Normal Vitamin B12 Metabolism Vitamin B12 is composed of A corrin nucleus which has 4 pyrrole rings bound to a central cobalt atom A 5,6 dimethylbenzimidazole group which is attached to the corrin ring and to the central cobalt atom Important cobalamins that are distinguished according to the ligand attached to the central cobalt atom are : cyanocobalamin, hydroxocobalmin, adenosylcoba lamin and methylcobalamin
Sources Liver, dairy products and seafish are major sources Although bacteria in the large intestine synthesize vitamin B12 it cannot be absorbed from this site Minimum amount required for an adult is 1 to 4 µg per day
Absorption of Vitamin B12 2 mechanism Active (75%) – requires the presence of intrinsic factor ( a glycoprotein produced by gastric mucosa) Passive – absorption occurs by diffusion and works when pharmacological doses of vitamin B12 are ingested
Vitamin B12 in food Stomach R-Binder B12-R-Binder complexDuodenum Intrinsic Factor (IF) IF-B12 complex + Freed R-Binder Receptor-IF-B12 TCII Epithelial cell of Degradation IF Receptor terminal iIeum B12-TCII Circulation
Transport of Vitamin B12 Following absorption by the ileal mucosal cells, vitamin B12 is carried in the plasma by various transporting proteins: Transcobalamin I Transcobalamin II Transcobalamin III
Transcobalamin I (TC I) is an alpha-globulinproduced by granulocytes. It functions as acirculating reserve store of B12. TC I carries mostlymethylcobalamin.Transcobalamin II (TC II) is a beta-globulin formedin the liver and is the dominant carrier of B12immediately after absorption. It is the main agentfor rapid transport of B12 to the body cells.Transcobalamin III (TC III) is an alpha-globulin.TC III may act as a defence mechanism bydepriving pathogens of B12 at sites of infection
Storage sites Total amount of vitamin in body is 2-5 mg ( adequate for 3 years ) Major site : liver Excreted through the bile and shedding of intestinal epithelial cells Most of the excreted vitamin B12 is again absorbed in the intestine (enterohepatic circulation)
Functions of Vitamin B12 Synthesis of methionine from homocysteine Conversion of methyl malonyl CoA to succinyl CoA
Role of Vitamin B12 and Folate in DNA synthesis VitB12 (Methylcobalamin) Homocysteine MethionineDietary folatesIntestinal cell Methyl FH4 FH4 Dihidrofolate Redutase Methylene FH4 FH2 dUMP dTMP Thymidylate Synthase DNA Synthesis
General Morphological Features OfMegaloblastic Anemia
PERIPHERAL BLOOD FINDINGS1. Hemoglobin – decreased2. Hematocrit – decreased3. RBC count – decreased/normal4. MCV - >100fl ( normal 82-98fl)5. MCH –increased6. MCHC – NORMAL7. Reticulocytopenia.8. Total WBC count – normal / low9. Platelet count – normal/ low10. Pancytopenia, especially if anaemia is severe.
PERIPHERAL SMEAR RBC: - Macro ovalocytes (macrocytic normochromic) [ macrocytosis is the earliest sign in Vit B12 deficiency and can be detected even before the onset of anaemia ] - In severe anaemia in addition to macrocytosis, marked anisopoikilocytosis, basophilic stippling, howell jolly bodies, Cabot’s rings may be found
Late or intermediate erythroblast with fine, open nuclear chromatin (megaloblast) may be seen in peripheral blood in severe anaemia
Marked macro-ovalocytosis (MCV 134 fl) in the peripheral blood smear of apatient with vitamin B12 deficiency.
PERIPHERAL SMEAR WBC Normal count or reduced count Hypersegmented neutrophils is one of the earliest sign of megaloblastic haematopoiesis and can be detected even in the absence of anaemia (when more than 5% of neutrophils show ≥ 5 lobes; 1% neutrophils with ≥ 6 lobes) PLATELETS: Normal or decreased (severe anaemia) Giant platelet can occur
BONE MARROW Markedly hypercellular Myeloid : erythroid ratio decreased or reversed. (Normally, there are three myeloid precursors for each erythroid precursor resulting in a 3:1 ratio, known as the M:E (myeloid to erythroid) ratio) Erythropoiesis : MEGALOBLASTIC
MEGALOBLAST1. Cell and nuclear size and amount of cytoplasm (deeply basophilic royal blue) are increased2. Nuclear chromatin is sieve like or stippled (open)3. Nuclear cytoplasmic asynchrony/dissociation4. Abnormally large precursor (promegaloblast and early megaloblast) are increased in BM – Maturation arrest5. Abnormal mitoses (increased)
Granulocytic series also display megaloblastic changes Most prominent change – giant metamyelocyte with horseshoe shaped nuclei and finer nuclear chromatin, and in band forms Megakaryocytes are often large with multiple nuclear lobes and paucity of cytoplasmic granules
BIOCHEMICAL FINDINGS Increase in serum unconjugated bilirubin- because of ineffective erythropoiesis Increase is LDH Normal serum iron and ferritin
Causes of Vit B12 deficiency Insufficient dietary intake (very rare) Strict vegetarians Deficient absorption Pernicious anaemia Total or partial gastrectomy Prolonged use of PPI or H2 blockers Diseases of small intestine Fish tapeworm infestation
PERNICIOUS ANEMIA Thomas Addison (1849) Disease of elderly – 5th to 8th decades (median age at diagnosis – 60 years) Genetic predisposition Tendency to form antibodies against multiple self antigens
PATHOGENESIS Immunologically mediated, autoimmune destruction of gastric mucosa CHRONIC ATROPHIC GASTRITIS – marked loss of parietal cells Three types of antibodies:a) Type I antibody- 75% - blocks vitamin B12 and IF bindingb) Type II antibody – prevents binding of IF-B12 complex with ileal receptorsc) Type III antibody – 85-90% patients – against specific structures in the parietal cell
Pathological changes are infiltration by mononuclear cells in submucosa and lamina propria of fundus and body of the stomach, progressive loss of parietal and chief cells, and their replacement by intestinal type mucous cells
Associated with other autoimmune disorders like Hashimoto’s, Graves’, vitiligo, diabetics mellitus, primary hyperparathyroidism, Addison’s and Myasthenia gravis Patients with pernicious anaemia have increase risk of gastric cancer
DIAGNOSTIC FEATURES1. Moderate to severe megaloblastic anemia2. Leucopenia with hypersegmented neutrophils3. Mild to moderate thrombocytopenia4. Mild jaundice due to ineffective erythropoiesis and peripheral hemolysis5. Neurologic changes6. Low levels of serum B12
7. Elevated levels of homocysteine8. Striking reticulocytosis after parenteral administration of vitamin B129. Serum antibodies to intrinsic factor (specific) and anti parietal cell antibodies in serum10. Abnormal Schilling test, pentagastrin-fast achlorhydria
GASTRECTOMY Total gastrectomy : Secondary to Vit B12 deficiency as it removes the site of synthesis of intrinsic factor Prophylactic vitamin B12 after surgery Partial gastrectomy Regular follow up after surgery for early detection of deficiency
DISEASES OF SMALL INTESTINE Tuberculosis, whipple’s disease, blind loop syndrome or resection of small intestine may interfere with absorption that occurs in the terminal ileum Blind loop syndrome – stasis of small intestine contents (diverticulum / stricture) may predispose to bacterial colonization and proliferation Utilization of most of the ingested Vit B12 by bacteria may lead to reduced or non avail of Vit for absorption
INFESTATION BY FISHTAPEWORM Diphyllobothrium latum (inadequately cooked fish) Vitamin deficiency by competing with the host for vitamin in food Diagnosis made by demonstration of ova in stool
CLINICAL FEATURES Anaemia, mild jaundice and sometimes neurological involvement Neurological involvement in the form of Peripheral neuropathy Subacute combined degeneration of spinal cord Cerebral changes (personality changes, dementia & psychosis) Patients can present with only neurological abnormalities without megaloblastic anaemia
LABORATORY FEATURES1. Morpholgical changes of megaloblastic anaemia in PS and BM2. Serum vitamin B12 assays3. Methylmalonic acid (MMA) and homocysteine in serum4. Schilling test5. Intrinsic factor antibodies in serum
1. SERUM VITAMIN B12 ASSAYSVarious methods are available, e.g.microbiological methods usingLactobacillus leichmannii or radio-isotopetechniques (RIA) using 57CoB12, coatedcharcoal and IF.
RADIO-ISOTOPE DILUTION ASSAY:A known amount of radioactive (hot) B12 isdiluted with the non-radioactive (cold) B12in the test serum, released from serumproteins by heat or chemical means.A measured volume of the hot and coldmixture is bound to intrinsic factor (IF)which is added in an amount insufficientto bind all the hot B12. The bound B12 isseparated from the free and itsradioactivity counted.
The count is inversely proportional to theB12 concentration in the test serum.The higher the serum B12 the greater willbe the dilution of the radioactive B12 andthus less radioactivity attached to the IF.By comparison with standards of knownB12 content, the B12 content of the testserum can be calculated.
In Vitamin B12 deficiency , Serum Vitamin B12 and red cell folate are depressed Serum folate is normal or increased ( accumulation of 5-methyl tetrahydrofolate ) [folate trap]
2. Methylmalonic acid (MMA) and homocysteine in serum Recent reports of S.methylmalonic acid and S.homocysteine are more sensitive for detection of Vitamin B12 than estimation of Vitamin B12 Raised early in tissue deficiency even before appearance of hematological changes
3. SCHILLING TEST For evaluation of absorption of vitamin B12 in the GIT Performed in 2 parts – part 1 and part 2 Part 1 : 0.5 to 1 µg of radiolabelled vitamin B12 is given orally After 2 hrs IM dose (1000 µg) of unlabelled vitamin B12 is given [ saturates binding sites of TC I and TC II and displaces any bound radiolabelled vitamin B12 (thus permitting urinary excretion of absorbed radiolabelled vitamin B12 )
Radioactivity is measured in subsequently collected 24 hr urine sample and expressed as a % of total oral dose In normal persons, > 7% of the oral dose of vitamin B12 is excreted in urine If excretion is less than normal it indicates impaired absorption, which may be due to either lack of IF or small intestinal malabsorption Part 2 performed if part 1 of test is abnormal
Part 2 : patient is orally administered radiolabelled vitamin B12 along with IF while remainder of test is carried out out as in part 1 Excretion becomes normal – lack of IF Excretion remains below normal – defective absorption in small intestine
4. INTRINSIC FACTOR ANTIBODIES IN SERUM Detection of anti-IF antibodies in serum is diagnostic of pernicious anemia
MANAGEMENT OF B12 DEFICIENCYWhen B12 deficiency is suspected a trial of B12 isessential. Failure of response can only bedetermined after careful follow-up over a period ofseveral months, particularly if the patient is non-anaemic.Standard therapy for all cases of B12 deficiency isby regular intramuscular injections of B12, usuallyin the form of hydroxycobalamin. In patients withinadequate dietary intake supplements may begiven by mouth. Underlying conditions should be
After initiation of therapy, reticulocyte count begins to increase around 3rd day – peak by 6th or 7th day – gradually returns to normal by end of 3rd week Hematocrit steadily rises and normalises in about 1- 2 months Blood transfusion is indicated in severely anaemic symptomatic patients or in patients with CCF
NOTE:Both B12 and folate are given to patients ifB12 deficiency has not been excluded.This is to prevent neurological damage, e.g.subacute combined degeneration of thespinal cord.