ludwig Lichtheim described a case of myelopathy.

Identification of liver as an anti-anemia food

During the 1920s, George Whipple discovered that ingesting large amounts of raw liver seemed to most rapidly cure the anemia of blood loss in dogs, and hypothesized that eating liver might treat pernicious anemia. Edwin Cohn prepared a liver extract that was 50 to 100 times more potent in treating pernicious anemia than the natural liver products. William Castle demonstrated that gastric juice contained an "intrinsic factor" which when combined with meat ingestion resulted in absorption of the vitamin in this condition. In 1934, George Whipple shared the 1934 Nobel Prize in Physiology or Medicine with William P. Murphy and George Minot for discovery of an effective treatment for pernicious anemia using liver concentrate, later found to contain a large amount of vitamin B₁₂.

Identification of the active compound

While working at the Bureau of Dairy Industry, U.S. Department of Agriculture, Mary Shaw Shorb was assigned work on the bacterial strain Lactobacillus lactis Dorner (LLD), which was used to make yogurt and other cultured dairy products. The culture medium for LLD required liver extract. Shorb knew that the same liver extract was used to treat pernicious anemia (her father-in-law had died from the disease), and concluded that LLD could be developed as an assay method to identify the active compound. While at the University of Maryland she received a small grant from Merck, and in collaboration with Karl Folkers from that company, developed the LLD assay. This identified "LLD factor" as essential for the bacteria's growth. Shorb, Folker and Alexander R. Todd, at the University of Cambridge, used the LLD assay to extract the anti-pernicious anemia factor from liver extracts, purify it, and name it vitamin B₁₂. In 1955, Todd helped elucidate the structure of the vitamin. The complete chemical structure of the molecule was determined by Dorothy Hodgkin based on crystallographic data and published in 1955 and 1956, for which, and for other crystallographic analyses, she was awarded the Nobel Prize in Chemistry in 1964. Hodgkin went on to decipher the structure of insulin.

George Whipple, George Minot and William Murphy were awarded the Nobel Prize in 1934 for their work on the vitamin. Three other Nobel laureates, Alexander R. Todd (1957), Dorothy Hodgkin (1964) and Robert Burns Woodward (1965) made important contributions to its study.

Commercial production

Industrial production of vitamin B₁₂ is achieved through fermentation of selected microorganisms. As noted above, the completely synthetic laboratory synthesis of B12 was achieved by Robert Burns Woodward and Albert Eschenmoser in 1972, though this process has no commercial potential, requiring more than 70 steps and having a yield well below 0.01%.

Society and culture

In the 1970s, John A. Myers, a physician residing in Baltimore, developed a program of injecting vitamins and minerals intravenously for various medical conditions. The formula included 1000 μg of cyanocobalamin. This came to be known as the Myers' cocktail. After his death in 1984, other physicians and naturopaths took up prescribing "intravenous micro-nutrient therapy" with unsubstantiated health claims for treating fatigue, low energy, stress, anxiety, migraine, depression, immunocompromised, promoting weight loss and more. However, other than a report on case studies there are no benefits confirmed in the scientific literature. Healthcare practitioners at clinics and spas prescribe versions of these intravenous combination products, but also intramuscular injections of just vitamin B₁₂. A Mayo Clinic review concluded that there is no solid evidence that vitamin B₁₂ injections provide an energy boost or aid weight loss.

There is evidence that for elderly people, physicians often repeatedly prescribe and administer cyanocobalamin injections inappropriately, evidenced by the majority of subjects in one large study either having had normal serum concentrations or had not been tested prior to the injections.

See also

• Adenosylcobalamin
• Cobalamin biosynthesis
• Cyanocobalamin
• Hydroxocobalamin
• Methylcobalamin
• Vitamins

Further reading

• Gherasim C, Lofgren M, Banerjee R (May 2013). "Navigating the B(12) road: assimilation, delivery, and disorders of cobalamin". J. Biol. Chem. 288 (19): 13186–13193. doi:10.1074/jbc.R113.458810. PMC 3650358. PMID 23539619.