The normal proliferation of cells
depends on adequate folate and vitamin B12. Folate is necessary
for efficient thymidilate synthesis and production of DNA.
B12 is needed to successfully incorporate circulating folic
acid into developing RBCs; retaining the folate in the RBC.
Review the metabolic mechanisms of B12 and folate (See Normal Hematopoiesis-B12
and Folate Metabolism)
Pyrimidines: thymine, cytosine, and uracil
Purines: adenine and guanine
Lack of folate or B12 leads to decreased dTTP synthesis, resulting
in a slowing of DNA synthesis. The role of folate is illustrated at
right. The mechanism by which B12 leads to decreased DNA synthesis is
unclear. Two likely explanations follow.
Methylfolate "trap" hypothesis. Lack of cobalamin slows the
methyltransferase reaction resulting in increased N5-methyl FH4. Thus
N5-methyl FH4 is "trapped" or unable to be metabolized to FH4. N5-methyl
FH4 can convert to N5, 10-methylene FH4, but poorly without new FH4
other forms of folate diminish leading to slowed synthesis of dTMP.
Formate "starvation" hypothesis. Lack of cobalamine slows
the methyltransferase reaction deceasing methionine production and
in turn depressing formate generation.
A deficiency of either vitamin Bl2 or folic
acid results in megaloblastic erythroid cells-megaloblasts.
These deficiencies result in a decrease in DNA synthesis which slows
and inhibits DNA replication (nuclear division). Nuclear maturation
is slowed whereas cytoplasmic maturation (largely dependent on RNA function
and unaffected by failure of thymidilate synthesis) is relatively unimpeded.
The impaired nuclear maturation is seen as open, loose, immature chromatin
In contrast to the nucleus, the cytoplasm of megaloblastic
cells is abundant with normal hemoglobinization. This disparity between
nucleus and cytoplasm is known as nuclear-cytoplasmic asynchrony.
Although most noticeable in erythroid cells failure of
DNA synthesis also affects myeloid and megakaryocytes.
Giant bands (right) and hypersegmented polymorphonuclear neutrophils
(below) are common.
Even megakaryocytes (right) may be hypersegmented.
The impaired RBC production and destruction of defective
RBCs in the marrow before release into the peripheral blood (ineffective
erythropoiesis) results in the anemia.
A bone marrow biopsy and aspirate reveal erythroid hyperplasia.
Erythrocytic precursors (promegaloblasts with open, immature chromatin)
Bone marrow biopsy from patient with megaloblastic anemia.
Arrowheads point to megaloblasts and arrow to RBC undergoing apoptosis.
Inhibition of thymidylate synthetase leads to decreased dTTP synthesis
and formation of excess dUTP. The dUTP is incorporated into DNA. Repair
of this abnormal DNA is blocked by lack of thymidine residues; the DNA
breaks apart (karyorrhexis) and the cell dies.
The megaloblastic changes are most apparent in the polychromatophilic
and orthochromatophilic stages. Multinucleate RBCs, abnormal karyorrhexis,
increased pyknosis, and Howell-Jolly bodies ( right) may be seen.
The peripheral blood reveals a pancytopenia (decreased RBCs, white
cells, and platelets), hypersegmented neutrophils (> five lobes),
and oval macrocytes.
Once a macrocytic anemia is identified (MCV >100) and medications
excluded, a PBS should be examined.
Round macrocytes suggest possible thyroid or liver disease, while oval
macrocytes suggest B12 or folate deficiency. The reticulocyte count
is usually normal or low (if increased: hemolysis or blood loss are
Megaloblastic anemia is most often due to a B12 or folate
deficiency, but the cause of the deficiency must be determined for proper
Megaloblastic anemia due to vitamin B12 deficiency caused by a lack
of intrinsic factor is specifically referred to as pernicious
Laboratory testing of B12 and folate are critical to establishing the
cause of a megaloblastic anemia.
B12 Absorption involves a series of several proteins and receptors.
Antibodies against the proteins of cells involved can be helpful in
the diagnosis of macrocytic anemia.
Anti-intrinsic factor antibodies, are fairly specific, but unfortunately,
Antiparietal cell antibodies while sensitive, are not specific
as they are seen in a number of other diseases.
The Schilling test tests for evidence of impaired vitamin
B12 absorption correctable by intrinsic factor.
The Schilling test
Radioactive cobalamin (Cbl*) is taken orally; followed by injection
of a saturating dose of non-radioactive cobalamin.
The level of Cbl* is measured in the urine. In pernicious anemia the
excreted levels of Cbl* are low.
If intrinsic factor is given with the Cbl* the Cbl* levels will correct
in PA, but not in ileal malabsorption.
Clinical findings include a yellowish- lemon skin, glossitis (smooth
tongue) and stomatitis in severe cases.
Neurologic abnormalities, secondary to defects in myelination, are
seen in Vitamin B12, but not folate deficiencies.
The mechanism for the demyelination is believed to be lack of methyl-B12
for conversion of homocysteine to methionine. This results in decreased
production of S-adenosylmethionine (SAM) needed for methylation of phosphatidylethanolamine
to phosphatidylcholine for incorporation into myelin.
Degeneration of the posterior columns and peripheral nerve damage leads
to numbness; "pins and needles" feeling; loss of position and vibratory
sense. Later, lack of coordination; weakness of the legs can be seen.
In long standing disorders an ataxic gait; +Babinski sign (lateral columns)
may be found.
Today neurologic manifestations are relatively rare (<20% of patients)
and usually mild.
Posterior and lateral degeneration typical of late stage disease.
B12 deficiency resulting in megaloblastic anemia may
also be caused by a complete gastric resection [remember parietal
cells (IF) are found in the gastric fundus]. Similarly, the IF-B12
complex is absorbed in the distal ileum, thus intestinal malabsorption
syndromes or ileal resection can result in B12 deficiency.
Antibiotics can allow cobalmin dependent bacterial over growth in the
intestine resulting in vitamin B12 depletion. [Similarly any intestinal
malformation (congenital or surgical) ie. blind loop syndroms, will
decrease peristalsis leading to statis; bacterial overgrowth and vitamin
As B12 is found solely in foods of animal origin, individuals on strict
vegetarian diets are susceptible to B12 deficiency.
Megaloblastic anemia secondary to folate deficiency can be
caused in several ways.
Most common is severe dietary deprivation of folate from chronic
alcohol abuse or malnuroishment.
Similarly, the demands of the fetus and poor maternal diet may combine
to produce folate deficiency in pregnancy.
Folate deficiency may also be caused by malabsorption secondary to
intestinal infection by Giardia or intestinal sprue.
Chemotherapy for malignancy such as methotrexate,
a folic acid antagonist, will cause megaloblastic anemia and requires
"rescue" with citrovorum (N5-formyl THF).
Dilantin and oral contraceptives may also cause folate deficiency.
A relative folate deficiency may occur when the demand for folate
increases due to accelerated erythropoiesis (ie. as a compensatory erythroid
hyperplasia in hemolytic anemia).
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