Megaloblastic anemia causes

1. FOLATE AND COBALAMIN
THEIR METABOLISM AND TESTS
Dr. K.C.Sharan

2. FOLATE

3. Tetrahydrofolate Triglutamate
Williams Hematology, 9E
• To form a functional compound,
folate must be reduced to
tetrahydrofolate. In this
reduction, dihydrofolate (FH2) is
an intermediate.
• A single enzyme, FH2 reductase
catalyzes both
 F→FH2 and
 FH2→FH4.

4. N5-methyltetrahydrofolate–homocysteine methyltransferase
reaction
Williams Hematology, 9E

5. PHYSIOLOGIC CYCLE OF ASSIMILATION-FOLATE

6. FOLATE ABSORPTION
 Principal site is the upper small intestine, and there is a steep fall-off in absorptive capacity in the lower
jejunum and ileum.
 The absorption of all forms is rapid, a rise in blood level occurring within 15–20 min of ingestion.
 The small intestine has a tremendous capacity to absorb folate mono-glutamates, as about 90% of a single
dose is absorbed regardless of whether this is small (100 μg) or large (15 mg).
 Absorption of pteroyl-glutamic acid occurs by a unsaturable (when folate luminal concentration exceeds 5-
10uM) and saturable process .
 Reasons of saturation:
 1) Limited capacity of the small intestine to hydrolyse these compounds.
 2) Limited transfer in the mucosal cell. (PCFT- Proton coupled folate transfer)
 On average, about 50% of food folates is absorbed.
-Hoffbrand, 7th edition

7. FOLATE ABSORPTION
 The polyglutamate (in this case, PteGlu7) is
hydrolyzed in the intestinal lumen or at the
brush-border..
 The resulting pteroylglutamate (PteGlu) is
transported into the intestinal cell, where it
is reduced and methylated, appearing in
the circulation chiely as N5-
methyltetrahydrofolate
Williams Hematology, 9E

8. FOLATE BINDERS AND RECEPTORS
High-affinity folate receptors in enterocyte surface
Concentrate folate in intracellular vesicles
From the vesicle into the cytosol
Retained by the cells partly through polyglutamylation
Intracellular folate-binding

9. FOLATE STORES AND TURNOVER
 Total body folate in the adult is about 10 mg,
 Liver contains the largest store.
 Daily adult requirements are about 100 μg.
 Up to 13 μg of folate is lost as such in the urine each day, but breakdown products of folate are also lost
in urine.
 Losses of folate also occur in sweat and skin;
 Faecal folate is largely derived from colonic bacteria.
 Stores are only sufficient for about 4 months in normal adults, so severe folate deficiency may develop
rapidly.
-Hoffbrand, 7th edition

10. COBALAMIN

11. COBALAMIN- ROLE IN METABOLISM
 The only two recognized cobalamin-dependent enzymes in human
cells-
 MeCbl-dependent N5-Methyltetrahydrofolate-Homocysteine
Methyltransferase
 AdoCbl-dependent Methylmalonyl Coenzyme A Mutase

12. COBALAMIN DEFICIENCY- FOLATE TRAP
 Cobalamin deficiency has a major impact on folate, homocysteine, and methionine metabolism by
impairing the methionine synthase reaction.
 MethylTHF has no alternative metabolic role or route- cobalamin deficiency thereby “traps” methylTHF
 Failure of methionine synthesis ultimately leads to formate “starvation,” with depletion of formylTHF,
methenylTHF, and methyleneTHF.

13. NORMAL COBALAMIN PHYSIOLOGY

14. PHYSIOLOGIC CYCLE OF ASSIMILATION- COBALAMIN

15. COBALAMIN ABSORPTION
 Cobalamin is released from food by pepsin at an
acid pH in the stomach and binds preferentially
to salivary TC I rather than IF at this pH (panel 1).
 Pancreatic secretions entering the duodenum
neutralize the pH and provide proteases to
degrade TC I. The released cobalamin, including
biliary cobalamin, thus becomes available to IF
(panel 2).
 The IF–cobalamin complex eventually attaches to
the cubilin receptor of CUBAM complex(panel 3).
Wintrobes Clinical Hematology, 13E

16. COBALAMIN ABSORPTION
 CUBAM complex: Cubilin + Amnionless
 Help in endocytosis and lysosomal degradation of IF-
coblamin complex.
 After endocytosis, the cubilin–IF–cobalamin complex
is split,
 Cobalamin exits the ileal cell into the bloodstream
several hours after its oral ingestion.

17. PLASMA TRANSPORT AND
CELLULAR UTILIZATION
 Some of the portal blood holo-TC II is
internalized by hepatocytes via TC II receptors.
 The remainder finds its way to other tissues for
calcium-dependent, TC II receptor-mediated
cellular endocytosis (panel 4).
 After lysosomal degradation of the TC II, its
cobalamin is released for attachment to
 Cytoplasmic methionine synthase and
conversion to methylcobalamin
 Mitochondria for conversion to
adenosylcobalamin.

18. MEGALOBLASTIC ANEMIA
 Disorders caused by impaired DNA synthesis
 Nuclear-cytoplasmic asynchrony
 Megaloblastic cells-morphologic hallmark
 Megaloblastic red cell precursors, granulocytic
precursors and megaloblastic megakaryocytes
 More severe the anemia-the more pronounced the
morphologic changes in the red cells.
 Slight macrocytosis often is the earliest sign of
megaloblastic anemia.

19.  In erythroblasts, no further DNA
synthesis takes place above a
haemoglobin concentration of 22%
MCHC.
 This concentration is considered to be
critical for the cessation of further DNA
replication in these cells.
 MCHC of the early polychromatic cells
of megaloblastic patients is able to
reach 22%.
 Hence preventing the further mitosis
and causing expansion of early large
forms (Megaloblasts) and
ineffective erythropoiesis.

20. Megaloblastic
anaemia
Vitamin B12
related
Folate-related
Vitamin B12- and
folate-
independent
CAUSES OF MEGALOBLASTIC ANEMIA

21. CAUSES
Williams Hematology, 9E

22. Diagnostic
Approach to
a Patient with
Macrocytic
Anemia
Wintrobes Clinical Hematology, 13E

23. Haemoglobin
RBC Decreased
Haematocrit
MCV Increased
MCH
MCHC Normal
Platelets Decreased/Normal
Retic Low for the degree of anemia
RDW Increased
Laboratory features

24. LABORATORY FEATURES
 Macrocytosis (May be
absent despite neuropathy;
many other causes)
 Macro-ovalocytosis
(characteristic but non-
specific)
 Hypersegmented
neutrophils
(Hypersegmentation of
neutrophils may be seen
early, at a stage when both
the Hb and MCV are within
the reference range)

25.  Platelets are slightly smaller than
normal and vary more widely in
size (increased PDW)
 Bicytopenia/ pancytopenia in
late stages
 Poikilocytosis at later stage-
teardrop cells, Howell-Jolly,
nRBC, Cabot ring, basophilic
stippling
HJ bodies
Cabot Ring

26. How do we confirm?
 Bone Marrow examination
 Vitamin B-12 and Folate
levels

27. BONE MARROW FINDINGS
 Hypercellular
 Erythroid hyperplasia
 Megaloblastic erythropoiesis
 Giant myelocytes and metamyelocytes
 Megakaryopoiesis reduced with hypersegmentation
 Bone marrow iron increased with abnormal sideroblasts

28. Marrow films. Megaloblastic anemia.

29. LABORATORY TESTS FOR FOLATE AND COBALAMIN

30. Diagnostic test Feature Exclude Pitfalls
Serum B12 <180 ng/l suggestive
of cobalamin def.
<180 ng/l with no S/S and
normal MMA,
homocysteine confirms
falsely low B12
May be due to folate def.
>180 ng/l + neuropathy
or strong clinical
suspicion requires
therapeutic trial or other
tests.
Serum folate Low Diurnal variation
RBC folate Low ( B12 def
excluded)
Low RBC folate and high
serum levels occur in
cobalamin def.
IF antibody test
( reflex test if
B12 reduced)
Positive in 50-60% of
pernicious anaemia, if
positive obviates
schilling test
False positive
Negative in 40-50% of
pernicious anaemia, if
negative prolong to
schilling test.
Schilling test(
Part I, II, III)
Part I and Part II
normal confirms
malabsorption with
lack of IF,
Invalid in renal failure
Part II may not correct in
PA if IF antibodies are
present in high conc in
gastric juice.

31. Diagnostic test Feature Exclude Pitfalls
Part I and II abnormal
suggest malabsorption not
resulting from IF deficiency
Upper GI endoscopy and
duodenal biopsy
Villous atrophy in coeliac
disease
Serum gastrin or gastric
juice pH
Raised serum gastrin and
gastric juice pH>6
confirms achlorhydria;
If not present PA is
suspected
Serum MMA and/or
plasma homocysteine,
before or 6 days after
treatment
Raised homocysteine in
folate and B12 def.
Raised MMA in B12 def.
Lack of significance of low
B12 if normal MMA,
homocysteine with no S/S
Both MMA and
homocysteine are
elevated in renal
impairement

32. LAB INVESTIGATIONS
 B12 work-up.
 Serum B12 levels.
 Intrinsic factor and anti- parietal cell antibodies.
 Holotranscobalamin II assays
 Deoxyuridine suppression test (DUST)
 Schilling Test
 Folate work up:
 Serum folate levels
 RBC folate levels
 Metabolite work-up.
 Serum/plasma/urine Methylmalonic acid
 Plasma homocysteine
 Therapeutic trial of B12 and folate.

33. SCHILLING TEST
 Identification of etiology
 Principle:
 capacity to absorb B12 measured by administering a dose of B12 labelled by
a radioisotope of cobalt and measuring the percentage that is excreted in
the urine
58Co (half-life 71 days) and 57Co (half-life 270 days)

34. Method:
 Part 1-
 Oral dose of 1.0 μ g of radioactive B12 (cyanocobalamin) to a patient (fasted overnight)
 At the same time, 1 mg of nonradioactive cyanocobalamin intramuscularly as a flushing dose
 Collect all the urine for 24 hours
 Measure the radioactivity of this urine
 Percentage dose excreted = Counts/min in urine x 100
Counts/min in std

35. INTERPRETATION OF RESULTS-PART 1
 Normal urinary excretion >10% of the test dose in the first 24 hrs
 Excretion <5% - pernicious anaemia or with B12 deficiency assoc with intestinal malabsorption

36. PART 2 AND PART 3
Part 2-
 Second test dose with IF given 48 hours after the first
INTERPRETATION-
 Absorption increased- Pernicious anaemia
 Failure of correction- intestinal defect absorption (bacterial overgrowth)
Part 3-
 Antibiotics
INTERPRETATION-
 Absorption increased- intestinal defect absorption

37. D/D BASED ON SCHILLING TEST
Condition Part I Part II Part III
Normal >8%
Vegan diet >8% >8%
Pernicious anemia <8% >8%
Blind loop syndrome <8% No change >8%
Malabsorption Usually <8% No change No change
Absence of ileum or ileal fistula <8% No change No change
Excretion of labelled vitamin B12

38. SERUM METHYLMALONATE AND HOMOCYSTEINE LEVEL
 Sensitive methods for measuring MMA and homocysteine in serum have been
introduced and recommended for the early diagnosis of cobalamin deficiency.
 Methylmalonic acid (MMA) measured by Gas chromatographic mass
spectrometry (GC- MS).
 Homocysteine measured by Enzyme immunoassays /HPLC

40. DIAGNOSIS OF FOLATE DEFICIENCY
 The earliest indicator of folate deficiency is a low serum folate.
 Serum folate follows folate intake closely, so a low serum folate (less than approximately 3µg/ml)
may indicate only a drop in folate intake over the preceding few days
 Similarly, a low serum folate rises quickly on refeeding.
 A better indicator of the tissue folate status is the red cell folate, which remains relatively
unchanged while a red cell is circulating and thus reflects folate turnover over the preceding 2
to 3 months

41. DIAGNOSIS OF FOLATE DEFICIENCY
 This is measured by an Enzyme Linked Immunosorbent Assay (ELISA).
 The serum folate level is low in all folate deficient patients.
 In most laboratories, the normal range is from 2.0 μ g/L to about 15 μ g/L.
 The serum folate is markedly affected by recent diet; inadequate intake for as little as 1 week
may cause the level to become subnormal.

42. URINARY FORMIMINOGLUTAMIC (FIGLU) ACID
 Folic acid coenzymes are required for the conversion of FIGLU to glutamic acid in the
catabolism of histidine.
 When oral histidine is given FIGLU will appear in increased amounts in the urine if folate
deficiency is present.
 The test is useful in patients with megaloblastic anemia or due to antifolate drugs.
 These patients have normal serum folate levels but greatly decreased tissue coenzyme levels.

43. THERAPEUTIC TRIAL OF B12 AND FOLATE
 Appropriate response with appropriate therapy
 Raise in retic:
 Starts in 2- 3 days (Raise in MCV)
 Peak at 5 to 8 days
 MCV stabilizes in 25 to 78 days
 Raise in RBC count:
 Start in 1 week
 Normalize in 4 to 8 weeks.