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Absorption, transport and metabolism of cyanocobalamin

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Presentation on theme: "Absorption, transport and metabolism of cyanocobalamin"— Presentation transcript:

1 Absorption, transport and metabolism of cyanocobalamin
Domina Petric, MD

2 Absorption I. November 23, 2018 Combs GF. The Vitamins. Fundamental Aspects in Nutrition and Health. Elsevier Inc

3 Digestion The naturally occurring vitamin B12 in foods is bound in coenzyme form to proteins. The vitamin is released from such complexes on heating, gastric acidification and/or proteolysis (especially by the action of pepsin). Impaired gastric parietal cell function, as in achlorhydria, impairs vitamin B12 utilization. November 23, 2018 Combs GF. The Vitamins. Fundamental Aspects in Nutrition and Health. Elsevier Inc

4 Protein binding in the gut
Free vitamin B12 is bound to proteins secreted by the gastric mucosa. November 23, 2018 Combs GF. The Vitamins. Fundamental Aspects in Nutrition and Health. Elsevier Inc

5 R proteins The binding of vitamin B12 to these glycoproteins may be adventitious. They are found in human gastric juice, intestinal contents and several other bodily fluids. November 23, 2018 Combs GF. The Vitamins. Fundamental Aspects in Nutrition and Health. Elsevier Inc

6 R proteins show structural and immunologic similarities.
Their differencies in electrophoretic mobility are due to differing carbohydrate contents. November 23, 2018 Combs GF. The Vitamins. Fundamental Aspects in Nutrition and Health. Elsevier Inc

7 R proteins The R proteins in the intestine are not necessary for the enteric absorption of vitamin B12, as they are normally digested proteolytically in the alkaline conditions of the small intestine, whereupon they release their ligands to be bound by intrinsic factor (IF). November 23, 2018 Combs GF. The Vitamins. Fundamental Aspects in Nutrition and Health. Elsevier Inc

8 R proteins Because vitamin B12 binds preferentially to R proteins rather than to IF under the acidic conditions of the stomach, R proteins can interfere with the absorption of vitamin B12. Patients with pancreatic exocrine insufficiency, and consequent deficiencies of proteolytic activities in the intestinal lumen, can achieve high concentrations of R proteins that cause poor absorption of the vitamin. November 23, 2018 Combs GF. The Vitamins. Fundamental Aspects in Nutrition and Health. Elsevier Inc

9 Intrinsic factor The intrinsic factor (IF) is synthesized and secreted by the gastric parietal cells in response to histamine, gastrin, pentagastrin and the presence of food. Individuals with loss of gastric parietal cell function may be unable to use dietary vitamin B12, as these cells produce both IF and acid, both of which are required for the enteric absorption of the vitamin. November 23, 2018 Combs GF. The Vitamins. Fundamental Aspects in Nutrition and Health. Elsevier Inc

10 Intrinsic factor Geriatric patients, many of whom are hypoacidic, may be at risk of low vitamin B12 status. November 23, 2018 Combs GF. The Vitamins. Fundamental Aspects in Nutrition and Health. Elsevier Inc

11 Intrinsic factor IF binds the four cobalamins with comparable, high affinities under alkaline conditions: methylcobalamin, adenosylcobalamin, cyanocobalamin and aquocobalamin. Intrinsic factor also binds a specific receptor in the ileal mucosal brush border. Cobalamin binding appears to have an allosteric effect on the ileal receptor-binding center of IF, causing the protein complex to dimerize and increasing its binding to the receptor. November 23, 2018 Combs GF. The Vitamins. Fundamental Aspects in Nutrition and Health. Elsevier Inc

12 Intrinsic factor Formation of the IF-vitamin B12 complex protects the vitamin from catabolism by intestinal bacteria and protects IF from hydrolytic attack by pepsin and chymotrypsin. November 23, 2018 Combs GF. The Vitamins. Fundamental Aspects in Nutrition and Health. Elsevier Inc

13 Mechanisms of absorption
November 23, 2018 Combs GF. The Vitamins. Fundamental Aspects in Nutrition and Health. Elsevier Inc

14 Active transport The carrier-mediated absorption of vitamin B12 is highly efficient and quantitatively important at low doses (1-2 µg). Such doses appear in the blood within 3-4 hours of consumption. The active transport of vitamin B12 depends on the interactions of the IF-vitamin B12 complex with a specific receptor in the microvilli of the ileum. November 23, 2018 Combs GF. The Vitamins. Fundamental Aspects in Nutrition and Health. Elsevier Inc

15 Active transport The ileal IF receptor, a glycoprotein, binds the IF-vitamin B12 complex, but little, if any, free IF or free vitamin B12. November 23, 2018 Combs GF. The Vitamins. Fundamental Aspects in Nutrition and Health. Elsevier Inc

16 Active transport Receptor binding occurs at neutral pH and depends on Ca2+, in the presence of which it forms a stable IF-vitamin B12-IF receptor complex. The receptor is anchored to the brush border membrane and effects the enteric absorption of vitamin B12 through the endocytotic internalization of the receptor-bound complex. November 23, 2018 Combs GF. The Vitamins. Fundamental Aspects in Nutrition and Health. Elsevier Inc

17 Active transport The absorption of vitamin B12 by the enterocyte involves the cellular uptake of the dissociated vitamin, with the release of the unbound IF to the intestinal lumen. Upon entering the enterocyte, the vitamin is bound to an intracellular protein that is immunologically similar to IF, and eventually transferred to the portal circulation bound to a specific carrier protein, transcobalamin II (TCII). November 23, 2018 Combs GF. The Vitamins. Fundamental Aspects in Nutrition and Health. Elsevier Inc

18 Active transport Human patients who lack IF have very low abilities to absorb vitamin B12, excreting in the feces % of oral doses (versus the 30-60% fecal excretion rates of individuals with adequate IF). November 23, 2018 Combs GF. The Vitamins. Fundamental Aspects in Nutrition and Health. Elsevier Inc

19 Simple diffusion Diffusion of the vitamin occurs with low efficiency (1%) throughout the small intestine and becomes significant only at higher doses. Such doses appear in the blood within minutes of consumption. This passive mechanism is utilized in therapy for pernicious anemia, in which patients are given high doses (>500µg/day) of vitamin B12 per os. The vitamin must be given an hour before or after a meal. November 23, 2018 Combs GF. The Vitamins. Fundamental Aspects in Nutrition and Health. Elsevier Inc

20 Transport II. November 23, 2018 Combs GF. The Vitamins. Fundamental Aspects in Nutrition and Health. Elsevier Inc

21 Transport proteins On absorption from the intestine, vitamin B12 is initially transported in the plasma, most of which is bound as the adenosylcobalamin and methylcobalamin to an R protein called transcobalamin I (TCI). Most of the remainder is bound to another binding protein transcobalamin II (TCII) synthesized in several tissues, including the intestinal mucosa, liver, seminal vesicles, fibroblasts, bone marrow and macrophages. November 23, 2018 Combs GF. The Vitamins. Fundamental Aspects in Nutrition and Health. Elsevier Inc

22 Transcobalamin I (TCI)
It is a 60-kDa α-glycoprotein. This is an R protein, also referred to as haptocorrin. Vitamin B12 bound to this protein appears to turn over very slowly (half-life is 9-10 days), becoming available for cellular uptake only over fairly long time frames. TCI occurs at very high concentrations in saliva, breast milk, tears and other secretions. November 23, 2018 Combs GF. The Vitamins. Fundamental Aspects in Nutrition and Health. Elsevier Inc

23 Transcobalamin II (TCII)
It is smaller (38 kDa). TCII serves as the chief transport protein of the vitamin, binding it stoichiometrically in a 1:1 molar ratio. Only 10-25% of plasma vitamin B12 is bound to this transporter. The rapid turnover (half-life is 90 min) of the protein-ligand complex renders TCII the only functional source of the vitamin for cellular uptake. November 23, 2018 Combs GF. The Vitamins. Fundamental Aspects in Nutrition and Health. Elsevier Inc

24 Transcobalamin III (TCIII)
It is electrophoretically similar to TCI, but antigenically similar to TCII. Its metabolic role is less clear. November 23, 2018 Combs GF. The Vitamins. Fundamental Aspects in Nutrition and Health. Elsevier Inc

25 Transport proteins The movement of vitamin B12 from the intestinal mucosal cells into the plasma appears to depend on the formation of the TCII-vitamin B12 complex (the vitamin is shuttled from IF to TCII). This complex turns over rapidly: half-life is about 6 min. TCII is necessary for normal cellular maturation of the hematopoietic system. Because cobalamin is lost within days from TCII, the amount bound to that protein can be a useful parameter of early-stage vitamin B12 deficiency. November 23, 2018 Combs GF. The Vitamins. Fundamental Aspects in Nutrition and Health. Elsevier Inc

26 Transcobalamin receptor
Membrane-bound receptor proteins for TCII occur in all cells. The TCII receptor is structurally similar to TC. It is a 50-kDa glycoprotein with a single binding site for the TCII-vitamin B12 complex. The binding is of high affinity and requires Ca2+. The cellular uptake of vitamin B12 might involve such TCII receptors mediating the pinocytotic entrance of the vitamin-TCII complex into the cell. November 23, 2018 Combs GF. The Vitamins. Fundamental Aspects in Nutrition and Health. Elsevier Inc

27 Role of R proteins Most recently absorbed vitamin B12 is transferred to the plasma R protein TCI, which binds approximately three-quarters of the circulating vitamin B12. Owing to the specificity of the TCI for methylcobalamin, that vitamer predominates in the circulation of humans. Congenital deficiency of this R protein results in low concentrations of vitamin B12 in the plasma, but not in detectable losses in function, as cobalamins bound to TCI do not appear to be available for cellular uptake. November 23, 2018 Combs GF. The Vitamins. Fundamental Aspects in Nutrition and Health. Elsevier Inc

28 Intracellular protein binding
After its cellular uptake, the TCII-receptor complex is degraded in the lysosome to yield the free vitamin, which can be converted to methylcobalamin in the cytosol. Virtually all of the vitamin within the cell is bound to two vitamin B12-dependent enzymes: ● Methionine synthetase (also called methyl-FH4 methyltransferase) in the cytosol ● Methylmalonyl-CoA mutase in mitochondria November 23, 2018 Combs GF. The Vitamins. Fundamental Aspects in Nutrition and Health. Elsevier Inc

29 Distribution in tissues
Vitamin B12 is the best stored of the vitamins. Under conditions of non-limiting intake, the vitamin accumulates to very appreciable amounts in the body, mainly in the liver (about 60% of the total body store) and muscles (about 30% of the total). November 23, 2018 Combs GF. The Vitamins. Fundamental Aspects in Nutrition and Health. Elsevier Inc

30 Distribution in tissues
Total hepatic reserve is about 1,5 mg. Mean total body stores of vitamin B12 are in the range of 2-5 mg. Pituitary gland, kidneys, heart, spleen and brain also contain substantial amounts: µg of vitamin B12. November 23, 2018 Combs GF. The Vitamins. Fundamental Aspects in Nutrition and Health. Elsevier Inc

31 Distribution in tissues
The great storage and long biological half-life ( days) of the vitamin provide substantial protection against periods of deprivation. The low reserve of the human infant (25 µg) is sufficient to meet physiological needs for about a year. November 23, 2018 Combs GF. The Vitamins. Fundamental Aspects in Nutrition and Health. Elsevier Inc

32 Distribution in tissues
The predominant form in plasma is methylcobalamin (60-80% of the total), owing to the presence of TCI that selectively binds that vitamer. November 23, 2018 Combs GF. The Vitamins. Fundamental Aspects in Nutrition and Health. Elsevier Inc

33 Distribution in tissues
The vitamin B12 concentration of human milk varies widely ( pg/ml) and is particularly great (10-fold that of mature milk) in colostrum. Although those products contain TCII, most of the vitamin (mainly methylcobalamin) is bound to R proteins. November 23, 2018 Combs GF. The Vitamins. Fundamental Aspects in Nutrition and Health. Elsevier Inc

34 Metabolism III. November 23, 2018 Combs GF. The Vitamins. Fundamental Aspects in Nutrition and Health. Elsevier Inc

35 Activation to coenzyme forms
Vitamin B12 is delivered to cells in the oxidized form, hydroxycob(III)alamin, where it is reduced by thiol- and reduced flavin-dependent reduction of the cobalt center of the vitamin (to Co+) to form cob(I)amin, also called vitamin B12s. November 23, 2018 Combs GF. The Vitamins. Fundamental Aspects in Nutrition and Health. Elsevier Inc

36 Activation to coenzyme forms
The vitamin is active in metabolism only as derivatives that have either a methyl group (methylcobalamin) or 5′-deoxyadenosyl group (adenosylcobalamin) attached covalently to the cobalt atom. The conversion to these coenzyme forms involves two different enzymatic steps. November 23, 2018 Combs GF. The Vitamins. Fundamental Aspects in Nutrition and Health. Elsevier Inc

37 Generation of methylcobalamin
This step is catalyzed by the cytosolic enzyme 5-methyl-FH4: homocysteine methyltransferase. By producing methylcobalamin, it renders the vitamin a carrier for the single carbon unit in the regeneration of methionine from homocysteine. November 23, 2018 Combs GF. The Vitamins. Fundamental Aspects in Nutrition and Health. Elsevier Inc

38 Generation of adenosylcobalamin
The adenosylation of the vitamin occurs in the mitochondria due to the action of vitamin B12 coenzyme synthetase, which catalyzes the reaction of cob(II)amin with a deoxyadenosyl moiety derived from ATP. This step depends on the entry of hydroxycobalamin into the mitochondria and its subsequent reduction in sequential, one electron steps involving NADH and NADPH linked aquacobalamin reductases to yield cob(II)alamin. November 23, 2018 Combs GF. The Vitamins. Fundamental Aspects in Nutrition and Health. Elsevier Inc

39 Catabolism Vitamin B12 is excreted as the intact cobalamin.
Only the free cobalamins (not the methylated or adenosylated forms) in the plasma are available for excretion. November 23, 2018 Combs GF. The Vitamins. Fundamental Aspects in Nutrition and Health. Elsevier Inc

40 Excretion Vitamin B12 is excreted via both renal and biliary routes at the daily rate of about 0,1-0,2% of total body reserves. Glomerular filtration of the vitamin is minimal: <0,25 µg/day. Urinary excretion of the vitamin after a small oral dose can be used to assess vitamin B12 status: this is called the Schilling test. November 23, 2018 Combs GF. The Vitamins. Fundamental Aspects in Nutrition and Health. Elsevier Inc

41 Excretion The biliary excretion of the vitamin is substantial, accounting for the secretion into the intestine of 0,5-5 µg/day. Most (65-75%) of this amount is reabsorbed in the ileum by IF-mediated active transport. This enterohepatic circulation constitutes a highly efficient means of conservation, with biliary vitamin B12 contributing only a small amount to the feces. November 23, 2018 Combs GF. The Vitamins. Fundamental Aspects in Nutrition and Health. Elsevier Inc

42 Literature Combs GF. The Vitamins. Fundamental Aspects in Nutrition and Health. Elsevier Inc November 23, 2018


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