2. Introduction .. Characterized by defective synthesis of
deoxyribonucleic acid (DNA) in all proliferating cells Most
commonly result from lack of folic acid or vitamin B12 3.
MEGALOBLASTIC ANAEMIA[ VITAMIN B12 DEFICIENCY ] 4. Normal Vitamin
B12 Metabolism Vitamin B12 is composed of A corrin nucleus which
has 4 pyrrole rings bound toa central cobalt atom A 5,6
dimethylbenzimidazole group which isattached to the corrin ring and
to the central cobaltatom Important cobalamins that are
distinguished according to the ligand attached to the central
cobalt atom are : cyanocobalamin, hydroxocobalmin, adenosylcoba
lamin and methylcobalamin 5. Sources Liver, dairy products and
seafish are majorsources Although bacteria in the large intestine
synthesizevitamin B12 it cannot be absorbed from this site Minimum
amount required for an adult is 1 to 4 gper day 6. 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 7. Vitamin B12 in
food Stomach R-Binder B12-R-Binder complexDuodenum Intrinsic Factor
(IF) IF-B12 complex + Freed R-Binder Receptor-IF-B12 TCIIEpithelial
cell of Degradation IFReceptorterminal iIeumB12-TCIICirculation 8.
Transport of Vitamin B12 Following absorption by the ileal mucosal
cells, vitamin B12 is carried in the plasma by various transporting
proteins:Transcobalamin ITranscobalamin IITranscobalamin III 9.
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 10. 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 ofintestinal
epithelial cells Most of the excreted vitamin B12 is againabsorbed
in the intestine (enterohepaticcirculation) 11. Functions of
Vitamin B12 Synthesis of methionine from homocysteine Conversion of
methyl malonyl CoA to succinyl CoA 12. Role of Vitamin B12 and
Folate in DNA synthesisVitB12 (Methylcobalamin)Homocysteine
MethionineDietary folatesIntestinal cell Methyl FH4 FH4
Dihidrofolate RedutaseMethylene FH4FH2 dUMPdTMPThymidylate Synthase
DNA Synthesis 13. General Morphological Features OfMegaloblastic
Anemia 14. 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. 15.
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, Cabots rings may be found 16. Late
or intermediate erythroblast with fine, open nuclear chromatin
(megaloblast) may be seen in peripheral blood in severe anaemia 17.
Marked macro-ovalocytosis (MCV 134 fl) in the peripheral blood
smear of apatient with vitamin B12 deficiency. 18. PERIPHERAL SMEAR
WBC Normal count or reduced count Hypersegmented neutrophils is one
of the earliest sign ofmegaloblastic haematopoiesis and can be
detected evenin the absence of anaemia (when more than 5%
ofneutrophils show 5 lobes; 1% neutrophils with 6 lobes) PLATELETS:
Normal or decreased (severe anaemia) Giant platelet can occur 19.
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 20. 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) 21. 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 22.
BIOCHEMICAL FINDINGS Increase in serum unconjugated
bilirubin-because of ineffective erythropoiesis Increase is LDH
Normal serum iron and ferritin 23. 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 24. PERNICIOUS ANEMIA Thomas
Addison (1849) Disease of elderly 5th to 8th decades (medianage at
diagnosis 60 years) Genetic predisposition Tendency to form
antibodies against multiple selfantigens 25.
PATHOGENESISImmunologically mediated, autoimmune destruction of
gastric mucosaCHRONIC ATROPHIC GASTRITIS marked loss of parietal
cellsThree 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 26.
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 27. Associated with other
autoimmune disorders like Hashimotos, Graves, vitiligo, diabetics
mellitus, primary hyperparathyroidism, Addisons and Myasthenia
gravis Patients with pernicious anaemia have increase risk of
gastric cancer 28. 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 29. 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 30.
GASTRECTOMY Total gastrectomy : Secondary to Vit B12 deficiency as
it removes thesite of synthesis of intrinsic factor Prophylactic
vitamin B12 after surgery Partial gastrectomy Regular follow up
after surgery for early detection of deficiency 31. DISEASES OF
SMALL INTESTINE Tuberculosis, whipples disease, blind loop syndrome
orresection of small intestine may interfere with absorptionthat
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 maylead to reduced or non avail of Vit
for absorption 32. INFESTATION BY FISHTAPEWORM Diphyllobothrium
latum (inadequately cooked fish) Vitamin deficiency by competing
with the host forvitamin in food Diagnosis made by demonstration of
ova in stool 33. CLINICAL FEATURES Anaemia, mild jaundice and
sometimesneurological 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 34. 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 35. 1. SERUM VITAMIN B12 ASSAYSVarious methods
are available, e.g.microbiological methods usingLactobacillus
leichmannii or radio-isotopetechniques (RIA) using 57CoB12,
coatedcharcoal and IF. 36. 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. 37. 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. 38. 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] 39. 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 40. 3. SCHILLING TEST For
evaluation of absorption of vitamin B12 in theGIT Performed in 2
parts part 1 and part 2 Part 1 : 0.5 to 1 g of radiolabelled
vitamin B12 is givenorally After 2 hrs IM dose (1000 g) of
unlabelled vitaminB12 is given [ saturates binding sites of TC I
and TCII and displaces any bound radiolabelled vitamin B12(thus
permitting urinary excretion of absorbedradiolabelled vitamin B12 )
41. 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 42.
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 defectiveabsorption in small intestine 43. 4. INTRINSIC
FACTOR ANTIBODIES IN SERUM Detection of anti-IF antibodies in serum
is diagnostic of pernicious anemia 44. 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 45. After initiation of therapy, reticulocyte count
begins toincrease 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 anaemicsymptomatic patients or in patients with CCF 46.
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.
