PERNICIOUS ANEMIA AND OTHER MEGALOBLASTIC ANEMIAS. 

Method of
Victor Herbert, M.D., J.D., M.A.C.P., F.R.S.M. (London)
Mount Sinai & Bronx V.A. Medical Centers
New York, NY.

    Megaloblastic (Greek: megalo=giant; blast=germ cell) anemias are those anemias resulting from pathologic slowing, by any cause, of the doubling of the nuclear DNA necessary for bone marrow nucleated cells to divide, with only minor slowing of the synthesis of cytoplasmic RNA.  This nuclear-cytoplasmic dissociation becomes greater with each daughter germ cell division, until either the cells die or omit terminal division, surviving in the marrow as end stage giant red cells, white cells, and platelets.  These are extruded into the peripheral blood as macroovalocytes, hypersegmented neutrophils, and giant platelets, all with a shortened life span, hence the pancytopenia (anemia, leucopenia, and thrombopenia), characteristic of full-blown megaloblastic anemia.   
   
The most common causes of megaloblastic anemia are deficiencies of vitamin B12 (cobalamin) and/or folate (folic acid).  As with any nutrient deficiency, these two B-vitamin deficiencies may arise from any of 3 inadequacies (inadequate ingestion, absorption, or utilization), or any of 3 excesses (excessive requirement, destruction, or excretion).  
   
Pernicious anemia is the descriptor for that insidiously progressive B12 deficiency resulting from genetically predisposed  and age-expressed gastric atrophy, which first produces inability to free B12 from food peptide bonds due to loss of gastric cells secreting acid and digestive enzymes, and eventually results in inability to absorb free crystalline B12 by the physiologic intrinsic factor-dependent mechanism due to loss of gastric cells secreting intrinsic factor.  By age 65, approximately 50% of the U.S. population has low serum holotranscobalamin (holo TC; B12 TC) due to inadequate B12 absorption, and approximately 60% of that 50% has vasculotoxically (and neurotoxically) high serum homocysteine due to B12 deficiency (with low serum holo TC but total serum B12 still >400 pg/ml).  (See Table 1).       
     Because of the above, I recommend that, starting at age 50, everyone should take a daily 100 μg pill of vitamin B12 on arising each morning, a half hour before breakfast, so it does not bind to the breakfast protein and thereby become unabsorbable.  Starting at age 50, everyone should also take a daily pill of 400μg of folic acid (For further details on both these recommendations, see www.victorherbert.com). 

LABORATORY FINDINGS

    Erythrocytic macrocytosis is obvious when the mean cell volume (MCV) is greater than 100 fL. It is easily determined using an automatic electronic particle counter, which gives an accurate and reproducible value for the MCV.  The finding of macrocytic indices diagnoses macrocytosis, not megaloblastosis, which requires visual inspection of the blood smear to determine that the erythrocytes are oval. If they are not oval, but round, the diagnosis is one of the conditions listed in table 2. If they are both oval and round, the diagnosis is most likely vitamin B12 and/or folate deficiency plus one of the conditions listed in table 2. A classic morphologic mess is the anemia of alcoholic liver disease, in which the peripheral blood smear shows the macrocytic target cells of liver disease mixed with the macroovalocytes of folic acid deficiency (alcoholics rarely waste money on fruits and vegetables) and the microcytes of iron deficiency (due to blood loss from G.I. bleeding).  An elevated MCV and macroovalocytosis may precede by months or years the onset of overt anemia.  Megaloblastosis (and macrocytosis) may be masked in the presence of conditions which produces microcytosis, such as chronic infection, inflammatory disease, or iron deficiency.  In all these combined situations, the MDR (range of red cell diameters) is well above normal in both directions.  

TABLE 1.

Sequence of Events in Progression of Inadequate B12 Absorption

1.       Low holotranscobalamin

2.      Loss of short-term memory  (Where did I park my car? What was the name of that person I just met?)

3.      Low (<400 pg/ml) serum B12

4.      Low red cell folate (<140 pg/ml)

5.      Granulocyte hypersegmentation

6.      High homocysteine (>15)

7.      Elevated MCV

8.      Early posterolateral column neuropathy (loss of index toe position sense and 256 vps vibration sense; great toe position sense and 128 vps vibration sense still intact); high methylmalonate

9.      Anemia

    The earliest sign of megaloblastosis reflected in the peripheral blood is hypersegmentation of the polymorphonuclear leukocytes rapidly followed by the appearance of oval macrocytes. Round macrocytes suggest liver disease (they are often žtarget cellsÓ), reticulocytosis (basophilic round macrocytes), and other conditions (see table 2).  A bone marrow examination is useful in cases in which the peripheral blood findings are equivocal, but it (like the peripheral blood) cannot distinguish vitamin B12 deficiency from folic acid deficiency.

TABLE 2.

Differential Diagnosis of Macrocytosis

Actual Macrocytosis

Liver disease

Reticulocytosis

Myeloproliferativediseases(leukemia, myelofibrosis)

Hypothyroidism

Aplastic anemia

Drugs (post chemotherapy, alcohol)

Spurious Macrocytosis

Autoagglutination/cold agglutination disease

 

DIAGNOSTIC WORKUP  

    The initial laboratory evaluation for cobalamin deficiency should always include a complete blood cell count with examination of the peripheral smear.  Total serum cobalamin, serum holotranscobalamin (holo TC; B12 TC), and serum and red cell folate levels should simultaneously be obtained.  The diagnosis of inadequate cobalamin absorption is made from the finding of low holo TC, with or without low (<400 pg/ml) total serum cobalamin. Serum folate levels will be normal to high, and red cell folate normal to low.   
    Holo TC is a surrogate Schilling test; if low it means inadequate B12 absorption.  A bone marrow examination which shows megaloblastic maturation can be useful if the peripheral blood picture is equivocal.  Before serum holo TC was commercially available, we used a Schilling test to demonstrate inadequate vitamin B12 absorption. This test determines cobalamin absorption by quantitating the radioactivity in the 24-hour urine collection after an oral dose of radio-labeled cobalamin.  At zero time, a parenteral loading dose of 1000μg cobalamin (vitamin B12) is administered to bind to all blood and tissue binding sites so that when the radioactive cobalamin is absorbed it has nowhere to bind, and so goes out in the urine. Since this loading dose corrects the laboratory and hematologic evidence of B12 deficiency, blood must be drawn for these studies before the loading dose of B12 is given.  Measurement of serum levels of methylmalonic acid and total homocysteine may also be useful in equivocal cases.  These amino acids are increased in cobalamin-deficient states because enzymes responsible for their conversion are cobalamin dependent.  These amino acid levels may be elevated before the falling total serum cobalamin and diagnostic blood and bone marrow morphologic findings are obvious.   Confounding variables are that renal disease elevates methylmalonate and homocysteine elevation occurs equally in B12 and folate deficiency.  

TREATMENT  
Cobalamin Deficiency  
(Pernicious Anemia)
 

    Therapy is initially directed at rapidly reversing the sequelae of the deficiency, such as the neurologic abnormalities and anemia, and subsequently at maintenance of the remission and prevention of relapse.  Without initial and maintenance treatment, the patient develops progressive neurologic disease (cognitive dysfunction, peripheral neuropathy, and subacute combined degeneration of the spinal cord) and further hematologic damage (severe pancytopenia).  

 Initial Treatment  

    The aim is to rapidly correct the deficiency and to replenish the stores.  Because the disorder is caused by cobalamin malabsorption in most cases, parenteral cobalamin supplementation with large doses (100 to 1000 μg B12) by deep subcutaneous or intramuscular routes given immediately after blood is drawn for the diagnostic tests.  Up to 80% of such large doses are excreted in the urine; so my initial therapy is with several large doses (100 to 1000μg) of cobalamin injected 1 to 2 days apart, which replenish stores about as rapidly as daily injections.  I then administer 100-μg injections of cobalamin weekly for a month, and monthly thereafter.  Patients or family members can be taught to inject the vitamin deep subcutaneously in the outer aspect of the upper arm themselves, using a 25 gauge needle and a 1 ml insulin syringe. Injections must be monthly for life, or, after 2 months, oral therapy with 100μg daily for life. 
    Administration of cobalamin is complete therapy for uncomplicated deficiency. Because the underlying cause of the cobalamin deficiency cannot usually be corrected, therapy is generally lifelong and the patient must be vigorously informed that therapy must never be stopped. 
    The clinical response to initial therapy is often dramatic. Decades ago, we produced rapid reversal of pancytopenia and oral mucosal changes with minimal parenteral doses as low as 1μg, since we had found the minimal daily absorbed requirement of vitamin B12 to sustain normality is only 0.1μg.  The patients often describe an immediate sense of well-being, and within a week the mucosal changes reverse.  Hematologic response is noted within 3 days with a sharp reticulocytosis, and improvement in the hemoglobin 5 to 7 days after treatment.  Disappearance of macroovalocytic red cells, correction of the MCV, and loss of hypersegmentation may take several weeks.  During initial therapy, it is advisable to monitor daily serum potassium levels for at least 4 days because rejuvenation of new cells may produce dangerous hypokalemia owing to movement of plasma potassium into the rapidly proliferating hematopoietic cells. Patients who are on diuretic therapy and have an initial hypokalemia, patients with a history of cardiac disease, and those on digitalis therapy should be especially carefully monitored.  Potassium, in a dose of 40 mEq/d, should be administered promptly to patients manifesting hypokalemia.          Coexistent iron deficiency or marginal bone marrow iron stores can limit the speed and completeness of recovery and should be treated with 300 mg of ferrous sulfate three times a day. 
   
As stated above, the second aim of vitamin replacement therapy is to maintain the vitamin stores so that relapse does not occur.  Good clinical remission can be maintained even with partial repletion of the stores. After initial repletion of the stores and in situations in which the underlying malabsorption is irreversible as in pernicious anemia, lifelong maintenance must be given. (Monthly injections of 100 μg of cobalamin, or daily oral 100 μg). Cyanocobalamin is the most widely used, shelf-stable, and inexpensive form of vitamin B12 available in the United States.  Another parenteral preparation, hydroxocobalamin, which is a little less shelf-stable, is preferred in Great Britain. It is more physiologic (the body has to convert cyano to hydroxo before using it) and has a longer biologic half-life after injection than cyanocobalamin. 
    Although patients can be instructed in self-administration of the injections and the oral therapy, they should still be followed (if no acute situation develops) at least twice a year by a physician. I see my patients twice a year, at which time they have a general examination, and complete blood studies as when first seen.  Clinical evaluation also includes three serial stool guaiac determinations because there is a 2% to 3% incidence of gastric carcinoma and a higher incidence of gastric carcinoid tumors (usually benign) in patients with pernicious anemia. Laboratory evaluation of thyroid status in patients with suggestive symptoms is also performed, because there is an association between pernicious anemia and autoimmune thyroid disorders.
       

   Pernicious anemia itself has autoimmune components, including circulating antibody to parietal cells and to intrinsic factor; these 2 antibodies should be sought on the initial blood sample and each 6-month follow-up.  Circulating antibody to H. pylori should also be sought.  
   
The need for continuous monitoring should be stressed to these patients because it is essential they understand that lifelong therapy is mandatory to prevent recurrence.  Proper patient education requires reinforcement of this fact, at each 6-month visit, inasmuch as relapse in pernicious anemia is not uncommon, with many patients who discontinue therapy not coming back until they have developed irreversible neurologic damage.  In the 60Ūs we published that these are usually patients who got 1000 μg monthly, got no žliftÓ from the monthly injection, and concluded they are žjust being ripped offÓ for unnecessary doctor visits and shots, but no longer need them. Patients who get a monthly injection of 100 μg get a perceptible euphoric žliftÓ from the injection, so they žknow it is workingÓ and religiously come back for their next monthly injection.
 

Other Cobalamin Deficiency States  

    Treatment of nonpernicious vitamin B12 deficiency anemia obviously depends on the underlying cause.  Patients who have had their source of intrinsic factor removed, such as those who have had a gastrectomy, require lifelong therapy. My program for these patients and for those who have had an ileal resection or have regional enteritis (the ileum is the site of physiologic [IF-dependent] B12 absorption) is identical to the management of patients with pernicious anemia. Vegans who are deficient in vitamin B12 because of negligible intake (such as vegans) can usually be adequately treated with small dose oral cyanocobalamin supplements or food fortificants.  Doses of 25 μg daily are sufficient, but I use and recommend 100 μg daily because some vegans go on to gastric atrophy. Alternatively, these patients may be given injections of cyanocobalamin every 3 months.  Follow-up of serum levels of holo TC and of serum total B12 every 6 months is desirable.   

Folic Acid Deficiency            

The hematologic findings of folic acid deficiency are essentially identical to those of B12 deficiency. These patients have no neurologic symptoms except for loss of recent (where did I park my car?) memory, and that is due to slowed synthesis of the DNA of the new brain cells we make each day of our lives to store new information which came in that day.

The laboratory hematology findings are also essentially identical to those found in pernicious anemia, with the complete blood cell count, peripheral blood smear, and bone marrow examination being indistinguishable from those in cobalamin deficiency states. The diagnosis is made by determining low serum and red cell folate levels.  Caveat: serum folate levels can be normal in the presence of low red cell folate stores immediately after a high dietary folate intake; this is why red blood cell folate is more sensitive for assessment of chronic folic acid deficiency.  Serum folate represents folate intake in the last 24 hours; red cell folate reflects folate intake over the last 4 months (the red cell life span).  
    Treatment of folic acid deficiency, which usually occurs as a result of decreased dietary intake, is aimed at reversal of the initial effects of the deficiency, replenishment of folate stores, and then maintenance of sufficient dietary intake to ensure adequate folate nutrition.   
    Folate absorption occurs primarily in the upper half of the small intestine. Megaloblastic anemia from folic acid deficiency responds readily to daily oral folate doses as low as 100 to 200 μg of PGA (pteroylglutamic acid).  In general, however, 400 μg to 1000μg (1mg) is administered and is usually sufficient to correct deficiency, even in malabsorption states.  My general approach is to give 1 mg daily for the first week, except in situations of severe malabsorption, when I administer 2 to 5 mg daily for the first week.  I ensure that cobalamin deficiency is not coexistent and may initially co-administer 100 μg of cobalamin. After the first week, I treat with 100 μg orally daily. Full replenishment of folate stores can be achieved within several weeks of oral therapy.  Maintenance should be with 400μg folic acid orally daily.  If the underlying cause can be reversed, maintenance therapy can be stopped. 
    Adverse effects can occur from folate treatment with 1 or more mg of PGA daily. This is because at such doses substantial amounts of PGA, which is an oxidized, shelf-stable and thus preferred pharmaceutical form of folic acid, are absorbed by diffusion without being first reduced as is desirable by alimentary tract enzymes to human-cell-usable tetrahydrofolic acid. This absorbed unreduced PGA then blocks cell uptake of tetrahydrofolate, and can actually produce folate deficiency, as Prof. John ScottŪs group at Trinity Medical College in Dublin, Ireland showed in a large and excellent study of fertile Irish females.  Also, giving folic acid to cobalamin-deficient patients will conceal the hematologic abnormalities of the B12 deficiency while allowing subacute combined degeneration of the spinal cord to proceed untreated to irreversibility. One must therefore always look for coexistent vitamin B12 deficiency in all patients for whom folate therapy is proposed. 
    A good understanding as to the need for folate replacement must be taught to every patient with nutritional deficiency. Patients should be educated about which foods contain the best sources of folate, such as green leafy vegetables, and about the fact that folic acid is a heat-sensitive vitamin and can easily be destroyed by overcooking or boiling.   
    In pregnancy, folic acid deficiency can occur due to the enhanced requirement by both mother and fetus. Prevention and treatment of megaloblastic anemia of pregnancy can be accomplished with 400 μg of folic acid orally daily administered from day 1 of pregnancy throughout pregnancy, and during the period of lactation.  
In alcoholics, poor nutrition is compounded by poor compliance and by the fact that alcohol per se is an antagonist to biologic folate cofactors.  I treat these patients with doses of 1 mg PGA daily, since compliance is the major limiting factor and the potential for adverse effects of up to 1 mg PGA daily in them is remote. 
V.H. CV #839       

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