Chemical Properties
Crystalline
Usage
Prototype of the family of naturally occurring cobalt coordination compounds knows as corrinoids. Analogs of vitamin B12 which differ only in the β-ligand of the cobalt are termed cobalamins. Synthesi
zed almost exclusively by bacteria. Dietary sources include fish, meat, liver, and dairy products; plants have little or no cobalamins. Converted by the body into its bioactive forms, methylcobalamin
and cobamamide, which serve as enzyme cofactors. Severe deficiency may result in megaloblastic anemia and/or neurological impairment.
Usage
vitamin, coenzyme B12
Originator
Berubigen,Upjohn,US,1949
History
VITAMIN B12 (Cobalamin), Sometimes also called cyanocobalamin, this vitamin is one of the more recent of the major B complex vitamins to be fully identified, with its structure not definitized (by Hodkin et al.)
Production Methods
Vitamin B12 dietary supplements are often prepared commercially by the fermentation of S. griseus, S. aureofaciens, Propionibacterium; or as a by-product of antibiotic production.
Manufacturing Process
The following is taken from US Patent 3,057,851. Milorganite was extracted
with water to obtain an aqueous extract containing vitamin B12 active
substances. This aqueous extract was purified by treatment with an ion
exchange resin according to the following method. An aqueous extract of
milorganite, 100 ml containing 300 μg of vitamin B12 active substances and
4.5 grams of total solids, was combined with 0.5 gram of sodium nitrite and
0.4 gram of potassium cyanide. The resulting solution was adjusted to pH 4.0
with hydrochloric acid and heated to boiling. The boiled solution was filtered
through a Super-Cel filter surface, and the filter was then washed with water.
The filtrate was obtained in a total volume of 130 ml including the washings.
Amerlite XE-97, an ion exchange resin of the carboxyl type (Rohm and Haas),
was classified to an average wet particle size of 100 to 150 mesh. The
classified resin was utilized in the hydrogen form, and was not buffered during
the ion exchange fractionation. The classified resin, in the amount of 35 ml,
was packed into a glass column having a diameter of 25 mm and a height of
250 mm. The cyanide-treated aqueous extract of milorganite was infused
gravitationally into the ion exchange bed at a rate of 3 ml per minute.
The effluent was discarded and the resin bed was then washed with the
following solutions in the specified sequence: (1) 120 ml of an aqueous 0.1 N
hydrochloric acid solution; (2) 75 ml of an aqueous 85% acetone solution;
and (3) 70 ml of an aqueous 0.1 N hydrochloric acid solution. After washing,
the resin bed was eluted with an aqueous 60% dioxane solution containing
0.1 N of hydrochloric acid. In this elution, 8 ml of colored eluate was
collected. This portion of the eluate was found to contain 295 μg of
cyanocobalamin and 9 mg of total solids.
Brand name
Nascobal (QOL).
Therapeutic Function
Hematinic
General Description
Pharmaceutical secondary standards for application in quality control, provide pharma laboratories and manufacturers with a convenient and cost-effective alternative to the preparation of in-house working standards.
Cyanocobalamin, also known as vitamin B12, belongs to a group of organic compounds important for the normal growth and development of human and animal bodies. It is used for the treatment of trigeminal neuralgia and multiple sclerosis.
Health Hazard
Deficiency diseases or disorders include retarded growth; pernicious anemia; megaloblastic anemia; macrocytic, hyperchromic anemia; glossitis; spinal cord degeneration; and sprue.
Biological Activity
Principal physiological functions include: (1) Coenzyme in nucleic acid, protein, and lipid synthesis; (2) maintains growth; (3) participates in methylations; (4) maintains epithelial cells and nervous system (myelin sheath); (5) erythropoiesis (with folic acid); (6) leukopoiesis.
Biochem/physiol Actions
Vitamin B12 is metabolized to its active form, methylcobalamin. Hindered vitamin B12 uptake is observed in pernicious anemia. Vitamin B12 is essential for erythroblast development. Pregnancy reduces the levels of vitamin B12. Aging reduces vitamin B12 absorption. It works as a coenzyme in fat, carbohydrate and protein metabolism. It is required for growth, genetic stability and survival of cells in vitro. Functions to support one-carbon metabolism. Present in many classical and serum-free formulations.
Veterinary Drugs and Treatments
Cyanocobalamin is used for treating deficiencies of vitamin B12.
Malabsorption of the nutrient secondary to gastrointestinal tract
disease, or dietary chromium deficiencies (in ruminants) can be associated
with dietary deficiencies of vitamin B12. As there appears
to be a high percentage of cats with exocrine pancreatic insufficiency
or gastrointestinal disease that are deficient in cobalamin, there is
considerable interest in evaluating serum cobalamin (vitamin B12)
in these patients. Giant schnauzers may have a genetic defect affecting
the location of the cobalamin-intrinsic factor, causing cobalamin
deficiency. Dogs with inflammatory bowel disease may also
develop cobalamin deficiency.
Purification Methods
Vitamin B12 crystallises from de-ionized H2O, with a solubility in H2O of 1g/80g, and is dried under vacuum over Mg(ClO4)2. The dry red crystals are hygroscopic and can absorb 12% of 2O. A solution at pH 4.5-5 can be autoclaved for 20minutes at 120o without decomposition. Aqueous solutions are stabilised by addition of (NH4)2SO4. [Golding Comprehensive Organic Chem Vol 5 (Ed. Haslam; Pergamon Press, NY, 1979) pp 549584.]
Alternatively an aqueous solution of the coenzyme can be concentrated, if necessary in a vacuum at 25o or less, until the concentration is 0.005 to 0.01M (as estimated by the OD at 522nm of an aliquot diluted with 0.01M K-phosphate buffer pH 7.0). If crystals begin to form on the walls of the container, they should be re-dissolved with a little H2O. The concentrated solution is placed in a glass stoppered flask and diluted with 5volumes of Me2CO. After 2-3hours at 3o it is centrifuged (10,000xg/10minutes) in Me2CO-insoluble plastic tubes to remove some amorphous precipitate. The clear supernatant is inoculated with a small crystal of the vitamin and allowed to crystallise overnight at 3o. Crystals are formed on the walls and the bottom of the container. A further 2volumes of Me2CO are added and set aside at 3o to further crystallise. Crystallisation is followed by observing the OD522 of the supernatant. When the OD falls to 0.27, then ca 94% of the crystals have separated. The supernatant is decanted (saved for obtaining a second crop), and the crystals are washed with a little cold 90% aqueous Me2CO (2x), 100% Me2CO (2x), Et2O (2x) at which time the crystals separate from the glass walls. Allow them to settle and remove residual Et2O with a stream of dry N2. The process can be repeated if necessary. The crystals can be dried in air or in a vacuum for 2hours over silica gel at 100o with an 8-9% weight loss. [Barker et al. Biochemical Preparations 10 33 1963.] This material gives a single spot on paper chromatography [see Weissbach et al. J Biol Chem 235 1462 1960.] The vitamin is soluble in H2O (16.4mM at 24o, 6.4mM at 1o), in EtOH and PhOH but insoluble in Me2CO, Et2O, CH2Cl2 and dioxane. UV: max at 260, 375 and 522nm ( 34.7 x 106, 10.9 x 106 and 8.0 x 106 / mole) in H2O. The dry crystals are stable for months in the dark, but aqueous solutions decompose on exposure to VIS or UV light or alkaline CN-, but are stable in the dark at pH 6-7. The vitamin is inactivated by strong acids or alkalies. [Barker et al. J Biol Chem 235 480 1960, see also Vitamin B12 (Zagalak & Friedrich Eds) Walter de Gruyter, Berlin 1979, Beilstein 2 6 IV 3117.]
