Cobalmin & Folate Metabolism

Cyanocobalamin (Vitamin B12) is one of a family of cobalamins consisting of 1) a corrin ring around a cobalt atom and 2) a nucleotide group of a base (5,6dimethylbenzimidazole) and a phosphorylated ribose with 1-amino,2-propanol. This complex is linked to a -group. The -group is: cyanide in cyanocobalamin (at right), an adenosyl group in adenosylcobalamin, a methyl group in methylcobalamin, and a hydroxyl group in hydroxocobalamin. Conversion between forms readily takes place in body tissues.

Cobalamin is synthesized exclusively by bacteria. Cobalamin is temporarily bound to R-binding proteins, but released by duodenal digestive enzymes. The newly freed cobalamin is bound by intrinsic factor (IF) secreted by gastric parietal cells. This IF-cobalamin complex binds to receptors on the brush border of the ileal mucosa and is absorbed over a period of several hours. Most absorbed cobalamin (90%) is transfered to transcobalamin II, the primary transport protein of cobalamin.

Total body stores of cobalamin measure 2-5 mg. The average daily diet in the US contains about 5-30 g, where as daily requirements are only 2-5 g/day. Cobalamin, in the form of adenosylcobalamin, is stored primarily in the liver. Adenosylcobalamin is key to conversion of methylmalonyl CoA to succinyl CoA (Krebs cycle). Methylcobalamin is an essential part of the folate-cobalamin reaction necessary for DNA synthesis. Folic acid (pteroyl monoglutamic acid) occurs in nature as relatively insoluble aggregates - polyglutamates.

Leafy vegetables (broccoli,spinach,asparagus,lettuce), fruit, milk, eggs, liver, and yeast contain folic acid. Some intestinal bacteria also form folic acid. The average US diet contains about 1,000 g of folate. The daily requirement is 100-200 g/day and body stores average 5,000g. Additional folate is required during pregnancy and growth. Liver is a major storage site, but releases folate only into the bile for reaborption in the jejunum and ileum. This process, refered to as the enterohepatic circulation, is necessary for maintainance of plasma folate levels. Alcohol interferes with the enterohepatic circulation of folate and can lower plasma folate levels within hours. Folate is absorbed in the proximal jejunum and ileum although the mechanism is unclear. Conjugases along the brush border break polyglutamates into monoglutamates for absorption. Folate circulates free or albumin bound in the plasma as N5-methyl FH4. Absorbed N5-methyl FH4 hands off a methyl group to synthesize methionine from homocysteine in a step requiring cobalamin and generates FH4 (tetrahydrofolate) which is reconjugated to N5,10-methylene FH4 or other FH4-(Glu)n for use in thymidilate and purine synthesis.

Vit B12

Cofactor for methionine synthase Methylcobalamin is required for the function of the folate-dependent enzyme, methionine synthase. This enzyme is required for the synthesis of the amino acid, methionine, from homocysteine. Methionine in turn is required for the synthesis of S-adenosylmethionine, a methyl group donor used in many biological methylation reactions, including the methylation of a number of sites within DNA and RNA (3). Methylation of DNA may be important in cancer prevention. Inadequate function of methionine synthase can lead to an accumulation of homocysteine, which has been associated with increased risk of cardiovascular diseases

S-adenosylhomocysteine is formed during Sadenosylmethionine-dependent methylation reactions, and the hydrolysis of Sadenosylhomocysteine results in homocysteine. Homocysteine may be remethylated to form methionine by a folatedependent reaction that is catalyzed by methionine synthase, a vitamin B12-dependent enzyme. Alternately, homocysteine may be metabolized to cysteine in reactions catalyzed by two vitamin B6-dependent enzymes.

Cofactor for L-methylmalonyl-CoA mutase 5-Deoxyadenosylcobalamin is required by the enzyme that catalyzes the conversion of L-methylmalonylCoA to succinyl-CoA. This biochemical reaction plays an important role in the production of energy from fats and proteins. Succinyl CoA is also required for the synthesis of hemoglobin, the oxygen carrying pigment in red blood cells (3). Symptoms of vitamin B12 deficiency Vitamin B12 deficiency results in impairment of the activities of B12-requiring enzymes. Impaired activity of methionine synthase may result in elevated homocysteine levels, while impaired activity of Lmethylmalonyl-CoA mutase results in increased levels of a metabolite of methylmalonyl-CoA called methylmalonic acid (MMA). Individuals with mild vitamin B12 deficiency may not experience symptoms, although blood levels of homocysteine and/or MMA may be elevated (12). Megaloblastic anemia Diminished activity of methionine synthase in vitamin B12 deficiency inhibits the regeneration of tetrahydrofolate (THF) and traps folate in a form that is not usable by the body (diagram), resulting in symptoms of folate deficiency even in the presence of adequate folate levels. Thus, in both folate and vitamin B12 deficiencies, folate is unavailable to participate in DNA synthesis. This impairment of DNA synthesis affects the rapidly dividing cells of the bone marrow earlier than other cells, resulting in the production of large, immature, hemoglobin-poor red blood cells. The resulting anemia is known as megaloblastic anemia and is the symptom for which the disease, pernicious anemia, was named (3). Supplementation with folic acid will provide enough usable folate to restore normal red blood cell formation. However, if vitamin B12 deficiency is the cause, it will persist despite the resolution of the anemia. Thus, megaloblastic anemia should not be treated with folic acid until the underlying cause has been determined (5).

Vitamin B12 and Nucleic Acid Metabolism

5,10-Methylene tetrahydrofolate (TH4) is required for the synthesis of nucleic acids, while 5-methyl TH4 is required for the formation of methionine from homocysteine. Methionine, in the form of S-adenosylmethionine, is required for many biological methylation reactions, including DNA methylation. Methylene TH4 reductase is a flavin-dependent enzyme required to catalyze the reduction of 5,10-methylene TH4 to 5methyl TH4.

Megaloblastic Anemia The normal proliferation of cells depends on adequate folate and vitamin B12. Folate is necessary for efficient thymidilate synthesis and production of DNA. B12 is needed to successfully incorporate circulating folic acid into developing RBCs; retaining the folate in the RBC. Review the metabolic mechanisms of B12 and folate (See Normal Hematopoiesis-B12 and Folate Metabolism) Pyrimidines: thymine, cytosine, and uracil Purines: adenine and guanine Lack of folate or B12 leads to decreased dTTP synthesis, resulting in a slowing of DNA synthesis. The role of folate is illustrated at right. The mechanism by which B12 leads to decreased DNA synthesis is unclear. Two likely explanations follow.

Methylfolate "trap" hypothesis. Lack of cobalamin slows the methyltransferase reaction resulting in increased N5-methyl FH4. Thus N5-methyl FH4 is "trapped" or unable to be metabolized to FH4. N5-methyl FH4 can convert to N5, 10-methylene FH4, but poorly without new FH4 other forms of folate diminish leading to slowed synthesis of dTMP. Formate "starvation" hypothesis. Lack of cobalamine slows the methyltransferase reaction deceasing methionine production and in turn depressing formate generation.

A deficiency of either vitamin Bl2 or folic acid results in megaloblastic erythroid cells-megaloblasts. These deficiencies result in a decrease in DNA synthesis which slows and inhibits DNA replication (nuclear division). Nuclear maturation is slowed whereas cytoplasmic maturation (largely dependent on RNA function and unaffected by failure of thymidilate synthesis) is relatively unimpeded. The impaired nuclear maturation is seen as open, loose, immature chromatin (cut-salami pattern).

In contrast to the nucleus, the cytoplasm of megaloblastic cells is abundant with normal hemoglobinization. This disparity between nucleus and cytoplasm is known as nuclear-cytoplasmic asynchrony.

Although most noticeable in erythroid cells failure of DNA synthesis also affects myeloid and megakaryocytes. Giant bands (right) and hypersegmented polymorphonuclear neutrophils (below) are common.

Even megakaryocytes (right) may be hypersegmented.

The impaired RBC production and destruction of defective RBCs in the marrow before release into the peripheral blood (ineffective erythropoiesis) results in the anemia. A bone marrow biopsy and aspirate reveal erythroid hyperplasia. Erythrocytic precursors (promegaloblasts with open, immature chromatin) are increased.

Bone marrow biopsy from patient with megaloblastic anemia. Arrowheads point to megaloblasts and arrow to RBC undergoing apoptosis.

Inhibition of thymidylate synthetase leads to decreased dTTP synthesis and formation of excess dUTP. The dUTP is incorporated into DNA. Repair of this abnormal DNA is blocked by lack of thymidine residues; the DNA breaks apart (karyorrhexis) and the cell dies.

The megaloblastic changes are most apparent in the polychromatophilic and orthochromatophilic stages. Multinucleate RBCs, abnormal karyorrhexis, increased pyknosis, and Howell-Jolly bodies ( right) may be seen.

The peripheral blood reveals a pancytopenia (decreased RBCs, white cells, and platelets), hypersegmented neutrophils (> five lobes), and oval macrocytes.

Once a macrocytic anemia is identified (MCV >100) and medications excluded, a PBS should be examined. Round macrocytes suggest possible thyroid or liver disease, while oval macrocytes suggest B12 or folate deficiency. The reticulocyte count is usually normal or low (if increased: hemolysis or blood loss are likely).

Megaloblastic anemia is most often due to a B12 or folate deficiency, but the cause of the deficiency must be determined for proper treatment. Megaloblastic anemia due to vitamin B12 deficiency caused by a lack of intrinsic factor is specifically referred to as pernicious anemia.

Laboratory testing of B12 and folate are critical to establishing the cause of a megaloblastic anemia.

B12 Absorption involves a series of several proteins and receptors. Antibodies against the proteins of cells involved can be helpful in the diagnosis of macrocytic anemia. Anti-intrinsic factor antibodies, are fairly specific, but unfortunately, not sensitve. Antiparietal cell antibodies while sensitive, are not specific as they are seen in a number of other diseases.

The Schilling test tests for evidence of impaired vitamin B12 absorption correctable by intrinsic factor. The Schilling test Radioactive cobalamin (Cbl*) is taken orally; followed by injection of a saturating dose of non-radioactive cobalamin. The level of Cbl* is measured in the urine. In pernicious anemia the excreted levels of Cbl* are low. If intrinsic factor is given with the Cbl* the Cbl* levels will correct in PA, but not in ileal malabsorption.

Clinical findings include a yellowish- lemon skin, glossitis (smooth tongue) and stomatitis in severe cases. Neurologic abnormalities, secondary to defects in myelination, are seen in Vitamin B12, but not folate deficiencies. The mechanism for the demyelination is believed to be lack of methyl-B12 for conversion of homocysteine to methionine. This results in decreased production of S-

Posterior and lateral degeneration typical of late stage disease.

adenosylmethionine (SAM) needed for methylation of phosphatidylethanolamine to phosphatidylcholine for incorporation into myelin. Degeneration of the posterior columns and peripheral nerve damage leads to numbness; "pins and needles" feeling; loss of position and vibratory sense. Later, lack of coordination; weakness of the legs can be seen. In long standing disorders an ataxic gait; +Babinski sign (lateral columns) may be found. Today neurologic manifestations are relatively rare (<20% of patients) and usually mild.

B12 deficiency resulting in megaloblastic anemia may also be caused by a complete gastric resection [remember parietal cells (IF) are found in the gastric fundus]. Similarly, the IF-B12 complex is absorbed in the distal ileum, thus intestinal malabsorption syndromes or ileal resection can result in B12 deficiency. Antibiotics can allow cobalmin dependent bacterial over growth in the intestine resulting in vitamin B12 depletion. [Similarly any intestinal malformation (congenital or surgical) ie. blind loop syndroms, will decrease peristalsis leading to statis; bacterial overgrowth and vitamin B12 deficiency.] As B12 is found solely in foods of animal origin, individuals on strict vegetarian diets are susceptible to B12 deficiency. Megaloblastic anemia secondary to folate deficiency can be caused in several ways. Most common is severe dietary deprivation of folate from chronic alcohol abuse or malnuroishment. Similarly, the demands of the fetus and poor maternal diet may combine to produce folate deficiency in pregnancy. Folate deficiency may also be caused by malabsorption secondary to intestinal infection by Giardia or intestinal sprue. Chemotherapy for malignancy such as methotrexate, a folic acid antagonist, will cause megaloblastic anemia and requires "rescue" with citrovorum (N5-formyl THF). Dilantin and oral contraceptives may also cause folate deficiency. A relative folate deficiency may occur when the demand for folate increases due to accelerated erythropoiesis (ie. as a compensatory erythroid hyperplasia in hemolytic anemia).