Vitamin B₁₂

The U.S. Recommended Dietary Allowance (RDA) for pregnancy is 2.6 µg/day, for lactation 2.8 µg/day. Determination of these values was based on the RDA of 2.4 µg/day for non-pregnant women plus what will be transferred to the fetus during pregnancy and what will be delivered in breast milk.[10][35]^: 972 However, looking at the same scientific evidence, the European Food Safety Authority (EFSA) sets adequate intake (AI) at 4.5 μg/day for pregnancy and 5.0 μg/day for lactation.[36] 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.[37][35] During pregnancy the placenta concentrates B₁₂, so that newborn infants have a higher serum concentration than their mothers.[10] 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.[10][38] 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.[38][35]

Low vitamin concentrations in human milk occur in families with low socioeconomic status or low consumption of animal products.[35]^{: 971, 973} 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.[39] 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.[35]^: 973 Breast milk B₁₂ decreases over months of nursing in both well-nourished and vitamin-deficient mothers.[35]^{: 973–974} 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.[35]^{: 974–975} Risk of deficiency persists if the post-weaning diet is low in animal products.[35]^{: 974–975} Signs of low vitamin levels in infants and young children can include anemia, poor physical growth and neurodevelopmental delays.[35]^: 975 Children diagnosed with low serum B₁₂ can be treated with intramuscular injections, then transitioned to an oral dietary supplement.[35]^: 976

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.[40][41][42] For post-operative oral supplementation, 1000 μg/day may be needed to prevent vitamin deficiency.[42]

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."[43] 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.[2] 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.[2] However, elevated MMA is not conclusive, as it is seen in people with B₁₂ deficiency, but also in elderly people who have renal insufficiency,[25] and elevated homocysteine is not conclusive, as it is also seen in people with folate deficiency.[44] In addition, elevated methylmalonic acid levels may also be related to metabolic disorders such as methylmalonic acidemia.[45] If nervous system damage is present and blood testing is inconclusive, a lumbar puncture may be carried out to measure cerebrospinal fluid B₁₂ levels.[46]

Severe vitamin B₁₂ deficiency is corrected with frequent intramuscular injections of large doses of the vitamin, followed by maintenance doses of injections or oral dosing at longer intervals. In the UK, standard initial therapy consists of intramuscular injections of 1000 μg of hydroxocobalamin three times a week for two weeks or until neurological symptoms improve, followed by 1000 μg every two or three months.[47] Injection side effects include skin rash, itching, chills, fever, hot flushes, nausea and dizziness.[47]

For cyanide poisoning, a large amount of hydroxocobalamin may be given intravenously and sometimes in combination with sodium thiosulfate.[48][49] 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.[50]

Vitamin B₁₂ is produced in nature by certain bacteria, and archaea.[59][60][61] 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.[62] 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.[62][63] Other mammalian species (examples: rabbits, pikas, beaver, guinea pigs) consume high-fibre plants which pass through the intestinal system and undergo bacterial fermentation in the cecum and large intestine. The first-passage of feces produced by this hindgut fermentation, called "cecotropes", are re-ingested, a practice referred to as cecotrophy or coprophagy. Re-ingestion allows for absorption of nutrients made available by bacterial digestion, and also of vitamins and other nutrients synthesized by the gut bacteria, including vitamin B₁₂.[63] 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.[63] For gut bacteria to produce vitamin B₁₂ the animal must consume sufficient amounts of cobalt.[64] Soil that is deficient in cobalt may result in B₁₂ deficiency, and B₁₂ injections or cobalt supplementation may be required for livestock.[65]

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.[51][2][66] For humans, the bioavailability from eggs is less than 9%, compared to 40% to 60% from fish, fowl and meat.[67] Insects are a source of B₁₂ for animals (including other insects and humans).[66][68] 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.[6][51][69]

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.[32]

Natural plant and algae sources of vitamin B₁₂ include fermented plant foods such as tempeh[70][71] and seaweed-derived foods such as nori and laverbread.[72][73][74] Methylcobalamin has been identified in Chlorella vulgaris.[75] 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,[5] or in the case of fermented plant foods, from bacterial fermentation.[70]

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.[69] 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.[76]

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.[39]

Vegan advocacy organizations, among others, recommend that every vegan consume B₁₂ from either fortified foods or supplements.[6][34][77][78]

A blister pack of 500 µg methylcobalamin tablets

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.[2]

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.[79] 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.[79]

Injection of hydroxycobalamin is often used if digestive absorption is impaired,[2] but this course of action may not be necessary with high-dose oral supplements (such as 0.5–1.0 mg or more),[80][81] 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.[82]

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.[83]

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.[84]

Pseudovitamin-B₁₂ refers to B₁₂-like analogues that are biologically inactive in humans.[19] 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.[20][85] These pseudo-vitamin compounds can be found in some types of shellfish,[19] in edible insects,[86] and at times as metabolic breakdown products of cyanocobalamin added to dietary supplements and fortified foods.[87]

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.[19]

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.[88]

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.[89]

Reduced serum levels of vitamin B₁₂ occur in up to 30% of people taking long-term anti-diabetic metformin.[90][91] 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.[92]

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.[93] 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.[94] In addition, certain drugs may interfere with laboratory tests for the vitamin, such as amoxicillin, erythromycin, methotrexate and pyrimethamine.[93]

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.[97] 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.[67] However, it is no longer the reference method due to the high measurement uncertainty of vitamin B₁₂.[98] Furthermore, this assay requires overnight incubation and may give false results if any inactive vitamin B₁₂ analogues are present in the foods.[99] Currently, radioisotope dilution assay (RIDA) with labelled vitamin B₁₂ and hog IF (pigs) have been used to determine vitamin B₁₂ content in food.[67] 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.[67][97]

Vitamin B₁₂ functions as a coenzyme, meaning that its presence is required in some enzyme-catalyzed reactions.[10][16] 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.[100]

2. Methyltransferases

   Methyl (–CH₃) group transfers between two molecules. These use the MeB₁₂ (methylcobalamin) form of the vitamin.[101]

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.[102][103]

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:

Simplified schematic diagram of the folate methionine cycle. Methionine synthase transfers the methyl group to the vitamin and then transfers the methyl group to homocysteine, converting that to methionine.

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.[100] 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.[4] 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.[104]

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.[101] 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.[101] Other cobalamin-requiring methyltransferase enzymes are also known in bacteria, such as Me-H₄-MPT, coenzyme M methyltransferase.[105]

Food B₁₂ is absorbed by two processes. The first is a vitamin B₁₂-specific intestinal mechanism using intrinsic factor through which 1–2 micrograms can be absorbed every few hours, by which most food consumption of the vitamin is absorbed. The second is a passive diffusion process.[10] The human physiology of active vitamin B₁₂ absorption from food is complex. Protein-bound vitamin B₁₂ must be released from the proteins by the action of digestive proteases in both the stomach and small intestine. Gastric acid releases the vitamin from food particles; therefore antacid and acid-blocking medications (especially proton-pump inhibitors) may inhibit absorption of B₁₂. After B₁₂ has been freed from proteins in food by pepsin in the stomach, R-protein (also known as haptocorrin and transcobalamin-1), a B₁₂ binding protein that is produced in the salivary glands, binds to B₁₂. This protects the vitamin from degradation in the acidic environment of the stomach.[106]

The pattern of B₁₂ transfer to a special binding protein secreted in a previous digestive step, is repeated once more before absorption. The next binding protein for B₁₂ is intrinsic factor (IF), a protein synthesized by gastric parietal cells that is secreted in response to histamine, gastrin and pentagastrin, as well as the presence of food. In the duodenum, proteases digest R-proteins and release their bound B₁₂, which then binds to IF, to form a complex (IF/B₁₂). B₁₂ must be attached to IF for it to be efficiently absorbed, as receptors on the enterocytes in the terminal ileum of the small bowel only recognize the B₁₂-IF complex; in addition, intrinsic factor protects the vitamin from catabolism by intestinal bacteria.[10]

Absorption of food vitamin B₁₂ thus requires an intact and functioning stomach, exocrine pancreas, intrinsic factor, and small bowel.[10] Problems with any one of these organs makes a vitamin B₁₂ deficiency possible. Individuals who lack intrinsic factor have a decreased ability to absorb B₁₂. In pernicious anemia, there is a lack of IF due to autoimmune atrophic gastritis, in which antibodies form against parietal cells. Antibodies may alternately form against and bind to IF, inhibiting it from carrying out its B₁₂ protective function. Due to the complexity of B₁₂ absorption, geriatric patients, many of whom are hypoacidic due to reduced parietal cell function, have an increased risk of B₁₂ deficiency.[107] This results in 80–100% excretion of oral doses in the feces versus 30–60% excretion in feces as seen in individuals with adequate IF.[107]

Once the IF/B₁₂ 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. Hereditary defects in production of the transcobalamins and their receptors 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 TC-II/B₁₂ complex must bind to a cell receptor, and be endocytosed. The transcobalamin II is degraded within a lysosome, and free B₁₂ is finally released into the cytoplasm, where it may be transformed into the proper coenzyme, by certain cellular enzymes (see above).[10][108]

Investigations into the intestinal absorption of B₁₂ point out that the upper limit of absorption per single oral dose, under normal conditions, is about 1.5 µg. The passive diffusion process of B₁₂ absorption—normally a very small portion of total absorption of the vitamin from food consumption[10]—may exceed the R-protein and IF mediated absorption when oral doses of B₁₂ are very large (a thousand or more µg per dose) as commonly happens in dedicated-pill oral B₁₂ supplementation. This allows pernicious anemia and certain other defects in B₁₂ absorption to be treated with oral megadoses of B₁₂, even without any correction of the underlying absorption defects.[109] See the section on supplements above.

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.[10] In the absence of enterohepatic reabsorption, only months to a year of vitamin B₁₂ are stored.[110]

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.[111][112] Later steps, especially the incorporation of the additional methyl groups of its structure, were investigated using ¹³C 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.[113][114]

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.[115][116]

Industrial production of B₁₂ is achieved through fermentation of selected microorganisms.[117] Streptomyces griseus, a bacterium once thought to be a fungus, was the commercial source of vitamin B₁₂ for many years.[118] The species Pseudomonas denitrificans and Propionibacterium freudenreichii subsp. shermanii are more commonly used today.[117] These are grown under special conditions to enhance yield. Rhone-Poulenc improved yield via genetic engineering P. denitrificans.[119] 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.[120]

The total world production of vitamin B₁₂ in 2008 was 35,000 kg (77,175 lb).[121]

The complete laboratory synthesis of B₁₂ was achieved by Robert Burns Woodward[122] and Albert Eschenmoser in 1972.[123][124] 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%.[125] Although there have been sporadic synthetic efforts since 1972,[124] the Eschenmoser–Woodward synthesis remains the only completed (formal) total synthesis.

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.[126]

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.[127] 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.[126] 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.[126][128]

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.[129] 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.[130] In 1955, Todd helped elucidate the structure of the vitamin, for which he was awarded the Nobel Prize in Chemistry in 1957. The complete chemical structure of the molecule was determined by Dorothy Hodgkin, based on crystallographic data in 1956, for which for that and other crystallographic analyses she was awarded the Nobel Prize in Chemistry in 1964.[131][132] Hodgkin went on to decipher the structure of insulin.[132]

Five people have been awarded Nobel Prizes for direct and indirect studies of vitamin B₁₂: George Whipple, George Minot and William Murphy (1934), Alexander R. Todd (1957), and Dorothy Hodgkin (1964).[133]

Industrial production of vitamin B₁₂ is achieved through fermentation of selected microorganisms.[117] 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 almost 70 steps and having a yield well below 0.01%.[125]