B12 and folate deficiency

Megaloblastic anemia: overview and mechanism

• Both B12 and folate are required for DNA synthesis. Deficiency causes impaired DNA replication while cytoplasmic (RNA, hemoglobin) maturation continues, producing nuclear-cytoplasmic desynchrony: impaired nuclear maturation with abundant cytoplasm and large marrow progenitors.
• Defective DNA synthesis leads to intramedullary cell death (ineffective erythropoiesis) with ↑↑ LDH and ↑ indirect bilirubin (jaundice from ↑ heme catabolism), mimicking hemolysis.
• B12 and folate deficiency give similar peripheral blood and marrow morphology; the neurologic syndrome and the metabolite pattern (MMA) distinguish them.

Differential diagnosis of macrocytosis

• Normal newborn infant.
• Trisomy 21.
• Brisk reticulocytosis (reticulocytes are large).
• Marrow failure.
• Medications (anti-epileptic drugs and others).
• Hypothyroidism, liver disease.
• Megaloblastic anemia (B12, folate deficiency).

Vitamin B12 (cobalamin) physiology

• Sources: animal products only (meat, fish, dairy, eggs). Humans are completely dependent on dietary sources; strict vegans require supplementation.
• Absorption:
  • Stomach: dietary B12 released from food by gastric acid and pepsin; binds R-protein (haptocorrin) from saliva.
  • Duodenum: pancreatic enzymes degrade R-protein, then B12 binds intrinsic factor (IF, secreted by gastric parietal cells).
  • Terminal ileum: IF-B12 complex absorbed via cubilin receptor. Disease there causes B12 malabsorption.
• Stores: liver stores 3 to 5 years' worth, so deficiency develops slowly and gradually (vs folate, 3 to 4 months).
• Function: cofactor for (1) methionine synthase (homocysteine to methionine, requires methyl-THF) and (2) methylmalonyl-CoA mutase (methylmalonyl-CoA to succinyl-CoA). B12 deficiency therefore raises BOTH MMA and homocysteine.

Causes of B12 deficiency

• Pernicious anemia (PA): autoimmune destruction of gastric parietal cells, causing ↓ intrinsic factor plus achlorhydria. Anti-IF Ab (specific, ~70% sensitivity); anti-parietal cell Ab (sensitive ~80%, less specific). ↑ risk of gastric carcinoid and adenocarcinoma. Often with other autoimmune disease (autoimmune polyglandular syndrome, vitiligo, Hashimoto thyroiditis, type 1 DM).
• Atrophic gastritis (especially elderly): age-related parietal cell loss; H. pylori-related.
• Gastric/duodenal surgery: gastric bypass (Roux-en-Y), gastrectomy, partial gastrectomy, causing ↓ IF and ↓ acid.
• Terminal ileal disease (cubilin receptor): Crohn disease, ileal resection, tropical sprue, intestinal TB, lymphoma.
• Bacterial overgrowth: blind loop, diverticula (bacteria consume B12 before absorption).
• Fish tapeworm (Diphyllobothrium latum): rare; consumes B12.
• Drugs: metformin (chronic use, ↓ B12 by ~10 to 30%), PPIs and H2 blockers (chronic, mild), nitrous oxide (acute: oxidizes the cobalt of B12 and inactivates methionine synthase; recreational abuse causes acute megaloblastic anemia plus neuropsychiatric findings), neomycin, colchicine.
• Dietary insufficiency: strict vegans (no animal products), and the breastfed infant of a vegan mother; rare in others given 3 to 5 year stores.
• HIV: malabsorption plus ↓ intake.

Inherited and congenital B12 disorders

• Juvenile pernicious anemia: autosomal recessive, rare; mutation in the gastric IF gene, giving low serum B12 and an abnormal Schilling test that IS corrected by IF. NOT associated with autoimmune disorders (unlike adult PA). Treat with IM B12.
• Imerslund-Grasbeck syndrome: autosomal recessive, usually presents in infancy to early childhood (variable onset); mutations in AMN or CUBN (encode amnionless and cubilin). B12 malabsorption from inability of the IF/B12 complex to bind in the terminal ileum; abnormal Schilling test NOT corrected by IF. Associated with proteinuria.
• Transcobalamin II deficiency: autosomal recessive, early infancy; pancytopenia, diarrhea, failure to thrive. Plasma cobalamin and folate levels are NORMAL, but marrow is floridly megaloblastic; serum TC-II is low or absent.

Clinical features of B12 deficiency

• Hematologic: macrocytic anemia (MCV often >115); pancytopenia in severe cases; hypersegmented neutrophils (≥6 lobes, or ↑ proportion of 5-lobed forms); ↑ LDH, ↑ indirect bilirubin (intramedullary hemolysis from ineffective erythropoiesis); reticulocytopenia.
• Neurologic (KEY: does NOT occur with folate deficiency):
  • Subacute combined degeneration of the spinal cord: posterior columns (loss of vibration, proprioception) plus lateral columns (upper motor neuron signs, spasticity, hyperreflexia, positive Babinski).
  • Peripheral neuropathy: symmetric, length-dependent, sensorimotor.
  • Cognitive/neuropsychiatric: dementia, mood disturbance, psychosis ("megaloblastic madness"), psychomotor retardation, seizures, ataxia.
  • Optic neuropathy: rare.
  • Neuro findings can occur WITHOUT anemia and may be irreversible if prolonged.
• GI: glossitis (smooth, beefy red tongue, Hunter glossitis), anorexia, weight loss.

Diagnosis

• Serum B12: <200 pg/mL diagnostic; 200 to 300 borderline (interpret with clinical scenario plus metabolites). The B12 level is variable and less reliable unless quite low.
• MMA (methylmalonic acid): ↑ in B12 deficiency (specific). Useful when serum B12 is borderline.
• Homocysteine: ↑ in BOTH B12 and folate deficiency.
• Anti-IF antibodies: specific for PA (~70% sensitivity).
• Anti-parietal cell antibodies: sensitive (~80%) but less specific.
• Gastrin: ↑↑ in PA (achlorhydria drives hypergastrinemia).
• Schilling test: obsolete (replaced by anti-IF antibodies and MMA).

Treatment of B12 deficiency

• Parenteral (IM or deep SC) for severe deficiency, neurologic symptoms, or malabsorption (PA): 1,000 mcg 1 to 3 times per week (or once daily for 1 week), then 1,000 mcg once weekly for 4 to 8 weeks, then monthly thereafter (lifelong if PA). If neurologic symptoms are present, may give 1,000 mcg every other day for up to 3 weeks or until no further improvement, then transition to weekly (Means 2021; Mechanick 2020; Stabler 2013).
• Oral high-dose (1,000 to 2,000 mcg/day): effective even in PA via passive absorption (~1% absorbed without IF). Convenient long-term option.
• Treat B12 BEFORE folate if both might be deficient. Folate alone can correct the hematologic picture while neurologic deterioration progresses.
• Response: reticulocytosis within 1 week (brisk); Hb normalizes over 6 to 8 weeks; neuro recovery is slower (months) and may not fully reverse if treatment was delayed.
• Hypokalemia: brisk erythropoiesis can drop potassium; monitor and replete.

Folate deficiency

• Sources: leafy greens, legumes, fortified grains. Much less common than B12 deficiency.
• Stores: only 3 to 4 months, so deficiency develops faster than B12.
• Causes:
  • Decreased intake (most common): alcoholism, elderly, "tea and toast" diet, infant/child drinking goat's milk.
  • Increased need: pregnancy, lactation, chronic hemolytic anemia (sickle cell, HS, thalassemia, PK deficiency), exfoliative skin disease, hemodialysis.
  • Drugs: methotrexate, sulfasalazine, phenytoin, trimethoprim, pyrimethamine, triamterene.
  • Malabsorption: celiac disease, tropical sprue, IBD (Crohn), jejunal resection.

Folate deficiency: clinical features and diagnosis

• Hematologic: same as B12 (macrocytic anemia plus hypersegmented neutrophils), indistinguishable morphologically.
• no subacute combined degeneration (key distinction from B12; rare sensory axonal neuropathy reported).
• Diagnosis: serum folate first-line (RBC folate, which reflects tissue stores with less daily variation, is rarely needed); serum folate <2 ng/mL diagnostic and >4 makes deficiency unlikely; homocysteine ↑, MMA NORMAL.

Folate deficiency: treatment

• Folic acid 1 to 5 mg PO daily for 1 to 4 months until hematologic recovery.
• Pregnancy and pre-conception: all women of reproductive age 0.4 mg/day; 4 mg/day if prior neural tube defect or on anti-epileptics.
• Chronic hemolytic anemia (sickle cell, HS, PK deficiency): lifelong folate 1 mg/day to support increased erythropoiesis.
• Always rule out B12 deficiency BEFORE giving folate: folate can correct the anemia of B12 deficiency while permitting neurologic deterioration.

Metabolite pattern: B12 vs folate deficiency

Copper deficiency (a megaloblastic-anemia mimic)

• Combined hematologic and neurologic manifestations: anemia, neutropenia, and myeloneuropathy. Often misdiagnosed as MDS.
• History of bariatric or other GI surgery, or excess zinc intake (zinc-induced copper deficiency).
• Smear/marrow: iron-containing plasma cells, hyposegmented and hypogranular neutrophils, dyserythropoiesis with cytoplasmic vacuoles, and ringed sideroblasts.
• Treat with copper supplementation.

High yield

• B12 deficiency: MMA ↑ AND homocysteine ↑. Folate deficiency: only homocysteine ↑ (MMA normal).
• Pernicious anemia: anti-IF Ab (specific); anti-parietal cell Ab (sensitive). ↑ gastric cancer and carcinoid risk; associated with other autoimmune disease.
• Treat B12 BEFORE folate if both could be deficient.
• Oral high-dose B12 (1 to 2 mg/day) is effective even for PA via passive absorption.
• Subacute combined degeneration: posterior plus lateral columns; B12 only.
• Nitrous oxide abuse causes acute megaloblastic anemia plus neuropsychiatric findings (inactivates methionine synthase).
• Metformin (chronic) reduces B12 absorption.
• Folate 4 mg preconception if prior neural tube defect or on anti-epileptics; sickle cell disease: lifelong folate 1 mg/day.
• Hypersegmented neutrophils (any with ≥6 lobes, or >5% with 5 lobes): early marker; typically resolves within about 10 to 14 days of replacement, before MCV and Hb normalize (about 8 weeks) in megaloblastic anemia.
• Copper deficiency (GI surgery, excess zinc) can mimic MDS with anemia, neutropenia, myeloneuropathy, and ringed sideroblasts.