Folate and vitamin B12 (cobalamin) play key roles in cell metabolism and the production of DNA. A deficiency of either can lead to megaloblastic anemia, where red blood cells are abnormally large with impaired DNA synthesis. Cobalamin deficiency specifically results in an intracellular folate deficiency through a "folate trap" mechanism. Pernicious anemia, caused by autoimmune destruction of gastric parietal cells and a lack of intrinsic factor, is the most common cause of cobalamin deficiency. Symptoms of megaloblastic anemia include fatigue, palpitations, and shortness of breath.

1. Folate, Cobalamin
and Megaloblastic
anemia
Grerk Sutamtewagul, M.D.
PGY-3
Morning Report 7/30/2013

2. Key Concepts
 Folate and Cobalamin (vitamin B12) play key
roles in the metabolism of all cells, particularly
proliferating cells.
 Folate in its tetrahydro form is a transporter of
one-carbon fragments which is an important step
in biosynthesis of purines, thymidine, and
methionine.
 Cobalamin is required for 2 reactions:
intramitochondrial conversion of methylmalonyl
coenzyme A to succinyl CoA and cytosolic
conversion of homocysteine to methionine.

3. Key Concepts
 The Megaloblastic anemia of cobalamin
deficiency results from an intracellular folate
deficiency.
 Absorption of cobalamin is a highly complex
process involving haptocorrin binder, intrinsic
factor (from gastric parietal cells), receptor-
mediated endocytosis, transcobalamin (serum
transporter).
 Most common cause of folate deficiency usually
nutritional in origin: alcoholics, elderly, patient
with hyperalimentation, hemolytic anemia,
hemodialysis, tropical/non-tropical sprue.

4. Key Concepts
 The most common cause of cobalamin
deficiency is pernicious anemia (autoimmune
destruction of gastric parietal cell).
 Pernicious anemia increases risk of gastric cancer
by 2-3 times.
 Other causes of cobalamin deficiency include
gastric resection, stasis of intestinal content (blind
loops, strictures, hypomotility), terminal ileum
resection/disease, vegan diet.

5. Key Concepts
 “Acute” megaloblastic anemia:
 Nitrous oxide
 Severe hemolytic anemia
 Other causes of megaloblastic anemia:
 Drugs (hydroxyurea, nucleoside analogues)
 Certain inborn errors of metabolism

6. Folate

7. Folate
 Source: vegetables, fruits, liver, folate fortification
(in the US)
 Daily requirement: 50 mcg minimum, RDA: 0.4 mg
 Increased requirement in
 Hemolytic anemia, Leukemia
 Other malignant diseases
 Alcoholism
 Growth
 Pregnancy and lactation (3-6 times)

8. Folate Metabolism
 Tetrahydrofolate is an
intermediate in reactions
involving the transfer of one-
carbon units.
 Metabolic systems requiring
folate coenzymes
 Serine-Glycine conversion
 Thymidylate synthesis
 Histidine catabolism
 Methionine synthesis
 Purine synthesis
 Pyrimidine synthesis

9. Folate Metabolism
 Intracellular folates exist primarily as
polyglutamate conjugates (75%).
 Intracellular Folylmonoglutamates leak out of the
cells at a fairly rapid rate whereas
Polyglutamates do not. Polyglutamate form is
more active and will retain in the cell.

10. Folate deficiency
 Dietary deficiency
 Impaired absorption
 Increased requirement

11. Folate deficiency
 Dietary deficiency
 Inadequate dietary intake (before 1990s) –
decreased dramatically after folate fortification
 Infant raised with goat milk
 Excessive cooking
 Impaired absorption
 Non-tropical sprue (Celiac disease)
 Tropical sprue
 Other intestinal disorders: scleroderma, amyloidosis,
DM, systemic bacterial infection

12. Folate deficiency
 Increased requirement
 Hemodialysis (folate loss in dialysate)
 Pregnancy (transfer to growing fetus)
 Difficult to diagnose due to physiologic anemia
and macrocytosis (mean MCV 104).
 Serum and rbc folate fall steadily even in well
nourished women.
 Hypersegmented neutrophil is a reliable clue.
 Increased cell turnover: Hemolytic anemia, chronic
exfoliative dermatitis, psoriasis

13. Diagnosis
of Folate deficiency
 History and laboratory finding indicating folate
deficiency
 Absence of the neurological signs of cobalamin
deficiency
 Full response to physiologic dose of folate

14. Laboratory Findings
 Serum Folate
 Earliest specific finding
 Correlate well (and varies) with recent intake (within
few days)
 RBC Folate
 Better indicator of tissue folate status
 Remain unchanged for 2-3 months
 Also falls in cobalamin deficiency – not use for
differentiate folate from cobalamin deficiency
 Serum Homocysteine
 Increase homocysteine may precede a fall in folate
level but is non-specific.

15. Laboratory Findings
 Macrocytosis
 Differential diagnosis of macrocytosis without
megaloblastic anemia
 Alcoholism
 Liver disease
 Hypothyroidism
 Aplastic anemia
 Certain myelodysplasia
 Pregnancy
 Reticulocytosis

16. Cobalamin

17. Cobalamin
 Vitamin B12 (cyanocobalamin – therapeutic
form)
 4 Forms of cobalamin in animal cell metabolism:
cyanocobalamin, hydroxocobalamin, adenosylc
obalamin, and methylcobalamin (major
circulating form)

18. Source of Cobalamin
 Animals cannot produce cobalamin.
 Animals depend on microbial synthesis or animal
product intake for cobalamin supply.
 Cobalamin has not been found in plants.
 Species from the following genera are known to
synthesize B12:
Acetobacterium, Aerobacter, Agrobacterium, Alcaligenes, Azotobacter, Bacillus,
Clostridium,Corynebacterium, Flavobacterium, Lactobacillus, Micromonospora,
Mycobacterium, Nocardia, Propionibacterium, Protaminobacter, Proteus,
Pseudomonas, Rhizobium, Salmonella, Serratia, Streptomyces, Streptococcus and
Xanthomonas.

19. Body composition of
cobalamin
 Total body cobalamin is around 2-5 mg.
 1 mg is in the liver.
 Daily loss of cobalamin is 0.1% of total body pool.
Several years is required to develop deficiency
state.

20. Cobalamin Metabolism
 There are only 2 recognized cobalamin-
dependent enzymes in human:
 Mitochondrial Adenosylcobalamin-dependent
Methylmalonyl CoA mutase
 Cytosolic Methylcobalamin-dependent
N5-Methyltetrahydrofolate-homocysteine
methyltransferase

21. Methylmalonyl CoA mutase
 Methylmalonyl CoA
(from proprionate) is
changed to Succinyl
CoA and enters Krebs
cycle.

22. N5-Methyltetrahydrofolate-
homocysteine methyltransferase
 Synthesis of Methionine
 Also involves in demethylation of
N5-methyltetrahydrofolate to tetrahydrofolate
which is needed for conjugation to
polyglutamate. THF-polyglutamate will retain in
the cell.
 Nitrous oxide (N2O) can
impair methyltransferase
Acute megaloblastic
anemia

23. Folate-cobalamin
relationship
 Folate can, at least, temporarily
correct megaloblastic anemia
from cobalamin deficiency.
 Cobalamin cannot correct
megaloblastic anemia from
folate deficiency.
 Megaloblastic anemia from
cobalamin deficiency is actually
an abnormality in folate
metabolism
(Folate trap hypothesis).

24. Cobalamin transport
Stomach
 Peptic digestion
liberates cobalamin
from foods.
 Cobalamin is bound
to Haptocorrin(HC)-
like protein with
more avidity than
intrinsic factor in
stomach pH.
Terminal Ilium
 Pancreatic protease
releases cobalamin
from HC complex.
 Cobalamin is then
bound to intrinsic
factor, forming a
complex which is
very resistant to
digestion.
Duodenum
 Cobalamin-IF
complex undergo
receptor-mediated
endocytosis via IF
receptor, Cubilin.
 IF is degraded in the
lyzosome, releasing
cobalamin into
cytoplasm.
 Transcobalamin forms
complex with
cobalamin  blood.

26. Cobalamin transport
 Like the folates, the cobalamins undergo
appreciable enterohepatic recycling.
 If the absorption is intact, a very long time – as
long as 20 years – is required for a clinically
significant cobalamin deficiency to develop from
strictly vegan diet.

27. Cobalamin deficiency -
outline
 Decreased uptake caused by impaired
absorption
 Intestinal diseases
 Blind Loop syndrome
 AIDS
 Pancreatic disease
 Dietary cobalamin deficiency

28. Cobalamin deficiency –
pernicious anemia
 Decreased uptake – impaired absorption
 Pernicious anemia (most common)
 Failure of gastric intrinsic factor production, gastric
mucosal atrophy, autoimmune
 Age of onset usually > 40 yrs
 Anti-intrinsic factor antibody/Anti-Cbl-IF complex
are very specific.
 Anti-parietal cell Ab (90% in PA, 60% in atrophic
gastritis)
 Related to other autoimmune diseases:
thyrotoxicosis, Hashimoto thyroiditis, DM type
1, Addison disease, postpartum
hypophysitis, infertility

29. Cobalamin deficiency -
others
 Gastrectomy syndrome (total, partial)
 Removal of intrinsic factor
 Zollinger-Ellison syndrome
 High acid prevent a transfer of cobalamin from the
HC complex to IF
 Diseases of terminal Ileum
 Extensive ileal resection
 IBD, lymphoma, XRT
 Hypothyroidism, medication
 Diphyllobothrium latum infestation

30. Cobalamin deficiency –
Blind loop syndrome
 Blind Loop syndrome
 Intestinal stasis from
 Anatomic lesions
(strictures, diverticula, anastomoses, surgical blind
loops)
 Impaired motility (scleroderma, amyloidosis)
 Treatment: antibiotics Cefalexin 250 mg QID plus
Metronidazole 250 mg TID for 10 days

31. Cobalamin deficiency –
Laboratory findings
 Serum Cobalamin level
 Low in most but not all patients with cobalamin
deficiency
 Low in normal subjects
(vegetarian, pregnancy, taking large dose ascorbic
acid)
 Serum Holotranscobalamin
 Functional fraction of serum bound cobalamin
 Urine Methylmalonic acid
 Very reliable indicator of cobalamin deficiency

32. Cobalamin deficiency –
Laboratory findings
 Serum Methylmalonic acid and Homocysteine
 Elevated MMA and Homocysteine levels are
indicators of tissue cobalamin deficiency.
 MMA is more sensitive and specific, persists several
days after treatment.
 MMA elevation is seen only in cobalamin deficiency
whereas Homocysteine elevation can be seen in
folate/pyridoxine deficiency and hypothyroidism.

33. Megaloblastic Anemias
 Disorders caused by impaired synthesis of DNA.
 Megaloblastic cells: Erythroid
 large cells with immature-appearing nuclei
 Increasing hemoglobinization of the cytoplasm
 “Nuclear-cytoplasmic asynchrony”
 Megaloblastic granulocytic cells
 Large giant band neutrophil in bone marrow
 Hypersegmented neutrophil in the marrow and blood
 Other rapidly dividing cells may also showed
cytologic abnormalities.

34. Pathogenesis of
Megaloblastic anemia
 Ineffective erythropoiesis
 Intramedullary destruction of red cell precursors
 Hypercellular marrow with apoptosis of late
precursors
 Ineffective granulopoiesis and thrombopoiesis
also present and can result in neutropenia and
thrombocytopenia.
 Mild hemolysis with shortening of red cell half-life.

35. Clinical features of
Megaloblastic anemia
 Anemia develops gradually and patient is usually
able to adapt to very low Hb level.
 Fatigue, palpitation, lightheadedness, shortness
of breath

36. Peripheral blood smear of
Megaloblastic anemia
 Hypersegmented neutrophil

37. Bone marrow smear of
Megaloblastic anemia
 Erythroid hyperplasia with
marked nuclear/cytoplasmic
dysynchrony noted at all stages
of erythroid maturation
 Giant Band neutrophil in the
bone marrow