Vitamin B12 (Cyanocobalamin) acts as a cofactor for two key enzymes: 1. Methylmalonyl-CoA mutase (required for the conversion of methylmalonyl-CoA to succinyl-CoA in branched-chain amino acid and odd-chain fatty acid metabolism; no IC50/Ki values reported). 2. Methionine synthase (catalyzes the conversion of homocysteine to methionine, regenerating tetrahydrofolate; no IC50/Ki values reported).

Out of all the B vitamins, vitamin B12 is one. In most cases, it affects DNA synthesis and regulation, but it also plays a role in energy production, fatty acid synthesis (particularly odd chain fatty acid synthesis), and the metabolism of every cell in the human body. Yet, since vitamin B12 is required for the body to produce folate again, folic acid (vitamin B9) in enough amounts can substitute for many, if not all, of the effects of vitamin B12. When the body lacks sufficient folic acid to produce thymine due to methyl trapping, poor synthesis of DNA occurs. Therefore, the majority of symptoms associated with vitamin B12 deficiency are actually symptoms of folate deficiency. This includes all the effects of megaloblastosis and pernicious anemia. All known B12-related deficiency syndromes return to normal when adequate folic acid is available, with the exception of those that are specifically linked to the growth of their respective substrates, homocysteine and methylmalonic acid, and the vitamin B12-dependent enzymes methylmalonyl Coenzyme A mutase and 5-methyltetrahydrofolate-homocysteine methyltransferase (MTR), also called methionine synthase. The reactive C-Co bond in Coenzyme B12 contributes to three main categories of enzyme-catalyzed reactions[1][2].

---

- Enzymatic Activity: - Reference [2]: In in vitro assays, Vitamin B12 (as methylcobalamin) is essential for the activity of methionine synthase. The enzyme requires methylcobalamin to transfer a methyl group from methyltetrahydrofolate to homocysteine, forming methionine. Similarly, adenosylcobalamin (a form of B12) is required for methylmalonyl-CoA mutase activity, facilitating the rearrangement of methylmalonyl-CoA to succinyl-CoA. These reactions are critical for proper cellular metabolism but were not quantified with IC50/EC50 values in the review.

- Metabolic Role: - Reference [2]: In animal models, Vitamin B12 deficiency leads to impaired methylmalonyl-CoA mutase activity, causing accumulation of methylmalonic acid and subsequent neurological dysfunction. Methionine synthase deficiency disrupts homocysteine metabolism, leading to hyperhomocysteinemia and DNA hypomethylation. These effects were observed in studies of B12-deficient rats and humans but were not quantified with specific in vivo efficacy metrics (e.g., dose-response curves).

- Methylmalonyl-CoA Mutase Activity Assay: - Reference [2]: The activity of methylmalonyl-CoA mutase is typically measured using radiolabeled substrates. For example, [14C]-methylmalonyl-CoA is incubated with enzyme and adenosylcobalamin in buffer containing MgCl2 and dithiothreitol. The reaction produces [14C]-succinyl-CoA, which is separated by thin-layer chromatography and quantified by scintillation counting. The assay confirms the requirement of adenosylcobalamin for enzyme activity but does not provide IC50 values for B12 itself (as it is a cofactor, not an inhibitor).
- Methionine Synthase Activity Assay: - Reference [2]: Methionine synthase activity is measured by monitoring the conversion of [3H]-homocysteine to [3H]-methionine in the presence of methyltetrahydrofolate and methylcobalamin. The reaction mixture includes Tris-HCl buffer (pH 7.5), EDTA, and reducing agents. The product is separated by ion-exchange chromatography and quantified. Again, no IC50 values for B12 were reported in the review.

Absorption, Distribution and Excretion
Vitamin B12 is rapidly absorbed after intramuscular (IM) and subcutaneous (SC) injections; peak plasma concentrations are reached approximately 1 hour after IM injection. Orally administered vitamin B12 binds to intrinsic factor (IF) as it passes through the stomach. In the terminal ileum, in the presence of calcium, vitamin B12 dissociates from intrinsic factor and is subsequently absorbed by the gastrointestinal mucosal cells. It is then transported via transcobalamin-binding proteins. Vitamin B12 can also passively diffuse across the intestinal wall, but this requires high doses (i.e., >1 mg). After oral doses below 3 mcg, peak plasma concentrations are reached 8 to 12 hours later due to temporary retention of the vitamin in the lower ileum wall. The drug is partially excreted in the urine. According to one clinical study, approximately 3–8 micrograms of vitamin B12 are secreted into the gastrointestinal tract daily via bile. In patients with adequate intrinsic factor levels, all but approximately 1 microgram of vitamin B12 is reabsorbed. When vitamin B12 doses are too high, reaching saturation in plasma proteins and the liver, unbound vitamin B12 is rapidly excreted in the urine. The amount of vitamin B12 stored in the body is positively correlated with the dose. Cobalamin is distributed in various tissues, primarily stored in the liver and bone marrow. During vitamin loads, the kidneys accumulate large amounts of unbound vitamin B12. The drug is partially cleared by the kidneys, but the multiligand receptor megalin promotes the reabsorption of vitamin B12 in the body. In mice intravenously injected with vitamin B12, the vitamin rapidly accumulates in the placenta and slowly transfers to the fetus. The fetal vitamin B12 concentration peaks 24 hours after administration, and the amount accumulated in the fetus is positively correlated with the dose. In mice, vitamin B12 exhibits an anomalous placental transfer pattern; even with a maternal dose of only 0.20 micrograms, the average fetal concentration is 130 times higher than that of the mother. This strongly suggests that vitamin B12 has a specific transport mechanism, possibly similar to its gastrointestinal absorption mechanism…
In rats, placental transport of vitamin B12 increases during pregnancy. Although the daily transport amount is proportional to fetal weight, the amount transported per gram of placenta increases tenfold from day 10 to day 19 of gestation.
After oral administration, the absorption of vitamin B12 in the distal small intestine is irregular. Dietary vitamin B12 binds to proteins, and this binding requires proteolysis and the action of gastric acid for absorption. In the stomach, free vitamin B12 binds to intrinsic factor; intrinsic factor is a glycoprotein secreted by the gastric mucosa and is essential for the active absorption of vitamin B12 in the gastrointestinal tract. The vitamin B12-intrinsic factor complex enters the intestine, and most of the complex is temporarily retained at specific receptor sites on the lower ileum wall before a portion of the vitamin B12 is absorbed into the systemic circulation.
For more complete data on the absorption, distribution, and excretion of cyanocobalamins (9 in total), please visit the HSDB record page.

---

Metabolism/Metabolites
Vitamin B12 or cyanocobalamin ingested from food first binds to haptoglobin, a protein in saliva with a high affinity for vitamin B12. This forms a haptoglobin-vitamin B12 complex. Cyanocobalamin passes through the stomach, where it is protected from degradation by gastric acid due to its binding to haptoglobin. In the duodenum, trypsin releases cobalamin from ingested vitamin B12-containing proteins, including the haptoglobin-vitamin B12 complex. Subsequently, cobalamin binds to another glycoprotein, intrinsic factor, promoting its uptake by terminal ileal mucosal cells via cuboprotein/AMN receptor-mediated endocytosis. After intrinsic factor is absorbed by intestinal cells, it is broken down in lysosomes, and cobalamin is subsequently released into the bloodstream. The transport protein ABCC1, located on the basolateral membrane of intestinal epithelial cells and other cells, transports cobalamin bound to transcobalamin out of the cells. Cyanocobalamin then enters the systemic circulation via the portal vein of the liver. The active forms of cyanocobalamin are methylcobalamin and adenosylcobalamin. Vitamin B12 is thought to be converted into a coenzyme form in the liver and may be stored in tissues in this form. Intracellular vitamin B12 exists in two active coenzyme forms: methylcobalamin and deoxyadenosylcobalamin. Biological half-life: Approximately 6 days (400 days in the liver). The half-life of intravenously administered cyanocobalamin in serum is approximately 6 days. Absorption: Reference [1]: Absorption of vitamin B12 requires intrinsic factor (IF) secreted by parietal cells of the stomach. IF binds to B12 in the stomach, and the complex is absorbed in the ileum via cuboprotein-mediated endocytosis. Oral bioavailability is approximately 50% at low doses (<1 μg) but decreases to approximately 1% at high doses (>1 mg).
- Distribution: - Reference [1]: Vitamin B12 is transported in plasma in the form of transcobalamin II (TCII) and stored in the liver (accounting for about 50% of the total body storage).
- Excretion: - Reference [1]: Unabsorbed vitamin B12 is excreted in feces; excess vitamin B12 is excreted in urine. Due to the efficient enterohepatic circulation, its biological half-life is about 3-5 years.

Effects During Pregnancy and Lactation
◉ Overview of Lactation Use
Vitamin B12 is a normal component of breast milk. The recommended daily intake for breastfeeding women is 2.8 micrograms, and for infants 6 months and under, it is 0.4 micrograms. Some authorities recommend a daily intake of 5.5 micrograms for breastfeeding women. Supplementation with vitamin B12 may be necessary to reach the recommended daily intake or to correct a known vitamin B12 deficiency. Low doses (1 to 10 micrograms) of vitamin B12 found in B vitamins or prenatal vitamins will only slightly increase the vitamin B12 content in breast milk. If the mother has a vitamin B12 deficiency, a higher daily dose of 50 to 250 micrograms may be required. In this case, the breastfed infant will not ingest excessive vitamin B12, and their vitamin B12 levels should improve if they were previously deficient. Infants deficient in vitamin B12 may experience anemia, abnormal skin and hair development, seizures, hypotonia, growth retardation, intellectual disability, and possible abnormal motor development, among other adverse health consequences. Recognized high-risk groups include infants whose mothers are vitamin B12 deficient due to minimal or no animal-based food intake, or whose mothers suffer from pernicious anemia due to maternal vitamin B12 malabsorption, and whose infants are exclusively breastfed. Vitamin B12 supplementation during pregnancy and lactation can improve infant vitamin B12 levels. Even if the opportunity for vitamin B12 supplementation was missed during pregnancy, mothers deficient in vitamin B12 should be encouraged to supplement during early breastfeeding, as the infant's vitamin B12 levels are closely correlated with the vitamin B12 levels in breast milk for breastfed infants up to 6 months of age. Although there are reports that adequate maternal vitamin B12 supplementation alone can improve the biochemical and clinical symptoms of vitamin B12 deficiency in exclusively breastfed infants, direct vitamin B12 supplementation for infants is still recommended if possible.
◉ Efficacy of Breastfed Infants
Twelve exclusively breastfed infants aged 4 to 11 months with biochemical, hematological, and clinical presentations consistent with a diagnosis of vitamin B12 deficiency. Their mothers received a single intramuscular injection of 50 micrograms of vitamin B12. Within 5 to 8 days after administration, the infants showed significant increases in hemoglobin and reticulocyte counts, normal erythropoiesis, improved mental status, regression of abnormal skin pigmentation, and reduction in tremors.
In India, 366 pregnant women received 50 micrograms of vitamin B12 or a placebo capsule daily from early pregnancy until 6 weeks postpartum. Among the 218 infants who underwent neurodevelopmental testing at 30 months of age, those whose mothers were randomly assigned to the vitamin B12 group had higher expressive language scores than those in the placebo group after adjusting for maternal baseline vitamin B12 deficiency. There were no differences in cognitive, receptive language, and motor scores between the two groups. Subsequent neurophysiological assessments at 6 years of age showed no differences in brain activity measurements between the two groups.
◉ Effects on Lactation and Breast Milk
As of the revision date, no relevant published information was found.

---

Protein Binding
Very high (binds to a specific plasma protein called transcobalamin); the binding rate of hydroxycobalamin is slightly higher than that of cyanocobalamin [FDA label].

---

Interactions
Aminoglycoside antibiotics, colchicine, sustained-release potassium tablets, aminosalicylic acid and its salts, anticonvulsants (e.g., phenytoin sodium, phenobarbital, primidone), small intestinal cobalt irradiation, and excessive alcohol consumption lasting more than 2 weeks can all reduce gastrointestinal absorption of vitamin B12.
Oral neomycin significantly reduces gastrointestinal absorption of vitamin B12. Colchicine administration appears to exacerbate neomycin-induced vitamin B12 malabsorption.
The reduced vitamin B12 absorption caused by aminosalicylic acid may be due to mild malabsorption syndrome in some patients receiving aminosalicylic acid (PAS) treatment.
Patients with pernicious anemia...receive poorer response to vitamin B12 treatment if chloramphenicol is taken concurrently.
For more (complete) data on interactions of cyanocobalamins (7 in total), please visit the HSDB record page.

---

Toxicity Overview
Cobalamin poisoning or overdose is generally not a problem, and there is no antidote for cobalamin. Vitamin B12 exists in the body in two forms: methylcobalamin and 5-deoxyadenosylcobalamin. Methionine synthase requires methylcobalamin as a cofactor. This enzyme is involved in the conversion of the amino acid homocysteine to methionine. Methionine is essential for DNA methylation. 5-deoxyadenosylcobalamin is a cofactor required by this enzyme, which converts L-methylmalonyl-CoA to succinyl-CoA. This conversion is an important step in the extraction of energy from proteins and fats. In addition, succinyl-CoA is essential for the synthesis of hemoglobin (the substance that carries oxygen in red blood cells).

---

-Acute toxicity:- Reference [1]: Vitamin B12 has low toxicity. The median lethal dose (LD50) in humans has not been determined. High doses (e.g., 1 mg/day) are generally well tolerated with very few reports of allergic reactions (rash, itching) or mild gastrointestinal symptoms. - Chronic toxicity: - Reference [1]: Long-term use of high doses of vitamin B12 does not cause significant organ toxicity. However, excessive intake of folic acid may mask folic acid deficiency or interact with certain drugs (e.g., metformin, antacids).

[1]. http://en.wikipedia.org/wiki/Vitamin_B12
[2]. The many faces of vitamin B12: catalysis by cobalamin-dependent enzymes. Annu Rev Biochem, 2003. 72: p. 209-47.

Therapeutic Uses
Important Vitamin B12 is used to treat pernicious anemia and other vitamin B12 deficiencies. …Cyanocobalamin… is generally indicated for patients with vitamin B12 malabsorption, such as those with tropical or non-tropical stomatitis (idiopathic steatorrhea, gluten-induced enteropathy); partial or total gastrectomy; regional enteritis; gastrointestinal anastomosis; ileal resection; or malignancies, granulomas, strictures, or anastomoses involving the ileum. Vitamin B12 absorption may be reduced when gastric mucosal damage (e.g., after ingestion of corrosive substances or in patients with extensive gastrointestinal tumors) leads to decreased intrinsic factor secretion, or when gastric atrophy is caused by multiple sclerosis, certain endocrine disorders, or iron deficiency, or when anti-intrinsic factor antibodies are present in gastric juice, and cyanocobalamin supplementation may be necessary. Vitamin B12 malabsorption can also be caused by bacteria (blind loop syndrome), Diphyllobothrium latum, or certain medications that compete with vitamin B12. For patients with simple pernicious anemia presenting only with mild or moderate anemia, vitamin B12 supplementation alone is sufficient to achieve good results. Patients with neurological changes, severe leukopenia, or thrombocytopenia accompanied by infection or bleeding require emergency treatment. Elderly patients with severe anemia (hematocrit less than 20%) may experience tissue hypoxia, cerebrovascular insufficiency, and congestive heart failure. Effective treatment should not wait for detailed diagnostic test results. …Patients should receive an intramuscular injection of 100 mcg cyanocobalamin and 1 to 5 mg of folic acid. For more complete data on the therapeutic uses of cyanocobalamin (13 items in total), please visit the HSDB record page.

---

Drug Warnings
Intramuscular or deep subcutaneous injection of cyanocobalamin is very safe, but it should never be administered intravenously.
Cyanocobalamin should not be used in patients with early-stage Leber's disease (hereditary optic atrophy) because there have been reports of rapid optic atrophy in these patients after taking this medication. Vitamin B12 is contraindicated in patients with hypersensitivity to vitamin B12 or cobalt. /“Shotgun”/...Vitamin therapy can be dangerous when treating...deficiency. ...Adequate folic acid administration may lead to hematologic recovery, but this may mask persistent vitamin B12 deficiency and may develop or worsen nerve damage if it is already present. Maternal use generally compatible with breastfeeding: Vitamin B12: Infant-reported signs or symptoms or effects on lactation: None. /Excerpt from Table 6/
Serious potassium levels should be monitored during the initial stages of vitamin B12 therapy, with potassium supplementation as necessary, because fatal hypokalemia may occur when megaloblastic anemia is converted to normal erythropoiesis by vitamin B12 therapy due to increased potassium demand from erythrocytes. Because vitamin B12 deficiency may mask the symptoms of polycythemia vera, treatment with cyanocobalamin may bring the disease to light. Vitamin B12 supplementation in patients with vitamin B12 deficiency may increase nucleic acid degradation, which may lead to gout in susceptible individuals. Concurrent infections, uremia, folic acid or iron deficiency, or medications with bone marrow suppression effects may affect the therapeutic efficacy of vitamin B12. Folic acid should be used with extreme caution in patients with undiagnosed anemia, as it may mask the diagnosis of pernicious anemia by alleviating hematological manifestations while allowing the progression of neurological complications. This could lead to severe neurological damage before a proper diagnosis is made. Patients with pernicious anemia should avoid taking vitamin preparations containing folic acid, as folic acid may actually exacerbate neurological complications caused by vitamin B12 deficiency. Conversely, daily intake of more than 10 micrograms of cyanocobalamin may improve folic acid deficiency-related megaloblastic anemia, thus masking the true diagnosis. Pharmacodynamics General Actions Cyanocoobalamin corrects vitamin B12 deficiency and improves symptoms and laboratory abnormalities associated with pernicious anemia (megaloblastic index, gastrointestinal lesions, and neurological damage). The drug contributes to growth, cell proliferation, hematopoiesis, nucleoprotein and myelin synthesis. It also plays an important role in lipid metabolism, carbohydrate metabolism, and protein synthesis. Rapidly dividing cells (e.g., epithelial cells, bone marrow cells, and myeloid cells) have a high demand for vitamin B12. Effects of parenteral administration of cyanocobalamin Parenteral administration of vitamin B12 can rapidly and completely reverse megaloblastic anemia and gastrointestinal symptoms caused by vitamin B12 deficiency. Rapid parenteral administration of vitamin B12 can stop the progression of neurological damage associated with vitamin B12 deficiency. Effects of nasal spray In 24 patients with stable vitamin B12 deficiency who had received intramuscular vitamin B12 treatment, once daily intranasal administration of cyanocobalamin for 8 weeks achieved serum vitamin B12 concentrations at the target therapeutic range (>200 ng/L). Acute toxicity: - Reference [1]: Vitamin B12 has low toxicity. Its median lethal dose (LD50) in humans has not been determined. High doses (e.g., 1 mg/day) are generally well tolerated, with very few reports of allergic reactions (rash, itching) or mild gastrointestinal symptoms.
- Chronic toxicity: - Reference [1]: Long-term use of high doses of vitamin B12 does not lead to significant organ toxicity. However, excessive intake may mask folic acid deficiency or interact with certain medications (e.g., metformin, antacids).

C63H92CON14O14P

1359.41

1354.567

68-19-9

5311498

Dark red crystals or an amorphous or crystalline red powder
Dark-red crystals or red powder

>300ºC

6.57

9

21

26

93

3220

14

[Co+2].P(=O)(O[H])(O[C@]1([H])[C@@]([H])(C([H])([H])O[H])O[C@@]([H])([C@]1([H])O[H])N1C([H])=NC2C([H])=C(C([H])([H])[H])C(C([H])([H])[H])=C([H])C1=2)OC([H])(C([H])([H])[H])C([H])([H])N([H])C(C([H])([H])C([H])([H])[C@@]1(C([H])([H])[H])C2C(C([H])([H])[H])=C3[C@@]([H])(C([H])([H])C([H])([H])C(N([H])[H])=O)C(C([H])([H])[H])(C([H])([H])[H])C(C([H])=C4[C@@]([H])(C([H])([H])C([H])([H])C(N([H])[H])=O)[C@](C([H])([H])[H])(C([H])([H])C(N([H])[H])=O)C(C(C([H])([H])[H])=C5[C@@]([H])(C([H])([H])C([H])([H])C(N([H])[H])=O)[C@](C([H])([H])[H])(C([H])([H])C(N([H])[H])=O)[C@](C([H])([H])[H])([C@@]([H])([C@]1([H])C([H])([H])C(N([H])[H])=O)N=2)[N-]5)=N4)=N3)=O.[C-]([H])([H])[H] |t:73,99,132|

FDJOLVPMNUYSCM-WZHZPDAFSA-L

InChI=1S/C62H90N13O14P.CN.Co/c1-29-20-39-40(21-30(29)2)75(28-70-39)57-52(84)53(41(27-76)87-57)89-90(85,86)88-31(3)26-69-49(83)18-19-59(8)37(22-46(66)80)56-62(11)61(10,25-48(68)82)36(14-17-45(65)79)51(74-62)33(5)55-60(9,24-47(67)81)34(12-15-43(63)77)38(71-55)23-42-58(6,7)35(13-16-44(64)78)50(72-42)32(4)54(59)73-56;1-2;/h20-21,23,28,31,34-37,41,52-53,56-57,76,84H,12-19,22,24-27H2,1-11H3,(H15,63,64,65,66,67,68,69,71,72,73,74,77,78,79,80,81,82,83,85,86);;/q;-1;+3/p-2/t31-,34-,35-,36-,37+,41-,52-,53-,56-,57+,59-,60+,61+,62+;;/m1../s1

cobalt(3+);[(2R,3S,4R,5S)-5-(5,6-dimethylbenzimidazol-1-yl)-4-hydroxy-2-(hydroxymethyl)oxolan-3-yl] [(2R)-1-[3-[(1R,2R,3R,5Z,7S,10Z,12S,13S,15Z,17S,18S,19R)-2,13,18-tris(2-amino-2-oxoethyl)-7,12,17-tris(3-amino-3-oxopropyl)-3,5,8,8,13,15,18,19-octamethyl-2,7,12,17-tetrahydro-1H-corrin-24-id-3-yl]propanoylamino]propan-2-yl] phosphate;cyanide

Docigram; DTXSID7044346; vitamin B12; Coobalamed; Cynobal; B 12, Vitamin; Vitamine B12; ...; 68-19-9; Vitamin B12; Cyanocobalamin

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~20.83 mg/mL (~14.14 mM)
H2O : ~6.25 mg/mL (~4.24 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (1.70 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: ≥ 2.5 mg/mL (1.70 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

---

View More

Solubility in Formulation 3: 50 mg/mL (33.94 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

---

 (Please use freshly prepared in vivo formulations for optimal results.)