Folate, Cobalamin and Megaloblastic anemiaGrerk Sutamtewagul, M.D.PGY-3Morning Report 7/30/2013

Key ConceptsFolate and Cobalamin (vitamin B12) play key

roles in the metabolism of all cells, particularly proliferating cells.

Folate in its tetrahydro form is a transporter of one-carbon fragments which is an important step in biosynthesis of purines, thymidine, and methionine.

Cobalamin is required for 2 reactions: intramitochondrial conversion of methylmalonyl coenzyme A to succinyl CoA and cytosolic conversion of homocysteine to methionine.

Key ConceptsThe Megaloblastic anemia of cobalamin

deficiency results from an intracellular folate deficiency.

Absorption of cobalamin is a highly complex process involving haptocorrin binder, intrinsic factor (from gastric parietal cells), receptor-mediated endocytosis, transcobalamin (serum transporter).

Most common cause of folate deficiency usually nutritional in origin: alcoholics, elderly, patient with hyperalimentation, hemolytic anemia, hemodialysis, tropical/non-tropical sprue.

Key ConceptsThe most common cause of cobalamin

deficiency is pernicious anemia (autoimmune destruction of gastric parietal cell).

Pernicious anemia increases risk of gastric cancer by 2-3 times.

Other causes of cobalamin deficiency include gastric resection, stasis of intestinal content (blind loops, strictures, hypomotility), terminal ileum resection/disease, vegan diet.

Key Concepts“Acute” megaloblastic anemia:

Nitrous oxide Severe hemolytic anemia

Other causes of megaloblastic anemia: Drugs (hydroxyurea, nucleoside analogues) Certain inborn errors of metabolism

Folate

FolateSource: vegetables, fruits, liver, folate

fortification (in the US)

Daily requirement: 50 mcg minimum, RDA: 0.4 mg

Increased requirement in Hemolytic anemia, Leukemia Other malignant diseases Alcoholism Growth Pregnancy and lactation (3-6 times)

Folate MetabolismTetrahydrofolate is an

intermediate in reactions involving the transfer of one-carbon units.

Metabolic systems requiring folate coenzymes Serine-Glycine conversion Thymidylate synthesis Histidine catabolism Methionine synthesis Purine synthesis Pyrimidine synthesis

Folate Metabolism Intracellular folates exist primarily as

polyglutamate conjugates (75%).

Intracellular Folylmonoglutamates leak out of the cells at a fairly rapid rate whereas Polyglutamates do not. Polyglutamate form is more active and will retain in the cell.

Folate deficiencyDietary deficiency

Impaired absorption

Increased requirement

Folate deficiencyDietary deficiency

Inadequate dietary intake (before 1990s) – decreased dramatically after folate fortification

Infant raised with goat milk Excessive cooking

Impaired absorption Non-tropical sprue (Celiac disease) Tropical sprue Other intestinal disorders: scleroderma,

amyloidosis, DM, systemic bacterial infection

Folate deficiency Increased requirement

Hemodialysis (folate loss in dialysate) Pregnancy (transfer to growing fetus)

Difficult to diagnose due to physiologic anemia and macrocytosis (mean MCV 104).

Serum and rbc folate fall steadily even in well nourished women.

Hypersegmented neutrophil is a reliable clue. Increased cell turnover: Hemolytic anemia, chronic

exfoliative dermatitis, psoriasis

Diagnosisof Folate deficiencyHistory and laboratory finding indicating folate

deficiency

Absence of the neurological signs of cobalamin deficiency

Full response to physiologic dose of folate

Laboratory Findings Serum Folate

Earliest specific finding Correlate well (and varies) with recent intake (within

few days)

RBC Folate Better indicator of tissue folate status Remain unchanged for 2-3 months Also falls in cobalamin deficiency – not use for

differentiate folate from cobalamin deficiency

Serum Homocysteine Increase homocysteine may precede a fall in folate

level but is non-specific.

Laboratory FindingsMacrocytosis

Differential diagnosis of macrocytosis without megaloblastic anemia Alcoholism Liver disease Hypothyroidism Aplastic anemia Certain myelodysplasia Pregnancy Reticulocytosis

Cobalamin

CobalaminVitamin B12 (cyanocobalamin – therapeutic

form)

4 Forms of cobalamin in animal cell metabolism: cyanocobalamin, hydroxocobalamin, adenosylcobalamin, and methylcobalamin (major circulating form)

Source of CobalaminAnimals cannot produce cobalamin.

Animals depend on microbial synthesis or animal product intake for cobalamin supply.

Cobalamin has not been found in plants.

Species from the following genera are known to synthesize B12: Acetobacterium, Aerobacter, Agrobacterium, Alcaligenes, Azotobacter, Bacillus, Clostridium,Corynebacterium, Flavobacterium, Lactobacillus, Micromonospora,  Mycobacterium, Nocardia, Propionibacterium, Protaminobacter, Proteus, Pseudomonas, Rhizobium, Salmonella, Serratia, Streptomyces, Streptococcus and Xanthomonas.

Body composition of cobalaminTotal body cobalamin is around 2-5 mg.

1 mg is in the liver.

Daily loss of cobalamin is 0.1% of total body pool. Several years is required to develop deficiency state.

Cobalamin MetabolismThere are only 2 recognized cobalamin-

dependent enzymes in human: Mitochondrial Adenosylcobalamin-dependent

Methylmalonyl CoA mutase Cytosolic Methylcobalamin-dependent

N5-Methyltetrahydrofolate-homocysteine methyltransferase

Methylmalonyl CoA mutaseMethylmalonyl CoA

(from proprionate) is changed to Succinyl CoA and enters Krebs cycle.

N5-Methyltetrahydrofolate-homocysteine methyltransferase

Synthesis of Methionine

Also involves in demethylation of N5-methyltetrahydrofolate to tetrahydrofolate which is needed for conjugation to polyglutamate. THF-polyglutamate will retain in the cell.

Nitrous oxide (N2O) can impair methyltransferase Acute megaloblastic anemia

Folate-cobalamin relationshipFolate can, at least, temporarily

correct megaloblastic anemia from cobalamin deficiency.

Cobalamin cannot correct megaloblastic anemia from folate deficiency.

Megaloblastic anemia from cobalamin deficiency is actually an abnormality in folate metabolism (Folate trap hypothesis).

Cobalamin transport

Stomach Peptic digestion

liberates cobalamin from foods.

Cobalamin is bound to Haptocorrin(HC)-like protein with more avidity than intrinsic factor in stomach pH.

Terminal Ilium Pancreatic protease

releases cobalamin from HC complex.

Cobalamin is then bound to intrinsic factor, forming a complex which is very resistant to digestion.

Duodenum Cobalamin-IF

complex undergo receptor-mediated endocytosis via IF receptor, Cubilin.

IF is degraded in the lyzosome, releasing cobalamin into cytoplasm.

Transcobalamin forms complex with cobalamin blood.

Cobalamin transportLike the folates, the cobalamins undergo

appreciable enterohepatic recycling.

If the absorption is intact, a very long time – as long as 20 years – is required for a clinically significant cobalamin deficiency to develop from strictly vegan diet.

Cobalamin deficiency - outlineDecreased uptake caused by impaired

absorption

Intestinal diseases

Blind Loop syndrome

AIDS

Pancreatic disease

Dietary cobalamin deficiency

Cobalamin deficiency – pernicious anemiaDecreased uptake – impaired absorption

Pernicious anemia (most common) Failure of gastric intrinsic factor production,

gastric mucosal atrophy, autoimmune Age of onset usually > 40 yrs Anti-intrinsic factor antibody/Anti-Cbl-IF complex

are very specific. Anti-parietal cell Ab (90% in PA, 60% in atrophic

gastritis) Related to other autoimmune diseases:

thyrotoxicosis, Hashimoto thyroiditis, DM type 1, Addison disease, postpartum hypophysitis, infertility

Cobalamin deficiency - othersGastrectomy syndrome (total, partial)

Removal of intrinsic factor

Zollinger-Ellison syndrome High acid prevent a transfer of cobalamin from the

HC complex to IF

Diseases of terminal Ileum Extensive ileal resection IBD, lymphoma, XRT Hypothyroidism, medication

Diphyllobothrium latum infestation

Cobalamin deficiency – Blind loop syndromeBlind Loop syndrome

Intestinal stasis from Anatomic lesions (strictures, diverticula,

anastomoses, surgical blind loops) Impaired motility (scleroderma, amyloidosis)

Treatment: antibiotics Cefalexin 250 mg QID plus Metronidazole 250 mg TID for 10 days

Cobalamin deficiency – Laboratory findingsSerum Cobalamin level

Low in most but not all patients with cobalamin deficiency

Low in normal subjects (vegetarian, pregnancy, taking large dose ascorbic acid)

Serum Holotranscobalamin Functional fraction of serum bound cobalamin

Urine Methylmalonic acid Very reliable indicator of cobalamin deficiency

Cobalamin deficiency – Laboratory findingsSerum Methylmalonic acid and Homocysteine

Elevated MMA and Homocysteine levels are indicators of tissue cobalamin deficiency.

MMA is more sensitive and specific, persists several days after treatment.

MMA elevation is seen only in cobalamin deficiency whereas Homocysteine elevation can be seen in folate/pyridoxine deficiency and hypothyroidism.

Megaloblastic Anemias Disorders caused by impaired synthesis of DNA.

Megaloblastic cells: Erythroid large cells with immature-appearing nuclei Increasing hemoglobinization of the cytoplasm “Nuclear-cytoplasmic asynchrony”

Megaloblastic granulocytic cells Large giant band neutrophil in bone marrow Hypersegmented neutrophil in the marrow and blood

Other rapidly dividing cells may also showed cytologic abnormalities.

Pathogenesis of Megaloblastic anemia Ineffective erythropoiesis

Intramedullary destruction of red cell precursors Hypercellular marrow with apoptosis of late

precursors

Ineffective granulopoiesis and thrombopoiesis also present and can result in neutropenia and thrombocytopenia.

Mild hemolysis with shortening of red cell half-life.

Clinical features of Megaloblastic anemiaAnemia develops gradually and patient is

usually able to adapt to very low Hb level.

Fatigue, palpitation, lightheadedness, shortness of breath

Peripheral blood smear of Megaloblastic anemiaHypersegmented neutrophil

Bone marrow smear of Megaloblastic anemiaErythroid hyperplasia with

marked nuclear/cytoplasmic dysynchrony noted at all stages of erythroid maturation

Giant Band neutrophil in the bone marrow