Megaloblastic Anemia

March

Megaloblastic anemia (MA) refers to a group of anemias caused by impaired deoxyribonucleic acid (DNA) synthesis, primarily due to deficiencies in folic acid and/or vitamin B₁₂, as well as acquired DNA synthesis impairments resulting from genetic or drug-related factors. This condition is typically characterized by macrocytic anemia, along with the presence of megaloblasts in the bone marrow, including erythroid cells, granulocytes, and megakaryocyte lineages. Due to defective DNA synthesis in erythroid precursor cells, some scholars also refer to this condition as erythroid dysplasia-related anemia.

Based on the type of deficiency, the disease can be classified into folic acid deficiency anemia, vitamin B₁₂ deficiency anemia, and combined folic acid and vitamin B₁₂ deficiency anemia. It can further be categorized based on the underlying cause:

Insufficient intake, such as inadequate consumption of folic acid or vitamin B₁₂.
Malabsorption, caused by gastrointestinal disorders, medication interference, or the formation of intrinsic factor antibodies (pernicious anemia).
Metabolic abnormalities, involving issues such as liver disease or the effects of certain anticancer drugs.
Increased demand, as seen during pregnancy or lactation.
Impaired utilization, caused by intrinsic abnormalities in purine or pyrimidine synthesis or the effects of chemotherapy drugs.

Etiology and Pathogenesis

Folic Acid Metabolism and Causes of Deficiency

Folic Acid Metabolism and Physiological Role

Folic acid, classified as a B-vitamin, is composed of pterin, para-aminobenzoic acid, and L-glutamic acid. It is abundant in fresh fruits, vegetables, and meats. However, prolonged cooking can lead to a loss of 50%–90% of dietary folic acid. Folic acid is mainly absorbed in the duodenum and proximal jejunum, with a daily dietary requirement of approximately 200 μg.
Food-derived polyglutamate folates are depolymerized by intestinal mucosal enzymes into monoglutamate or diglutamate forms before being absorbed by intestinal epithelial cells. These are subsequently converted by the action of folate reductase and reduced nicotinamide adenine dinucleotide phosphate (NADPH) into dihydrofolate (FH₂) and tetrahydrofolate (FH₄). FH₄ is then further converted into its active form, 5-methyltetrahydrofolate (N⁵-FH₄), which is transported to the liver via the portal vein. Some N⁵-FH₄ is excreted into the small intestine via bile and reabsorbed, forming the enterohepatic circulation of folic acid. In plasma, N⁵-FH₄ is transported to target tissues bound to albumin and interacts with folate receptors.

Within cells, N⁵-FH₄ is converted back into FH₄ by methionine synthase, a vitamin B₁₂-dependent enzyme. FH₄ is then used to donate one-carbon groups, such as methyl (—CH₃), methylene (—CH₂—), and formyl (—CH=O), for DNA synthesis. Additionally, FH4 is converted into its polyglutamate form by folylpolyglutamate synthetase and functions as an intracellular coenzyme. The human body stores 5–20 mg of folic acid, nearly half of which is in the liver. Folic acid is primarily excreted through urine and feces, with a daily excretion of 2–5 μg.

Causes of Folic Acid Deficiency

These causes include:

Reduced Intake: The primary cause is improper food preparation, such as prolonged cooking or high temperatures, which destroy significant amounts of folic acid. Other factors include imbalanced diets with reduced consumption of vegetables, meat, and eggs.
Increased Demand: During pregnancy, folic acid requirements rise to 400–600 μg per day. Increased demands are also observed in children, adolescents undergoing growth spurts, patients with chronic hemolysis, leukemia, malignancies, hyperthyroidism, or those on long-term dialysis due to chronic renal failure. Inadequate supplementation under these conditions can lead to folic acid deficiency.
Malabsorption: Conditions such as diarrhea, intestinal inflammation, tumors, surgery, or the action of certain drugs (e.g., anticonvulsants, sulfasalazine, ethanol) can impair folic acid absorption.
Impaired Utilization: Antinucleotide synthesis drugs, including methotrexate, trimethoprim, pyrimethamine, aminopterin, and ethylenediamine, may interfere with folic acid utilization. Congenital enzyme deficiencies, such as methyl-FH₄ transferase, N⁵,N¹⁰-methylene-FH₄ reductase, FH₂ reductase, and formimino transferase deficiencies, also impair folic acid metabolism.
Increased Excretion: Hemodialysis and chronic alcohol consumption can increase the excretion of folic acid.

Vitamin B₁₂ Metabolism and Causes of Deficiency

Vitamin B₁₂ Metabolism and Physiological Role

Vitamin B₁₂ exists in the human body as methylcobalamin in plasma and as 5-deoxyadenosylcobalamin in the liver and other tissues. The daily requirement for vitamin B₁₂ is approximately 1 μg, with primary dietary sources including animal liver, kidney, meat, fish, eggs, and dairy products. In food, vitamin B₁₂ is bound to proteins and is separated through digestion by gastric acid and pepsin. Once released, it binds to R-protein (produced by gastric mucosal parietal cells), forming the R-protein-vitamin B₁₂ complex (R-B₁₂). In the duodenum, R-protein is degraded by pancreatic protease, allowing vitamin B₁₂ to bind to intrinsic factor (IF), also secreted by gastric mucosal epithelial cells, forming the IF-B₁₂ complex. Intrinsic factor protects vitamin B₁₂ from degradation by gastrointestinal secretions. The IF-B₁₂ complex then binds to receptors on the brush border of ileal epithelial cells in the terminal ileum and is absorbed into enterocytes before entering the liver through the portal vein.

The human body stores about 2–5 mg of vitamin B₁₂, with 50–90% stored in the liver. Vitamin B₁₂ is primarily excreted through feces and urine.

Causes of Vitamin B₁₂ Deficiency

These causes include:

Reduced Intake: Vitamin B₁₂ deficiency may occur in individuals following strict vegetarian diets. Under normal conditions, 5–10 μg of vitamin B₁₂ is released daily into the intestinal lumen via bile, and intrinsic factor secreted by the gastric wall facilitates its reabsorption. As a result, vitamin B12 deficiency in vegetarians typically develops over 10–15 years.
Malabsorption: Malabsorption is the most common cause of vitamin B₁₂ deficiency and may include the following:
- Intrinsic factor deficiency, as seen in pernicious anemia, gastrectomy, or atrophic gastritis.
- Deficiency of gastric acid or pepsin.
- Deficiency of pancreatic proteases.
- Intestinal disorders.
- Congenital intrinsic factor deficiency or impaired vitamin B₁₂ absorption.
- Medication effects, including those of para-aminosalicylic acid, neomycin, metformin, colchicine, and phenformin.
- Intestinal parasites (e.g., Diphyllobothrium latum infection) or bacterial overgrowth, which can consume vitamin B₁₂.
Impaired Utilization: Congenital transcobalamin II (TCII) deficiency can disrupt vitamin B₁₂ transport. The use of the anesthetic nitrous oxide can oxidize cobalamin and inhibit methionine synthase activity.

Pathogenesis

Various active forms of folic acid, including N⁵-methyltetrahydrofolate (N⁵-FH₄) and N⁵,N¹⁰-methylenetetrahydrofolate (N⁵,N¹⁰-FH₄), serve as cofactors that provide one-carbon groups for DNA synthesis. Of primary importance is the methylation of dUMP to form dTMP through the action of thymidylate synthase, which subsequently forms dTTP. Folic acid deficiency results in reduced dTTP synthesis and impaired DNA synthesis, which delays DNA replication. In contrast, RNA synthesis is less affected, leading to an increased intracellular RNA/DNA ratio. This results in cell enlargement, with nuclear development lagging behind cytoplasmic development, leading to megaloblastic changes. Megaloblastic changes are observed in erythroid, granulocytic, and megakaryocytic cells in the bone marrow, with abnormalities in differentiation and maturation. Many of these cells undergo premature apoptosis in the bone marrow, resulting in pancytopenia. Impaired DNA synthesis also affects mucosal epithelium, leading to impaired function of the oral and gastrointestinal tract.

Vitamin B₁₂ deficiency disrupts the conversion of homocysteine to methionine by methionine synthase, a reaction that requires N⁵-FH₄ as a methyl donor. This interference blocks the conversion of N⁵-FH₄ back to FH₄, resulting in reduced synthesis of N⁵,N¹⁰-FH₄. Since N⁵,N¹⁰-FH₄ is required as a methyl donor for dUMP methylation to dTTP, impaired N⁵,N¹⁰-FH₄ synthesis leads to further reductions in dTTP synthesis and worsened DNA synthesis deficits.

Additionally, vitamin B₁₂ deficiency can lead to neurological and psychiatric abnormalities. The mechanism involves dysfunction in two vitamin B₁₂-dependent enzymes: L-methylmalonyl-CoA mutase and methionine synthase. Deficiency in the former affects myelin synthesis and results in the incorporation of odd-chain or branched-chain fatty acids into the myelin sheath. Deficiency in the latter compromises methylation reactions in nerve cells.

Medications that interfere with nucleotide synthesis can also cause megaloblastic anemia.

Clinical Manifestations

Hematological Manifestations

The onset is gradual, often accompanied by symptoms of anemia such as pallor, fatigue, reduced endurance, dizziness, lightheadedness, and palpitations. Severe cases may present with pancytopenia, recurrent infections, and bleeding. Mild jaundice may occur in a small number of patients.

Gastrointestinal Manifestations

The oral mucosa and lingual papillae may become atrophic, with recurrent glossitis, a smooth tongue, and disappearance of the lingual papillae, giving the appearance of a "beefy tongue," often accompanied by tongue pain. Atrophy of gastrointestinal mucosa may result in anorexia, nausea, abdominal distension, diarrhea, or constipation.

Neurological and Psychiatric Manifestations

Symmetrical distal limb numbness and impaired deep sensation may be observed, along with ataxia or an unsteady gait. Reductions in taste and smell, positive pyramidal tract signs, increased muscle tone, hyperreflexia, vision loss, and episodes of scotoma may occur. Severe cases may involve urinary and fecal incontinence. Folate deficiency may lead to irritability and delusional psychiatric symptoms. Vitamin B₁₂ deficiency may result in depression, insomnia, memory loss, delirium, hallucinations, delusions, and even mental confusion or psychosis.

Laboratory Tests

Peripheral Blood Examination

Features include macrocytic anemia, with elevated mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) but normal mean corpuscular hemoglobin concentration (MCHC). Reticulocyte counts may be normal or slightly elevated. Severe cases often show pancytopenia. Blood smears may reveal anisocytosis, disappearance of central pallor, macro-ovalocytes, and stippled red blood cells. Neutrophils often show hypersegmentation (with >5% having five lobes or more or the presence of six-lobed nuclei). Giant band neutrophils may also be observed.

Bone Marrow Examination

Bone marrow may exhibit hyperactive or markedly hyperactive erythropoiesis. Erythroid cells are significantly increased with megaloblastic changes, characterized by large cell size, relatively mature cytoplasm compared to the nucleus ("nuclear-cytoplasmic asynchrony"), and granulocytic series showing megaloblastic changes with hypersegmented mature granulocytes. Megakaryocytes are often larger with excessive lobulation. Bone marrow iron staining often suggests increased stored iron.

Measurement of Serum Vitamin B₁₂, Serum Folate, and Red Blood Cell Folate

Serum vitamin B₁₂ levels below 74 pmol/L (100 ng/mL) indicate vitamin B₁₂ deficiency. Serum folate levels below 6.8 nmol/L (3 ng/mL) or red blood cell folate levels below 227 nmol/L (100 ng/mL) indicate folate deficiency.

Other Tests

Gastric acid levels may be reduced, with positive intrinsic factor antibodies and Schilling's test (indicating impaired absorption of radiolabeled vitamin B₁₂) observed in pernicious anemia.

Elevated 24-hour urinary excretion of homocysteine suggests vitamin B₁₂ deficiency.

Slightly increased serum unconjugated bilirubin may be present.

Diagnosis

Diagnosis is made based on the following:

Identification of etiologies and clinical manifestations of folate or vitamin B₁₂ deficiency.
Peripheral blood smear showing macrocytic anemia and hypersegmentation of neutrophils.
Bone marrow examination yielding classic megaloblastic changes without evidence of other dysplastic hematopoiesis.
Low serum folate and/or vitamin B₁₂ levels.
Improvement with therapeutic intervention, particularly a rise in reticulocytes within about one week of folate or vitamin B₁₂ treatment, strongly indicative of deficiency.

Differential Diagnosis

Hematologic Neoplasms

Conditions such as acute erythroleukemia or myelodysplastic syndromes can mimic megaloblastic anemia (MA) with dysplastic hematopoiesis, including megaloblastic changes, but folate and vitamin B₁₂ levels are not reduced, and supplementation is ineffective.

Disorders with Autoimmune Hemolysis

Diseases such as warm autoimmune hemolytic anemia, Evans syndrome, or immune-related pancytopenia can lead to increased red cell size at certain stages due to antibody attachment. These disorders may also present with elevated unconjugated bilirubin. In some cases, patients may exhibit positive intrinsic factor antibodies, leading to confusion with folate or vitamin B₁₂ deficiency-induced MA. Key distinguishing features include autoimmune disease characteristics and anemia that significantly improves only with immunosuppressive therapy.

Anemias with Hyperviscosity Syndrome

In conditions such as multiple myeloma, M-protein adheres to red blood cells, causing them to form rouleaux. This can result in artificially elevated MCV readings on automated hematology analyzers. However, specific features of multiple myeloma differentiate it from MA.

Non-Hematologic Disorders

Other conditions, such as hypothyroidism or chemotherapy-induced myelosuppression, can also be considered in the differential diagnosis.

Treatment

Treatment of Underlying Conditions

For megaloblastic anemia (MA) caused by underlying diseases such as gastrointestinal disorders or autoimmune diseases, efforts should focus on treating the primary condition. For MA induced by certain medications, discontinuation of the drug may be necessary when appropriate.

Nutrient Supplementation

Folate Deficiency

Folate supplementation can be provided orally at doses of 5–10 mg three times daily until anemia is completely resolved. Maintenance therapy is generally unnecessary in the absence of underlying conditions. If vitamin B₁₂ deficiency is also present, concurrent administration of vitamin B₁₂ injections is required to avoid further neurological damage.

Vitamin B₁₂ Deficiency

For vitamin B₁₂ supplementation, intramuscular injections of 500 μg are administered twice weekly. For individuals without absorption disorders, oral vitamin B₁₂ at a dose of 500 μg daily may be used. In cases involving neurological symptoms, treatment should continue for 6 months to 1 year. For patients with pernicious anemia, lifelong therapy is necessary.

Prevention

Education on nutritional knowledge should be promoted, encouraging a balanced diet and discouraging poor cooking habits and alcohol abuse. Preventive measures may be taken for high-risk populations. For infants, complementary feeding should be introduced in a timely manner. Adolescents and pregnant women should consume more fresh vegetables and may also benefit from low-dose folate or vitamin B₁₂ supplementation. Patients undergoing treatment with medications that interfere with nucleotide synthesis should receive concurrent folate and vitamin B₁₂ supplementation.

Prognosis

The prognosis of megaloblastic anemia largely depends on the underlying condition. Most patients respond quickly to appropriate treatment, with rapid improvement in clinical symptoms. Neurological symptoms tend to resolve more slowly and, in some cases, may not fully recover. Reticulocyte counts typically begin to increase 5–7 days after treatment initiation, and hemoglobin levels can return to normal within 1–2 months. In most cases, the overall prognosis is favorable.