Vitamin B12 (Cobalamin) - mohsinalsaleh.commohsinalsaleh.com/wp-content/uploads/2017/03/Vitamin-B12-Mohsen.pdf · Vitamin B 12: Structure Vitamin B 12: is a group of chemically related

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.

Questions?

You can ask ? Or choose to have vitamin B12 which help to release the Melatonin (sleep hormone)