Subject Review. The Biochemical Basis of Cobalamin Deficiency

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1 Subject Review The Biochemical Basis of Cobalamin Deficiency AYALEW TEFFERI, M.D., AND RAJIV K. PRUTHI, M.D. Objective: In this report, our goal was to summarize the current knowledge of the biochemical basis for the impaired DNA synthesis and neuropathy associated with vitamin B12 deficiency. Material and Methods: We reviewed the pertinent literature and our clinical experience with cobalamin deficiency. Results: Studies have established that the megaloblastic hematopoiesis associated with vitamin B]2 and folate deficiency is secondary to impaired DNA synthesis. Two mechanisms of impairment of DNA synthesis have been proposed: the "methylfolate trap hypothesis" and the "formate starvation hypothesis." One possibility is that both hypotheses may be contributory that is, incoming dietary folate may be inaccessible for polyglutamation in accordance with the methylfolate trap hypothesis, whereas the formate starvation hypothesis may explain the failure to use already polyglutamated forms of folate. Conclusion: Although the pathophysiologic mechanisms of vitamin B12 and folate deficiency are not completely understood, nutritional anemias offer suitable models for the study of the biochemical basis of disease. (Mayo Clin Proc 1994; 69: ) Ado = adenosyl; C = carbon; Cbl = cobalamin; CN = cyanide; CoA = coenzyme A; IF = intrinsic factor; Me = methyl; MeMaCoA = methylmalonyl-coenzyme A; OH = hydroxyl; PGA = pteroylglutamic acid (folic acid); SAM = S-adenosylmethionine; SGMT = serine-glycine hydroxymethyltransferase; TC = transcobalamin; THF = tetrahydrofolate The megaloblastic anemias result from partially impaired DNA synthesis during hematopoiesis, which eventuates in morphologically evident larger-than-normal precursor cell's with delayed nuclear maturation. The impaired DNA synthesis is attributable to a perturbation of the enzymatic DNA repair or synthetic pathways. Ineffective enzymatic activity may be caused by either a deficiency or an inhibition of a cofactor activity. The latter mechanism is being exploited in cancer chemotherapy. Two of the cofactors necessary for DNA synthesis are vitamin B]2 and folate. A knowledge of their interdependent metabolic pathways is essential for understanding the pathophysiologic aspects of megaloblastic anemia caused by vitamin B12 or folate deficiency. VITAMIN B12 Vitamin B12 was first isolated in and synthesized in It is structurally classified as a corrinoid.3 The corrinoids are a family of compounds with a corrin ring (Fig. 1 ). The corrin ring consists of four reduced pyrrole subrings joined in a macrocyclic ring linked by the a positions of the pyrrole subrings in a planar configuration (corrin nucleus). The pyrrole subrings are linked to a central cobalt atom. The From the Division of Hematology and Internal Medicine, Mayo Clinic Rochester, Rochester, Minnesota. Address reprint requests to Dr. Ayalew Tefferi, Division of Hematology, Mayo Clinic Rochester, 200 First Street SW, Rochester, MN term "corrin" was initially proposed because this structure forms the core of the vitamin B]2 molecule, not because it contains cobalt.3 Subfamilies of the corrinoids are formed by the addition of various substituents on the cobalt atom, above and below the plane of the corrin nucleus. For example, the cobalamins (Cbls) are corrinoids in which 5,6-dimethylbenzimidazole (a nucleotide) is linked to the cobalt atom below the plane of the corrin nucleus (Fig. 1). Furthermore, the addition of various anionic group ligands, linked to the cobalt atom above the plane of the corrin nucleus, yields the various forms of the Cbls (Fig. 1). Accordingly, the addition of cyanide (CN) gives rise to cyano-cbl (CN-Cbl), methyl (Me) to Me-Cbl, adenosyl (Ado) to Ado-Cbl, and hydroxyl (OH) to OH-Cbl. Animal products are the primary dietary source of Cbls. Meat contains OH-Cbl and Ado-Cbl, and dairy products contain OH-Cbl and Me-Cbl.4 Crystalline vitamin B12, which is used for treating Cbl deficiency, is CN-Cbl. For the rest of our discussion, vitamin B12 will be referred to as CN- Cbl. The coenzymatically active forms are Me-Cbl and Ado-Cbl the respective cofactors for methionine synthase (Fig. 2 A) and methylmalonyl-coenzyme A (MeMaCoA) mutase5 (Fig. 2 B). (These cofactors will be discussed in more detail subsequently in this report.) OH-Cbl and CN- Cbl are converted into the coenzymatically active forms (Me-Cbl and Ado-Cbl) in human tissue. Mayo Clin Proc 1994; 69: Mayo Foundation for Medical Education and Research

2 182 BIOCHEMISTRY OF COBALAMIN DEFICIENCY Mayo Clin Proc, February 1994, Vol 69 Fig. 1. Chemical structure of cobalamins. Chanarin I. The Megaloblastic Anaemias. Blackwell, By permission.) Anionic ligand CN CH3 OH *Ado Coffin ring Anucteotide (dimethyl benzimidazole) (Modified from 2nd ed. Oxford: Cbl in food is bound to protein and must undergo peptic digestion in the acidic environment of the stomach to be released. The freed Cbl initially binds to R-binder (a cobalophilin with a rapid electrophoretic mobility [hence the "R"], found in saliva and gastric juice), which is later degraded by pancreatic enzymes in the duodenum; this process releases, once again, free Cbl. Thereafter, the Cbl binds tó parietal cell-derived intrinsic factor (IF). This relationship is a prerequisite for absorption of Cbl at the terminal ileum by means of IF receptors (Fig. 3). Absorbed Cbl is transported from the enterocyte to Cblrequiring tissues by transcobalamin (TC) II. Approximately 80% of plasma Cbl, however, is bound to other cobalophilins (TC I and the granulocyte-derived TC III) which, unlike TC II, also bind Cbl analogues and transport them to the liver.6 The holotranscobalamin complex (TC II-Cbl complex) binds to specific cellular receptors and is internalized (Fig. 3). FOLATE Unlike the Cbls, the dietary sources of folate are both animal products and leafy vegetables. Structurally, folic acid (pteroylglutamic acid [PGA]) consists of a pteridine moiety linked to a para-aminobenzoic acid residue attached to the glutamate side chain (Fig. 4). Although the synthetic vitamin is a monoglutamate (PGA,), the naturally occurring folates, including the dietary forms, are polyglutamates (PGAn).7 The adult daily requirement is approximately 200 μg, but the recommended amount increases during pregnancy and lactation.8 The monoglutamate form (PGA,) is necessary for transport across cell membranes (Fig. 5). PGA, is formed from PGAn by hydrolysis of the glutamate side chain at the jejunal brush-border surface by folate hydrolase (a deconjugase).9 Interaction with membrane-associated folate-binding proteins may result in internalization of the receptor-pga, complex, as has been demonstrated in isolated nonintestinal cell systems.10 Within the enterocyte, PGA, is polyglutamated to PGAn for two reasons: (1) to promote folate-dependent reactions (the active coenzyme forms are polyglutamates) and (2) to maintain a concentration gradient for the uptake of monoglutamates.i0 Subsequently, PGAn are reconverted into the monoglutamate form for release into the portal circulation after it undergoes methylation and reduction into 5- methyltetrahydrofolate (Me-THF,)" (Fig. 5). In the plasma, a third of the folate is free, and the rest is nonspecifically bound to albumin. Intracellular uptake may be mediated by folate-binding receptors.10 INTRACELLULAR COFACTOR ACTIVITIES OF Cbl AND FOLATE As previously discussed, the target cell (for example, an erythrocyte precursor) is provided with Me-THF, and a Cbl derivative. Coparticipation of these two compounds in inter- (Homocysteine) CH2 CH2 CH COOH SH NH2 (Propionyl CoA) CH3^- CH2 «0 = C SCoA B Methionine synthase (MeMaCoA) : CH3-C COOH 0=C SCoA (Methionine) CH2 CH2 CH COOH I I S NH2 Cbl (enzyme bound) THF MeMaCoA (Succinyl CoA) mutase CH2 CH2 COOH 0=C-SCoA Cofactor = Ado Cbl Fig. 2. A, Cofactor activity of methylcobalamin (CH3-Cbl) in methionine synthesis. THF = tetrahydrofolate. B, Cofactor activity of adenosylcobalamin (Ado-Cbt) in succinyl-coenzyme A {CoA) synthesis. MeMaCoA = methylmalonyl-coenzyme A.

3 Mayo Clin Proc, February 1994, Vol 69 BIOCHEMISTRY OF COBALAMIN DEFICIENCY 183 Intestinal Lumen Mucosa Blood Stomach Duodenum Distal ileum H IF-CW - Cbl + TCn -» TCn-Cbl complex Fig. 3. Enteric processing and absorption of cobalamin (Cbl). IF = intrinsic factor; R-binder = a cobalophilin with a rapid (compared with IF) electrophoretic mobility; TCu = transcobalamin II. mediary metabolic pathways leads to effective DNA synthesis. Although these pathways have not been precisely characterized, several prior investigations have provided a framework on which reasonable postulates can be constructed and discussed. The primary intracellular function of folates is to transfer one-carbon (1-C) units, at the oxidation levels of methyl (-CH 3 ), méthylène (-CH 2 -), or formyl (HCO), to facilitate DNA synthesis. To achieve this coenzymatic activity, the folate analogues must be in both a reduced form (THF) and the polyglutamated form (THF n ). 12 The primary function of Cbl is to provide coenzymatic activity for the synthesis of methionine and succinyl-coenzyme A (CoA) (Fig. 2). For this function, the Cbl derivatives must be converted to Me- or Ado-Cbl, and the Cbl molecule must be in a reduced state for optimal enzyme binding. 13 In DNA synthesis, the formation of methionine is the "central" reaction (Fig. 6). Both Cbl and folate are necessary for this reaction. Me-Cbl is the cofactor for methionine synthase in the conversion (by methylation) of homocysteine to methionine (Fig. 2 A and 6). The original methyl donor for the reaction is Me-THF in either the monoglutamate form (Me-THF,, obtained primarily from the diet) or the polyglutamate form (Me-THF n, obtained primarily from intracellular recycling of folate intermediates). 13 The methyl group is first transferred from Me-THF to the enzyme-bound Cbl to form Me-Cbl, which then transfers the methyl group onto homocysteine to generate methionine (Fig. 6). This central reaction provides two products essential for DNA synthesis THF n for 1-C unit transport and methionine, which, although controversial, may facilitate the availability of the appropriate folate intermediary in the process. FORMATION OF INTERMEDIATE FOLATE ANALOGUES Polyglutamation (by means of folylpolyglutamate synthase) of folate analogues is necessary for cellular retention 12 and use in 1-C transfer pathways during DNA synthesis. Furthermore, Me-THF, must be demethylated before it can be polyglutamated. 12 This sequence of events offers a partial explanation for the shortage of THF n during Cbl deficiency. Of note, the poor substrate activity for polyglutamation is restricted to Me-THF, and does not involve other monoglutamate forms, including methylene-thf, and formyl-thf,. 14 / V I H // Vi II N (' N >C-NH-C CH, Substituted pteridine moiety Para-amino benzoic acid residue (PABA) Fig. 4. Chemical structure of folic acid. c COOH CH CH 2 ÇH 2 COOH L-glutamic acid residue

4 184 BIOCHEMISTRY OF COBALAMIN DEFICIENCY Mayo Clin Proc, February 1994, Vol 69 Jejunal Lumen (Polyglutamated folate) PGAn' Brush-border folate hydrolase PGA, (Monoglutamated folate) Mucosa Reduction & methylation PGA - - Me-THF, PGA, Folylpoly- ' glutamate synthase Blood Me-THF, (No specific blood transport protein) Fig. 5. Enteric processing and absorption of folates. Me = methyl; PGA t and PGA n = pteroylglutamic acid (folic acid) monoglutamated and polyglutamated forms; THF I = monoglutamated form of tetrahydrofolate. A méthylène group is added to THF n by serine-glycine hydroxymethyltransferase (SGMT) during the conversion of serine to glycine (Fig. 6). This is a reversible reaction in that methylene-thf can be converted back to THF.' 2 Alterna- J n tively, certain data suggest that, in vivo, methylene-thf may be produced by the oxidation of Me-THF, 15 a reaction catalyzed by méthylène reductase (Fig. 6). Pertinent to a later discussion herein, the reduction of methylene-thf to Me-THF (by the same enzyme) is inhibited by methionine derivatives.' 5 Methylene-THF n is a key intermediate and may be used in one of three ways: ( 1 ) it can provide the méthylène group to convert deoxyuridylate into thymidylate, (2) it can be oxidized to formyl-thf n, which provides 1-C fragments in purine synthesis, and (3) it can be reduced back to Me-THF n to provide a methyl group during methionine synthesis (Fig. 6). Formyl-THF is the other folate analogue that is directly involved with DNA synthesis (purine synthesis). Formyl- THF may be produced by oxidation of methylene-thf and vice versa (Fig. 6). Alternatively, formyl-thf may be produced by direct formate transfer (through formyl-thf synthase) 16 from THF n (Fig. 6). For the latter reaction, the sources of formate include methionine, which may independently facilitate the formate transfer without being the formate donor. 16 The foregoing discussions support one point of view, which maintains that methionine may be important in promoting the availability of the appropriate folate analogues (methylene-thf and formyl-thf), which are essential for n DNA synthesis. Methionine inhibits the reduction of methylene-thf to Me-THF 15 and facilitates formate transfer in the formation of formyl-thf n from THF n. 16 Therefore, when the production of methionine is directly curtailed by Cbl deficiency, DNA synthesis is indirectly affected. MECHANISM OF IMPAIRED DNA SYNTHESIS IN Cbl DEFICIENCY The "Methylfolate Trap Hypothesis." As an extension of the foregoing material, méthylène- and formyl-thf can be considered directly involved in thymidylate and purine synthesis, respectively. At least two sources of methylene-thf n are available, one of which is the circulating Me-THF!. As discussed previously, Me-THF! cannot be demethylated in the absence of Cbl and thus is inaccessible for polyglutamation. 12 The second source involves a recycling of THF n as a by-product of the 1-C transfer reactions. Some of the methylene-thf n is reduced to Me-THF n (a reaction that is normally inhibited by a methionine derivative [Sadenosylmethionine or SAM]; see Figure 6), which could be "trapped" at the methionine synthase reaction stage the basis for the methylfolate trap hypothesis. 17 Observations that contradict the methylfolate trap hypothesis include the reversal of the defect by methionine, 18 the possibility that Me-THF can be directly oxidized to methylene-thf,' 5 and the inability of demethylated THF to correct the defect. 19 These observations have been used as evidence to support an alternative hypothesis (see subsequent section) that considers the availability and utilization of formate the main defect in Cbl deficiency. 20

5 Mayo Clin Proc, February 1994, Vol 69 BIOCHEMISTRY OF COBALAMIN DEFICIENCY 185 Methionine synthase Adenosyl transferase i Homocysteine _^- ^_»Methionine'» S-adenosylmethionine - s (SAM) CrVCW CW \ / N / I Φ CHrTHF, THF, ï Folylpolyglutamate synthase Θ / I è Serlne^lyclne _ Serine TJFn ^ F/ methyl transferase Glyci 5^jj5^ Oxidation.<«ss$$^ Deoxyuridylate Thymidylate CH3-THFn Méthylène y^reductase purine synthesis H Central reaction S Key Intermediates Enzymes Fig. 6. Intracellular interdependent cofactor activity of cobalamin (Cbl) and folate. CH} = methyl group; THF and THF = monoglutamated and polyglutamated forms of tetrahydrofolate. Although the methylfolate trap hypothesis can explain both plasma and tissue accumulations of Me-THF in Cbl deficiency, alternative explanations also exist. Plasma Me- THF accumulations in Cbl deficiency can result from reduced cellular uptake21 rather than leakage of trapped cellular Me-THF. Similarly, the lack of methionine and its derivative (SAM) in Cbl deficiency results in excess Me-THF because of lack of inhibition, by SAM, of methylene-thf reductase, which converts methylene-thf to Me-THF15 (Fig. 6). The "Formate Starvation Hypothesis." An alternative hypothesis, the formate starvation hypothesis, considers the lack of methionine (as a result of Cbl deficiency) the most important causative factor in impaired DNA synthesis.20 This theory is based on several observations, including the substantial but incomplete reversal of Cbl deficiency states with methionine18 and the lack of formyl-thf generation and increased formate accumulation during Cbl deficiency, emphasizing the importance of methionine in formate transfer.16 The incomplete reversal of clinical features with methionine may reflect the failure of methionine to improve cellular uptake of folates rather than suboptimal coenzymatic activity in formate delivery and utilization.19 In addition, methionine may be adenosylated (SAM) and may be used as a source of methyl and formate groups20 22 (Fig. 6). With methionine deficiency, concentrations of SAM decline, a situation that promotes methylene-thf reduction to Me-THF15 (SAM inhibits the forward conversion of methylene-thf to Me-THF; see Figure 6). Furthermore, an effort to conserve SAM for essential methylation might result in decreased SGMT activity and lead to reduced generation of methylene-thfn23 (SGMT adds a méthylène group to THFn during conversion of serine to glycine; see Figure 6). Moreover, the inability of THF, but not formyl-thf, to correct folate-dependent reactions in Cbl deficiency has been cited as further evidence in support of the formate starvation hypothesis.20 A recent study, however, has demonstrated that thymidylate synthesis in Cbl deficiency can be corrected by THF, although with less efficiency than formyl- THF.24 Could Both Hypotheses Be Contributory? One possibility is that circulating methylfolate (Me-THF^ may be handled differently than recycled cellular polyglutamated methylfolate (Me-THFn). Thus, incoming dietary folate (Me-THFj) may be inaccessible for polyglutamation in accordance with the methylfolate trap hypothesis, whereas the formate starvation hypothesis may explain the failure to use already polyglutamated forms of folate. Nevertheless, neither hypothesis can satisfactorily explain the reversal of megaloblastic hematopoiesis in some patients treated with pharmacologie doses of folic acid.

6 186 BIOCHEMISTRY OF COBALAMIN DEFICIENCY Mayo Clin Proc, February 1994, Vol 69 BIOCHEMICAL BASIS OF NEUROPATHY IN Cbl DEFICIENCY The absence of neuropathy in patients with folate deficiency suggested that methionine synthesis may not be causally related to Cbl-associated neuropathy, and attention initially focused on the other Cbl-dependent reaction, which is the conversion of MeMaCoA to succinyl-coa catalyzed by MeMaCoA mutase 5 (Fig. 2 ß). For this reaction, Ado-Cbl is the cofactor, and the oxidative state and intracellular site of the Cbl molecule differ 13 from those of Me-Cbl, factors that may explain the initial absence of methylmalonic aciduria in N 2 0 poisoning. 20 Earlier studies showed that excess MeMaCoA inhibited long-chain fatty acid synthesis by being incorporated into terminal positions and resulting in the formation of abnormal branched-chain fatty acids. 25 Similarly, an excess of propionyl-coa, the immediate precursor of MeMaCoA, results in odd-chained fatty acid synthesis. 26 Therefore, investigators have presumed that these abnormal fatty acids participated in abnormal myelin formation. Recent observations, however, do not support the aforementioned hypothesis. First, hereditary MeMaCoA mutase deficiency and defects in Ado-Cbl synthesis do not cause Cbl neuropathy, 13 whereas N 2 0 poisoning, which inactivates methionine synthase, does not appreciably affect MeMaCoA mutase and yet causes neuropathy. Second, hereditary defects of the methionine synthesis reaction cause Cbl neuropathy, 23 and the administration of methionine to Cbldeficient animals ameliorates the neuropathy. 27 Therefore, dysfunction of methionine synthesis rather than succinyl- CoA synthesis may be responsible for Cbl neuropathy. Methionine is adenosylated into SAM, which along with its derivatives is needed in transmethylation reactions and polyamine synthesis, including those that occur in the central nervous system. Although defective amine turnover in the central nervous system is thus a possibility, 28 the exact mechanism by which reduced methionine causes demyelination remains to be elucidated. A recent communication suggested that cultured human glial cells may be suitable for study of the biochemical basis of Cbl neuropathy. 29 REFERENCES 1. Rickes EL, Brink NG, Koniuszy FR, Wood TR, Folkers K. Crystalline vitamin B 2. Science 1948; 107: Maugh TH II: Vitamin B 12 : after 25 years, the first synthesis. 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