Saturday, January 31, 2015

Megaloblastic Anemia: Folate and Vitamin B12 Metabolism, Causes, diagnosis and therapy

Fig. Megaloblastic anemia showing megaloblastics cells

An increased MCV can be due to a number of reasons but careful review of the patient's history and blood smear can narrow the diagnostic possibilities.  The differential can be divided into two broad categories based on RBC morphology.

Round macrocytosis-due to abnormal lipid composition of the eryth­rocyte membrane.  Common etiologies include:
            1. Alcoholism.
            2. Liver Disease.
            3. Renal Disease.
            4. Hypothyroidism ("myxedema of the red cell").

Oval macrocytosis (macroovalocytes) is a sign of problem with cell DNA replica­tion.  The developing red cell has difficulty in undergoing cell division but RNA contin­ues to be translated and transcribed into protein leading to growth of the cytoplasm while the nucleus lags behind.  Often one or more cell division are skipped leading to a larger than normal cell.  Common causes are:

1.  Drug effect including cytotoxic chemotherapy.
2.  Megaloblastic Anemias-Folate Deficiency or Vitamin B12 deficiency - Patients will have hypersegmented neutrophils on review of the peripheral smear.
3. Myelodysplasia - Patients often have hyposegmented neutrophils and abnormal        platelet morphology.

Patients with increased reticulocyte counts can also have an increase MCV due to the large size of the reticulocyte (MCV = 160).


Folate: The body stores very little folate (several weeks) and maintenance of folate stores is dependent on adequate dietary intake.  Folate is found in green leafy vegetables, and liver.  Folate is absorbed in the small bowel and circulates in a free form or loosely bond to albumin.
Vitamin B12: In contrast to folate the body stores copious amounts of vitamin B12 (2-6 years).  This is fortunate as the absorption of vitamin B12 is complex and can be interrupted by a variety of mechanisms.  Vitamin B12 is synthesized by microbes and the major dietary source is animal protein.  When animal protein is ingested, vitamin B12 is freed from the protein and binds to "R proteins".  The R protein-vitamin B12 complex travels to the duodenum where pancreatic enzymes destroy the R protein.  This allows intrinsic factor (IF) to bind to vitamin B12.  This IF-vitamin B12 complex is absorbed only in the last 1-2 feet of terminal ileum.  Vitamin B12 binds to transcobalamin II and is delivered to tissues.
Fig. Vitamin B12 and folate metabolic pathway

Both vitamin B12 and folate are key components in the synthesis of DNA due to their role in conversion of uridine to thymidine.  When methyltetrahydrofolate loses a methyl group to form tetrahyrodrofolate, vitamin B12 "shuttles" the methyl group to homocysteine converting it to methionine.  Tetrahydrofolate is eventually converted to methylenetetrahydrofolate which is required for thymidine synthase. Vitamin B12 other role is a co-factor in the conversion of methymalonyl-CoA to succinyl-CoA.


When vitamin B12 or folate is deficient, thymidine synthase function is impaired and DNA synthesis is interrupted.  As described above this leads to megaloblastic changes in all rapidly dividing cells.  The inability to synthesized DNA leads to ineffectual erythropoiesis.  There is often erythroid hyperplasia in the marrow but most of these immature cells die before reaching maturity.  This process, intramedullary hemolysis, leads to the classic biochemical picture of hemolysis-elevated LDH and indirect bilirubinemia.  The LDH level is often in the 1,000's in patients with megaloblastic anemia.  The lack of DNA synthesis affects the neutrophils leading to nuclear hypersegmentation.  The anemia is of gradual onset and is often very well tolerated despite low hematocrits.  Often a mild pancytopenia is seen but thrombocytopenia can be severe.
Other rapidly dividing tissue are influenced by the megaloblastic process.  In the GI tract this can lead to atrophy of the luminal lining and further malabsorption.  This also leads to the classic sign of tongue smoothing.
As discussed further below, only vitamin B12 deficiency leads to neurological damage.  The mechanism is unknown.


Decreased intake- The average intake of folate in the diet is only 2-300 ug/day which is less than the estimated daily requirement.  Thus, for most people a poor diet or decrease eating will lead to folate deficiency.

Increased requirements-Patients who are pregnant, have hemolytic anemia, or psoriasis have increased needs for folate which can cause them to rapidly develop folate deficiency if intake is not kept up.


Drugs - Patient with underlying mild folate deficiency are more susceptible to trimethoprim/sulfa,
pyrimethamine and methotrexate toxicity.  Oral contraceptive and anticonvulsants lead to increase consumption of folate.

Alcohol- Alcohol affects several aspects of folate metabolism.   Alcoholics have poor intake of folate.  In addition, folate metabolism is interfered with leading to a functional folate deficiency.  Alcoholics have an inability to mobilize folate stores and can have depleted tissue stores with normal serum levels of folate.


Inadequate intake is rare but seen in very strict vegins.

Abnormal gastric events include being unable to dissociated vitamin B12 from food due to lack of stomach acid or enzymes.  This is a recently recognized group of patients which may compose a very large subset of patients with vitamin B12 deficiency.  10-30% percent of patients with partial gastrectomy will develop vitamin B12 deficiency.

Deficient intrinsic factor most commonly occurs due to destruction of parietal cells by autoantibodies (pernicious anemia).

Abnormal small bowel events include pancreatic insufficiency, blind loops syndromes (bacterial absorbing vitamin B12-IF complexes) and patients infested with Diphyllobothrium latum.

Abnormal mucosal events including malabsorption syndromes and surgical removal of the terminal ileum.

Drugs - Metformin, PPIs


            1. Recognizing that a megaloblastic anemia is present.
            2. Diagnosing vitamin B12 and or folate deficiency
            3. Determining the underlying cause.
            4. Therapy


It turns out that simply measuring serum levels of B12 or folate is very inadequate to diagnosis deficiency.  Up to 30% of people with low normal B12 levels will be deficient and many people with low B12 stores have normal tissue stores.  A more reliable method is to assay for the metabolic products that accumulate in B12 deficiency.  Since B12 is involved in conversion of homocysteine to methionine, lack of B12 will lead to elevated homocysteine level.  Also B12 is involved in conversion of methylmalonic acid to succinyl so in B12 deficiency, methylmalonic acid accumulates.  Both homocysteine and methylmalonic acid assays are widely available and should be the first line tests for B12 deficiency.

Serum folate levels are also very unreliable.  Since folate is needed for conversion of homocysteine to methionine, serum homocysteine will also accumulate in folate deficiency and is a more sensitive marker of tissue folate stores.










Increased Homocysteine level
B12 or folate deficiency, renal failure (kidney significant organ for homocysteine metabolism).

1)  Marker for possible nutritional deficiency
2) Elevated levels of homocysteine are associated with an increased risk of atherosclerosis or venous thrombosis.
3) Increased levels of homocysteine (reflecting lack of folic acid) in pregnant women is a risk factor for neural tubes defects.

Increased Methylmalonic Acid

B12 deficiency, renal failure (MMA renal excreted), methylmalonic aciduria (rare)

B12 deficiency.


In the majority of patients with folate deficiency, one can determine the underlying cause by history.  The key concern in vitamin B12 deficiency is determining a which point in the complex pathway of vitamin B12 absorption the "lesion" is.  The Schilling test is a test of vitamin B12 absorption.  Patients are given radiolabeled vitamin B12 orally and a large dose of vitamin B12 is given intravenously.  The IV dose of vitamin B12 prevents binding any absorbed labeled vitamin B12 and this is excreted.  The amount of excreted vitamin B12 is reflective of vitamin B12 absorption.  The Schilling test is NOT a test of vitamin B12 deficiency but a tool to determine the etiology of the deficiency.  The tradition Schilling test is call "stage I".  If less than 8% of the labeled vitamin B12 is excreted then one can perform the Schilling test with a variety of diagnostic maneuvers to pinpoint the lesion.  This includes giving intrinsic factor, pancreatic enzymes, or antibiotics.
The Schilling test has several shortcomings.  One is it require patient cooperation in collecting the 24 hour urine sample.  As noted above patients can have secondary malabsorption due to vitamin B12 deficiency.  The classic Schilling test will not detect abnormalities in patients with difficulties in disassociating vitamin B12 from food.
Patients with pernicious anemia can be detected by assaying for autoantibodies but these tests can lack diagnostic specificity.  Antibodies to IF are specific but not sensitive and antibodies to parietal cells are sensitive but not specific for pernicious anemia.


Patients with severe megaloblastic anemia need immediate therapy.  One should quickly obtain serum vitamin B12 and red cell folate levels and then give 1-5 mg of folate and 1000 ug IM of vitamin B12.  Patients should be treated daily with folate.  Schedules for vitamin B12 replacement vary but a common approach to all is daily therapy for one week to rapidly build up stores and supply vitamin B12 to tissues, then weekly for a month, then monthly life-long.  Patients with severe anemia should have increased reticulocyte by day three and increased hematocrit by day 5.  Patients with alcoholism and folate deficiency can take up to three weeks to respond to folate therapy.  It used routine to use IM injection to replace vitamin B12.  Oral therapy with 1-2000 ug/day has been tested and has been found to be just as reliable as IM therapy and is becoming more widely used.
Although patients will megaloblastic anemia often present with severe anemia, transfusion therapy is rarely indicated.  Since the anemia is rapidly reversible with therapy there is little justification for exposing the patient to the risk of transfusion except if the patient is having life-threatening symptoms such as severe ischemia.  A further hazard of transfusion is since some of these patients have high-output heart failure, overzealous transfusion may lead to pulmonary edema.

Recently it has become clear the patients can have neurological damage due to vitamin B12 deficiency without anemia.  In fact as many as 30% of patients with neurological disease due to vitamin B12 deficiency will have no or only subtle hematological symptoms.  Patients with the most severe neurological manifestation often have mild hematological disease.  Thus it is appearing that vitamin B12 deficiency may exhibit two different types of disease states in humans - hematological or neurological.  Neurological symptoms are reversible if found early but those present for over a year slowly, if ever, improve.        

The neurological symptoms include:
o   Paresthesias-most often in fingers and toes.  The most common symptom of vitamin B12 deficiency.
o   Diminished vibratory sense
o   Gait ataxia
o   Increases deep tendon reflexes
o   Memory loss
o   Personality change
o   Orthostatic hypotension

On routine screening as many as 10-23% of elderly patients will have low vitamin B12 levels.  One study found that 14.5% had levels below 300 pg/ml with 56% of these patients having increased levels of homocysteine and methylmalonic acid indicative of tissue vitamin B12 deficiency.  The most common mechanism is inability to absorb vitamin B12 from food.  It is speculated the rapid rise in the use of H2 blockers will increase this problem in this patient population. Patients with dementia have lower levels of vitamin B12 then those without but treatment with vitamin B12 is often not effective, perhaps due to the long duration of the neurological damage.  Studies are underway to examine the relationship of vitamin B12 deficiency to neurological disease in the elderly and the effects of early intervention.


Folate Deficiency

Vitamin B12 Deficiency

Most common cause

Dietary deficiency


Time to development



Neurologic abnormalities



Response to treatment with vitamin B12



Response to treatment with folate


Hematologic - Yes
Neurologic - No

Thomas G. DeLoughery, MD FACP
Professor of Medicine, Pathology, and Pediatrics
Oregon Health Sciences University
Portland, Oregon
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