Vitamin B12 Deficiency:Causes, Manifestation and Management

Dr Jeetam singh Jr 3 Internal medicine MLN Medical college

Moderator:Dr. Manoj Mathur(MD)Assistant Professor PG Department of MedicineMLN Medical college

Synonyms

• Cyanocobalamine• Anti Pernicious anemia factor• Extrinsic factor of Castle

Structure

• Cobalamin is analogous to heme in its structure having as its base a tetrapyrrole ring.

• Instead of iron as a metal cofactor for heme, cobalamin has cobalt in a coordination state of six with a benzimidazole group nitrogen coordinated to one axial position, the four equatorial positions coordinated by the nitrogens of the four pyrrole groups.

Structure of Vitamin B12

oThe sixth position occupied by either a deoxyadenosine group, a methyl group or a CN– group in the commercially available form in vitamin tablets.

Forms of Cobalamin• Cobalamin (vitamin B12) exists in a number of different

chemical forms. • All have a cobalt atom at the center of a corrin ring. • In nature, the vitamin is mainly in the 2-deoxyadenosyl

(ado) form, which is located in mitochondria.• The other major natural cobalamin is methylcobalamin,

the form in human plasma and in cell cytoplasm. • There are also minor amounts of hydroxocobalamin to

which methyl- and adenosyl cobalamin are rapidly converted by exposure to light.

Dietary Sources

• Cobalamin is synthesized solely by microorganisms. • Ruminants obtain cobalamin from the foregut, but

the only source for humans is food of animal origin, e.g. meat, fish,eggs and dairy products.

• Vegetables, fruits, and other foods of non-animal origin are free from cobalamin unless they are contaminated by bacteria.

• Strict vegetarians are at risk of developing B12 deficiency.

Dietary Sources of Vitamin B12

Requirements of vitamin B12

• A normal Western diet contains between 5 and 30 μg of cobalamin daily.

• Adult daily losses (mainly in the urine and feces) are between 1 and 3 μg (~0.1% of body stores) and, as the body does not have the ability to degrade cobalamin, daily requirements are also about 1 to 3 μg.

• Body stores are of the order of 2 to 3 mg, sufficient for 3 to 4 years if supplies are completely cut off.

RDA OF VITAMIN B12

• Age Male Female Pregnant Lactating • 0-6months0.4µg 0.4µg• 7-12months 0.5µg 0.5µg• 1-3 years 0.9µg 0.9µg • 4-8 years 1.2µg 1.2µg • 9-13 years 1.8µg 1.8µg• 14+ years 2.4µg 2.4µg 2.6µg 2.8µg

Absorption

• Two mechanisms exist for cobalamin absorption. • Passive absorption-occurring equally through

buccal, duodenal and ileal mucosa, it is rapid but extremely inefficient, <1% of an oral dose being absorbed by this process.

• Active absorption-The normal physiologic mechanism is active, it occurs through the ileum and is efficient for small (a few micrograms) oral doses of cobalamin and is mediated by gastric intrinsic factor (IF).

Absorption

• Dietary cobalamin is released from protein complexes by enzymes in the stomach, duodenum, and jejunum

• It combines rapidly with a salivary glycoprotein that belongs to the family of cobalamin-binding proteins known as haptocorrins (HCs)/Cubophilin.

• In the intestine, the haptocorrins are digested by pancreatic trypsin and the cobalamin transferred to intrinsic factor(IF).

Absorption and the role of Intrinsic factor

• Intrinsic factor (IF) is produced in the gastric parietal cells of the fundus and body of the stomach, its secretion parallels that of hydrochloric acid.

• The IF-cobalamin complex passes to the ileum, where IF attaches to a specific receptor (Cubulin) on the microvillus membrane of the enterocytes.

• Cubulin with its ligand IF-cobalamin complex is endocytosed.

• The cobalamin-IF complex enters the ileal cell where IF is destroyed.

Absorption of Vitamin B12 and the role of Intrinsic factor

Intrinsic factor deficiency

• In the absence of the intrinsic factor inadequate amounts of cobalamin are absorbed (the dietary requirement is approximately 200 ng/day).

• When the root cause of the resultant Megaloblastic anemia is absence of or inadequate amounts of intrinsic factor the condition is called pernicious anemia.

Transportation of Cobalamin

• Three plasma transport proteins have been identified.

• Transcobalamine I and III (differing only in carbohydrate structure) are secreted by white blood cells.

• Although approximately 90 percent of plasma vitamin B12 circulates bind to these proteins, only transcobalamine II is capable of transporting vitamin B12 into cells.

Storage of Cobalamin

• The liver contains 2000 to 3000 mcg of stored vitamin B12.

• Since daily losses are 1 to 3 mcg/day, the body usually has sufficient stores of vitamin B12 so that vitamin B12 deficiency develops more than 3 years after vitamin B12 absorption ceases.

Metabolic Role of Cobalamin

1)Cobalamin plays a vital role in the catabolism of odd-chain fatty acids, threonine, methionine, and the branched-chain amino acids (leucine, isoleucine, and valine).

• The degradation of each of these compounds produces the same metabolite, Propionyl-CoA.

Fate of Propionyl CoA

Fate of Propionyl CoA in B12 deficiency

• In cases of cobalamin deficiency these reactions of utilization of propionyl co A are compromised leading to an accumulation of methylmalonyl-CoA in serum, which has been suggested as a possible source of neurologic defects seen in cobalamin deficiency by decreasing lipid synthesis.

• Excess of MMCoA converted into MMA which lead to synthesis of abnormal fatty acid instead of myelin.

• These FA incorporated into neuronal lipid leading to fragile myelin sheath.

• Excess methylmalonyl-CoA in B12 deficiency gets excreted in urine causing methylmalonic aciduria

2.Role of cobalamin in DNA synthesis and the biochemical basis of Megaloblastic anemia

• The cause of megaloblastic anemia seen in strict vegetarians is attributed to the effects of cobalamin deficiency on DNA synthesis, specifically the thymidylate synthase reaction which converts dUMP→ dTMP.

Implications of Inadequate Thymidylate synthesis

• Inadequate dTMP restricts DNA but not RNA synthesis leading to the appearance of large erythroid cells with small nuclei containing a high ratio of RNA to DNA.

• These cells are removed from the circulation, thus stimulating erythrogenesis and giving rise to anemia with an elevated presence of megaloblasts.

3. Role of cobalamin in methionine metabolism

• Cobalamin is required for the conversion of homocysteine into methionine.

• Cobalamin must first undergo methyl transfer to form methyl cobalamin.

• It receives the methyl group from N5-methyltetrahydrofolate thus regenerating tetrahydrofolate to participate in other one-carbon transfers in purine metabolism or pyrimidine remodeling.

• This N5-MethylTHF provided through diet.

Role of Methionine

• Methionine help in formation of monoamine neurotransmitter eg: Dopamine, Noradrenaline, Serotonine.

• That’s why the def of b12 lead to def. of methionine which ultimately lead to psychiatric symptoms like delusion, hallucination, depression, cognitive changes, dementia.

• Methionine also help in the formation of myelin sheath, so in the absence of methionine there is myelin degenration.

Folate trap• In cobalamin deficiency, the methionine synthase reaction

cannot occur, N5-methyltetrahydrofolate accumulates and the other C-1 donor forms of tetrahydrofolate cannot be formed.

• The methionine synthesis from homocysteine ceases allowing the “trapping” of the folate pool as N5-methyltetrahydrofolate, diminishing levels of N5, N10-methylenetetrahydrofolate

• N5,N10-methylenetetrahydrofolate, is required for the methylation of dUMP to dTMP, thus in it’s deficiency ,the thymidylate synthase reaction is slowed and dTMP levels drops and hence DNA synthesis is also slowed down due to non availability of deoxy ribonucleotides

Roles of cobalamin and folic acid in methionine metabolism

Vitamin B12 deficiencyLevel of vit B12 in blood (ng/L)

Severity

160-200 to 1000 Normal

100 to 200 Borderline/Mild deficiency

<100 Severe deficiency

Vitamin B12 deficiencyCauses of Vitamin B12 Deficiency1. Nutritional Def.: Dietary deficiency:- Vegan (rare)

2. Inherited:a) Transcoblamine deficiency.b) Intrinsic factor deficiency: Pernicious Anaemia.

3. Malabsorption:• Gastrectomy• Pancreatic insufficiency• Fish tapeworm (rare)• Helicobacter pylori infection

B12 deficiency cause cont..

• Crohn’s disease.• Tropical sprue and coeliac sprue.• Ileal Surgical resection.• Decreased ileal absorption of vitamin B12.• Competition for vitamin B12 in gut Blind loop

syndrome.• Drug: Neomycin,Metformin (biguanides),PPI,Nitric

oxide (inhibits methionine synthase).

B12 deficiency cont….

Manifestation:

Anemia• Anemia is because of bone marrow suppression &

ineffective erythropoesis.• Patient mostly presented with features of anemia:Weakness, fatigue, loss of appetite, anorexia, wt loss, Dyspnea on exertion, giddiness, lack of concentration etc.Suppression of epithelial surface proliferation:• The megaloblastic state also produces changes in mucosal

cells, leading to glossitis, angular cheilosis, mucositis, stomatitis,sore or burning mouth, glossitis,glossodynia, red tongue,apthous ulcer as well as other vague gastrointestinal disturbances such as anorexia and diarrhea, constipation.

Neurological changes in B12 deficiency1. Cerebrum: Complex neuropsychiatric symptoms: Delusion, illusion, hallucination, cognitive impairment, dementia, optic atrophy.2. Spinal cord: Subacute combined degenration of spinal cord Post column: Diminished vibration sensation and proprioception. Corticospinal tract: Upper motor neuron sign.3. Peripheral Nerve: Tingling & numbness. Glove and stocking paraesthesia. Loss of ankle reflex. • Peripheral nerves are usually affected first, and patients complain initially of

paresthesias.• The posterior columns next become impaired, and patients complain of

difficulty with balance.

Cardiovascular disease: Vit B12 deficiency lead to hyperhomocysteinemia which is

predispose to hypercoagulable state which ultimately increase cardiovascular risks.

There is increase risk of ischemic heart disease, peripheral arterial disease, venous thromboembolism, cerebrovascular disease.

Pregnancy related complication: Gonads are also affected, and infertility is common in both

men women . Both folate and coblamin deficiency implicated inrecurrent

fetal loss and neural tube defect.

Other manifestation: Thrombocytopenia :- Petaechial rashes, easy bruising,

hemetemesis, malena, bleeding gums. Leucopenia :- Recurrent respiratory and urinary tract

infections. Jaundice:- haemolytic jaundice due to increase haemolysis of

immature precursor cells. Reversible melanin skin hyperpigmentation.

Laboratory Findings The peripheral blood smear:• It is usually strikingly abnormal, with anisocytosis and

poikilocytosis. A characteristic finding is the macro-ovalocyte, but numerous other abnormal shapes are usually seen. The neutrophils are hyper segmented.

• The MCV is usually strikingly elevated >100 fl.• The reticulocyte count is reduced(<0.5%). • Because vitamin B12 deficiency affects all hematopoietic cell

lines, in severe cases the white blood cell count and the platelet count are reduced, and pancytopenia is present.

Peripheral blood smear in Megaloblastic anemia

Blood film in vitamin B12deficiency showing macrocytic red cells and a hyper segmented neutrophil.

Laboratory Findings Bone marrow morphology :• It is characteristically abnormal. • Marked erythroid hyperplasia is present as a response to

defective red blood cell production (ineffective erythropoiesis), the cells are larger than normoblast.

• Megaloblastic changes in the erythroid series include abnormally large cell size and asynchronous maturation of the nucleus and cytoplasm—i.e. cytoplasmic maturation continues while impaired DNA synthesis causes retarded nuclear development.

• Giant metamyelocytes(in myeloid series) & enlarged hyperpolyploid megakaryocytes are characteristically seen.

Laboratory Findings

• Other laboratory abnormalities include elevated serum lactate dehydrogenase (LDH) and a modest increase in indirect bilirubin.

• These two findings are a reflection of intramedullary destruction of developing abnormal erythroid cells and are similar to those observed in peripheral hemolytic anemias.

Laboratory FindingsSerum cobalamin level: The diagnosis of vitamin

B12 deficiency is made by finding an abnormally low vitamin B12 (cobalamin) serum level.

• The normal vitamin B12 level is > 200 ng/L,• Most patients with overt vitamin B12 deficiency

can have serum levels < 170 ng/mL, with symptomatic patients usually having levels < 100 ng/ml.

• A level of 100 to 200 ng/L is borderline.

Laboratory Findings

Estimation of serum methylmalonic acid levels:• In patients with coblamine deficiency sufficient to

cause anemia or neuropathy serum MMA level is raised.

• When the serum level of vitamin B12 is borderline, the diagnosis is best confirmed by finding an elevated level of serum methylmalonic acid (> 4.7ug/dL).

• However, elevated levels of serum methylmalonic acid can also be due to renal insufficiency.

• There is also increase level of Homocysteine in blood.• The MMA and homocysteine level start rising even

before decrease in cobalamin level in blood so both can be use as a screening marker for cobalamin deficiency.

• The Schilling test is now rarely used.

Schilling test:

• Why the Schilling test is performed?The test can help to determine whether stomach is

producing “intrinsic factor’’ or not.And where is the pathology(stomach/ pancreas/

intestine).

Procedure:Stage 1: oral vitamin B12 plus intramuscular vitamin B12 • Oral dose: patient is given radiolabeled Vit B12 – The most

commonly used radiolabels are 57Co and 58Co. • An intramuscular injection of unlabeled vitamin B12 is given

an hour later. • The patient's urine is then collected over the next 24 hours to

assess the absorption. • A normal result shows at least 10% of the radiolabeled vitamin B12 in the urine over the first 24 hours. • In patients with impaired absorption, less than 10% of the radiolabeled vitamin B12 is detected .

Stage 2: Vitamin B12 + IF If an Stage-I is abnormal: • The test is repeated with additional oral intrinsic factor • If this second urine collection is normal, this shows a

lack of intrinsic factor production, or pernicious anemia. • A low result on the second test implies “Malabsorption” • –Coeliac disease. • –Biliary disease.• –Whipple's disease.• –Fish tapeworm infestation (Diphyllobothrium latum). • –Liver disease.• –Immerslund syndrome.

Stage 3: vitamin B12 and antibiotics:• This stage is useful for identifying patients with

bacterial overgrowth syndrome.

Stage 4: vitamin B12 and pancreatic enzymes:• This stage, in which pancreatic enzymes are

administered, can be useful in identifying patients with pancreatitis.

Interpretation of schilling test

Differential Diagnosis

• Causes of macrocytic anemia:Vit B12 deficiency.Folic acid deficiency.Hypothyroidism.Thiamine deficiency. Liver disease- CLD. Lithium toxicity.Hereditary orotic aciduria.

Treatment of vitamin B12 deficiency

The indications for starting cobalamin therapy are :• A well-documented Megaloblastic anemia • or other hematological abnormalities • or neuropathy due to the deficiency.• Pt with total gastrectomy or ileal resection.Duration: It is necessary to treat patients who have develoved

coblamine deficiency with lifelong regular coblamin injections.

Treatment of vitamin B12 deficiency• Patients with pernicious anemia have historically been

treated with parenteral therapy. • Intramuscular injections of 1000 mcg of vitamin B12 are

adequate for each dose.• Replacement is usually given daily for the first week, weekly

for the first month, and then monthly for life. • It is a lifelong disorder, and if patients discontinue their

monthly therapy the vitamin deficiency will recur. • Oral cobalamin may be used instead of parenteral therapy

and can provide equivalent results. The dose is 1000 mcg/day and must be continued indefinitely.

• Sublingual therapy has also been proposed for those in whom injection are difficult because of bleeding tndency.

Injection content Amount(per ml)

Vitcofol(10ml) Nicotinamide+folic acid+cyanocob. 200mg+15mg+500ug

Nurokind plus(2ml) Nicotinamide+B6+methylcob. 50mg+50mg+500ug

Nurokind gold(2ml) same 50mg+50mg+750ug

Neurobion RF(2ml) B6+methylcobalamin 50mg+500mg

Eldervit-12(2ml) Nicotinamide+folic acid+b12+vit C 75mg+6mg+1000mg+0.7mg

Sublingual tablets:

Coblamin transdermal patches: Used once a day and a day in a week. Easy to use, better compliance but efficacy is like that of oral

formulation but lesser than injectable formulation. It is also costlier than all other foemulation.

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Questions

1. Macrocytic anaemia seen in all except:a)Liver diseaseb)Copper deficiencyc)Thiamine deficiencyd)Orotic aciduria

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• Ans B• Causes of Macrocytic anemia1. Vit b12 def.2. Folic acid def.3. Orotic aciduria.4.Nitrous oxide inhalation.5. Liver disease.6. Hypothyroidism.7. Thiamine Def.

2. Which of the following not found in vit b12 def.a) Moeller’s glossitisb) Sore tonguec) Macroglossiad) Atrophic glossitise) Median Rhomboid glossitis

• Ans. E• Median rhomboid tongue:- This condition is

characterised by persistent erythematous, rhomboidal depappilated lesion in central area of dorsum of tongue just in front of circumvallate papillae. It is a type of oral candidiasis seen in immunosuppresant patients.

Tongue feature seen in vit b12 deficiency:Macroglossia- Tongue may be large.Atrophic glossitis- Smooth tongue due to loss of papillae.Moeller’s glossitis-Red patches on red line on ventrum of

tongue.Sore/Beefy tongue- Red and inflamed tongue.

3. Megaloblastic anemia due to folic acid deficiency is commonly due to:

a) Inadequate dietary intake.b) Defective intestinal absorption.c) Absence of folic acid binding protein in serum.d) Absence of glutamic acid in the intestine.

Ans.A

4. Megaloblastic anemia due to B12 def. is most commonly due to:

a) Pernicious anemiab) Defective intestinal absorptionc) Inadequate dietry intaked) Absence of transcoblamin

Ans. B

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5. Which of the following is incorrect about pernicious anemia

a) Lack of intrinsic factorb) Peak age of onset is <20 yrsc) Life expectency is more in women then mend) Gastric biopsy shows atrophy of all layer of body

and fundus.

Ans. B Peak age of onset is 60 yrs.

6. All of the following is true about folic acid prophylaxis except

a) It prevent NTD.b) Folic acid prophylaxis prevent against colon

adenomac) Prophylaxis in preg reduces the subsequent

incidence of ALLin childhoodd) Supplementation of folic acid in homocystenemia

prevent or reduce cardiovascular events.

Ans D

7.Which of the following incorrect about Imerslund’s syndrome

a) Selective malabsorption of vit b12b) Associated with proteinuria.c) Autosomal recessived) There is decrease secretion of gastric acid

Ans. D. Imerslund’s syndrome/Imerslund-grasbecksyndrome/congenital cobalamin malabsorption:This is autosomal recessive disease most common

cause of megaloblastic anemia due to cobalamin def. in western countries.

Patients secrete normal amount of IF and gastric acid but are unable to absorb cobalamin.

Over 90% of the pts shows nonspecific proteinuria but renal function is otherwise normal and renal biopsy has not shown any consistent defect.

8. Megaloblastic anemia should be treated with both folic acid and vit b12 because

a) It is a cofactor b) It is enzymec) Folic acid causes improvement of hematological

symptoms but worsening of neurological symptomsd) None of the above

• Ans. CMegaloblastic anemia may be caused by a deficiency of

vit b12 or folate def. Unless it is clear that which of two def causes anemia treatment should include administration of both folic acid and vit b12 . If only folic acid is administered in a patient with megaloblastic anemia due to vit b12 def , worsening of neurological symptoms (cobalamin neuropathy) is seen despite an improvement in hematological symptoms.

9. Cubilin receptor (receptor for IF-Cobalamin complex )Found in all of the following excepta) Intestineb) Yolk sacc) Myelin sheathd) Renal proximal tubule

• Ans. Myelin sheath

10. 65 yrs old man present with anemia, tingling, numbness, posterior column dysfunction which of the following is likely aetiology

a) SACDb) Vit b1 deficiencyc) Multiple sclerosisd) Vit b12 deficiency

• Ans DSACD: Condition of spinal cord characterised by demyelination of its neurons due to

deficiency of vit b12 which result in deficient myelination. Such demyelination occurs predominantly in1) Posterior coloumn of spinal cord2) Pyramidal tract of spinal cord

Degenration of Post column Degenration of Lateral column/Pyramidal tract

Paraesthesia: tingling,numbness,pins,needle sensation

Motor defect such as weakness & spasticity

Loss of vibration sense Increased DTR’s, Clonus

Ataxic gait Plantar extensor

Position sense involve to alesser extent Spastic gait(initially it is ataxic become but later become both spastic and ataxic)