Vitamin b12

B12 metabolism..................................... and role of various proteins in b12 metabolism..... necessity of supplementation..........................................

1. Vitamin B12

2. B12 is also known as Red vitamin because it exists as a dark red crystalline compounds. (Color is due to Co metal ions ) Vitamin B12 is composed of – * Tetrapyrrole ring surrounding Cobalt atom. * 5,6-dimethyl-benzimidazole. * R group. R group may be – - Cyanide in Cyanocobalamin (present in supplements, not a physiological form, more stable) - Methyl in methyl cobalamin - 5’- deoxyadenosine in adenosylcobalamin (active forms) -OH in hydroxocobalamin . (natural form, produced by bacteria, used in supplementation tablets and injections ) Attached to Cobalt

3. Sources Main dietary sources are meat products (especially liver), fish, egg , dairy products and fortified cereals. Vitamin B12 can be synthesized by microbes only. These microbes are present in the gut normally. ( e.g. Saccharomyces cerevisiae , Red star T6335+ etc.) Animals must obtain vitamin B12 directly or indirectly from microbes. RDA : 3 mcg/day B12 is not present in vegetables.

4. Cbl-cobalamin R-R protein IF- intrinsic factor TCII- transcobalamin II Digestion and absorption of B12 CMAJ.  2004 Aug 3;171(3):251-9.

5. Various proteins associated with vitamin B12 metabolism R protein (aka Haptocorrin or TCN I ) Intrinsic factors Cubilin receptors Transcobalamin II Cell surface receptors for TCNII-B12 complex Enzymes involved in formation of- adenosyl and methyl cobalamin forms.

6. Intrinsic factor G astric intrinsic factor  (GIF ) is a glycoprotein  produced by the parietal cells of the stomach . It is necessary for the absorption of vitamin B 12 . E ncoded by the  GIF   gene located at 11q12.1 chromosome. T wo-domain   protein containing 417 amino acids (45KDa). Overall fold  of the molecule is that of  an alpha/alpha   barrel and the Cobalamin is bound  at the interface of the  domains. {Crystal structure of human IF- cobalamin complex}

7. IF deficiency may be due to defect in GIF gene or GIF antibodies. Juvenile cobalamin deficiency (JCD) {potentially fatal megaloblastic anemia in western world} is due to GIF mutation. Andrea et al. (2008) in their study on individuals of African ancestry found a frameshift mutation (183_186delGAAT) in the intrinsic factor gene. Identification of such mutations in different type of populations allows for quick and easy genetic testing in a disease that is difficult to diagnose but easy to treat.

8. Haptocorrin Haptocorrin (HC)  also known as transcobalamin-1 (TC-1 ) or R-protein is encoded by the TCN1 gene located on 11q12.1 near GIF gene.   G lycoprotein - 433 amino acids (48KDa) and 30% carbohydrates . The essential function of haptocorrin is protection of the acid sensitive vitamin B12  while it moves through the stomach . 80% of the B12 in circulation is bound to HC, f unction of which is not known clearly (may be circulatory storage form). Polymorphism (rs191904676, rs184157997 etc. ) at gene level is observed for TCN I gene but its effect on vitamin B12 level or other clinical significance has not been observed yet.

9. Cubilin receptor Also known as Intrinsic Factor- Cobalamin Receptor encoded by CUBN gene located on 10p12.31. Cubilin protein and aminonless (AMN) protein forms a Cubam complex which helps in vitamin B12 absorption. Cubilin recognizes the IF-B12 complex and AMN helps inreceptor mediated endocytosis. Cubilin is a co-transporter protein having 3623 amino acids (398 KDa ) and transportation requires calcium. Apart from B12 it also facilitates uptake of lipoprotein and Iron. Autosomal recessive mutation in CUBN and AMN leads to B12 deficiency megaloblastic anemia which is known as Imerslund-Grasbeck syndrome. ( prevalence about 1 in 200,000)

10. Transcobalamin II Transcobalamin II (TCN2 ) gene is located on 22q12.2 and encodes for TCN2 protein (427 amino acids, 47 KDa ). It doesn’t have carbohydrate content. In enterocyte B12 is liberated from IF and appears in blood bound to TC2 ( holotranscobalamin ) which carries B12 to various cells. 20% of total B12 is present in holotranscobalmin form which is supposed to be the form available for cellular uptake. P olymorphism in TCN2 gene (C776G, G1196A etc.) has been observed to reduce plasma TC II concentration and causes decrease in cellular availability of B12 and increased homocysteine levels.

11. Cell surface receptors for TCNII-B12 complex V itamin B12-TCII complex is transported to target cells and undergoes receptor-mediated endocytosis using the transcobalamin II receptor (TCII-R), a specific vitamin B12 cell surface receptor. TCII-R (282 amino acids, 29KDa) is encoded by CD320 gene which is located on 19p13.3-p13.2. Mutation in CD320 are associated with TCII-R functional defect and methylmalonic acidurias .. Cancer cells have increased demand for B12 for DNA synthesis. It has been observed in immunohistochemical analysis that expression of TCII-R is increased on tumor cell surface.

12. Enzymes involved in formation of- active cobalamin forms Adenosylcobalamin deficiency ( methylmalonicaciduria ) - failure to translocate cobalamin to mitochondria for AdenosylCobalamin formation ( CblA ) failure to convert cob(I) alamin to adenosylcobalamin ( CblB ) Methylcobalamin deficiency ( homocystinuria and hypomethioninemia ) - Impaired methylation of cob(I) alamin due to defect in apoenzyme methionine synthase ( CblG ) Defect in reduction of cob(III) alamin to cob(I) alamin prior to methylation. ( CblE ) CblC and CblD are the diseases having combined adenosyl and methyl cobalamin deficiencies. In CblF there is defect in release of vitamin B12 from lysosomes.

13. B12 and osteoporosis B12 deficiency reduces osteoblastic activity B12 deficiency indirectly stimulates osteoclstic activity mediated by elevated levels of homocysteine and methylmalonic acid. So during B12 deficiency there is increased chances of osteoporosis and fractures. Immunomodulatory effect of B12 Vitamin B12 supplementation has been observed to increase the total leucocyte count including CD8+ cells. It also enhances NK-cell activity. High dose supplementation with B12 shifts the immune response from Th2 to Th1 which downregulate the IgE production in allergic individuals. Role of B12 in various system disorders

14. B12 and Alzheimer’s A dequate levels of vitamin B12 is necessary for the brain's myelin sheath. Methylmalonate , a marker of vitamin B12 deficiency, is associated with a reduction of brain volume and so may contribute to cognitive problems. Homocysteine , an amino acid associated with low B12 levels as well as folate, was linked to thinking problems through a different mechanism involving abnormal white matter signals. B12 and Atherosclerosis B12 deficiency causes hyperhomocysteinemia Vitamin B12 deficiencies are reported to cause hypomethylation in DNA of arterial intima cells resulting in mutation and proliferation of smooth-muscle cells which leads to atherosclerosis.

15. B12 and cancer Vitamin B12 concentrations have been reported to be negatively associated with DNA damage. Supplementation with vitamin B12 also reduced DNA damage in younger subjects. Vitamin B12 deprivation has been found to increase both uracil misincorporation and global DNA hypomethylation in the colonic mucosa of rats. A positive correlation between vitamin B12 concentrations and markers of genotoxicity has been noted in smokers.

16. Vitamin B12 deficiency definition Serum cobalamin levels <150 pmol /L on 2 separate occasion OR Serum cobalamin levels <150 pmol /L and Total serum homocysteine level >13 μ mol /L or methylmalonic acid >0.4 μ mol /L (in the absence of renal failure and folate and B6 deficiencies) Four Stages of Vitamin B12 Deficiency Serum B12 concentration low; no clinical or metabolic abnormalities. Plasma and cells stores B12 become depleted. (Normally 2-3mg B12 is stored in the Liver which is sufficient to fulfill the cellular demand in a healthy individual for upto 3-4 years. ) Increased level of HCY and MMA and low holotranscobalamin levels. Clinical signs become recognizable .

17. Vitamin B12 deficiency: Causes Stage of Cobalamin metabolism Cause of Cobalamin deficiency Food ingestion Strict vegetarianism without fortification & supplementation Digestion Gastrectomy, Gastric atrophy, H. pylori infection, Use of antacids (H2 receptor blockers or proton pump inhibitors etc.) Absorption Ileal resection, malabsorption syndromes, pernicious anemia, fish tapeworm infestation, pancreatic exocrine failure, drugs interfering absorption ( metphormin , cholchicine , neomycin, ethanol etc.) Transportation Congenital deficiency or defect in transcobalamin II ( C776G, G1196A polymorphisms ) Intracellular metabolism Congenital deficiency in various intracellular enzymes required for conversion to its active forms

18. Deficiency manifestation

19. Diagnosis of B12 deficiency Clinical examination Peripheral smear Serum B12 levels Serum MMA, Hcy levels Holotranscobalamin II levels Schlling’s test Anti IF antibody Molecular diagnostics

20. Mass Supplementation with B12 ???? In view of association of B12 deficiency with many disorders, supplementation of B12 in high doses seems a logical step. Moreover, B12 is a water soluble vitamin and toxic effects are not thought of. However, there are certain issues which needs to be addressed, before advising mass B12 supplementation.

21. Long term administration of cobalamin was associated with the risk of subsequently diagnosed cancer notably in prostate cancer . Increasing plasma levels of vitamin B12 were statistically significantly associated with increased prostate cancer risk, with an odds ratio 2.63 (95% CI = 1.61-4.29; p(trend) < 0.001) for vitamin B12 for highest vs. lowest quartile. ( Int J Cancer. 2005 Feb) In a cohort study conducted on more than 3,00,000 people it was observed that people with higher cobalamin levels had higher risk of subsequent development of various cancers. ( J Natl Cancer Inst  2013 ) Cyanocobalamin should not be used in patients with early Leber's disease (hereditary optic nerve atrophy), since rapid optic nerve atrophy has been reported following admin of the drug to these patients. Vitamin B12 is contraindicated in patients who have experienced hypersensitivity reactions to the vitamin or to cobalt.