Vitamin B 12 (Cobalamin) By: Mohsen Al-Saleh ID #: M020944
Vitamin B12 (Cobalamin)
By: Mohsen Al-Saleh
ID #: M020944
Outlines: • Introduction • Dietary Sources and RDA • Biosynthesis pathway of vitamin B12
• Vitamin B12 binding proteins • Absorption, metabolism and storage of vitamin B12 • Deficiency of vitamin B12 & pernicious anemia • Laboratory assessment of vitamin B12 • Vitamin B12 treatments and cardiovascular disease • Summary of main points • References
Vitamin B12: Structure
Vitamin B12: is a group of chemically related metabolically equiactive compounds with a central “corrin” ring system, surrounding a cobalt atom
Corrin or porphyrin ring
Cobalt Ion
Nucleotide Purine Ring
Active Group: -OH or -CN or -CH3 or -Cl or Adenosyl- or -H2O or -NH2 or –NO2
Pyrrole ring
Ribose
Vitamin B12: Derivatives
1. Cyanocobalamin CN-B12 (Digested form)
2. Hydroxycobalamin (form by accepting –OH.)
3. Chlorocobalamin Cl-B12
4. Methylcobalamin (Serum and breast milk)
5. Adenosylcobalamin (Physiologically useful)
6. (5’-deoxyadenosylcobalamin) (Cellular form)
Vitamin B12: Nomenclature
-Aquocobalamin = H2O -Nitrocobalamin = NO2
Active Form of Vitamin B12
Vitamin B12: Physical & Chemical Properties
• Red crystalline & stable at room temperature
• Molecular mass ~ 1,355.37 g/mol (-CN)
• Chemical formula: C63H88CoN14O14P
• In neutral form, vitamin B12 is bound to protein
• Freely soluble in water and alcohol
• Liable in strong acid, alkali, heat, and light
• Decompose in the presence of vitamin C, thiamine and nicotinamide
Vitamin B12: Introduction
• Isolated in 1948 but structure was discovered in 1926
• Store mainly in the liver (taken 2 decades to identify)
• Help to correct pernicious anaemia & activate folate
• Enables electrical impulses in the brain & protects the nerve
• Formation of RBCs & aids tissue growth
• Involved in amino acid, fatty acid, and nucleic acid metabolism
Sources of Vitamin B12
• Fish
• Eggs
• Meat (liver, Kidney)
• Dairy Products
B12 Requirement & Intake
• US average is 3-5 ug/day • RDA for adult: 2.4 ug/day (0.2-0.4↑ during
pregnancy) • Normal reference range (at SQUH) is from 133 – 675 pmol/L
• RDA > 51 years: 2.4 ug/day mainly in
fortification/supplementation • Body store of 5 mg is enough for 5 years
One of the most complicated biosynthetic pathways
Involves over 30 enzymes
Two pathways:
1. Aerobic
2. Anaerobic
16 intermediates between uroporphyrinogen III and adenosylcobalamin (aerobic pathway)
Biosynthesis of Vitamin B12
Discovery of the Biosynthetic Pathway
the Techniques
• 13C labeling (1990)
• NMR spectroscopy
• Enzymology
• Molecular genetics
Vitamin B12 Binding Proteins
Transcobalamin I
R-type binding protein
33% is carbohydrate
Molecular weight = 125,000-150,000
Beta globulin
Carries ~80% of vitamin B12 in blood
Vitamin B12 has half-life of 10-12 days when bound to it
Transcobalamin II
Molecular weight = 38,000 Alpha globulin and NOT a glycoprotein Carries less than 25% of Vitamin B12 in blood Vitamin B12 has half-life of under 1 ½ hours when bound to it Encourages absorption in a number of tissues Degenerates once B12 is released Transcobalamin II deficiency results in pernicious anemia
Transcobalamin III
R-type binding protein
33% is carbohydrate
Molecular weight = 125,000-150,000
Alpha globulin
Released from granulocytes
Vitamin B12 Absorption – Oral Phase
CN-Cobalamin
Has high affinity to Cobalamin
Vitamin B12 Absorption – Gastric Phase
HCl
Vitamin B12 Absorption – Intestinal Phase
As TC-I
-OH -CN
-CN
Stay in cytosol
Move to Mito.
GSH
Vitamin B12 affects on 2 Major
Pathways: (co-factors)
Homocysteine Methionine
Methylmalonyl CoA Succinyl CoA
Methylation
CH3-Cobalamin
Adenosyl-Cobalamin
Conversion of
homocysteine to methionine
Homocysteine
Methionine
Methylcobalamin
Methionine Synthase
5-methyltetrahydrofolate tetrahydrofolate Tetrahydrofolate methyltransferase
Vitamin B12
In cytoplasm of cells
Conversion of
Methylmalonyl CoA to Succinyl CoA
L-Methylmalonyl CoA
Succinyl CoA
Adenosylcobalamin
Methylmalonyl CoA mutase
In mitochondria
Vitamin B12 Deficiency
• homocysteine and methylmalonyl CoA:
1. Increase in methylmalonyl CoA:
– Increased enzyme activity in fatty acid synthesis
– Build up of odd fatty acids around peripheral nerves
– (values >0.4 mmol/L) indicates vitamin B12 deficiency
– Highly specific to vitamin B12 deficiency
2. Increase in homocysteine:
– Vascular/nervous problems
– Poor specificity may influence by low folate & vitamin B6
Vitamin B12 Deficiency
• Excess homocysteine & MMA excreted in urine:
– Diagnosis for cobalamin deficiency
• Methylmalonyl CoA mutase & Methionine
synthase affect amino acid metabolism:
– Amino acid metabolism inhibited by deficiency
Vitamin B12 deficiency
Cobalamin level in blood = below 200 pg/mL
Deficiency may develop within 2-30 years RDA, 1989
Common in elderly (50% of elders have low intake) Baik HW, 1999
Elders with low B12 status frequently lack symptoms Chernoff R, 1999
Causes of Vitamin B12 Deficiency
Malabsorption (inability to absorb food containing cobalamin) Inability to separate cobalamin from food in stomach Proton pump inhibitors Gastritis and Stomach/bowel resection >100 cm (Low intrinsic factor in the stomach) Chron’s disease Pancreatitis (Low proteases secretion) Gastric lymphoma Myeloma and HIV Antibiotics & Laxatives Excess Vitamin C Nitric Oxide Long term vegan vegetarianism (bacteria which is only found in meat products) Lack of calcium Defective gene for intrinsic factor)
Symptoms/Effects of Vitamin B12 Deficiency
Pernicious Anemia (Vitamin B12 is necessary for RBC production) Weight loss Weakness & fatigue Neuro-psychiatric disorders (Dementia) Leucopenia Thrombocytopenia Depression Alzheimer’s disease & loss of memory Increased liver weight Fat accumulation around heart, liver, peripheral nerves
Sensitive skin
High folate may mask B12 deficiency allowing devastating neurologic symptoms.
(Russell, R.M., 1999)
Pernicious Anemia
• Most common cause of cobalamin deficiency
• Caused by the absence of IF
– Atrophy of the mucosa
– Autoimmune destruction of parietal cells
• Seen in individuals of northern European descent and African Americans
• Men and women are equally affected
• Disease of the elderly, the average patient presenting near age 60
Symptoms/Effects of Vitamin B12 Deficiency
Increase:
Homocysteine
MMA
Bilirubin excretion
LDH
Liver glycogen
Hepatic citrate synthase
Succinate dehydrogenase
Amino Acids
Cell metabolism
Protein synthesis
Fatty acid synthesis enzymes
Decrease:
Transcobalamin II
Intrinsic factor
B12 deficiency
Note difference in growth and condition Pig at top has not grown as well and has rougher skin and hair coat
Clinical Manifestations of Vitamin B12 Deficiency
• Hematologic:
– Macrocytic anemia
• Gastrointestinal:
– Diarrhea
• Neurologic (found in 3/4th of individuals with pernicious anemia):
– Numbness and weakness – Disturbances of mentation (mental activity) – Neuronal death (Last stage is irreversible)
Large immature cells
Hypersegmented
Diagnosis of Vitamin B12 Deficiency
• Macrocytosis
• Peripheral blood smear
• Cobalamin levels (SQUH-Clinical Biochemistry)
• Elevated serum methylmalonic acid and homocysteine levels
• Schilling Test
Measurement of Vitamin B12
Measurement of Vitamin B12 Direct Heterogenous Immunoassay Techniques (wash step)
Treatment
• Supplements (Usual daily dose) :
– Oral pills (effectives)
– Intramuscular injections (effectives)
– Intranasal
• Overgrowth bacteria convert B12 into B12
analogues & could be reversed by antibiotic
treatment with tetracycline Chernoff R, 1999
Treatments
• Active form is most successful
• Must be supplied with folic acid
• Fish should be emphasized in diet
• 6 months → one year to recover
Vitamin B12 & CVD
• Low B12 intake is one of the determinants of high homocysteine (CVD) Stolzenberg-Solomoin RZ, 1999
• Evidence confirms: low vitamin B12 levels predispose to elevated homocysteine levels Waldmann A, Koschizke JW, et.al. 2004
• Homocysteine risk factor for (CVD) Lonn E, et.al. 1999
• Increased incidence and progression of CVD in patients with diabetes when homocysteine levels are >11.21 umol/L was found in a study of Polish men aged <55 Skibinska E, Sawicki R, et.al. Kardiol Pol. 2004 Mar;60(3):197-205.
Summary • Synthesized by bacteria and stored in animal
• Commercially available as CN- , OH- and CH3
• Stored in the liver as transcobalamin I
• Transported in a tissue as transcobalamin II
• Its absorption require Intrinsic factor
• RDA for adult is 2.4 ug/day
• No known toxicity
• Present in liver, fish, eggs & absent in vegetables
References: (Article Reviews)
• Waldmann A, Koschizke JW, et.al. HCY and cobalamin status in German vegans. Public Health Nutr. 2004 May;7(3):467-72.
• Flood VM, Webb KL, Smith W, et.al. Prevalence of low serum folate, red cell folate, serum vitamin B12 and elevated HCY. Asia PacJ Clin Nutr. 2004;13(Suppl):S85.
• Brosnan JT, Jacobs RL, et.al. Methylation demand: a key determinant of homocysteine metabolism. Acta Biochim Pol. 2004;51(2):405-13.
• Selhub J, Jacques PF, et.al. Vitamin Status and intake as primary determinants of HCYemia in an elderly population. JAMA 1993; 270: 2693-2698.
References: (Books)
1. Suzanne, K. et al.,(1991) Vitamin Intake and Health-Scientific Review, 1st ed.270 Madison Avenue, New York -USA: Marcel Dekker, Inc.
2. Wilhelm, F. (1988) Vitamins, 1st ed. Berlin. New York: Walter de Gruyter. 3. John, M. (1985) the vitamins – their role in medical practice. 1st ed.
Lancaster, England-Falcon House-UK. MTP press limited. 4. Sareen, S. et al., (2005) Advance Nutrition and Human Metabolism. 4th ed.
10 Davis Drive Belmont, CA 94002-3098, USA. Thomson-Wadsworth. 5. Maurice E. et al., (2006) Modern Nutrition in Health and Disease. 10th ed.
530 Walnut Street, Philadelphia, PA 19106, USA. Lippincott Williams & Wilkins.
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