Vitamin B12 – Chemistry, functions and clinical significance

Professor(Dr.) Namrata Chhabra

Biochemistry for medics- Lecture notes

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Synonyms

• Cyanocobalamine

• Anti Pernicious anemia factor

• Extrinsic factor of Castle

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Structure

• Cobalamin is analogous to heme in its structure having as its base a tetrapyrrole ring.

• Instead of iron as a metal cofactor for heme, cobalamin has cobalt in a coordination state of six with

o a benzimidazole group nitrogen coordinated to one axial position,

o the four equatorial positions coordinated by the nitrogens of the four pyrrole groups and

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Structure of Vitamin B12

oThe sixth position occupied by either a deoxyadenosine group, a methyl group or a CN– group in the commercially available form in vitamin tablets.

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Forms of Cobalamin

• Cobalamin (vitamin B12) exists in a number of different chemical forms.

• All have a cobalt atom at the center of a corrin ring.

• In nature, the vitamin is mainly in the 2-deoxyadenosyl (ado) form, which is located in mitochondria

• The other major natural cobalamin is methylcobalamin, the form in human plasma and in cell cytoplasm.

• There are also minor amounts of hydroxocobalamin to which methyl- and adenosyl cobalamin are rapidly converted by exposure to light.

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Dietary Sources

• Cobalamin is synthesized solely by microorganisms.

• Ruminants obtain cobalamin from the foregut, but the only source for humans is food of animal origin, e.g. meat, fish, and dairy products.

• Vegetables, fruits, and other foods of non-animal origin are free from cobalamin unless they are contaminated by bacteria.

• Strict vegetarians are at risk of developing B12deficiency.

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Sources of vitamin B12

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Requirements of vitamin B12

• A normal Western diet contains between 5 and 30 μg of cobalamin daily.

• Adult daily losses (mainly in the urine and feces) are between 1 and 3 μg (~0.1% of body stores) and, as the body does not have the ability to degrade cobalamin, daily requirements are also about 1 to 3 μg.

• Body stores are of the order of 2 to 3 mg, sufficient for 3 to 4 years if supplies are completely cut off.

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Absorption

• Two mechanisms exist for cobalamin absorption.

• Passive absorption-occurring equally through buccal, duodenal and ileal mucosa; it is rapid but extremely inefficient, <1 percent of an oral dose being absorbed by this process.

• Active absorption-The normal physiologic mechanism is active; it occurs through the ileum and is efficient for small (a few micrograms) oral doses of cobalamin and is mediated by gastric intrinsic factor (IF).

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Absorption

• Dietary cobalamin is released from protein complexes by enzymes in the stomach, duodenum, and jejunum

• It combines rapidly with a salivary glycoprotein that belongs to the family of cobalamin-binding proteins known as haptocorrins (HCs).

• In the intestine, the haptocorrins are digested by pancreatic trypsin and the cobalamin transferred to intrinsic factor(IF).

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Absorption of Vitamin B12 and the role of Intrinsic factor

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Absorption and the role of Intrinsic factor

• Intrinsic factor (IF) is produced in the gastric parietal cells of the fundus and body of the stomach, its secretion parallels that of hydrochloric acid.

• The IF-cobalamin complex passes to the ileum, where IF attaches to a specific receptor (Cubulin) on the microvillus membrane of the enterocytes.

• Cubulin with its ligand IF-cobalamin complex is endocytosed.

• The cobalamin-IF complex enters the ileal cell where IF is destroyed.

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Intrinsic factor deficiency

• In the absence of the intrinsic factor inadequate amounts of cobalamin are absorbed (the dietary requirement is approximately 200 ng/day) resulting in Megaloblastic anemia.

• When the root cause of the resultant Megaloblastic anemia is absence of or inadequate amounts of intrinsic factor the condition is called pernicious anemia.

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Transportation of Cobalamin

• Three plasma transport proteins have been identified.

• Transcobalamine I and III (differing only in carbohydrate structure) are secreted by white blood cells.

• Although approximately 90 percent of plasma vitamin B12 circulates bind to these proteins, only transcobalamine II is capable of transporting vitamin B12 into cells.

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Storage of Cobalamin

• The liver contains 2000 to 5000 mcg of stored vitamin B12.

• Since daily losses are 1 to 3 mcg/day, the body usually has sufficient stores of vitamin B12 so that vitamin B12 deficiency develops more than 3 years after vitamin B12 absorption ceases.

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Metabolic Role of Cobalamin

1)Cobalamin plays a vital role in the catabolism of odd-chain fatty acids, threonine, methionine, and the branched-chain amino acids (leucine, isoleucine, and valine).

• The degradation of each of these compounds produces the same metabolite, Propionyl-CoA.

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Fate of Propionyl CoA

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Fate of Propionyl CoA in B12 deficiency

• In cases of cobalamin deficiency these reactions of utilization of propionyl co A are compromised leading to an accumulation of methylmalonyl-CoA in serum, which has been suggested as a possible source of neurologic defects seen in cobalamin deficiency by decreasing lipid synthesis.

• Excess methylmalonyl-CoA in B12 deficiency gets excreted in urine causing methylmalonic aciduria

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2.Role of cobalamin in DNA synthesis and the biochemical basis of Megaloblastic anemia

• The cause of megaloblastic anemia seen in strict vegetarians is attributed to the effects of cobalamin deficiency on DNA synthesis, specifically the thymidylate synthesize reaction which converts dUMP→ dTMP.

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Reaction catalyzed by Thymidylate synthase

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Implications of Inadequate Thymidylate synthesis

• Inadequate dTMP restricts DNA but not RNA synthesis leading to the appearance of large erythroid cells with small nuclei containing a high ratio of RNA to DNA.

• These cells are removed from the circulation, thus stimulating erythrogenesis and giving rise to anemia with an elevated presence of megaloblasts.

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3. Role of cobalamin in methionine metabolism

• Cobalamin is required for the conversion of homocysteine into methionine.

• Cobalamin must first undergo methyl transfer to form methyl cobalamin.

• It receives the methyl group from N5-methyltetrahydrofolate thus regenerating tetrahydrofolate to participate in other one-carbon transfers in purine metabolism or pyrimidine remodeling.

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Folate trap

• In cobalamin deficiency, the methionine synthase reaction cannot occur, N5-methyltetrahydrofolate accumulates and the other C-1 donor forms of tetrahydrofolate cannot be formed.

• The methionine synthesis from homocysteine ceases allowing the “trapping” of the folate pool as N5-methyltetrahydrofolate, diminishing levels of N5, N10-methylenetetrahydrofolate

• N5,N10-methylenetetrahydrofolate, is required for the methylation of dUMP to dTMP, thus in it’s deficiency ,the thymidylate synthase reaction is slowed and dTMP levels drops and hence DNA synthesis is also slowed down due to non availability of deoxy ribonucleotides

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Roles of cobalamin and folic acid in methionine metabolism

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Vitamin B12 deficiency

Causes of Vitamin B12 Deficiency

• Dietary deficiency (rare)

• Decreased production of intrinsic factor

• Pernicious anemia

• Gastrectomy

• Pancreatic insufficiency

• Fish tapeworm (rare)

• Helicobacter pylori infection

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Vitamin B12 deficiency

• Crohn’s disease

• Surgical resection

• Decreased ileal absorption of vitamin B12

• Transcobalamine II deficiency (rare)

• Competition for vitamin B12 in gut Blind loop syndrome

• The most common cause of vitamin B12 deficiency is associated with pernicious anemia

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Vitamin B12 deficiency

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Megaloblastic anemia

Clinical Findings• The hallmark of symptomatic vitamin B12 deficiency is

megaloblastic anemia. • However, subclinical cobalamin deficiency is an increasingly

recognized condition, especially in those with predisposing conditions such as ileal disease or gastric surgery.

• In advanced cases, the anemia may be severe, with hematocrit as low as 10 to 15 percent, and may be accompanied by leucopenia and thrombocytopenia.

• The megaloblastic state also produces changes in mucosal cells, leading to glossitis, as well as other vague gastrointestinal disturbances such as anorexia and diarrhea.

• Patients are usually pale and may be mildly icteric

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Peripheral blood smear in Megaloblastic anemia

Blood film in vitamin B12deficiency showing macrocytic red cells and a hyper segmented neutrophil.

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Neurological changes in B12 deficiency

• Vitamin B12 deficiency also leads to a complex neurologic syndrome.

• Peripheral nerves are usually affected first, and patients complain initially of paresthesias.

• The posterior columns next become impaired, and patients complain of difficulty with balance.

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Neurological changes in B12 deficiency

• In more advanced cases, cerebral function may be altered as well, and on occasion dementia and other neuropsychiatric changes may precede hematologic changes.

• Neurologic examination may reveal decreased vibration and position sense but is more commonly normal in early stages of the disease.

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Laboratory Findings

• The MCV is usually strikingly elevated, between 110 and 140 fL.

• The peripheral blood smear is usually strikingly abnormal, with anisocytosis and poikilocytosis. A characteristic finding is the macro-ovalocyte, but numerous other abnormal shapes are usually seen. The neutrophils are hyper segmented.

• The reticulocyte count is reduced. • Because vitamin B12 deficiency affects all

hematopoietic cell lines, in severe cases the white blood cell count and the platelet count are reduced, and pancytopenia is present.

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Laboratory Findings

• Bone marrow morphology is characteristically abnormal.

• Marked erythroid hyperplasia is present as a response to defective red blood cell production (ineffective erythropoiesis).

• Megaloblastic changes in the erythroid series include abnormally large cell size and asynchronous maturation of the nucleus and cytoplasm—i.e. cytoplasmic maturation continues while impaired DNA synthesis causes retarded nuclear development.

• In the myeloid series, giant metamyelocytes are characteristically seen.

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Laboratory Findings

• Other laboratory abnormalities include elevated serum lactate dehydrogenase (LDH) and a modest increase in indirect bilirubin.

• These two findings are a reflection of intramedullary destruction of developing abnormal erythroid cells and are similar to those observed in peripheral hemolytic anemias

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Laboratory Findings

• Serum cobalamin level: The diagnosis of vitamin B12 deficiency is made by finding an abnormally low vitamin B12 (cobalamin) serum level.

• The normal vitamin B12 level is > 240 pg/ml,

• Most patients with overt vitamin B12 deficiency can have serum levels < 170 pg/ml, with symptomatic patients usually having levels < 100 pg/ml.

• A level of 170 to 240 pg/ml is borderline.

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Laboratory Findings

Estimation of serum methylmalonic acid levels

• When the serum level of vitamin B12 is borderline, the diagnosis is best confirmed by finding an elevated level of serum methylmalonic acid (> 1000 nmol/L

• However, elevated levels of serum methylmalonic acid can also be due to renal insufficiency.

• The Schilling test is now rarely used.

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Essentials of diagnosis

• Essentials of diagnosis are macrocytic anemia.

• Macro-ovalocytes and hyper segmented neutrophils on peripheral blood smear, and

• serum vitamin B12 level less than 100 pg/ml.

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Differential Diagnosis

• Vitamin B12 deficiency should be differentiated from folic acid deficiency, the other common cause of megaloblastic anemia, in which red blood cell folate is low while vitamin B12 levels are normal.

• The distinction between vitamin B12 deficiency and myelodysplasia (the other common cause of macrocytic anemia with abnormal morphology) is based on the characteristic morphology and the low vitamin B12 level

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Pernicious Anemia

• Pernicious anemia is a chronic illness caused by impaired absorption of vitamin B12 because of a lack of intrinsic factor (IF) in gastric secretions.

• The disease was named pernicious anemia because it was fatal before treatment became available

• The term pernicious is no longer appropriate, but it is retained for historical reasons.

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Pernicious Anemia

• Pernicious anemia occurs as a relatively common adult form of anemia that is associated with gastric atrophy and a loss of IF production and

• as a rare congenital autosomal recessive form in which IF production is lacking without gastric atrophy.

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Clinical manifestations in Pernicious anemia

• General findings: Weight loss of 10 to 15 pounds occurs in about 50 percent of patients and probably is due to anorexia, which is observed in most patients.

• Anemia: The anemia often is well tolerated in pernicious anemia, and many patients are ambulatory with hematocrit levels in the mid teens.

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Clinical manifestations in Pernicious anemia

Gastrointestinal findings: • Approximately 50 percent of patients have a smooth tongue with

loss of papillae. The tongue may be painful and beefy red. These symptoms may be associated with changes in taste and loss of appetite.

• Patients may report either constipation or having several semisolid bowel movements daily. This has been attributed to megaloblastic changes of the cells of the intestinal mucosa.

• Nonspecific gastrointestinal symptoms include anorexia, nausea, vomiting, heartburn, flatulence, and a sense of fullness.

• Rarely, patients present with severe abdominal pain associated with abdominal rigidity; this has been attributed to spinal cord pathology.

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Clinical manifestations in Pernicious anemia

Nervous system:

• Neurological symptoms can be elicited in most patients with pernicious anemia, and the most common symptoms are paresthesias, weakness, clumsiness, and an unsteady gait.

• These neurological symptoms are due to myelin degeneration and loss of nerve fibers in the dorsal and lateral columns of the spinal cord and cerebral cortex.

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Clinical manifestations in Pernicious anemia

Genitourinary system:

• Urinary retention and

• Impaired micturition may occur because of spinal cord damage.

• This can predispose patients to urinary tract infections.

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Laboratory Studies

• The peripheral smear shows oval macrocytes, hyper segmented granulocytes, and anisopoikilocytosis.

• In severe anemia, red blood cell inclusions may include Howell-Jolly bodies, Cabot rings, and punctate basophilia.

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Laboratory Studies

• Gastric secretions: Total gastric secretions are decreased to about 10 percent of the reference range.

• Most patients with pernicious anemia are achlorhydric, even with histamine stimulation.

• IF is either absent or markedly decreased.

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Laboratory Studies

• Serum Cbl levels: The serum Cbl is low in patients with pernicious anemia; however, it may be within the reference range in certain patients with other forms of Cbl deficiency.

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Laboratory Studies

• Schilling test: The Schilling test measures Cbl absorption by increasing urine radioactivity after an oral dose of radioactive Cbl.

• The test is useful in demonstrating that the anemia is caused by an absence of IF and is not secondary to other causes of Cbl deficiency.

• It is used to identify patients with classic pernicious anemia, even after they have been treated with vitamin B12

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Laboratory Studies

• Serum: The indirect bilirubin may be elevated because pernicious anemia is a hemolytic disorder associated with increased turnover of bilirubin.

• The serum lactic dehydrogenase usually is markedly increased

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Histological Findings

• The bone marrow biopsy and aspirate usually are hyper cellular and show trilineage differentiation.

• Erythroid precursors are large and often oval.

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Complications

• If patients are not treated early in the disease, neurological complications can become permanent.

• Severe anemia can cause congestive heart failure or precipitate coronary insufficiency.

• The incidence of gastric adenocarcinoma is 2-to 3-fold greater in patients with pernicious anemia than in the general population of the same age.

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Prognosis

• Early recognition and treatment of pernicious anemia provides a normal, and usually uncomplicated, lifespan.

• Delayed treatment permits progression of the anemia and neurological complications. The mental and

• neurological damage can become irreversible without therapy.

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Treatment of vitamin B12 deficiency

• The indications for starting cobalamin therapy are :

• A well-documented Megaloblastic anemia

• or other hematological abnormalities

• or neuropathy due to the deficiency.

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Treatment of vitamin B12 deficiency

• Patients with pernicious anemia have historically been treated with parenteral therapy.

• Intramuscular injections of 100 mcg of vitamin B12 are adequate for each dose.

• Replacement is usually given daily for the first week, weekly for the first month, and then monthly for life.

• It is a lifelong disorder, and if patients discontinue their monthly therapy the vitamin deficiency will recur.

• Oral cobalamin may be used instead of parenteral therapy and can provide equivalent results. The dose is 1000 mcg/day and must be continued indefinitely.

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Further reading

• A case oriented approach towards Biochemistry- By Namrata Chhabra

http://www.jaypeedigital.com/(X(1)S(vclizd4r0zry5eoz45exstdx))/Book/BookDetail?isbn=9789350901885

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