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
Attached to
Cobalt
(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)

3. Sources
B12 is not present in vegetables.
• 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

4. Digestion and absorption of B12
Cbl-cobalamin
R-R protein
IF- intrinsic factor
TCII-transcobalamin II
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
• Gastric intrinsic factor (GIF) is a
glycoprotein produced by the parietal
cells of the stomach. It is necessary for
the absorption of vitamin B12.
• Encoded by the GIF gene located at
11q12.1 chromosome.
• Two-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.
• Glycoprotein - 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, function 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 andAMN 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.
• Polymorphism 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
• Vitamin 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) -
(i) failure to translocate cobalamin to mitochondria for AdenosylCobalamin formation
(CblA)
(ii) failure to convert cob(I)alamin to adenosylcobalamin (CblB)
• Methylcobalamin deficiency (homocystinuria and hypomethioninemia) -
(i) Impaired methylation of cob(I)alamin due to defect in apoenzyme methionine
synthase (CblG)
(ii) 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. Role of B12 in various system disorders
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.

14. B12 and Alzheimer’s
• Adequate 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 μmol/L or methylmalonic acid >0.4 μmol/L
(in the absence of renal failure and folate and B6 deficiencies)
Four Stages of Vitamin B12 Deficiency
I. Serum B12 concentration low; no clinical or metabolic abnormalities.
II. 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. )
III. Increased level of HCY and MMAand low holotranscobalamin levels.
IV. 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

19. Deficiency manifestation
Blood: pancytopenia, macrocytosis, hypersegmented neutrophils
Neurological,: myelopathy, paresthesia, loss proprioception, ataxia,
spasticity or autonomic dysfunction
Tongue: glossitis (red beefy tongue), taste impairment.
Bone marrow: hyperplasia, megaloblastic features
Brain : impaired cognition, depression, psychosis, delusion, irritability
Cardiac: cardiomyopathy
Reproduction: infertility

20. 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

21. 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.

22. • 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.

23. Thank you… 