Megaloblastic Anemias

Nutritional and Other Causes

Ralph Green, MD, PhD*, Ananya Datta Mitra, MD

KEYWORDS
 Anemia  Megaloblastic  Vitamin B12  Folate  Pernicious anemia
 Methylmalonic acid  Homocysteine  Transcobalamin

KEY POINTS
 Vitamin B12 and folate deficiencies are the most important causes of megaloblastic
anemia.
 The main cause of vitamin B12 deficiency that results in megaloblastic anemia is perni-
cious anemia caused by autoimmune destruction of gastric parietal cells.
 The prevalence of folate deficiency has decreased because of widespread food folate
fortification, but it still occurs among the poor, elderly, and alcoholics, particularly in coun-
tries that do not fortify the diet. It can also result from malabsorption or increased cell turn-
over and increased demand.
 Nuclear-cytoplasmic asynchrony from the ineffective DNA synthesis causes megalo-
blastic changes in the bone marrow with resulting anemia and cytopenias.

MEGALOBLASTIC ANEMIAS: NUTRITIONAL AND OTHER CAUSES

Introduction
Ineffective DNA synthesis in hematopoietic precursor cells is the primary mechanism
that leads to megaloblastic anemia. The most frequent causes of megaloblastic ane-
mia are deficiencies of either vitamin B12 or folate within a long list that includes defi-
ciency of other micronutrients, congenital disorders, myelodysplastic syndromes, and
acquired DNA synthesis defects as seen in the settings of chemotherapy (Box 1).1 The
ineffective hematopoiesis resulting from the asynchrony between nuclear and cyto-
plasmic development is most evident on Wright-Giemsa–stained bone marrow aspi-
rate smears. Megaloblastic erythroid precursors are larger than normal and their
nuclei are larger and appear immature with granular chromatin. During the initial
stages of cellular differentiation, the slow condensation of chromatin results in an
open sievelike vesicular nucleus. In subsequent stages there is more cytoplasm in

Disclosures: The authors have nothing to disclose.

Department of Pathology and Laboratory Medicine, UC Davis Medical Center, University of Cal-
ifornia Davis Health System, 4400 V. Street, Sacramento, CA 95817, USA
* Corresponding author.
E-mail address: rgreen@UCDAVIS.EDU

Med Clin N Am 101 (2017) 297–317

http://dx.doi.org/10.1016/j.mcna.2016.09.013 medical.theclinics.com
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298 Green & Datta Mitra

Box 1
Causes of megaloblastic anemia

Vitamin deficiencies
Deficiency of folate
1. Decreased intake
a. Nutritional deficiency (elderly, alcoholics, poverty)
b. Diet induced: goat’s milk, synthetic diets
c. Infants with prematurity
d. Hyperalimentation
2. Decreased absorption
a. Celiac disease and tropical sprue
3. Increased demand
a. Pregnancy
b. Puberty
c. Chronic hemolytic anemia
d. Exfoliative dermatitis
e. Hemodialysis
Deficiency of vitamin B12
1. Impaired absorption
a. Pernicious anemia
b. After gastrectomy or ileal resection
c. Zollinger-Ellison syndrome
d. Blind loop syndrome
e. Fish tapeworm infestation
f. Pancreatic insufficiency
2. Decreased intake
a. Vegans
b. Vegetarians
Other causes
1. Drugs (eg, antibiotics, anticancer agent, anticonvulsants and oral contraceptives)
2. Inborn errors of metabolism
3. Acute megaloblastic anemia
a. Nitrous oxide exposure
b. Acute illness
4. Idiopathic (congenital dyserythropoietic anemia, erythroleukemia, and refractory
megaloblastic anemia)
5. Thiamine-responsive megaloblastic anemia

the megaloblastic erythroid precursors relative to the size of the nucleus. The granu-
locytic precursors also show nuclear-cytoplasmic dyssynchrony with the develop-
ment of so-called giant metamyelocytes and bands, which have a characteristic
horseshoe-shaped nucleus and open chromatin. The development of megakaryo-
cytes is also affected, as reflected by abnormal large polylobated megaloblastic
megakaryocytes with a lack of cytoplasmic granules. Corresponding changes in the
blood smear include anemia with oval macrocytes, anisocytosis, poikilocytosis, leuko-
penia with hypersegmented polymorphonuclear cells, and thrombocytopenia.

Pathogenesis of Megaloblastic Anemia

Deficiencies of folate and vitamin B12 are the leading causes of megaloblastic anemia
worldwide. In the era of food folic acid fortification, there is a decreasing trend of folate
deficiency prevalence in most developed countries. Classically, in megaloblastosis,

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 Megaloblastic Anemias 299

there is defective DNA synthesis in rapidly dividing hematopoietic precursors.2,3 To a

lesser extent, RNA and protein synthesis are impaired and these cells generally have
much more cytoplasm and RNA than do their normal counterparts, suggesting that
cytoplasmic constituents are synthesized faster than DNA. There is prolongation of
the S-phase of DNA synthesis because of the delay in migration of the DNA replication
fork and the connection of DNA fragments synthesized from the lagging strand (Okazaki
fragments).4,5 Evidence suggests that, because of this nuclear maturation arrest, unbal-
anced cell growth and impaired cell division occur.6 There is substantial loss of defec-
tive hematopoietic precursors through apoptosis.7 The metabolic interrelationship
between folate and B12 has been implicated in the development of the megaloblastic
anemia observed in either of the 2 vitamin deficiencies because B12 is necessary to
regenerate tetrahydrofolate (THF) through the methionine synthase reaction and this
is required for the production of methylene-THF, which is essential for thymidine and
hence DNA synthesis.3 Vitamin B12 is the cofactor for both the cytoplasmic methionine
synthase and the mitochondrial l-methylmalonyl–coenzyme A mutase reactions.8,9

Clinical Picture
Anemia develops gradually with sufficient time for cardiopulmonary and intraerythro-
cytic compensation to occur before symptoms develop.10 Symptoms generally
develop in severely anemic patients. The main symptoms include weakness, palpita-
tion, fatigue, light-headedness, and shortness of breath caused by low hematocrit.
Jaundice can occur from intramedullary and extravascular hemolysis. Leukopenia
or thrombocytopenia are generally present, but do not typically cause any clinical
concern. With B12 deficiency, there are often neurologic symptoms as well as auto-
nomic gastrointestinal disturbances. The neurologic symptoms result from symmetric
paresthesias, numbness, and impaired vibration and position sense leading to gait
disturbances.11,12 About 10% of B12-deficient patients show hyperpigmentation and
some patients with pernicious anemia have associated autoimmune vitiligo. There
may be cerebral manifestations in B12 deficiency, including mental confusion, para-
noia, dementia, and even frank psychosis.13 Other associated symptoms encountered
rarely with vitamin B12 deficiency include generalized malabsorption caused by intes-
tinal megaloblastosis, infertility, glossitis, and cerebral venous thrombosis.14,15 A
greater risk of thrombosis may occur as a result of hyperhomocysteinemia in severe
B12 deficiency.

Laboratory Features
Megaloblastic anemia is suspected based on consideration of the clinical features
described earlier in conjunction with the results of a blood count and examination of
the blood smear showing anemia, and increased red cell indices (mean corpuscular
volume [MCV], mean corpuscular hemoglobin) together with anisocytosis, poikilocyto-
sis, and hypersegmented neutrophils.16 However, the presence of hypersegmented
neutrophils is not diagnostic of megaloblastic anemia because they can also be
encountered in iron deficiency anemia. Thus studies for iron deficiency should also
be considered in cases of megaloblastic anemia.17 The initial tests that should be per-
formed to evaluate megaloblastic anemia are the measurement of plasma or serum
levels of vitamin B12 and folate as well as red cell folate.18 Because of folic acid forti-
fication programs in the United States and many other parts of the world, vitamin B12
deficiency is more commonly encountered than folate deficiency. For this reason, B12
deficiency is considered first.
B12 deficiency is suggested by a B12 level of less than 200 pg/mL. However, B12
levels may be low without underlying deficiency because of low or absent levels of

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300 Green & Datta Mitra

the major plasma B12 binding protein, haptocorrin,19 or can be spuriously normal in
patients with pernicious anemia if they have excessive levels of circulating intrinsic
factor antibodies. Intrinsic factor antibodies can interact with the intrinsic factor
binder that is used in most B12 assays.20 Borderline B12 levels (200–400 ng/L) should
be followed up with measuring metabolite levels that increase in B12 deficiency.21,22
Increased methylmalonic acid (MMA) levels are widely considered to be a highly sen-
sitive and specific test for identifying B12 deficiency and, when MMA levels are not
increased, this is strongly suggestive of normal B12 status.21,22 The levels of MMA
always decrease with vitamin B12 therapy when there is underlying B12 defi-
ciency.23,24 MMA can increase (300–700 nmol/L) in renal failure and is refractory to
B12 administration.24,25 B12 deficiency also leads to high plasma homocysteine
levels. However, this is not a specific indicator of B12 deficiency, because high homo-
cysteine levels are also caused by folate deficiency.18,21 The fraction of the B12 in cir-
culation that is bound to the B12 transport protein transcobalamin as opposed to the
other plasma B12-binding protein, haptocorrin, has been proposed as a more reliable
indicator of functional B12 status because it is responsible for cellular delivery and
uptake of the vitamin.26 However, because of problems with other indices of B12 sta-
tus assessment used alone, there has been a trend to combine the use of 2 or more
analytes, such as total B12 with either transcobalamin or MMA.27 More recently, use
of a combined indicator of B12 status, cB12 (where cB12 5 log10 [(hol-
otranscobalamin$B12)/(MMA$total homocysteine)]), has been successfully applied
in population studies to identify B12 deficiency. However, this approach awaits vali-
dation in patients with megaloblastic anemia suspected of being caused by B12 defi-
ciency.28 In B12 deficiency, serum folate levels are frequently increased because of
the trapping of folate in the form of methylfolate.29 Because megaloblastic anemia
is usually associated with considerable extravascular hemolysis, including intrame-
dullary destruction of erythroid precursors, serum bilirubin and lactate dehydroge-
nase (LDH) levels are typically increased.17
There are many causes of B12 deficiency and a good understanding of the physi-
ology of B12 absorption is necessary to evaluate the several possible underlying dis-
orders that can lead to B12-deficient megaloblastic anemia. Complete and detailed
considerations of the causes of B12 deficiency are well described elsewhere.3,22 In
general, causes of B12 deficiency may be divided into gastric and ileal causes,
because the key components of the normal pathway for B12 absorption require a func-
tioning stomach for production of the protein intrinsic factor and an intact cubilin re-
ceptor for the intrinsic factor–B12 complex in the terminal ileum.3,22 Preeminent
among the several causes of B12 deficiency is the disease pernicious anemia, caused
by autoimmune destruction of the gastric parietal cells that produce intrinsic factor.
The presence of circulating anti–intrinsic factor l antibodies is highly specific for the
diagnosis of pernicious anemia. However, the test has a poor sensitivity, being posi-
tive in only 60% of patients with pernicious anemia.30,31 Chronic atrophic gastritis,
which is associated with pernicious anemia, can be diagnosed by endoscopic biopsy,
an increased fasting serum gastrin level, and decreased serum pepsinogen I
levels.32,33 Causes of B12 deficiency are discussed in further detail later.
The initial tests for possible folate deficiency include a plasma or serum level of less
than 4 mg/L, which indicates folate deficiency. However, occasionally, a borderline
folate level (4–8 mg/L) might be associated with high plasma levels of homocysteine
consistent with underlying folate deficiency being responsible for megaloblastic
change in the marrow. In contrast, borderline serum folate levels with normal homo-
cysteine level are not considered deficient.21,34 An increased level of serum total ho-
mocysteine is less specific for folate deficiency because homocysteine level is also

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 Megaloblastic Anemias 301

increased in vitamin B12 deficiency,21,35,36 classic homocystinuria, hypothyroidism,

and renal failure. Although low red blood cell folate level is seen in folate deficiency,
it is not specific, because B12 deficiency can also be associated with a low red cell
folate level. Thus, red blood cell folate testing should be combined with serum or
plasma B12 and folate testing. The major advantage of measuring red blood cell folate
level is that it reflects average folate status of the individual during the 3-month to 4-
month period that corresponds with normal red cell lifespan and is not influenced by
recent dietary intake of folate.3,18
In the evaluation of megaloblastic anemias, clinical signs and symptoms should be
considered apart from the laboratory parameters. Although patients with folate defi-
ciency mainly present with hematologic symptoms, those with B12 deficiency often
have predominantly neurologic signs and symptoms. Also, long-term consequences
of folate deficiency can lead to cardiovascular, developmental, cerebral, and immuno-
logic defects.37

CONDITIONS THAT MAY CONFUSE OR OBSCURE THE PICTURE OF MEGALOBLASTIC

ANEMIA
Coexistent Microcytic Anemia
Coexisting microcytic anemias resulting from iron deficiency anemia, the anemia of
chronic disease, or thalassemia minor can obscure the megaloblastic features in
the bone marrow or peripheral blood.38,39 Peripheral blood may show a dimorphic pic-
ture with marked anisocytosis and the blood count showing a normal MCV but a mark-
edly increased red cell distribution width (RDW). Intermediate megaloblasts,40 which
look smaller than the usual megaloblastic cells, predominate in the marrow. However,
in these situations, there are still hypersegmented neutrophils in the blood and giant
metamyelocytes and bands in the marrow. Coexistence of a megaloblastic and micro-
cytic process can also be encountered in celiac disease,41 gastric bypass surgery for
morbid obesity,42 and chronic Helicobacter pylori infection.43,44
Acute Leukemia and Myelodysplasia
A hypercellular and dysplastic bone marrow with bizarre bone marrow morphology
seen in severe nutritional megaloblastic anemia can be mistaken for signs of acute
leukemia or myelodysplasia.17,45,46 Rarely, the erythroid series show little or no matu-
ration, and megaloblastic pronormoblasts predominate in the marrow with dysmor-
phic forms and prominent mitotic figures. There are often associated severe
cytopenias. This picture resembles erythroid leukemia and, because the treatment
and prognosis differ greatly between the conditions, a high level of awareness and
suspicion as well as suitable testing to exclude a possible megaloblastic anemia
should be undertaken if megaloblastic features are prominent before treatment of
acute leukemia is instituted.
Attenuated Megaloblastic Anemia
Megaloblastic changes may be attenuated if patients with advanced megaloblastic
anemia receive B12 or folate therapy or blood transfusion before marrow aspiration;
the anemia continues until the marrow has responded to the hematinic therapy but
the megaloblastic changes may be masked within 36 to 48 hours.
Acute Megaloblastic Anemia
A serious megaloblastic state can develop acutely within a few days because of tissue
folate or B12 deficiency. These patients present with thrombocytopenia and/or leuko-
penia with minimal effects in red cell counts because of the higher longevity of red

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302 Green & Datta Mitra

blood cells. The commonest cause of acute megaloblastic anemia is nitrous oxide
(N2O) anesthesia, which destroys the methylcobalamin form of B12, leading to a mega-
loblastic state in the bone marrow within 12 to 24 hours.47,48 Hypersegmented neutro-
phils generally appear after 5 days of exposure but then persist for several days.49 If
the source is removed, the effects of N2O generally disappear over a few days and
the removal can be expedited by the use of folinic acid or cobalamin.50 Sometimes
acute megaloblastic anemia can develop in seriously ill patients in intensive care
units,51 patients receiving massive transfusion at surgery,52 those on dialysis or total
parenteral nutrition, and those who are receiving folate antagonists such as trimetho-
prim or low-dose methotrexate. Levels of red cell folate and serum vitamin B12 might
be normal but the marrow shows megaloblastosis. There is always a prompt response
to therapeutic doses of parenteral folate and B12 in these patients.

CAUSES OF VITAMIN B12 DEFICIENCY

There are several causes and varying degrees of severity of B12 deficiency17,22 (see
Box 1).

Impaired Absorption
Pernicious anemia is an autoimmune gastritis, occurring in middle-aged to elderly
adults, leading to the destruction of gastric parietal cells and failure of intrinsic factor
production,53 resulting in achlorhydria.32,54 Susceptibility to pernicious anemia is
associated with human leukocyte antigen types A2, A3, B7, and B12,55 and with blood
group A.56

Helicobacter pylori Infection

Very commonly, H pylori infects the gastric mucosa and causes gastritis and peptic
ulcers. However, the role of H pylori in the pathogenesis of pernicious anemia is still
a mystery. Previous studies have shown a very low incidence of H pylori infection in
patients with pernicious anemia.57 An interesting proposition has been put forward,
that chronic infection with H pylori may trigger an autoimmune process directed
against the host H1/K1-ATPase protein.44,58

Gastrectomy Syndromes
Gastric resections for any cause as well Roux-en-Y gastric bypass surgery for morbid
obesity lead to multiple nutritional deficiencies.3 Iron deficiency anemia is the most
common anemia to occur after gastric surgery; however, B12 deficiency with megalo-
blastic anemia occurs occasionally. B12 deficiency usually develops 5 to 6 years post-
surgery because of the lack of intrinsic factor and the gradual depletion of the body’s
B12 stores.59 In classic B12 deficiency serum iron levels are usually high. In contrast,
postgastrectomy patients with low serum B12 levels typically have low serum iron
levels.60 After partial gastrectomy, B12 deficiency might develop because of mucosal
atrophy61 or bacterial overgrowth, classically known as blind loop syndrome.62 B12
malabsorption in blind loop syndrome occurs because of intestinal colonization with
bacteria, which thrive on cobalamin. This cause of B12 deficiency is therefore
amenable to antibiotic therapy.63

Hyperchlorhydria
Zollinger-Ellison syndrome, results from a gastrin-secreting tumor in the pancreas,
which stimulates the gastric mucosa to secrete immense amounts of hydrochloric

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 Megaloblastic Anemias 303

acid. The large quantities of acid acidify the duodenal contents, thus preventing the
binding of B12 to the intrinsic factor.64

Diseases of the Small Intestine

Because the terminal ileum is the eventual site for B12 absorption; intestinal disorders
such as ileal resection,65 ileitis of any cause (inflammatory bowel disease, radiation),66
lymphoma arising in the terminal ileum, drug-related or toxin-related disorders,67 or
tropical sprue68 can lead to B12 deficiency.

Inherited Disorders of Vitamin B12 Absorption

Imerslund-Graesbeck syndrome (IGS) is a rare genetic pediatric disorder resulting in
vitamin B12 malabsorption in the terminal ileum.69 Usually clinically evident during
the first 6 years of life, IGS usually results in megaloblastic anemia and may be accom-
panied by neurologic defects of variable severity with/without proteinuria. The disor-
der results from defects in one of 2 genes (cub or amn) that together code for the
cubilin protein, which comprises the receptor for the intrinsic factor–B12 complex.70
Hereditary intrinsic factor deficiency (HIFD) occurs because of recessive mutations
as well as a partial gene deletion in the intrinsic factor gene,71 and results in decreased
intrinsic factor leading to B12 malabsorption.
Definitive diagnosis of IGS and HIFD can only be confirmed by genetic testing.
Treatment consists of parenteral vitamin B12 supplementation, which should be
administered without confirmation of diagnosis.

Miscellaneous Causes
Infestation by the fish tapeworm Diphyllobothrium latum, arising from the ingestion of
uncooked or partially cooked fish, can lead to B12 deficiency caused by competition
between the worm and the host for ingested B12.72
Patients with acquired immunodeficiency syndrome sometimes present with low
serum B12 levels caused by B12 malabsorption.73 This malabsorption can have a com-
bination of intestinal and gastric causes.74,75
Patients with pancreatic diseases can have B12 deficiency caused by malabsorption
from pancreatic insufficiency.76 Pancreatic proteases, which are lacking in pancreatic
insufficiency, are required for digestion of the salivary B12-binding protein haptocorrin
and transfer of B12 to intrinsic factor for absorption.77
Dietary insufficiency of B12 is common among vegans and vegetarians and this can
lead to reduced B12 status, but true deficiency of B12, sufficient to cause megalo-
blastic anemia, is very rare among even strict vegetarians who do not consume milk
or eggs.78 Low serum B12 levels can be found in 50% to 60% of patients among
this group. Depletion of the body’s B12 store occurs slowly, caused by efficient reab-
sorption of biliary B12 by the enterohepatic pathway.79,80 It may take 10 to 20 years for
a vegan to manifest features of megaloblastic anemia caused by B12 deficiency.81,82
In addition, B12 deficiency may occur in severe general malnutrition. A megalo-
blastic anemia can develop without B12 deficiency in protein calorie malnutrition,
such as kwashiorkor or marasmus.83

Inherited Disorders of B12 Metabolism

Several inherited disorders of cobalamin metabolism involving various steps in the
cellular processing of B12 and its conversion to active forms have been identified
and designated as the cobalamin mutants. These mutants have been extensively
described in several reviews.84,85

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304 Green & Datta Mitra

Methylmalonic aciduria is an autosomal condition caused by complete or partial

deficiency of the enzyme methylmalonyl-coenzyme A mutase.85 Patients present early
in life with lethargy, vomiting, hypotonia, hypothermia, respiratory distress, ketoacido-
sis, hyperammonemia, neutropenia, and thrombocytopenia without megaloblastic
anemia and are fatal within the first 4 weeks of life.

Homocystinuria
The defect lies in the enzyme N5-methyltetrahydrofolate-homocysteine methyltrans-
ferase (MTR; methionine synthase), resulting in decreased production of methylcoba-
lamin.86 Patients present early in life, or rarely in adult life, with vomiting, mental
retardation, megaloblastic anemia, severe homocystinuria, and hyperhomocysteine-
mia without methylmalonic aciduria or methylmalonic acidemia. Prenatal diagnosis
in infants followed by B12 treatment usually results in normal development.

Methylmalonic Aciduria and Homocystinuria

When there is a defect in the reduction of the cobalt atom in the cobalamin molecule
from Co21 to Co11 this leads to combined hyperhomocysteinemia and methylma-
lonic acidemia. Onset of the manifestations ranges from the neonatal period to adoles-
cence with lethargy; failure to thrive; neurologic abnormalities in infants; and
psychological abnormalities, progressive dementia, and motor defect in older pa-
tients.85 Megaloblastic anemia occurs in about half the cases and patients are partially
responsive to B12.

CLINICAL FEATURES OF B12 DEFICIENCY

The main features include megaloblastic anemia, cytopenias, glossitis, cardiomyopa-

thy, yellow discoloration of skin, weight loss, neurologic abnormalities, and immuno-
logic defects.87,88 Other effects of B12 deficiency include combined degeneration of
the spinal cord, increased risk of vascular disease caused by hyperhomocysteinemia,
potential increase in breast cancer risk in premenopausal women,89 and
osteoporosis.90,91

Neurologic Abnormalities
The neurologic manifestations include gradual onset of paresthesias, involving the tips
of fingers and toes, along with lancinating pains caused by peripheral neuropathy. This
stage is followed by loss of vibratory sense and proprioception resulting in abnormal-
ities of gait. Progressive demyelination of the dorsal and lateral columns of the spinal
cord leads to spastic ataxia in untreated patients.92 There may also be development of
somnolence, dysgeusia, and visual disturbances caused by optic atrophy and demen-
tia of the Alzheimer type.93 There are also psychological disturbances resulting from
B12 deficiency that include psychotic depression, paranoid schizophrenia, and frank
psychosis.94

Concealed B12 Deficiency

Some patients have a B12-responsive neuropsychiatric disease with normal, border-
line, or low serum B12 levels.12 These patients present with peripheral neuropathy,
gait disturbance, memory loss, and psychiatric symptoms. Although their B12 levels
are within the normal range, they have increased levels of serum MMA and/or homo-
cysteine, suggesting a tissue B12 deficiency. These patients typically are responsive to
B12 therapy.

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 Megaloblastic Anemias 305

MANAGEMENT AND PROGNOSIS

The required daily allowance of vitamin B12 is 2.4 mg.95,96 Patients with clinical vitamin
B12 deficiency have a resolution of megaloblastic anemia and resolution of or improve-
ment in neurologic symptoms with adequate supplementation of B12; however, most
of these patients have malabsorption and require parenteral or high-dose oral replace-
ment therapy.95

Injected Vitamin B12

Injections of vitamin B12, known as cyanocobalamin in the United States and hydrox-
ocobalamin in Europe,95,97 are recommended in different dosage schedules. Depend-
ing on the dose, only approximately 10% of the injected dose of the drug is retained in
the body. Patients with severe abnormalities require frequent doses of 1000 mg of B12
numerous times per week for 1 to 2 weeks followed by once a week and then once a
month, after clinical improvement. Hematologic response is brisk, with an increase in
the reticulocyte count in 1 week and rapid improvement of megaloblastic erythroid
changes in the bone marrow, which returns to normal within 36 to 48 hours of treat-
ment. However, the abnormal giant granulocytic forms may persist for 1 to 2 weeks.
Neutropenia and thrombocytopenia, if present, generally resolve within a week. Total
recovery from the anemia occurs within 6 to 8 weeks. The effect of therapy on neuro-
logic disease depends on the severity and duration of the neurologic abnormalities
before initiation of treatment.11,12 Treatment of PA, or any B12 deficiency that is caused
by malabsorption, needs to be maintained lifelong. In patients in whom vitamin B12
therapy is discontinued after clinical recovery, neurologic symptoms generally relapse
within 6 months, and megaloblastic anemia reappears within 1 to several years.95,97

High-dose Oral Treatment

Studies have shown that high-dose oral (2000 mg of B12 daily) and parenteral therapy
are equally effective in producing excellent hematologic and neurologic remis-
sions.12,98 More information is required from long-term studies to assess whether
oral treatment is effective when doses are administered less frequently than daily. Pa-
tients must be informed of the advantages and disadvantages of oral versus paren-
teral therapy, and, irrespective of the form of treatment, those with pernicious
anemia or malabsorption should be made aware of the need for lifelong replacement.

Special Circumstances
B12 is always recommended after a total gastrectomy, although it is not required after
a partial gastrectomy, but patients should be followed diligently for any evidence of
B12 deficiency or anemia, because megaloblastic changes can be masked by post-
gastrectomy iron deficiency.99,100 Blind loop syndrome anemia can be treated with
parenteral B12 therapy following a week’s therapy with oral broad-spectrum antibi-
otics.101 Surgical correction of the anatomic lesion also cures the syndrome. Fish
tapeworm treatment consists of a single oral dose of a 50 mg/kg of niclosamide or
a dose of 5 to 10 mg/kg of praaziquantel, followed by B12 replacement.3

CAUSES OF FOLATE DEFICIENCY

Cause and Pathogenesis
The most common causes of folate deficiency are nutritional deficiency, impaired ab-
sorption, and loss or increased requirements. However, the prevalence of folate defi-
ciency has decreased dramatically as a result of mandated fortification of the food
supply with folic acid in many countries.

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306 Green & Datta Mitra

Dietary Deficiency
Before the mid-1990s, inadequate intake was one of the most common causes of defi-
ciency of folate. The feasibility of folic acid fortification of a staple grain or cereal,102
together with the discovery that correction of folate deficiency during pregnancy could
reduce the risk of neural tube defect pregnancies,103 led to the mandated fortification
of the diet with folic acid in many countries. Excessive cooking can destroy dietary
folate and aggravate folate deficiency. Because of limitation of folate stores, folate
deficiency develops rapidly in malnourished individuals and chronic alcoholics. Other
causes of folate deficiency include hyperalimentation104 and subtotal gastrectomy.105
Premature infants are at increased risk of folate deficiency during infection, diarrhea,
or hemolytic anemia.106 Children on synthetic diets107 and infants exclusively fed on
goat’s milk108 also develop folate deficiency. Megaloblastic anemia in alcoholic
cirrhosis is usually the result of folate deficiency,109 although megaloblastic changes
occur even when large doses of folate supplementation are given and alcohol con-
sumption is maintained, suggesting that there is a metabolic effect of alcohol beyond
its nutritional effects.110

Impaired Absorption
Folate deficiency can occur in nontropical sprue or celiac disease caused by chronic
inflammation of the proximal small intestinal mucosa caused by dietary intake of
gluten.111 The patients typically have weight loss, glossitis, diarrhea, steatorrhea,
iron deficiency, hypocalcemia, and other fat-soluble vitamin deficiencies. The serum
folate levels are low, resulting in megaloblastic anemia.112 Another entity that might
lead to folate deficiency is tropical sprue, an idiopathic malabsorptive disease
endemic to the Caribbean, south India, parts of southern Africa, and southeast
Asia.113 This condition can be promptly corrected by folate therapy together with sys-
temic antibiotics, suggesting an infectious source of the disease.114

Other Intestinal Defects

Regional enteritis,115 surgical resection of the small intestine,116 lymphoma or
leukemic infiltration of the small intestine,117 Whipple disease,117 scleroderma and
amyloidosis,118 and diabetes mellitus119 can all lead to impairment of folate
absorption.

Increased Requirements
Folate deficiency is one of the major cause of megaloblastic anemia of pregnancy,120
mostly in developing countries.121 In pregnancy, there is a 5-fold to 10-fold increased
requirement of folate caused by transmission of folate to the growing fetus.122,123 This
requirement intensifies with multiple pregnancies, poor nutrition, infection, concomi-
tant hemolytic anemia, or anticonvulsant medication. Folate deficiency is challenging
to diagnose in pregnancy because of the development of physiologic anemia associ-
ated with physiologic macrocytosis; MCV may increase to 120 fL.124 Serum and red
cell folate levels decrease gradually during pregnancy, even in healthy women not
on folic acid supplementation.125 Hypersegmented neutrophils, usually a consistent
indicator of early megaloblastic anemia, are less prominent in early megaloblastic ane-
mia of pregnancy.126 Folate deficiency also occurs during lactation and is aggravated
by protracted lactation.127 Folic acid fortification of the diet has been shown to be
effective in ameliorating the folic acid deficiency and hence the risk of megaloblastic
anemia during pregnancy.128

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 Megaloblastic Anemias 307

Increase in Cell Turnover

Folate requirement increases abruptly in chronic hemolytic anemia because of
increased bone marrow turnover.129 The bone marrow becomes megaloblastic within
days during episodes of acute hemolysis. Folate deficiency can also occur in chronic
exfoliative dermatitis, in which there is folate loss of 5 to 20 mg/d.130 Folate supplemen-
tation in patients with psoriasis before initiation of methotrexate therapy can prevent
the development of folate deficiency from methotrexate without diminishing its thera-
peutic effect.130 There can be significant folate loss in dialysis fluid during
hemodialysis.131

CLINICAL FEATURES OF FOLATE DEFICIENCY

Clinical manifestations of folate-deficient megaloblastic anemia typically include a his-

tory of nutritional deficiency with no neurologic signs and response to folate therapy.

LABORATORY FEATURES OF FOLATE DEFICIENCY

The initial test for folate deficiency is the presence of low serum or plasma folate levels
(<3 ng/mL). Although an increase in homocysteine level often precedes low plasma
folate levels, it is a less specific indicator.21 Another indicator that is useful for identi-
fying folate deficiency is the red cell folate.132 Red cell folate remains comparatively
unaffected by sudden changes in folate intake, because it reflects folate status over
the lifespan of red cells. However, red cell folate cannot reliably distinguish between
folate and B12 deficiency and also cannot detect acutely developing megaloblastic
anemia.133

OTHER EFFECTS OF FOLATE DEFICIENCY

Neural Tube Closure
Folate deficiency results in congenital anomalies of the fetus, most notably defects in
neural tube closure.134 This outcome may also be related to antibodies against folate
receptors, which have been detected in some women and may be overcome by higher
folate intake.134,135 Mutations in the enzymes of folate metabolism136 result in an in-
crease in the risk of congenital anomalies caused by the diminished conversion of
methylene-tetrahydrofolate to methyl-tetrahydrofolate, which is essential for embry-
onic development. Fortification of folate has been successful in reducing the incidence
of neural tube defects by between 20% and 50% in North America.137,138 In addition to
the hematological complications, there are other detrimental effects of folate defi-
ciency affecting the brain and vascular system, as well as cancer risk. These effects
have been reviewed elsewhere.37

INBORN ERRORS OF FOLATE METABOLISM

Hereditary Folate Malabsorption
Hereditary folate malabsorption is an uncommon congenital disorder in which there is
impairment in the intestinal absorption of folate, often associated with impaired folate
transportation through the choroid plexus into the cerebrospinal fluid (CSF).139,140 This
defect involves the proton-coupled folate transporter. Patients typically present with
severe megaloblastic anemia and central nervous system (CNS) symptoms (seizures
and mental retardation) with low serum folate and undetectable CSF folate levels.141
Although parenteral folate corrects the anemia it has no effect on CSF folate concen-
tration and neurologic symptoms. Daily folinic acid therapy restores CSF folate level
and reduces CNS manifestations.142

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308 Green & Datta Mitra

Dihydrofolate Reductase Deficiency

Deficiency of the enzyme dihydrofolate reductase results in folate-refractory megalo-
blastic anemia responsive to folinic acid within days or weeks of birth.143

Methionine Synthase (MTR) Deficiency

Diminished MTR activity results in megaloblastic anemia and mental retardation. The
anemia is refractory to folate, cobalamin, or pyridoxal phosphate.144

Methylene Tetrahydrofolate Reductase Deficiency

This is a rare autosomal recessive disorder characterized by severe hyperhomocystei-
nemia and homocystinuria without megaloblastic anemia or methylmalonic aciduria.
Patients generally present with neurologic and vascular complications.142

MANAGEMENT AND PROGNOSIS

Folic acid supplementation, 1 to 5 mg/d, given orally, is usually adequate to correct

anemia. Parenteral preparation (5 mg/mL) of folate should be used in patients with
malabsorption. Tropical sprue is usually treated with folate, together with antibiotics
and with additional doses of B12 if indicated. Treatment should be continued for a min-
imum of 2 years to prevent relapse. During pregnancy the required allowance of folate
is 400 mg/d because higher doses can obscure B12 deficiency.145 Mothers with nutri-
tional deficiency or with malabsorption may be given 1 mg of vitamin B12 parenterally
every 3 months to prevent any associated B12 deficiency during gestation. Evaluation
of serum folate and B12 levels is mandatory in the treatment of megaloblastic anemia
because monotherapy with folate may partly correct the hematologic abnormalities
caused by B12 deficiency, but may lead to progressive and catastrophic neurologic
consequences.146 In cases of emergency when identification of the cause of the defi-
ciency is ambiguous, both folate and B12 supplementation must be given, ideally after
obtaining serum samples.

DRUG-INDUCED MEGALOBLASTIC ANEMIA

The structural similarity of aminopterin and methotrexate to folic acid leads to the
entry of these drugs into the cells via the folate carrier.147 As suitable substrates
for the polyglutamyl synthase enzyme, these folate analogues then acquire a poly-
glutamate chain,148 which makes them strong inhibitors of dihydrofolate reductase,
resulting in the blocking of the conversion of dihydrofolate to tetrahydrofolate. This
inhibition of one-carbon metabolism causes a decrease in nucleotide (particularly
thymidine) biosynthesis that leads to a major derangement in DNA replication.149
Drug toxicity often presents with mouth and esophageal ulcerations, abdominal
pain, vomiting and diarrhea, ulcerations throughout the large and small intestines,
alopecia, hyperpigmentation, and megaloblastic anemia. Toxicity from the use of
folate antagonists is treated with folinic acid (N5-formyl tetrahydrofolate) 3 to
6 mg/d intramuscularly. This treatment is known as folinic acid rescue and is often
used in chemotherapy to rescue patients receiving high doses of methotrexate.150
Another antifolate compound, pemetrexed, used in the treatment of lung cancer
and mesothelioma, can cause megaloblastic anemia that is treatable with cobal-
amin and folate. Zidovudine (azidothymidine [AZT]), used in acquired immunodefi-
ciency syndrome therapy, is another agent that has the side effect of severe
megaloblastic anemia.151 Hydroxyurea is sometimes still used in the treatment of
myeloproliferative disorders, including chronic myelogenous leukemia,

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 Megaloblastic Anemias 309

polycythemia vera, and essential thrombocythemia, as well as psoriasis, rheumatoid

arthritis, and sickle cell disease, and inhibits conversion of ribonucleotides to deoxy-
ribonucleotides,152 resulting in megaloblastosis in the marrow. This condition de-
velops within a few days of initiating hydroxyurea therapy and is promptly
reversible after the withdrawal of the drug. Chronic use of proton pump inhibitors
causes inhibition of parietal cell function leading to low serum levels of B12.153
Trimethoprim is a microbial dihydrofolate reductase inhibitor that is reported to
have only a minimal effect on the human enzyme. However, trimethoprim does
induce a biochemical state of megaloblastosis154 and can aggravate a state of
folate deficiency in patients with borderline folate status.155

OTHER RARE CAUSES OF MEGALOBLASTIC ANEMIA

Congenital Dyserythropoietic Anemia
These are inherited dysplastic anemias affecting the erythroid lineage only, leading to
the development of multinucleated normoblasts. The underlying molecular defect in
these anemias is in the glycosylation of polylactosaminoglycans associated with
membrane proteins and ceramides.156
Refractory Megaloblastic Anemia
Sideroblastic anemia and myelodysplastic disorders can manifest as refractory mega-
loblastic anemia.157 Atypical megaloblastic changes are confined to the erythroid line-
age. Very rarely, some patients with sideroblastic anemia are responsive to pyridoxine
(200 mg/d).158
Acute Erythroid Leukemia
In this form of acute myelogenous leukemia159 nucleated red cells with marked macro-
cytosis appear in the peripheral blood. There is prominent erythroid hyperplasia in the
bone marrow with unusual vacuolated and multinucleated red cell precursors with
increased numbers of blasts.
Thiamine-responsive Megaloblastic Anemia
Thiamine-responsive megaloblastic anemia is an autosomal recessive disease and it
is typically associated with diabetes and deafness. It is thought to be caused by a
defect in the thiamine (vitamin B1) transport mechanism that results in reduced nucleic
acid production through impaired transketolase catalysis,160 which induces cell cycle
arrest or apoptosis in bone marrow. The condition generally responds to therapeutic
doses of thiamine, resulting in improvement of diabetes and correction of anemia.
However, the advanced sensorineural deafness is irreversible.
These unusual causes of megaloblastic anemia should be considered in the differ-
ential diagnosis in cases of megaloblastic anemia refractory to folate and B12 therapy
and when all the common and correctable causes are excluded.

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