Linda H. Goossen
Physiologic Roles of Vitamin B12 and Folate
Defect in Megaloblastic Anemia Due to Deficiency in Folate and Vitamin B12
Other Causes of Megaloblastosis
Systemic Manifestations of Folate and Vitamin B12 Deficiency
Causes of Vitamin Deficiencies
Vitamin B12 Deficiency
Specific Diagnostic Tests
Macrocytic Nonmegaloblastic Anemias
After completion of this chapter, the reader will be able to:
1. Discuss the relationships among macrocytic anemia, megaloblastic anemia, and pernicious anemia, and classify anemias appropriately within these categories.
2. Discuss the physiologic roles of folate and vitamin B12 in DNA production and the general metabolic pathways in which they act.
3. Describe the absorption and distribution of vitamin B12, including carrier proteins and the biologic activity of various vitamin-carrier complexes.
4. Describe the biochemical basis for development of anemia with deficiencies of vitamin B12 and folate, and explain the cause of the accompanying megaloblastosis.
5. Recognize individuals at risk for megaloblastic anemia by virtue of age, dietary habits, or physiologic circumstance such as pregnancy, drug regimens, or pathologic conditions.
6. Recognize complete blood count, reticulocyte count, red and white blood cell morphologies, and bone marrow findings consistent with megaloblastic anemia.
7. Given the results of tests measuring levels of serum vitamin B12, serum methylmalonic acid, serum folate, plasma or serum homocysteine, and antibodies to intrinsic factor, determine the likely cause of a patient’s deficiency.
8. Recognize results of bilirubin and lactate dehydrogenase tests that are consistent with megaloblastic anemia and explain why the test values are elevated in this condition.
After studying the material in this chapter, the reader should be able to respond to the following case study:
During a holiday visit, the children of a 76-year-old man noticed that he seemed more forgetful than usual and that he had difficulty walking. Concerned about the possibility of a mild stroke, the children insisted that he see his physician. The physician diagnosed a peripheral neuropathy affecting the father’s ability to walk. In addition, the physician noted that he was pale and slightly jaundiced and ordered routine hematologic studies. The results were as follows:
WBC differential: unremarkable with the exception of hypersegmentation of neutrophils
RBC morphology: moderate anisocytosis, moderate poikilocytosis, macrocytes, oval macrocytes, few teardrop cells
Because of these findings, additional tests were ordered, with the following results:
• Serum vitamin B12: decreased
• Serum folate: within reference interval
• Serum methylmalonic acid: increased
1. Which of the CBC findings led the physician to order the vitamin assays?
2. Is the patient’s reticulocyte response adequate to compensate for the anemia?
3. Based on the available test results, what can you conclude about the cause of the patient’s anemia?
4. What additional testing would be helpful to diagnose the specific cause of this patient’s anemia?
Impaired deoxyribonucleic acid (DNA) metabolism causes systemic effects by impairing production of all rapidly dividing cells of the body. These are chiefly the cells of the skin, the epithelium of the gastrointestinal tract, and the hematopoietic tissues. Because these all must be replenished throughout life, any impairment of cell production is evident in these tissues first. Patients may experience symptoms in any of these systems, but the blood provides a ready tissue for analysis. The hematologic effects, especially megaloblastic anemia, have come to be recognized as the hallmark of the diseases affecting DNA metabolism.
The root cause of megaloblastic anemia is impaired DNA synthesis. The anemia is named for the very large cells of the bone marrow that develop a distinctive morphology (see section on laboratory diagnosis) due to a reduction in the number of cell divisions. Megaloblastic anemia is one example of a macrocytic anemia. shows the classification of macrocytic anemias. Understanding the etiology of megaloblastic anemia requires a review of DNA synthesis with particular attention to the roles of vitamin BBox 21-112 (cobalamin) and folic acid (folate).
Classification of Macrocytic Anemias by Cause
Impaired absorption (e.g., inflammatory bowel disease)
Impaired use due to drugs
Excessive loss with renal dialysis
Vitamin B12 deficiency
Failure to split from food
Failure to split from haptocorrin
Lack of intrinsic factor
Helicobacter pylori infection
Hereditary intrinsic factor deficiency
General malabsorption (e.g., inflammatory bowel disease)
Inherited errors in absorption or transport
Competition for the vitamin
Diphyllobothrium latum (fish tapeworm) infection
Blind loop syndrome
Other causes of megaloblastosis
Acute erythroid leukemia
Congenital dyserythropoietic anemia
Reverse transcriptase inhibitors
Macrocytic, nonmegaloblastic anemias
Normal newborn status
Bone marrow failure
Physiologic roles of vitamin B12 and folate
Vitamin B12 (cobalamin) is an essential nutrient consisting of a tetrapyrrole (corrin) ring containing cobalt that is attached to 5,6-dimethylbenzimidazolyl ribonucleotide (Figure 21-1). Vitamin B12 is a coenzyme in two biochemical reactions in humans. One is isomerization of methylmalonyl coenzyme A (CoA) to succinyl CoA, which requires vitamin B12 (in the adenosylcobalamin form) as a cofactor and is catalyzed by the enzyme methylmalonyl CoA mutase (Figure 21-2). In the absence of vitamin B12, the impaired activity of methylmalonyl CoA mutase leads to a high level of serum methylmalonic acid (MMA), which is useful for the diagnosis of vitamin B12 deficiency (discussed in the section on laboratory diagnosis). The second reaction is the transfer of a methyl group from 5-methyltetrahydrofolate (5-methyl THF) to homocysteine, which thereby generates methionine. This reaction is catalyzed by the enzyme methionine synthase and uses vitamin B12 (in the methylcobalamin form) as a coenzyme (discussed later in this section). Methylcobalamin is synthesized through reduction and methylation of vitamin B12. This reaction represents the link between folate and vitamin B12 coenzymes and appears to account for the requirement for both vitamins in normal erythropoiesis.1, 2
FIGURE 21-1 Structure of vitamin B12 (cobalamin) and its analogs, hydroxycobalamin and cyanocobalamin (forms often found in food and supplements) and methylcobalamin and 5′-deoxyadenosylcobalamin (coenzyme forms). The basic structure of cobalamin includes a tetrapyrrole (corrin) ring with a central cobalt atom linked to 5,6-dimethylbenzimidazolyl ribonucleotide. Source: (From Scott JM, Browne P: Megaloblastic anemia. In Caballero B, Allen L, Prentice A, editors: Encyclopedia of human nutrition, ed 2, Oxford, 2006, Academic Press, p 113.)
FIGURE 21-2 Role of vitamin B12 in the metabolism of methylmalonyl coenzyme A (CoA). Vitamin B12, in the 5′-deoxyadenosylcobalamin form, is a coenzyme in the isomerization of methylmalonyl CoA to succinyl CoA. The reaction is catalyzed by the enzyme methylmalonyl CoA mutase.
Folate is the general term used for any form of the vitamin folic acid. Folic acid is the synthetic form in supplements and fortified food. Folates consist of a pteridine ring attached to para-aminobenzoate with one or more glutamate residues (Figure 21-3). The function of folate is to transfer carbon units in the form of methyl groups from donors to receptors. In this capacity, folate plays an important role in the metabolism of amino acids and nucleotides. Deficiency of the vitamin leads to impaired cell replication and other metabolic alterations. Folate circulates in the blood predominantly as 5-methyl THF.3 5-Methyl THF is metabolically inactive until it is demethylated to tetrahydrofolate (THF), whereupon folate-dependent reactions may take place.
FIGURE 21-3 Structure of synthetic folic acid and the naturally occurring forms of the vitamin. Source: (From Scott JM, Browne P: Megaloblastic anemia. In Caballero B, Allen L, Prentice A, editors: Encyclopedia of human nutrition, ed 2, Oxford, 2006, Academic Press, p 114.)
Folate has an important role in DNA synthesis. As seen in , within the cytoplasm of the cell, a methyl group is transferred from 5-methyl THF to homocysteine, which converts it to methionine and generates THF. This reaction is catalyzed by the enzyme methionine synthase and requires vitamin BFigure 21-412 in the form of methylcobalamin as a cofactor. THF is then converted to 5,10-methylenetetrahydrofolate (5,10-methylene THF); the methyl group for this reaction comes from serine as it is converted to glycine. The methyl group of 5,10-methylene THF is then transferred to deoxyuridine monophosphate (dUMP), which converts it to deoxythymidine monophosphate (dTMP). This reaction is catalyzed by thymidylate synthase and results in the conversion of 5,10-methylene THF to dihydrofolate (DHF). Deoxythymidine monophosphate is a precursor to deoxythymidine triphosphate (dTTP), which, like the other nucleotide triphosphates, is a building block of the DNA molecule. THF is regenerated by the conversion of DHF to THF by the enzyme dihydrofolate reductase. Because some of the folate is catabolized during the cycle, the regeneration of THF also requires additional 5-methyl THF from the plasma. Once in the cell, folate is rapidly polyglutamated by the addition of one to six glutamic acid residues. This conjugation is required for retention of THF in the cell, and it also promotes attachment of folate to enzymes.4
FIGURE 21-4 Role of folate and vitamin B12 in DNA synthesis. Folate enters the cell as 5-methyltetrahydrofolate (5-methyl THF). In the cell, a methyl group is transferred from 5-methyl THF to homocysteine, converting it to methionine and generating tetrahydrofolate (THF). This reaction is catalyzed by methionine synthase and requires vitamin B12 as a cofactor. THF is then converted to 5,10-methylene THF by the donation of a methyl group from serine. The methyl group of 5,10-methylene THF is then transferred to deoxyuridine monophosphate (dUMP), which converts it to deoxythymidine monophosphate (dTMP) and converts 5,10-methylene THF to dihydrofolate (DHF). This reaction is catalyzed by thymidylate synthase. dTMP is a precursor of deoxythymidine triphosphate (dTTP), which is used to synthesize DNA. THF is regenerated by the conversion of DHF to THF by the enzyme dihydrofolate reductase. A deficiency of vitamin B12 prevents the production of THF from 5-methyl THF; as a result, folate becomes metabolically trapped as 5-methyl THF. This constitutes the “folate trap.”
Defect in megaloblastic anemia due to deficiency in folate and vitamin B12
When either folate or vitamin B12 is missing, thymidine nucleotide production for DNA synthesis is impaired. Folate deficiency has the more direct effect, ultimately preventing the methylation of dUMP. The effect of vitamin B12 deficiency is more indirect, preventing the production of THF from 5-methyl THF. When vitamin B12 is deficient, progressively more and more of the folate becomes metabolically trapped as 5-methyl THF. This constitutes what has been called the folate trap as 5-methyl THF accumulates and is unable to supply the folate cycle with THF. Some 5-methyl THF also leaks out of the cell if it is not readily polyglutamated. This results in a decrease in intracellular folate.5 In addition, when either folate or vitamin B12 is deficient, homocysteine accumulates because methionine synthase is unable to convert it to methionine without vitamin B12 as a cofactor (see Figure 21-4).
In this state of diminished thymidine availability, uridine is incorporated into DNA.6, 7 The DNA repair process can remove the uridine, but without available thymidine, the repair process is unsuccessful. Although the DNA can unwind and replication can begin, at any point where a thymidine nucleotide is needed, there is essentially an empty space in the replicated DNA sequence, which results in many single-strand breaks. When excisions at opposing DNA strand sites coincide, double-strand breaks occur. Repeated DNA strand breaks lead to fragmentation of the DNA strand.4 The resulting DNA is nonfunctional, and the DNA replication process is incomplete. Cell division is halted, resulting in either cell lysis or apoptosis8 of many erythroid progenitor cells within the bone marrow. Cells that survive continue the abnormal maturation with a fewer number of red blood cells (RBCs) released into the circulation. This abnormal blood cell development is called ineffective hematopoiesis. The dependency of DNA production on folate has been used in cancer chemotherapy (Box 21-2).
Disruption of the Folate Cycle in Cancer Chemotherapy
Folate has a complex relationship with cancer. Folate deficiency leads to DNA strand breaks, which leave the DNA vulnerable to mutation. In this way, folate deficiency is a risk factor for the initiation of cancer. The central role of folate in cell division also makes it a target for chemotherapeutic drugs used to treat cancer. Folate analogues can be used to compete for folate in DNA production and result in impaired cell division. The cells in the cell cycle, such as cancer cells and other normally rapidly dividing cells such as epithelium and blood cells, are most susceptible to the drug interference. Methotrexate, used in the treatment of leukemia and arthritis, is an example of a folate antimetabolite drug. Methotrexate has a higher affinity for dihydrofolate reductase than does tetrahydrofolate. Thus methotrexate enters the folate cycle in preference to tetrahydrofolate, and the folate cycle is blocked by the drug. Methotrexate treatment typically is followed by what is known as leucovorin rescue. Leucovorin is a folic acid derivative that can be administered to counteract the effects of methotrexate or other folate antagonists.
In addition to the increased apoptosis of erythroid progenitor cells in the bone marrow discussed above, the remaining erythroid cells are larger than normally seen during the final stages of erythropoiesis, and their nuclei are immature-appearing compared with the cytoplasm. In contrast to the normally dense chromatin of comparable normoblasts, the nuclei of megaloblastic erythroid precursors have an open, finely stippled, reticular pattern.5 The nuclear changes seen in the megaloblastic cells are related to cell cycle delay, prolonged resting phase, and arrest in nuclear maturation. Electron microscopy has revealed that reduced synthesis of histones is also responsible for morphologic changes in the chromatin of megaloblastic erythroid precursors.9 Ribonucleic acid (RNA) function is not affected by vitamin B12 or folate deficiency because RNA contains uracil instead of thymidine nucleotides, so cytoplasmic development progresses normally. The slower maturation rate of the nucleus compared with the cytoplasm is callednuclear-cytoplasmic asynchrony. Together, the accumulation of cells with nuclei at earlier stages of development and cells with increased size and immaturity result in the appearance of erythroid cells in the bone marrow that are pathognomonic of megaloblastic anemia.8 Because ineffective hematopoiesis affects all three blood cell lineages, pancytopenia is also evident, with certain distinctive cellular changes (see section on laboratory diagnosis).
Other causes of megaloblastosis
Vitamin B12 and folate deficiency are not the only causes of megaloblastic erythrocytes. Dysplastic erythroid cells in myelodysplastic syndrome (MDS) can also have megaloblastoid features (Chapter 34). In MDS, however, the macrocytic erythrocytes and their progenitors characteristically show delayed cytoplasmic and nuclear maturation, including cytoplasmic vacuole formation, nuclear budding, multinucleation, and nuclear fragmentation, and thus may be distinguished from the megaloblastic RBCs seen in the vitamin deficiencies. In addition, nuclear-cytoplasmic asynchrony and megaloblastic RBCs may be seen in congenital dyserythropoietic anemia (CDA) types I and III (Chapter 22). The CDAs are rare conditions that usually manifest in childhood and may be distinguished from the acquired causes of megaloblastosis by clinical history and morphologic differences. In CDA I, internuclear chromatin bridging of erythroid cells or binucleated forms are observed, and in CDA III, giant multinucleated erythroblasts are present. Another rare condition in which RBC precursors have a megaloblastic appearance is acute erythroid leukemia, previously classified as FAB M6 (Chapter 35). In this condition, the cells are macrocytic, and the immature appearance of the nuclear chromatin is similar to the more open appearance of the chromatin in megaloblasts. There are usually other aberrant findings in erythroid leukemia, including an increase of myeloblasts in the bone marrow; however, an experienced morphologist can discern the subtle differences. Reverse transcriptase inhibitors, used to treat human immunodeficiency virus (HIV) infections, interfere with DNA production and may also lead to megaloblastic changes.10
Although the conditions described in this section are characterized by megaloblastic morphology, they are due to acquired or inherited mutations in progenitor cells or interference with DNA synthesis and are refractive to therapy with vitamin B12 or folic acid.
Systemic manifestations of folate and vitamin B12 deficiency
When DNA synthesis and subsequent cell division are impaired by lack of folate or vitamin B12, megaloblastic anemia and its systemic manifestations develop. With either vitamin deficiency, patients may experience general symptoms related to the anemia (fatigue, weakness, and shortness of breath) and symptoms related to the alimentary tract. The loss of epithelium on the tongue results in a smooth surface and soreness (glossitis). Loss of epithelium along the gastrointestinal tract can result in gastritis, nausea, or constipation.
Although the blood pictures seen with the two vitamin deficiencies are indistinguishable, the clinical presentations vary. In vitamin B12 deficiency, neurologic symptoms may be pronounced and may even occur in the absence of anemia.5 These include memory loss, numbness and tingling in toes and fingers, loss of balance, and further impairment of walking by loss of vibratory sense, especially in the lower limbs.11Neuropsychiatric symptoms may also be present, including personality changes and psychosis. These symptoms seem to be the result of demyelinization of the spinal cord and peripheral nerves, but the relationship of this demyelinization to vitamin B12 deficiency is unclear. The roles of increases in tumor necrosis factor-α, a neurotoxic agent, and decreases in epidermal growth factor, a neurotrophic agent, in the development of neurologic symptoms in vitamin B12-deficient patients are being researched.12, 13
At one time, folate deficiency was believed to be more benign clinically than vitamin B12 deficiency. Later research suggested that low levels of folate and the resulting high homocysteine levels were risk factors for cardiovascular disease.14 More recent research has provided mixed results, with studies both refuting this association15, 16 as well as substantiating the association between high circulating homocysteine levels and the risk of cardiovascular disease.17 Several studies suggest that high folate levels provide a cardioprotective effect in diabetic patients and certain ethnic populations.15, 18, 19 The evidence at this time is unclear as to whether persistent suboptimal folate status may have a significant long-term health impact. In addition, there is evidence of depression, peripheral neuropathy, and psychosis related to folate deficiency. Folate levels appear to influence the effectiveness of treatments for depression.23 Folate deficiency during pregnancy can result in impaired formation of the fetal nervous system, resulting in neural tube defects such as spina bifida,24 despite the fact that the fetus accumulates folate at the expense of the mother. Pregnancy requires a considerable increase in folate to fulfill the requirements related to rapid fetal growth, uterine expansion, placental maturation, and expanded blood volume.3 Insuring adequate folate levels in women of childbearing age is particularly important because many women are likely to be unaware of their pregnancy during the first crucial weeks of fetal development. Fortification of the U.S. food supply with folic acid in grain and cereal products was mandated by the Food and Drug Administration in 1998 to lower the risk of neural tube defects in the unborn.
Causes of vitamin deficiencies
In general, vitamin deficiencies may arise because the vitamin is in relatively short supply, because use of the vitamin is impaired, or because of excessive loss. Folate deficiency can be caused by all of these mechanisms.
Folate is synthesized by microorganisms and higher plants. Folate is ubiquitous in foods, but a generally poor diet can result in deficiency. Good sources of folate include leafy green vegetables, dried beans, liver, beef, fortified breakfast cereals, and some fruits, especially oranges.3, 25 Folates are heat labile, and overcooking of foods can diminish their nutritional value.3
Increased need for folate occurs during pregnancy and lactation when the mother must supply her own needs plus those of the fetus or infant. Infants and children also have increased need for folate during growth.3
Food folates must be hydrolyzed in the gut before absorption in the small intestine; however, only 50% of what is ingested is available for absorption.3 A rare autosomal recessive deficiency of a folate transporter protein (PCFT) severely decreases intestinal absorption of folate.5, 26 Once across the intestinal cell, most folate is transported in the plasma as 5-methyl THF unbound to any specific carrier.11 Its entry into cells, however, is carrier mediated.27
Folate absorption may also be impaired by intestinal disease, especially sprue and celiac disease. Sprue is characterized by weakness, weight loss, and steatorrhea (fat in the feces), which is evidence that the intestine is not absorbing food properly. It is seen in the tropics (tropical sprue), where its cause is generally considered to be overgrowth of enteric pathogens.28 Celiac disease (nontropical sprue) has been traced to intolerance of the gluten in some grains28 (gluten-induced enteropathy) and can be controlled by eliminating wheat, barley, and rye products from the diet. Surgical resection of the small intestine and inflammatory bowel disease can also decrease folate absorption.
Impaired use of folate
Numerous drugs impair folate metabolism ().Box 21-329, 30 Antiepileptic drugs are particularly known for this,31 and the result is macrocytosis with frank megaloblastic anemia. In most instances, folic acid supplementation is sufficient to override the impairment and allow the patient to continue therapy.32 Because folate deficiency results in inhibition of cell replication, several anticancer drugs, including methotrexate, are folate inhibitors.3
Some Drugs That May Lead to Impaired Use of Folate
Impair folate metabolism
• Methotrexate (Trexall): antiarthritic, chemotherapeutic
• 5-Fluorouracil (Adrucil): chemotherapeutic
• Hydroxyurea (Hydrea): antimetabolite
• Pyrimethamine (Daraprim): antibacterial
• Pentamidine (Pentam): antimicrobial
• Phenytoin (Dilantin): anticonvulsant
• Trimethoprim (Primsol): antimicrobial
Impair folate absorption
• Metformin (Glucophage): oral antidiabetic
• Cholestyramine (Questran): cholesterol lowering
Excessive loss of folate
Physiologic loss of folate occurs through the kidney. The amount is small and not a cause of deficiency. Patients undergoing renal dialysis lose folate in the dialysate, however; thus supplemental folic acid is routinely provided to these individuals to prevent megaloblastic anemia.11
Vitamin B12 deficiency
While true dietary deficiency of vitamin B12 is rare, this condition is possible for strict vegetarians (vegans) who do not eat meat, eggs, or dairy products. Although it is an essential vitamin for animals, plants cannot synthesize vitamin B12, and it is not available from vegetable sources. The best dietary sources are animal products such as liver, dairy products, fish, shellfish, and eggs.33 In contrast to the heat-labile folate, vitamin B12 is not destroyed by cooking.
Increased need for vitamin B12 occurs during pregnancy, lactation, and growth. Due to the vigorous cell replication, what would otherwise be a diet adequate in vitamin B12 can become inadequate during these periods.
Vitamin B12 in food is released from food proteins primarily in the acid environment of the stomach, aided by pepsin, and is subsequently bound by a specific salivary protein, haptocorrin, also known as R proteinor transcobalamin I (Figure 21-5). In the small intestine, vitamin B12 is released from haptocorrin by the action of pancreatic proteases, including trypsin. It is then bound by intrinsic factor, which is produced by the gastric parietal cells. Vitamin B12 binding to intrinsic factor is required for absorption by ileal enterocytes that possess receptors for the complex. These receptors are cubilin-amnionless complex, collectively known as cubam, which binds the vitamin B12–intrinsic factor complex, and megalin, a membrane transport protein.5, 33-36 Once in the enterocyte, the vitamin B12 is then freed from intrinsic factor and bound to transcobalamin (previously called transcobalamin II) and released into the circulation. In the plasma, only 10% to 30% of the vitamin B12 is bound to transcobalamin; the remaining 75% is bound to transcobalamin I and III, referred to as the haptocorrins.33, 37 The vitamin B12–transcobalamin complex, termed holotranscobalamin (holoTC), is the metabolically active form of vitamin B12. Holotranscobalamin binds to specific receptors on the surfaces of many different types of cells and enters the cells by endocytosis, with subsequent release of vitamin B12 from the carrier.38 The body maintains a substantial reserve of absorbed vitamin B12 in hepatocytes.4
FIGURE 21-5 Normal absorption of vitamin B12. Dietary vitamin B12 (cobalamin, CBL) is food protein (P) bound. In the stomach, pepsin and hydrochloric acid (HCl) secreted by parietal cells release CBL from P. CBL then binds haptocorrin (R protein, R), released from salivary glands, and remains bound until intestinal pancreatic proteases, including trypsin, catalyze its release. Parietal cells secrete intrinsic factor (IF), which binds CBL in the duodenum. Cubilin-amnionless (cubam) and megalin receptors in ileal enterocytes bind CBL-IF and release the CBL. Enterocytes produce transcobalamin (TC), which binds CBL and transports it through the portal circulation. Bone marrow pronormoblast membrane TC receptors (TC-R) bind CBL-TC and release the CBL, which is converted to methylcobalamin (methyl-CBL). Methyl-CBL is a coenzyme that supports homocysteine-methionine conversion. Hepatocyte TC-R receptors bind CBL-TC and release the CBL, which is moved to storage organelles or excreted through the biliary system.
The absorption of vitamin B12 can be impaired by (1) failure to separate vitamin B12 from food proteins in the stomach, (2) failure to separate vitamin B12 from haptocorrin in the intestine, (3) lack of intrinsic factor, (4) malabsorption, and (5) competition for available vitamin B12.
Failure to separate vitamin B12 from food proteins.
A condition known as food-cobalamin malabsorption is characterized by hypochlorhydria and the resulting inability of the body to release vitamin B12 from food or intestinal transport proteins for subsequent binding to intrinsic factor. Food-cobalamin malabsorption is caused primarily by atrophic gastritis or atrophy of the stomach lining that often occurs with increasing age.39 Because histamine 2 receptor blockers and proton pump inhibitors lower gastric acidity, the long-term use of these drugs for the treatment of ulcers and gastroesophageal reflux disease, and gastric bypass surgery also induce food-cobalamin malabsorption.39
Failure to separate vitamin B12 from haptocorrin.
Lack of gastric acidity or lack of trypsin as a result of chronic pancreatic disease can prevent vitamin B12 absorption because the vitamin remains bound to haptocorrin in the intestine and unavailable to intrinsic factor.11
Lack of intrinsic factor.
Lack of intrinsic factor constitutes a significant cause of impaired vitamin B12 absorption. It is most commonly due to autoimmune disease, as in pernicious anemia, but can also result from the loss of parietal cells with Helicobacter pylori infection, total or partial gastrectomy, or hereditary intrinsic factor deficiency.
Pernicious anemia is an autoimmune disorder characterized by impaired absorption of vitamin B12 due to a lack of intrinsic factor.40 This condition is called pernicious anemia because the disease was fatal before its cause was discovered. The incidence per year is roughly 25 new cases per 100,000 persons older than 40 years of age.5 Pernicious anemia most often manifests in the sixth decade or later, but can also be found in children. Patients with pernicious anemia have an increased risk of developing gastric tumors.4
In pernicious anemia, autoimmune lymphocyte-mediated destruction of gastric parietal cells severely reduces the amount of intrinsic factor secreted in the stomach. Pathologic CD4 T cells inappropriately recognize and initiate an autoimmune response against the H+/K+–adenosine triphosphatase embedded in the membrane of the parietal cells.41 A chronic inflammatory infiltration follows, which extends into the wall of the stomach.40 Over a period of years and even decades, there is progressive development of atrophic gastritis resulting in the loss of the parietal cells with their secretory products, H+ and intrinsic factor. The loss of H+ production in the stomach constitutes achlorhydria. Low gastric acidity was previously an important diagnostic criterion for pernicious anemia. Serum gastrin levels can be markedly elevated due to the gastric achlorhydria.4 The absence of intrinsic factor can also be detected using the Schilling test. However, because the test requires a 24-hour urine collection and the use of radioactive cobalt in vitamin B12 to trace absorption, safer diagnostic tests are currently used (see section on laboratory diagnosis).
Another feature of the autoimmune response in pernicious anemia is the production of antibodies to intrinsic factor42 and gastric parietal cells43 that are detectable in serum. The most common antibody to intrinsic factor blocks the site on intrinsic factor where vitamin B12 binds,40 which inhibits the formation of the intrinsic factor–vitamin B12 complex and prevents the absorption of the vitamin. These blocking antibodies are present in serum or gastric fluid in about 90% of patients with pernicious anemia.5 Parietal cell antibodies are detectable in the serum of about 90% of patients with pernicious anemia.5, 40
Other causes of lack of intrinsic factor.
A lack of intrinsic factor may also be related to H. pylori infection. Left untreated, colonization of the gastric mucosa with H. pylori progresses until the parietal cells are entirely destroyed, a process involving both local and systemic immune processes.44, 45 In addition, partial or total gastrectomy, which results in removal of intrinsic factor–producing parietal cells, invariably leads to vitamin B12 deficiency.
Impaired absorption of vitamin B12 can also be caused by hereditary intrinsic factor deficiency. This is a rare autosomal recessive disorder characterized by the absence or nonfunctionality of intrinsic factor. In contrast to the acquired forms of pernicious anemia, histology and gastric acidity are normal.35
General malabsorption of vitamin B12 can be caused by the same conditions interfering with folate absorption, such as celiac disease, tropical sprue, and inflammatory bowel disease.
Inherited errors of vitamin B12 absorption and transport.
Imerslund-Gräsbeck syndrome is a rare autosomal recessive condition caused by mutations in the genes for either cubilin or amnionless. This defect results in decreased endocytosis of the intrinsic factor–vitamin B12 complex by ileal enterocytes. Transcobalamin deficiency is another rare autosomal recessive condition resulting in a deficiency of physiologically available vitamin B12.35, 46
Competition for vitamin B12.
Competition for available vitamin B12 in the intestine may come from intestinal organisms. The fish tapeworm Diphyllobothrium latum is able to split vitamin B12 from intrinsic factor,47 rendering the vitamin unavailable for host absorption. Also, blind loops, portions of the intestines that are stenotic as a result of surgery or inflammation, can become overgrown with intestinal bacteria that compete effectively with the host for available vitamin B12.11 In both of these cases, the host is unable to absorb sufficient vitamin B12, and megaloblastic anemia results.
The tests used in the diagnosis of megaloblastic anemia include screening tests and specific diagnostic tests to identify the specific vitamin deficiency and perhaps its cause.
Five tests used to screen for megaloblastic anemia are the complete blood count (CBC), reticulocyte count, white blood cell (WBC) manual differential, serum bilirubin, and lactate dehydrogenase.
Complete blood count and reticulocyte count
Slight macrocytosis often is the earliest sign of megaloblastic anemia. Patients with uncomplicated megaloblastic anemia are expected to have decreased hemoglobin and hematocrit values, pancytopenia, and reticulocytopenia. Megaloblastic anemia develops slowly, and the degree of anemia is often severe when first detected. Hemoglobin values of less than 7 or 8 g/dL are not unusual.4 When the hematocrit is less than 20%, erythroblasts with megaloblastic nuclei, including an occasional promegaloblast, may appear in the peripheral blood. The mean cell volume (MCV) is usually 100 to 150 fL and commonly is greater than 120 fL, although coexisting iron deficiency, thalassemia trait, or inflammation can prevent macrocytosis. The mean cell hemoglobin (MCH) is elevated by the increased volume of the cells, but the mean cell hemoglobin concentration (MCHC) is usually within the reference interval because hemoglobin production is unaffected. The red blood cell distribution width (RDW) is also elevated.
The characteristic morphologic findings of megaloblastic anemia in the peripheral blood include oval macrocytes (enlarged oval RBCs) () and hypersegmented neutrophils with six or more lobes (Figure 21-6Figure 21-7).48 Impaired cell production results in a low absolute reticulocyte count, especially in light of the severity of the anemia, and polychromasia is not observed on the peripheral blood film. Additional morphologic changes may include the presence of teardrop cells, RBC fragments, and microspherocytes. These smaller cells further increase the RDW. The presence of schistocytes sometimes leads to a paradoxically lower mean cell volume (MCV) than is seen in less severe cases. These erythrocyte changes reflect the severity of the dyserythropoiesis and should not be taken as evidence of microangiopathic hemolysis. Nucleated RBCs, Howell-Jolly bodies, basophilic stippling, and Cabot rings may also be observed.
FIGURE 21-6 Oval macrocytes, teardrop cells (dacryocytes), other red blood cell abnormalities, and a small lymphocyte for size comparison in megaloblastic anemia (peripheral blood, ×500).
FIGURE 21-7 Hypersegmented neutrophil, oval macrocytes, and a Howell-Jolly body in megaloblastic anemia (peripheral blood, ×500). Source: (From Rodak BF, Carr JH: Clinical hematology atlas, ed 4, St. Louis, 2013, Elsevier, Saunders.)
White blood cell manual differential
Hypersegmentation of neutrophils is essentially pathognomonic for megaloblastic anemia. It appears early in the course of the disease49 and may persist for up to 2 weeks after treatment is initiated.5Hypersegmented neutrophils noted in the WBC differential report are a significant finding and require a reporting rule that can be applied consistently because even healthy individuals may have an occasional one. One such rule is to report hypersegmentation when there are at least 5 five-lobed neutrophils per 100 WBCs or at least 1 six-lobed neutrophil is noted.11 Some laboratories perform a lobe count on 100 neutrophils and then calculate the mean. In megaloblastic anemia, the mean lobe count should be greater than 3.4.11 The cause of the hypersegmentation is not understood, despite considerable investigation.50More recent advances in the understanding of growth factors and their impact on transcription factors may yet solve this mystery. Nevertheless, a search for neutrophil hypersegmentation on a peripheral blood film constitutes an inexpensive yet sensitive screening test for megaloblastic anemia.
Bilirubin and lactate dehydrogenase levels
Although generally considered a nutritional anemia, megaloblastic anemia is in one sense a hemolytic anemia. Because many RBC precursors die during division in the bone marrow, many RBCs never enter the circulation (ineffective hematopoiesis), so a decrease in reticulocytes occurs in the peripheral blood. The usual signs of hemolysis are evident in the serum, including an elevation in the levels of total and indirect bilirubin and lactate dehydrogenase (predominantly RBC derived).
The constellation of findings including macrocytic anemia, moderate to marked pancytopenia, reticulocytopenia, oval macrocytes, hypersegmented neutrophils, plus increased levels of total and indirect bilirubin and lactate dehydrogenase justifies further testing to confirm a diagnosis of megaloblastic anemia and determine its cause. Occasionally, the classic findings may be obscured by coexisting conditions such as iron deficiency, which makes the diagnosis more challenging. Most hematologic aberrations do not appear until vitamin deficiency is fairly well advanced ().Box 21-451
Sequence of Development of Megaloblastic Anemias
1. Decrease in vitamin levels
2. Hypersegmentation of neutrophils in peripheral blood
3. Oval macrocytes in peripheral blood
4. Megaloblastosis in bone marrow
Specific diagnostic tests
Bone marrow examination
Modern tests for vitamin deficiencies and autoimmune antibodies have made bone marrow examination an infrequently used diagnostic test for megaloblastic anemia. Nevertheless, it remains the reference confirmatory test to identify the megaloblastic appearance of the developing RBCs.
Megaloblastic, in contrast to macrocytic, anemia refers to specific morphologic changes in the developing RBCs. The cells are characterized by a nuclear-cytoplasmic asynchrony in which the cytoplasm matures as expected with increasing pinkness as hemoglobin accumulates. The nucleus lags behind, however, appearing younger than expected for the degree of maturity of the cytoplasm (Figure 21-8). This asynchrony is most striking at the stage of the polychromatic normoblast. The cytoplasm appears pinkish-blue as expected for that stage, but the nuclear chromatin remains more open than expected, similar to that in the nucleus of a basophilic normoblast. Overall, the marrow is hypercellular, with a myeloid-to-erythroid ratio of about 1:1 by virtue of the increased erythropoietic activity. The hematopoiesis is ineffective, however, and although cell production in the bone marrow is increased, the apoptosis of cells in the marrow results in peripheral pancytopenia.
FIGURE 21-8 Erythroid precursors in megaloblastic anemia. Note nuclear-cytoplasmic asynchrony (bone marrow, ×500). Source: (From Rodak BF, Carr JH: Clinical hematology atlas, ed 4, St. Louis, 2013, Elsevier, Saunders.)
The WBCs are also affected in megaloblastic anemia and appear larger than normal. This is most evident in metamyelocytes and bands, because in the usual development of neutrophils, the cells should be getting smaller at these stages. The effect creates “giant” metamyelocytes and bands ().Figure 21-9
FIGURE 21-9 Giant band (early) in megaloblastic anemia (bone marrow, original magnification ×1000).
Megakaryocytes do not show consistent changes in megaloblastic anemia. They may be either increased or decreased in number and may show diminished lobulation. The latter finding is not consistently seen, however, and even when present, it is difficult to assess.
Assays for folate, vitamin B12, methylmalonic acid, and homocysteine
Although bone marrow aspiration is confirmatory for megaloblastosis, the invasiveness of the procedure and its expense mean that other testing is performed more often than a bone marrow examination. Furthermore, the confirmation of megaloblastic morphology in the marrow does not identify its cause. Tests for serum levels of folate and vitamin B12 are readily available using immunoassay; serum vitamin B12may also be assayed by competitive binding chemiluminescence.52 However, there are a number of interferences with these assays that can cause false increased and decreased results5, 52 (Box 21-5); reflexive testing to methylmalonic acid and homocysteine (covered below) can increase diagnostic accuracy. RBC folate levels may also be measured. Unlike serum folate levels, which fluctuate with diet, RBC folate values are stable and may be a more accurate reflection of true folate status53; however, current RBC folate tests have less than optimal sensitivity and specificity and have not been validated in actual patients with normal and deficient folate levels. Thus the serum folate level is preferred over RBC folate level in the United States as the initial test for evaluation of folate deficiency.5
Causes of False Increases and Decreases of Vitamin B12 and Folate Assays5, 52
False increases in vitamin B12 assay results
Assay technical failure
Alcoholic liver disease
Increased transcobalamin I and II binders (e.g., myeloproliferative states, hepatomas, and fibrolamellar hepatic tumors)
Activated transcobalamin II–producing macrophages (e.g., autoimmune diseases, monoblastic leukemias, and lymphomas)
Release of cobalamin from hepatocytes (e.g., active liver disease)
High serum anti–intrinsic factor antibody titer
False decreases in vitamin B12 assay results
Plasma cell myeloma
Human immunodeficiency virus
Plasma cell myeloma
Transcobalamin I deficiency
Megadose vitamin C therapy
False increases in folate assay results
False decreases in folate assay results
Severe anorexia requiring hospitalization
Acute alcohol consumption
Some laboratories conduct a reflexive assay for methylmalonic acid if vitamin B12 levels are low. As indicated previously, in addition to playing a role in folate metabolism, vitamin B12 is a cofactor in the conversion of methylmalonyl CoA to succinyl CoA by the enzyme methylmalonyl CoA mutase (Figure 21-2). If vitamin B12 is deficient, methylmalonyl CoA accumulates. Some of it hydrolyzes to methylmalonic acid, and the increase can be detected in serum and urine. Because methylmalonic acid is also elevated in patients with impaired renal function, the test is not specific, and thus increased levels cannot be definitively related to vitamin B12 deficiency.3 Methylmalonic acid is assayed by gas chromatography–tandem mass spectrometry.
Homocysteine levels are affected by deficiencies in either folate or vitamin B12. 5-Methyl THF donates a methyl group to homocysteine in the generation of methionine. This reaction uses vitamin B12 as a coenzyme (Figure 21-4). Thus a deficiency in either folate or vitamin B12 results in elevated levels of homocysteine. Total homocysteine can be measured in either plasma or serum. Homocysteine may be assayed by gas chromatography–mass spectrometry, high-performance liquid chromatography, or fluorescence polarization immunoassay. Homocysteine levels are also elevated in patients with renal failure and dehydration. Figure 21-10 presents an algorithm of the analysis of these analytes in the diagnosis of vitamin B12 and folate deficiency.
FIGURE 21-10 Algorithm for the use of assays for serum folate, vitamin B12, methylmalonic acid, and homocysteine in the diagnosis of vitamin B12 and folate deficiency.5, 44 ↑, Increased;MCV, mean cell volume; MMA, methylmalonic acid; N, within reference interval.
Gastric analysis and serum gastrin
Gastric analysis may be used to confirm achlorhydria, an expected finding in pernicious anemia. Achlorhydria occurs in other conditions, however, including natural aging. When other causes of vitamin B12deficiency have been eliminated, a finding of achlorhydria is supportive, although not diagnostic, of pernicious anemia. The H+ concentration is determined by pH measurement.
As a result of the gastric achlorhydria, serum gastrin levels can be markedly elevated.4 Serum gastrin is measured by immunoassay, including chemiluminescent immunometric assays.
Antibodies to intrinsic factor and parietal cells can be detected in the serum of most patients with pernicious anemia. Various immunoassays can detect intrinsic factor–blocking antibodies; parietal cell antibodies can be detected by indirect fluorescent antibody techniques or enzyme-linked immunosorbent assays. Anti-IF antibodies are highly specific and confirmatory for pernicious anemia, but their absence does not rule out the condition. The test for parietal cell antibodies is nonspecific and not clinically useful for the diagnosis of pernicious anemia.5
presents an algorithm for the diagnosis of pernicious anemia using tests for serum vitamin BFigure 21-1112, methylmalonic acid, intrinsic factor blocking antibody, and serum gastrin levels.
FIGURE 21-11 Algorithm for the diagnosis of pernicious anemia. Source: (Adapted from Klee GG. Cobalamin and folate evaluation: Measurement of methylmalonic acid and homocysteine vs vitamin B12 and folate. Clin Chem 2000; 46, p. 1281.)
Holotranscobalamin is the metabolically active form of vitamin B12. Until recently, methods for measuring holotranscobalamin were manual and not suitable for use in clinical laboratories. Newer, more rapid immunoassays using monoclonal antibodies specific for holotranscobalamin have been developed in the past several years that are both sensitive and specific.54, 55 Recent studies suggest the specificity of holotranscobalamin to detect vitamin B12 deficiency is low; thus the adoption of holotranscobalamin in routine clinical testing is not supported.53
Deoxyuridine suppression test
The principle of the deoxyuridine suppression test is that the preincubation of normal bone marrow with deoxyuridine will suppress the subsequent incorporation of labeled thymidine into DNA because the normal cells can successfully methylate the uridine into thymidine. However, in patients with either a vitamin B12 or a folate deficiency, this suppression is abnormally low. By adding either vitamin B12 or folate to the test cells, one can determine whether the inadequate suppression is caused by vitamin B12 or folate deficiency.56-58 Although micromethods have been developed, the necessity of using bone marrow tissue and the complexity of the test make it impractical for clinical testing.
Stool analysis for parasites
When vitamin B12 is found to be deficient, a stool analysis for eggs or proglottids of the fish tapeworm D. latum may be part of the diagnostic workup.
contains a summary of laboratory tests used to diagnose vitamin BTable 21-112 and folate deficiency.
Laboratory Tests Used to Diagnose Vitamin B12 and Folate Deficiency
Vitamin B12 Deficiency
Complete blood count
↓HGB, HCT, RBCs, WBCs, PLTs
Same as Folate Deficiency
Manual differential count
Hypersegmented neutrophils, oval macrocytes, anisocytosis, poikilocytosis, RBC inclusions
Same as Folate Deficiency
Absolute reticulocyte count
Serum total and indirect bilirubin
Serum lactate dehydrogenase
Specific diagnostic tests
Bone marrow examination*
Erythroid hyperplasia (ineffective)
Same as Folate Deficiency
Presence of megaloblasts
Serum vitamin B12
N or ↑†
N or ↓†
Serum methylmalonic acid
Antibodies to intrinsic factor and gastric parietal cells
Present in pernicious anemia
Can be markedly elevated in pernicious anemia
Achlorhydria in pernicious anemia
Stool analysis for parasites
Diphyllobothrium latum may be the cause of deficiency
* Bone marrow examination and gastric analysis are not usually required for diagnosis.
† Without vitamin B12, the cell is unable to produce intracellular polyglutamated tetrahydrofolate; therefore, 5-methyltetrahydrofolate leaks out of the cell, which results in a decreased level of intracellular folate.
‡ Holotranscobalamin level is also decreased in transcobalamin deficiency.
↑, Increased; ↓, decreased; HGB, hemoglobin; HCT, hematocrit; MCH, mean cell hemoglobin; MCV, mean cell volume; N, within reference interval; PLT, platelet; RBC, red blood cell; WBC, white blood cell.
Macrocytic nonmegaloblastic anemias
The macrocytic nonmegaloblastic anemias are macrocytic anemias in which DNA synthesis is unimpaired. The macrocytosis tends to be mild; the MCV usually ranges from 100 to 110 fL and rarely exceeds 120 fL. Patients with nonmegaloblastic, macrocytic anemia lack hypersegmented neutrophils and oval macrocytes in the peripheral blood and megaloblasts in the bone marrow. Macrocytosis may be physiologically normal, as in the newborn (Chapter 45), or the result of pathology, as in liver disease, chronic alcoholism, or bone marrow failure. Reticulocytosis is a common cause of macrocytosis. Figure 21-12 presents an algorithm for the preliminary investigation of macrocytic anemias.
FIGURE 21-12 Source: Algorithm for preliminary investigation of macrocytic anemias. MCV, Mean cell volume.
Treatment should be directed at the specific vitamin deficiency established by the diagnostic tests and should include addressing the cause of the deficiency (e.g., better nutrition, treatment for D. latum), if possible. Vitamin B12 is administered intramuscularly to treat pernicious anemia to bypass the need for intrinsic factor. High-dose oral vitamin B12 treatment is increasingly popular in the treatment of pernicious anemia.39, 59,60 Regardless of the treatment modality, those with pernicious anemia or malabsorption must have lifelong vitamin replacement therapy. Folic acid can be administered orally. The inappropriate treatment of vitamin B12 deficiency with folic acid improves the anemia but does not correct or stop the progress of the neurologic damage, which may advance to an irreversible state.3 Thus proper diagnosis prior to treatment is important. Iron is often supplemented concurrently to support the rapid cell production that accompanies effective treatment.
When proper treatment is initiated, the body’s response is prompt and brisk and can be used to confirm the accuracy of the diagnosis. The bone marrow morphology will begin to revert to a normoblastic appearance within a few hours of treatment. A substantial reticulocyte response is apparent at about 1 week, with hemoglobin increasing toward normal levels in about 3 weeks.11 Hypersegmented neutrophils disappear from the peripheral blood within 2 weeks of initiation of treatment.5 Thus with proper treatment, hematologic parameters may return to normal within 3 to 6 weeks.
• Impaired DNA synthesis affects all rapidly dividing cells of the body, including the skin, gastrointestinal tract, and bone marrow. The effect on hematologic cells results in megaloblastic anemia.
• Vitamin B12 and folate are needed for the production of thymidine nucleotides for DNA synthesis. Deficiencies of either vitamin impair DNA replication, halt cell division, and increase apoptosis, which results in ineffective hematopoiesis and megaloblastic morphology of erythrocyte precursors.
• Vitamin B12 deficiency is associated with peripheral neuropathies and neuropsychiatric abnormalities as a result of demyelinization of nerves in the peripheral and central nervous system. Peripheral neuropathy and depression also may accompany folate deficiency. Folate deficiency in early pregnancy can lead to neural tube defects in the fetus.
• Lack of vitamin B12 leads to the accumulation of methylmalonic acid (MMA) and homocysteine. Folate deficiency, in particular, leads to elevation of homocysteine levels and possible risk of coronary artery disease.
• Folate deficiency may result from inadequate intake, increased need with growth or pregnancy, impaired absorption, impaired use, or excessive loss. The action of folate can be impaired by drugs such as those used to treat epilepsy or cancer. Renal dialysis patients experience significant folate loss to the dialysate.
• Vitamin B12 deficiency arises from inadequate intake, increased need, or inadequate absorption. Inadequate intake of vitamin B12, although possible, is uncommon because vitamin B12 is ubiquitous in animal products. Pregnancy, lactation, and growth create increased need for vitamin B12.
• Absorption of vitamin B12 depends on production of intrinsic factor by parietal cells of the stomach. Vitamin B12 bound to transcobalamin—holotranscobolamin—is the metabolically active form of the vitamin in the circulation.
• Impaired absorption of vitamin B12 can be caused by several mechanisms. Decrease in gastric acid production or lack of trypsin in the intestine causes vitamin B12 to be excreted in the stool rather than absorbed. Malabsorption can be caused by intestinal diseases, such as sprue, celiac disease, and inflammatory bowel disease. Competition for vitamin B12 can develop from an intestinal parasite (D. latum) or bacteria in intestinal blind loops. Lack of intrinsic factor may result from loss of gastric parietal cells with pernicious anemia, H. pylori infection, gastrectomy, or inherited intrinsic factor deficiency.
• Pernicious anemia is vitamin B12 deficiency resulting from an autoimmune disease that causes destruction of gastric parietal cells. H+ and intrinsic factor secretion is lost. Antibodies to parietal cells or intrinsic factor, or both, are detectable in the serum.
• Classic CBC findings in megaloblastic anemia include decreased hemoglobin, hematocrit, and RBC count; leukopenia; thrombocytopenia; decreased absolute reticulocyte count; elevated MCV (usually greater than 120 fL); elevated RDW and MCH; MCHC within the reference interval; and oval macrocytes and hypersegmented neutrophils observed on the peripheral blood film. Additional abnormal laboratory test findings may include elevated levels of total and indirect serum bilirubin and lactate dehydrogenase due to the intramedullary hemolysis of megaloblastic erythroid precursors.
• The bone marrow in megaloblastic anemia is hyperplastic with increased erythropoiesis; however, it is ineffective due to increased apoptosis of developing cells. RBC precursors show nuclear-cytoplasmic asynchrony, with the nuclear maturation lagging behind the cytoplasmic maturation. Giant metamyelocytes and bands are evident.
• The cause of megaloblastic anemia is determined using specific immunoassays for serum folate and vitamin B12. Immunoassays for antibodies to intrinsic factor and parietal cells can aid in the diagnosis of pernicious anemia. Additional tests for gastrointestinal disease or parasites may be needed.
• Treatment of megaloblastic anemia is directed at correcting the cause of the deficiency and supplementing the missing vitamin.
• For pernicious anemia, lifelong supplementation with vitamin B12 is necessary.
Now that you have completed this chapter, go back and read again the case study at the beginning and respond to the questions presented.
Answers can be found in the Appendix.
1. Which of the following findings is consistent with a diagnosis of megaloblastic anemia?
a. Hyposegmentation of neutrophils
b. Decreased serum lactate dehydrogenase level
c. Absolute increase in reticulocytes
d. Increased MCV
2. A patient has a clinical picture of megaloblastic anemia. The serum folate level is decreased, and the serum vitamin B12 level is 600 pg/mL (reference interval is 200–900 pg/mL). What is the expected value for the methylmalonic acid assay?
c. Within the reference interval
3. Which one of the following statements characterizes the relationships among macrocytic anemia, megaloblastic anemia, and pernicious anemia?
a. Macrocytic anemias are megaloblastic.
b. Macrocytic anemia is pernicious anemia.
c. Megaloblastic anemia is macrocytic.
d. Megaloblastic anemia is pernicious anemia.
4. Which of the following CBC findings is most suggestive of a megaloblastic anemia?
a. MCV of 103 fL
b. Hypersegmentation of neutrophils
c. RDW of 16%
d. Hemoglobin concentration of 9.1 g/dL
5. In the following description of a bone marrow smear, find the statement that is inconsistent with the expected picture in megaloblastic anemia. “The marrow appears hypercellular with a myeloid-to-erythroid ratio of 1:1 due to prominent erythroid hyperplasia. Megakaryocytes appear normal in number and appearance. The WBC elements appear larger than normal, with especially large metamyelocytes, although they otherwise appear morphologically normal. The RBC precursors also appear large. There is nuclear-cytoplasmic asynchrony, with the nucleus appearing more mature than expected for the color of the cytoplasm.”
a. Erythroid nuclei that are more mature than cytoplasm
b. Larger than normal WBC elements
c. Larger than normal RBCs
d. Normal appearance of megakaryocytes
6. Which one of the following findings would be inconsistent with elevated titers of intrinsic factor blocking antibodies?
a. Hypersegmentation of neutrophils
b. Low levels of methylmalonic acid
c. Macrocytic RBCs
d. Low levels of vitamin B12
7. Which of the following is the most metabolically active form of absorbed vitamin B12?
b. Intrinsic factor–vitamin B12 complex
d. Haptocorrin–vitamin B12 complex
8. Folate and vitamin B12 work together in the production of:
a. Amino acids
9. The macrocytosis associated with megaloblastic anemia results from:
a. Reduced numbers of cell divisions with normal cytoplasmic development
b. Activation of a gene that is typically active only in megakaryocytes
c. Reduced concentration of hemoglobin in the cells so that larger cells are needed to provide the same oxygen-carrying capacity
d. Increased production of reticulocytes in an attempt to compensate for the anemia
10. Which one of the following groups has the highest risk for pernicious anemia?
a. Malnourished infants
b. Children during growth periods
c. Persons older than 60 years of age
d. Pregnant women
1. Scott J.M. Folate and vitamin B12 . Proc Nutr Soc; 1999; 58:441-448.
2. Wickramasinghe S.N. The wide spectrum and unresolved issues of megaloblastic anemia. Semin Hematol; 1999; 36:3-18.
3. Miller J.W. Folic acid. In: Caballero B. Encyclopedia of human nutrition. 3rd ed. Amsterdam : Elsevier/Academic Press 2013; 262-269.
4. Carmel R. Megaloblastic anemias disorders of impaired DNA synthesis. In: Greer J.P, Foerster J, Rodgers G.M, et al. Wintrobe’s Clinical Hematology. 12th ed. Philadelphia : Wolters Kluwer Health/Lippincott Williams & Wilkins 2009; 1143-1172.
5. Antony A.C. Megaloblastic anemias. In: Hoffman R, Benz E.J, Silberstein L.E, et al. Hematology basic principles and practice 6th ed. Philadelphia : Churchill Livingstone 2013; 473-504.
6. Goulian M, Bleile B, Tseng B.Y. Methotrexate-induced misincorporation of uracil into DNA. Proc Natl Acad Sci U S A; 1980; 77:1956-1960.
7. Wickramasinghe S.N, Fida S. Bone marrow cells from vitamin B12 - and folate-deficient patients misincorporate uracil into DNA. Blood; 1994; 83:1656-1661.
8. Koury M.J, Horne D.W, Brown Z.A, et al. Apoptosis of late stage erythroblasts in megaloblastic anemia association with DNA damage and macrocyte production. Blood; 1997; 89:4617-4623.
9. Das K.C, Das M, Mohanty D, et al. Megaloblastosis from morphos to molecules. Med Princ Pract; 2006; 14:2-14.
10. Geené D, Sudre P, Anwar D, et al. Causes of macrocytosis in HIV-infected patients not treated with zidovudine. Swiss HIV Cohort Study. J Infect; 2000; 40:160-163.
11. Carmel R, Rosenblatt D.S. Disorders of cobalamin and folate metabolism. In: Handin R.I, Lux S.E, Stossel T.P. Blood: principles and practice of hematology. 2nd ed. Philadelphia : Lippincott Williams & Wilkins 2003; 1361-1398.
12. Solomon L.R. Disorders of cobalamin (vitamin B12 ) metabolism emerging concepts in pathophysiology, diagnosis and treatment. Blood Rev; 2007; 21:113-130.
13. Scalabrino G, Peracchi M. New insights into the pathophysiology of cobalamin deficiency. Trends Mol Med; 2006; 12:247-254.
14. Morrison H.I, Schaubel D, Desmeules M, et al. Serum folate and risk of fatal coronary heart disease. JAMA; 1996; 275:1893-1896.
15. Voutilainen S, Virtanen J.K, Rissanen T.H, et al. Serum folate and homocysteine and the incidence of acute coronary events the Kuopio Ischaemic Heart Disease Risk Factor Study. Am J Clin Nutr; 2004; 80:317-323.
16. de Bree A, Verschuren W.M, Blom H.J, et al. Coronary heart disease mortality, plasma homocysteine, and B-vitamins a prospective study. Atherosclerosis; 2003; 166:369-377.
17. Blom H.J, Smulders Y. Overview of homocysteine and folate metabolism. With special references to cardiovascular disease and neural tube defects. J Inherit Metab Dis; 2011; 346:75-81.
18. Soinio M, Marniemi J, Laakso M, et al. Elevated plasma homocysteine level is an independent predictor of coronary heart disease events in patients with type 2 diabetes mellitus. Ann Intern Med; 2004; 140:94-100.
19. Lindeman R.D, Romero L. J, Yau C.L, et al. Serum homocysteine concentrations and their relation to serum folate and vitamin B12 concentrations and coronary artery disease prevalence in an urban, bi-ethnic community. Ethn Dis; 2003; 13:178-185.
20. Bottiglieri T, Laundry M, Crellin R, et al. Homocysteine, folate, methylation, and monoamine metabolism in depression. J Neurol Neurosurg Psychiatry; 2000; 69:228-232.
21. Kronenberg G, Colla M, Endres M. Folic acid, neurodegenerative and neuropsychiatric disease. Curr Mol Med; 2009; 9:315-323.
22. Reynolds E.H, Rothfeld P, Pincus J.H. Neurological disease associated with folate deficiency. BMJ; 1973; 2:398-400.
23. Alpert J.E, Mischoulon D, Nierenberg A.A, et al. Nutrition and depression focus on folate. Nutrition; 2000; 16:544-546.
24. Blom H.J, Shaw G.M, den Heijer M, et al. Neural tube defects and folate case far from closed. Nat Rev Neurosci; 2006; 7:724-731.
25. Gallagher M.L. Vitamins. In: Mahan L.K, Escott-Stump S. Krause’s food, nutrition, and diet therapy. 11th ed. Philadelphia : Saunders 2004; 74-119.
26. Qui A, Jansen M, Sakaris A, et al. Identification of an intestinal folate transporter and the molecular basis for hereditary folate malabsorption. Cell; 2006; 127:917-928.
27. Dixon K.H, Lanpher B.C, Chiu J, et al. A novel cDNA restores reduced folate carrier activity and methotrexate sensitivity to transport deficient cells. J Biol Chem; 1994; 269(1):17-20.
28. Nath S.K. Tropical sprue. Curr Gastroenterol Rep; 2005; 7:343-349.
29. Waxman S, Metz J, Herbert V. Defective DNA synthesis in human megaloblastic bone marrow effects of homocysteine and methionine. J Clin Invest; 1969; 48:284-289.
30. Moran R.G, Keyomarsi K. Biochemical rationale for the synergism of 5-fluorouracil and folinic acid. NCI Monogr; 1987; 5:159-163.
31. Kishi T, Fujita N, Eguchi T, et al. Mechanism for reduction of serum folate by antiepileptic drugs during prolonged therapy. J Neurol Sci; 1997; 145:109-112.
32. Froscher W, Maier V, Laage M, et al. Folate deficiency, anticonvulsant drugs, and psychiatric morbidity. Clin Neuropharmacol; 1995; 18:165-182.
33. Green R. Vitamin B12 physiology, dietary sources, and requirements. In: Caballero B. Encyclopedia of human nutrition. 3rd ed. Amsterdam : Elsevier/Academic Press 2013; 351-356.
34. He Q, Madsen M, Kilkenney A, et al. Amnionless function is required for cubilin brush-border expression and intrinsic factor-cobalamin (vitamin B12 ) absorption in vivo. Blood; 2005; 106:1447-1453.
35. Morel C.F, Rosenblatt D.S. In: Sarafoglou K, Hoffman G.F, Roth K.S. Pediatric endocrinology and inborn errors of metabolism. New York : McGraw-Hill 2009; 195-210.
36. Fyfe J.C, Madsen M, Hojrup P, et al. The functional cobalamin (vitamin B12 )–intrinsic factor receptor is a novel complex of cubilin and amnionless. Blood; 2004; 103:1573-1579.
37. Afman L.A, Van Der Put N.M.J, Thomas C.M.G, et al. Reduced vitamin B12 binding by transcobalamin II increases the risk of neural tube defects. QJM; 2001; 94:159-166.
38. Seetharam B, Bose S, Li N. Cellular import of cobalamin (vitamin B-12). J Nutr; 1999; 129:1761-1764.
39. Dali-Youcef N, Andres E. An update on cobalamin deficiency in adults. QJM; 2009; 102:17-28.
40. Toh B.H, van Driel I.R, Gleeson P.A. Pernicious anemia. N Engl J Med; 1997; 337:1441-1448.
41. Karlsson F.A, Burman P, Loof L, et al. Major parietal cell antigen in autoimmune gastritis with pernicious anemia in the acid-producing H+,K+ -adenosine triphosphatase of the stomach. J Clin Invest; 1988; 81:475-479.
42. Bardhan K.D, Hall J.R, Spray G.H, et al. Blocking and binding autoantibody to intrinsic factor. Lancet; 1968; 1:62-64.
43. Strickland R.G, Hooper B. The parietal cell heteroantibody in human sera prevalence in a normal population and relationship to parietal cell autoantibody. Pathology; 1972; 4:259-263.
44. Kaferle J, Strzoda C.E. Evaluation of macrocytosis. Am Fam Physician; 2009; 79(3):203-208.
45. Kaptan K, Beyan C, Ural A.U, et al. Helicobacter pylori—is it a novel causative agent in vitamin B12 deficiency. Arch Intern Med; 2000; 160:1349-1353.
46. Morel C.F, Watkins D, Scott P, et al. Prenatal diagnosis for methylmalonic acidemia and inborn errors of vitamin B12 metabolism and transport. Mol Genet Metab; 2005; 86:160-171.
47. Nyberg W, Gräsbeck R, Saarni M, et al. Serum vitamin B12 levels and incidence of tape worm anemia in a population heavily infected with Diphyllobothrium latum. Am J Clin Nutr; 1961; 9:606-612.
48. Zittoun J, Zittoun R. Modern clinical testing strategies in cobalamin and folate deficiency. Semin Hematol; 1999; 36:35-46.
49. Herbert V. Experimental nutritional folate deficiency in man. Trans Assoc Am Physicians; 1962; 75:307-320.
50. Wickramasinghe S.N. The wide spectrum and unresolved issues in megaloblastic anemia. Semin Hematol; 1999; 36:3-18.
51. Carmel R. Introduction beyond megaloblastic anemia. Semin Hematol; 1999; 36:1-2.
52. Oberley M.J, Yang D.T. Laboratory testing for cobalamin deficiency in megaloblastic anemia. Am J Hematol; 2013; 88:522-526.
53. Piyathilake C.J, Robinson C.B, Cornwell P. A practical approach to red blood cell folate analysis. Anal Chem Insights; 2007; 2:107-110.
54. Brady J, Wilson L, McGregor L, et al. Active B12 a rapid, automated assay for holotranscobalamin on the Abbott AxSYM analyzer. Clin Chem; 2008; 54(3):567-573.
55. Ulleland M, Eilertsen I, Quadros E.V, et al. Direct assay for cobalamin bound to transcobalamin (holo-transcobalamin) in serum. Clin Chem; 2002; 48(3):526-532.
56. Metz J. The deoxyuridine suppression test. CRC Crit Rev Clin Lab Sci; 1984; 20:205-241.
57. Wickramasinghe S.N, Matthew J.H. Deoxyuridine suppress biochemical basis and diagnostic applications. Blood Rev; 1988; 2:168-177.
58. Carmel R, Bedros A.A, Mace J.W, et al. Congenital methylmalonic aciduria—homocystinuria with megaloblastic anemia observation on response to hydroxycobalamin and on the effect of homocysteine and methionine on the deoxyuridine suppression test. Blood; 1980; 55:570-579.
59. Butler C.C, Vidal-Alaball J, Cannings-John R, et al. Oral vitamin B12 versus intramuscular vitamin B12 for vitamin B12 deficiency a systematic review of randomized controlled trials. Fam Pract; 2006; 23:279-285.
60. Stabler S.P. Clinical practice. Vitamin B12 deficiency. N Engl J Med; 2013; 368:149-160.