CHAPTER 25 METABOLIC ASPECTS OF FOLIC ACID AND COBALAMIN
CHAPTER 25 METABOLIC ASPECTS OF FOLIC ACID AND COBALAMIN
BERNARD M. BABIOR
Folate in its tetrahydro form is a transporter of one-carbon fragments, which it can carry at any of three oxidation levels: methanol, formaldehyde, and formic acid. The oxidation levels of the folate-bound one-carbon fragments can be altered in oxidation and reduction reactions that require NADP and NADPH, respectively. The chief source of the folate-bound one-carbon fragments is serine, which is converted to glycine as it passes its terminal carbon to folate. The one-carbon fragments are used for the biosynthesis of purines, thymidine, and methionine. During the biosynthesis of purines and methionine, free folate is released in its tetrahydro form, but, during the biosynthesis of thymidine, the tetrahydrofolate is oxidized to the dihydro form and has to be re-reduced by dihydrofolate reductase in order to continue to function in one-carbon metabolism. Methotrexate acts as an anticancer agent because it is an exceedingly powerful inhibitor of dihydrofolate reductase.
In the cell, folates are conjugated by the addition of a chain of 6 or 7 glutamic acid residues. These serve to prevent the folates from leaking out of the cell. When folates are absorbed from the intestine, a process that takes place chiefly in the duodenum and proximal jejunum, all but one of the glutamates are removed by an enzyme known as conjugase. Folates travel in the blood stream and are taken up by the cells mainly in the form of unconjugated methyltetrahydrofolate. In the cell, the newly absorbed folates are rapidly reconjugated. If reconjugation is prevented, the folates leak back out of the cell, resulting in an intracellular deficiency of folate.
Cobalamin is required for two reactions: the conversion of methylmalonyl CoA (a product of catabolism of branched-chain amino acids) to succinyl CoA, a Krebs cycle intermediate, and the conversion of homocysteine to methionine, a reaction in which the methyl group of methyltetrahydrofolate is donated to the sulfur atom of homocysteine. In cobalamin deficiency, methyltetrahydrofolate accumulates, because for practical purposes donation of the methyl group to homocysteine is the only way to generate free tetrahydrofolate from methyltetrahydrofolate. Free tetrahydrofolate is an excellent substrate for the conjugase, but methyltetrahydrofolate is a poor substrate. Consequently, much of the methyltetrahydrofolate taken up by a cobalamin-deficient cell leaks out of the cell before it can be conjugated. The megaloblastic anemia of cobalamin deficiency is actually due to an intracellular folate deficiency that arises because of the limited ability of the cell to conjugate methyltetrahydrofolate.
The absorption of cobalamin is a highly complex process. On first arriving in the stomach, cobalamin is taken up by R-binder (also called haptocorrin or cobalophilin), a glycoprotein that is found in virtually all secretions. When the cobalamin-R binder complex enters the duodenum, the R binder is digested and the cobalamin is released into the intestinal lumen, where it is taken up by intrinsic factor, a protein secreted by the gastric parietal cells. The cobalamin-intrinsic factor complex is absorbed by cells in the ileum, where the cobalamin is released and transported to the blood stream. Here the vitamin binds to transcobalamin II, which delivers its cargo of cobalamin to cells throughout the body. Folic acid (pteroylglutamic acid) and cobalamin (vitamin B12) play key roles in the metabolic economy of proliferating cells.
Acronyms and abbreviations that appear in this chapter include: AICAR, 5-amino-4-imidazole carboxamide ribotide; ATP, adenosine 5’9-triphosphate; NADP, nicotinamide adenine dinucleotide phosphate; NADPH, nicotinamide adenine dinucleotide phosphate (reduced form); TC II, transcobalamin II.
Folic acid (pteroylglutamic acid) is composed of a pteridine derivative, a p-aminobenzoate residue, and an L-glutamic acid residue (Fig. 25-1a). The first two together are called pteroic acid.1 In nature, folic acid occurs largely as conjugates in which multiple glutamic acids are linked by peptide bonds involving their g-carboxyl groups (Fig. 25-1b). Conjugates are named according to the length of the glutamate chain (e.g., pteroylglutamate, pteroyldiglutamate, pteroylhexaglutamate). Therapeutic folic acid (abbreviated PteGlu, or F has one glutamic acid.
FIGURE 25-1 Folic acid. (a) Folic acid (pteroylglutamic acid) and its components. (b) Tetrahydrofolate triglutamate.
To form a functional compound, folate must be reduced to tetrahydrofolate (FH4) (see Fig. 25-1b). In this reduction, dihydrofolate (FH2) is an intermediate. A single enzyme, dihydrofolate reductase, catalyzes both F®FH2 and FH2®FH4.
The folate family consists largely of FH4 derivatives bearing a one-carbon substituent (symbolized as FH4-C). The varieties of FH4-C differ in the identity of the one-carbon unit and the site of its attachment to FH4 (Fig. 25-2). One-carbon substituents of biochemical significance include the following:
FIGURE 25-2 Derivatives of tetrahydrofolic acid (FH4), their interconversions, and the metabolic pathways in which they participate. One-carbon substituents are shown in blue.
These substituents are attached to FH4 through N5, N10, or both (see Fig. 25-2). Specific enzymes interconvert these various FH4 derivatives.
Reduced derivatives of folic acid are usually sensitive to air oxidation. An exception of clinical importance is N5-formyl FH4, also called citrovorum factor, leucovorin, and folinic acid.
There are many sources for folic acid. The richest vegetable sources are asparagus, broccoli, endive, spinach, lettuce, and lima beans; each contains more than 1 mg of folate per 100 g dry weight. The best fruit sources are lemons, bananas, and melons. Folates are also abundant in liver, kidney, yeast, and mushrooms. An average daily U.S. diet contains 400 to 600 µg of folate.2 Foods are readily depleted of their folate, however, by excessive cooking, especially with large amounts of water.
In the normal adult, the minimum daily requirement for folic acid is approximately 50 µg. The average diet contains many times this amount, but some may be unavailable. Accordingly, the officially recommended daily allowance of food folate for the adult is 0.4 mg.3 The body is thought to contain approximately 5 mg of folate4; when folate intake is reduced to 5 µg/day, megaloblastic anemia develops in about 4 months.5
Increased requirements for folic acid occur in hemolytic anemia, leukemia, and other malignant diseases; in alcoholism6; during growth; and in pregnancy and lactation, which increase requirements threefold to sixfold.7 For reasons described in Chap. 38, adequate folate supplies are particularly important in pregnant women, in whom the recommended intake is 400 µg per day.8
FH4 is an intermediate in reactions involving the transfer of one-carbon units from a donor X—C to an acceptor Y:
The metabolic systems of animal tissues known to require folic acid coenzymes are summarized in Table 25-1 and reviewed in references 3, 6, 25, and 26.
TABLE 25-1 METABOLIC SYSTEMS REQUIRING FOLIC ACID COENZYME IN ANIMAL CELLS
One-carbon units enter the folate pool principally by way of the serine hydroxymethyltransferase reaction9:
which requires pyridoxal phosphate as cofactor. In addition, the conversion of methionine to polyamines is accompanied by the production of one-carbon fragments that combine with folate at the oxidation level of formate10,11 (Fig. 25-3). Less important sources of one-carbon units are the catabolism of histidine12 and the ATP-dependent formation of N5-formyl FH4 (folinic acid) from formic acid and FH4.13
FIGURE 25-3 Formate production during polyamine biosynthesis.
Among the one-carbon transfers mediated by folic acid, the one that appears to be clinically the most important is the methylation of deoxyuridylate to thymidylate, catalyzed by the enzyme thymidylate synthase.14 This reaction is an essential step in the synthesis of DNA (Fig. 25-4). In carrying out this reaction, N5, N10-methylene FH4 simultaneously transfers and reduces a one-carbon group, itself serving as the hydrogen donor for the reduction.15 The reaction generates FH2 (Fig. 25-5), which must be reduced again to FH4 by dihydrofolate reductase and NADPH before it can again be utilized as a coenzyme:
FIGURE 25-4 Pathways of deoxynucleotide and DNA synthesis.
FIGURE 25-5 Dihydrofolate (FH2). The double bond formed when tetrahydrofolate loses two hydrogens is shown in blue.
Limitation of thymidylate synthesis in folic acid deficiency causes uracil to be incorporated into DNA instead of thymine.16
Deficiency of folate also diminishes purine biosynthesis by slowing (1) the folate-dependent formylation of glycinamide ribotide to N-formylglycinamide ribotide, the reaction that places the C-8 in the purine ring, and (2) the folate-dependent conversion of 5-amino-4-imidazole carboxamide ribotide (AICAR) to 5-formamido-4-imidazole carboxamide ribotide, the reaction that places the C-2 in the purine ring.17 The decrease in purine synthesis, however, is offset by the ability of AICAR to slow purine degradation by inhibiting both adenosine deaminase and adenylate deaminase.1 This may explain why no clinical manifestations have thus far been traced to the block in purine synthesis. Interference with the breakdown of histidine (Fig. 25-6) leads to the excretion of formininoglutamic acid (FIGlu) in the urine of folate-deficient patients.
FIGURE 25-6 The catabolism of histidine.
Additional pteridine-dependent reactions of potential metabolic importance are the hydroxylation of phenylalanine to tyrosine, the oxidation of long-chain alkyl ethers of glycerol to fatty acid, the hydroxylation of tryptophan to 6-hydroxytryptophan (a precursor of serotonin), the 17-a-hydroxylation of progesterone,1,17,18 and the production of nitric oxide (NO).19,20 and 21 The cofactor for these reactions is biopterin, a nonfolate pteridine derivative. Tetrahydrofolic acid is weakly active in some of these systems in vitro22,23; whether it plays any such role in vivo is not known.
SIGNIFICANCE OF FOLYLPOLYGLUTAMATES
Intracellular folates exist primarily as polyglutamate conjugates.24,25 and 26 About 75 percent of the folate in human erythrocytes and leukocytes is conjugated.27,28 Plasma folate, however, consists almost exclusively of the monoglutamate N5-methyl FH429 and is transported into the cells in this form. Inside the cells, the polyglutamate chain is added by an ATP-dependent folylpoly-g-glutamyl synthase.30,31 The activity of the human synthase depends strongly on the form of the folate substrate, declining in the order N5, N10-methylene FH4®N10-formyl FH4®N5-methyl FH4, toward which the enzyme is almost inert.32 In humans, conjugated folates carry on average seven to eight glutamyl residues33; the length of the polyglutamate chain may be determined by the ability of the higher folate polyglutamates to inhibit folylpolyglutamyl synthase.34 There is evidence that folylpolyglutamyl synthase is regulated within cells, its activity closely paralleling rates of DNA synthesis.35
Intracellular folylmonoglutamates leak out of the cells at a fairly rapid rate, while the polyglutamates do not, presumably because of the highly charged polyglutamate tail.36 Attachment of the polyglutamate chain is therefore essential for retaining folates within cells. Moreover, folylpolyglutamates are superior to monoglutamates as substrates for folate-dependent enzyme reactions.37,38
The proximal jejunum is the principal site of folate absorption. Absorption of a dose of either unconjugated or conjugated folate begins within minutes, and peak levels are reached in 1 to 2 h. Since only folylmonoglutamate appears in plasma, all folylpolyglutamates are deconjugated during absorption across the intestine.39,40
Deconjugating enzymes (“conjugases”) play an important but poorly understood role in the intestinal absorption of folate.40,41 Folylpolyglutamate may be hydrolyzed within the lumen of the intestine, and the monoglutamate product may be absorbed subsequently.42,43 Alternatively, hydrolysis may occur at the brush border of the intestinal cell (Fig. 25-7). A brush border conjugase purified from human jejunum catalyzes the Zn2+-dependent deconjugation of folate polyglutamates ranging from PteGlu2 to at least PteGlu7 (Km=µM for both substrates).44 It is an exopeptidase, successively removing single glutamate residues from the end of the polyglutamate chain to yield the folylmonoglutamate.
FIGURE 25-7 Digestion and absorption of folate polyglutamate by the intestine. The polyglutamate (in this case, PteGlu7) is hydrolyzed in the intestinal lumen or at the brush border. The resulting pteroylglutamate (PteGlu) is transported into the intestinal cell, where it is reduced and methylated, appearing in the circulation chiefly as N5-methyl FH4.
Conjugases are not found only in the intestine. Human plasma, for example, contains sufficient conjugase to convert polyglutamates containing more than three glutamyl residues to monoglutamates.45 Other conjugases appear to be lysosomal carboxypeptidases31,46 that have nothing to do with the absorption of folates from the intestine.
Once deconjugated, the folates are actively transported across the intestinal epithelium47,48 by a carrier-mediated mechanism (Km=1 to 2 µM) that is independent of Na+, K+, and transmembrane potential.49 This mechanism uses the pH gradient between the jejunal lumen (pH~6) and the interior of the epithelial cell to drive folate into the cell against a concentration gradient.50 Passive transport may also occur.51 In the intestinal cell, the absorbed folate monoglutamates are reduced if necessary, then converted to N5-methyl FH4 (some N10-formyl FH4 is made as well) and transported into the bloodstream without further change.29,52
Folate has been shown to undergo an enterohepatic cycle in which it is first secreted against a concentration gradient into the bile, appearing there chiefly as N5-methyl FH4 monoglutamate, and is then reabsorbed from the small intestine.29,53,54 Bile contains approximately 2 to 10 times the folate concentration of normal serum,29,54 with biliary excretion accounting for up to 0.1 mg of folate per day. This quantity is large enough that the interruption of the enterohepatic cycle by biliary diversion causes serum folate levels to fall by over 50 percent in less than a day.53 It has been proposed that the enterohepatic cycle serves to redistribute folate between hepatic stores and peripheral tissues according to the state of the exogenous folate supplies55; this view, however, has been disputed.56
Tritiated folylmonoglutamate (3H-F) administered intravenously is almost completely removed from the bloodstream in a few minutes.57 Uptake involves two classes of folate-binding proteins56,58: the high-affinity folate receptors59,60,61 and 62 that concentrate folate in intracellular vesicles and a membrane folate transporter that transports folate into the cytosol. The high-affinity receptors, which are attached to the outer surface of the cell membrane by glycosyl-phosphatidylinositol linkages,63 bind very tightly (Kd’s in the nM range) to most physiologic folate monoglutamates,56,58,64,65 and 66 in particular to N5-methyl FH4, the major circulating folate.67 Their very high affinity enables them to take up N5-methyl FH4 from the plasma even at its ambient concentration of approximately 10 nM. The membrane folate transporter is a probenecid-inhibitable organic anion carrier anion that among other things carries reduced folates and methotrexate (but not oxidized folate itself) in and out of the cytoplasm.56,58,67,68 Its Km for folate is in the µM range. These two classes of receptors cooperate in the following way to transport N5-methyl FH4 into the cell67,69,70: (1) A region of membrane containing a group of folate-loaded high-affinity receptors is internalized as a vesicle (the caveola), (2) the caveola is acidified, releasing the folate into the vesicle lumen, (3) the folate is passed from the caveola to the cytoplasm by the membrane folate transporter, and, finally, (4) the caveola recycles to the cell surface, where its high-affinity receptors take on another load of N5-methyl FH4.
Once internalized, the folates are retained by the cells partly through polyglutamylation, as was discussed above,71 but also through tight association with a set of intracellular folate-binding proteins.67,72,73 and 74 Three of these proteins are enzymes involved in methyl group metabolism: sarcosine dehydrogenase and dimethylglycine dehydrogenase (mitochondrial)75 and glycine N-methyl transferase (cytosolic).76 It is not known why these enzymes bind folate so avidly or whether this binding affects overall methyl group metabolism, although it has been speculated that glycine N-methyl transferase regulates methyl group metabolism by controlling the tissue concentration of S-adenosylhomocysteine, one of its reaction products and a potent inhibitor of most methyltransferases.
Folates have been found in all body tissues that have been analyzed. The principal form of the vitamin in tissues as well as blood appears to be the N5-methyl form.75,77,78 and 79 Human liver contains 0.7 to 17 µg of folate per gram.80
The total folate pool turns over very slowly.81 A portion of this turnover is accounted for by degradation; p-aminobenzoylglutamate has been identified as a breakdown product. The fate of the pteridine moiety is unknown.
FOLATE-BINDING PROTEINS OF SERUM AND MILK
The soluble folate-binding proteins of serum and milk are high-affinity folate receptors that have been released from cell membranes by proteolysis.56,82 These proteins can be detected in approximately 15 percent of normal individuals83 and are found at increased levels in some pregnant women, women on oral contraceptives, folate-deficient alcoholics (but not patients with cobalamin deficiency),84 and patients with uremia, hepatic cirrhosis, and chronic myelogenous leukemia.85,86 In normal subjects, the proteins are about two-thirds saturated and show a total folate-binding capacity of approximately 175 pg per milliliter of serum.87 Failure to detect the proteins in some subjects seems to be due to their prior saturation with unlabeled folate.88 The serum protein has an Mr of 40,000 and prefers oxidized to reduced folates.88
Folate-binding proteins have also been found in milk and in normal granulocytes.89,90 Folate bound to the milk folate binder is absorbed chiefly in the ileum91 rather than the jejunum, the principal site of absorption of free folate. The milk folate binder, a glycoprotein, also promotes folate transport into the liver via the asialoglycoprotein receptor.92 It has been speculated that the milk folate binder protects an infant’s folate supply by preventing bacteria from sequestering the vitamin away from the intestinal lumen.56 The folate-binding protein in granulocytes has been localized to the specific granules, from which it is released when the granulocytes are stimulated.93
Folates are both resorbed and secreted by the kidney. Resorption is accomplished by means of a membrane-bound high-affinity folate receptor (Km for N5-methyl FH4 = 0.4 nM) located in the brush borders of the proximal tubules.94 Filtered folate is carried rapidly by this receptor into the proximal tubule cell and from there makes its way slowly into the bloodstream.95,96 At the same time, folate is secreted into the proximal tubule by a nonspecific probenecid-sensitive organic anion carrier that is closely related or identical to the membrane folate transporter and is also responsible for the renal secretion of p-aminohippuric acid (which blocks renal folate secretion), penicillin, and uric acid.96 The net result of these two processes is the resorption of most of but not all the filtered folate.
In humans, intact folates and their cleavage products are excreted by the kidney at a rate of 2 to 5 µg/day.97 Folates given in doses lower than 15 µg/kg are excreted in the urine in reduced forms, particularly as N10-formyl FH4.98 With doses of folate greater than 15 µg/kg, large amounts are excreted unchanged.
A small percentage of parenterally administered 3H-F is recoverable in the feces. This mainly represents overflow from the entero-hepatic cycle discussed above.
ASSAY OF SERUM FOLATE
Microbiological assays for folate were in use for many years. Now, however, they have been largely supplanted by isotopic methods employing various folate binders. These isotopic folate assays are identical in principle to radioimmunoassays.
STRUCTURE AND NOMENCLATURE
The cobalamin molecule has two major portions: a porphyrinlike near-planar macrocycle known as corrin and a nucleotide that lies almost perpendicular to the corrin ring (Fig. 25-8). The corrin moiety contains four reduced pyrrole rings8,16,32,81 that bind a central cobalt atom whose two remaining coordination positions are occupied by a 5,6-dimethylbenzimidazolyl group (below the ring) and various ligands (above the ring; in this case, —CN).
FIGURE 25-8 I, structure of cyanocobalamin (CNCbl; vitamin B12). II, partial structure of CNCbl, showing the relationship between the corrin ring and the nucleotide.
Compounds containing the corrin ring are known as corrinoids. The cobalamins are corrinoids whose nucleotide contains 5,6-dimethylbenzimidazole. There are two connections between the corrin and the nucleotide: (1) a bond between the nucleotide phosphate and a side chain in ring D and (2) a bond between cobalt and a nitrogen atom of benzimidazole. The numbering and ring designations of the corrin system are summarized in Fig. 25-9.
FIGURE 25-9 The corrin ring, showing ring designations and standard numbering of the atoms.
The term vitamin B12 is sometimes employed as a generic term for the corrinoids. It is probably best reserved, however, as an alternative name for cyanocobalamin, the usual therapeutic corrinoid.
Four cobalamins are of importance in animal cell metabolism. Two are cyanocobalamin (CNCbl; vitamin B12) and hydroxocobalamin (OHCbl). The other two are alkyl derivatives that are synthesized from hydroxocobalamin and serve as coenzymes. In one, adenosylcobalamin (AdoCbl), a 5-deoxyadenosyl replaces OH as the cobalt ligand above the ring (Fig. 25-10).99 In the second, methylcobalamin (MeCbl), the upper ligand is a methyl group. Methylcobalamin is the major form of cobalamin in human blood plasma.100
FIGURE 25-10 Adenosylcobalamin (AdoCbl). R=CH2CONH2; R’=CH2CH2CONH2.
Cobalamin is synthesized only by certain microorganisms, and animals depend ultimately on microbial synthesis for their cobalamin supply. Foods that contain cobalamin are those of animal origin: meat, liver, seafood, and dairy products. Cobalamin has not been found in plants.
The average daily diet in Western countries contains 5 to 30 µg of cobalamin. Of this, 1 to 5 µg is absorbed.101 Less than 250 ng appears in the urine; the unabsorbed remainder appears in the feces. Total body content is 2 to 5 mg in an adult,102 with approximately 1 mg in the liver. The kidneys are also rich in cobalamin.103 Relative to the daily requirement, body reserves of cobalamin are much larger than those of folate.
Cobalamin has a daily rate of obligatory loss of approximately 0.1 percent of the total body pool, irrespective of the pool size. For this reason, a deficiency state will not develop for several years after the cessation of cobalamin intake. The officially recommended daily allowance for adults is 5 µg3; growth, hypermetabolic states, and pregnancy increase daily requirements. For infants during the first year the recommended daily allowance is 1 to 2 µg. In cobalamin-deficient subjects, a normal diet containing about 15 µg per day will replenish depleted body stores.104
ROLE IN METABOLISM
The only two recognized cobalamin-dependent enzymes in human cells are adenosylcobalamin-dependent methylmalonyl CoA mutase and methylcobalamin-dependent methyltetrahydrofolate-homocys-teine methyltransferase. The presence in humans of a third cobalamin-dependent enzyme, leucine 2,3-aminomutase, is controversial.104,105
METHYLMALONYL CoA MUTASE
Methylmalonyl CoA mutase is a mitochondrial enzyme that participates in the disposal of the propionate formed during the breakdown of valine and isoleucine. The enzyme is a homodimer of a 78-kD subunit that is encoded by a gene on chromosome 6.106,107 In the reaction catalyzed by methylmalonyl CoA mutase, methylmalonyl CoA, which is produced during the catabolism of propionate,108 is converted to succinyl CoA, a Krebs cycle intermediate. In the course of this reaction, a hydrogen on the methyl carbon of the substrate exchanges places with the —COSCoA group (Fig. 25-11).
FIGURE 25-11 Biosynthesis of AdoCbl.
The coenzyme serves as an intermediate hydrogen carrier, accepting the hydrogen from the substrate in the initial phase of the reaction and returning it to the product after the migration of —COSCoA. The place for the migrating hydrogen is created by the cleavage of the carbon-cobalt bond to form cob(II)alamin and the 5′-deoxyadenos-5′-yl radical at the active site of the enzyme. This is one of the few examples of an enzyme-catalyzed reaction mediated by an active-site free radical situated on an unactivated carbon.
Methylcobalamin participates in the cobalamin-dependent synthesis of methionine according to the scheme shown in Fig. 25-12.109,110 and 111 S-adenosylmethionine and methionine synthase reductase are required for methyltransferase activity, probably to reactivate enzyme molecules whose coenzyme has become inactivated by oxidation of the cobalt.112 The reductase converts the oxidized cobalt to the readily alkylatable Co1+, which then accepts a methyl group from S-adenosylmethionine, a powerful biological methylating agent, thereby restoring the activity of the methyltransferase. In humans this pathway also serves as a mechanism for converting N5-methyltetrahydrofolate to tetrahydrofolate. The demethylation of N5-methyl FH4 is a prerequisite for the attachment of the polyglutamate chain to newly acquired folate, which is largely taken up by the cell in the form of N5-methyl FH4 monoglutamate.29 Nitrous oxide (N2O) impairs the methyltransferase by oxidizing cob(I)alamin (a catalytic intermediate in the methyltransferase reaction) to cob(II)alamin.113 This depletes MeCbl and produces a cobalamin deficiency–like state.114
FIGURE 25-12 Incorporation of thymidine into DNA via the de novo and salvage pathways. (Adapted from Metz.219)
Since cobalamin has the capacity to bind cyanide, it is possible that it participates in the metabolism of this toxin in humans. Tobacco and certain foods (fruits, beans, and nuts) contain cyanide. Although the evidence is inconclusive, it is thought that cobalamin may play a role in neutralizing cyanide taken in via these substances.115
THE FOLATE-COBALAMIN RELATIONSHIP
In both folate deficiency and cobalamin deficiency, the megaloblastic anemias are fully corrected by treatment with the appropriate vitamin. The megaloblastic anemia of cobalamin deficiency is also largely corrected by folic acid supplementation even if no cobalamin is given, while, conversely, the anemia of folate deficiency is not helped at all by cobalamin. These clinical observations indicate that in cobalamin deficiency, the megaloblastic anemia is actually a result of an abnormality in folate metabolism.36 Further evidence that folate metabolism is deranged by cobalamin deficiency is provided by the observation that urinary excretion of FIGlu and AICAR, normally regarded as a sign of folate deficiency, is seen occasionally in pure cobalamin deficiency.116
Two explanations have been offered to account for the folate responsiveness of cobalamin-deficient megaloblastic anemia: the methylfolate trap hypothesis, which has been accepted by the majority of authorities, and the formate starvation hypothesis (Fig. 25-13).
FIGURE 25-13 How cobalamin deficiency causes intracellular folate levels to fall. Methyl FH4, the principal form of folate in the bloodstream, circulates in the unconjugated form (i.e., it has no polyglutamate side chain). This and other forms of unconjugated FH4 can be taken into cells but leak out again unless they are conjugated. Methyl FH4 is a poor substrate for the conjugating enzyme, so conjugation cannot take place until the methyl FH4 is converted to another form of folate. Cobalamin is necessary for this process, because it is the cofactor for the reaction that converts methyl FH4 to FH4. In cobalamin deficiency the conversion of methyl FH4 to FH4 is defective. Newly transported folate therefore remains in the form of methyl FH4, which cannot be conjugated and leaks back out of the cell. According to the methylfolate trap hypothesis (a), all forms of FH4 other than methyl FH4 can be conjugated, so methyl FH4 is the only folate species that leaks out of the cell. The formate deficiency hypothesis (b) differs from the methylfolate trap hypothesis solely in assuming that only the formylated folates (N10-formyl FH4 and/or N5,N10-methenyl FH4) can be conjugated, so newly transported methyl FH4,N5,N10-methylene FH4, and free FH4 all leak out of the cell. (CH2) FH4=N5,N10-methylene FH4; (CHO) FH4=N10-formyl FH4 or N5,N10-methenyl FH4.
The Methylfolate Trap Hypothesis The methylfolate trap hypothesis117,118 is based on the fact that the folate-requiring enzyme N5-methyl FH4 homocysteine methyltransferase is also dependent on cobalamin. This hypothesis states that in cobalamin deficiency, tissue folates are gradually diverted into the N5-methyl FH4 pool because of the slowing of the methyltransferase reaction,119,120 the only route out of that pool for folate. As N5-methyl FH4 levels increase, levels of the other forms of folate decline, with a consequent fall in the rates of reactions in which those forms participate. In particular, the synthesis of dTMP is slowed, and megaloblastic anemia ensues.
In its simplest form, this hypothesis predicts that in cobalamin deficiency, tissue levels of N5-methyl FH4 should be abnormally high and those of other forms of folate should be abnormally low. Although serum N5-methyl FH4 levels are elevated in cobalamin deficiency,118,121 tissue folate levels actually decline, while increases in the fraction of tissue folates in the form of N5-methyl FH4 may78,120 or may not25,122 occur. The folates whose relative levels do consistently fall as total folate levels decline are the polyglutamates.28,122,123 Their fall appears to be related to the substrate specificity of the folate-conjugating enzyme. This enzyme works very poorly with N5-methyl FH4 and is therefore unable to carry out normal g-glutamylation of newly internalized N5-methyl FH4 monoglutamate in cobalamin-deficient cells because the freshly acquired folate cannot be converted into a suitable substrate (i.e., free FH4 or formyl FH4). Thus, while sequestration of tissue folates in an expanded N5-methyl FH4 pool may account for some of the effects of the blockade in methyltransferase activity, the major problem seems to be a failure to convert newly acquired folate into a form that can be retained by the cell, the upshot being the development of a tissue folate deficiency as the unconjugated folate leaks out (Fig. 25-14). This whole process is aggravated by a drop in tissue levels of S-adenosylmethionine as the methionine supply is curtailed because of the diminished activity of the methyltransferase.36,124,125 S-adenosylmethionine, which is necessary for methyltransferase activity as discussed above, is also a powerful inhibitor of N5,N10-methylene FH4 reductase,36,124 the enzyme responsible for the production of N5-methyl FH4. The relief of this inhibition as S-adenosylmethionine levels fall accelerates the flow of folates toward N5-methyl FH4, further aggravating the metabolic imbalance that results from the impairment in methyltransferase activity.
FIGURE 25-14 The methylmalonyl CoA mutase reaction.
This problem could be overcome if N5-methyl FH4 could be converted into a substrate for the conjugating enzyme by another route. In theory, this could be accomplished by the reversal of the N5,N10-methylene FH4 reductase reaction or by the catabolism of N5-methyl FH4 via the methylation of biogenic amines.22,23,109 In fact, however, the N5,N10-methylene FH4 reductase reaction is for practical purposes irreversible in vivo,126 and the methylation of biogenic amines by N5-methyl FH4 is too slow to provide much relief.
The Formate Starvation Hypothesis This hypothesis holds that formate starvation is the basis for the folate-responsive megaloblastic anemia of cobalamin deficiency.124,127 This theory is based on the diminished capacity of cobalamin-deficient lymphoblasts to incorporate formaldehyde into purine and methionine125 and on experiments showing that N5-formyl FH4 is more effective than FH4 at correcting some of the abnormalities in folate metabolism seen in cobalamin deficiency.124,128,129 The hypothesis states that with the fall in methionine production in cobalamin-deficient states, the generation of formate is depressed (since normally the methyl group of excess methionine is rapidly oxidized to formate124,130,131), leading to a decline in the production of N5-formyl FH4. If N5-formyl FH4 but not FH4 is a substrate for the conjugating enzyme,128 then the low tissue folate levels seen in cobalamin deficiency cannot be due merely to impaired demethylation of N5-methyl FH4 by a cobalamin-deficient homocysteine methyltransferase but must reflect a decreased production of methionine, the source of the formate needed to produce the conjugable substrate, N5-formyl FH4.
INTESTINAL ABSORPTION: THE INTRINSIC FACTOR MECHANISM
Intrinsic factor is one of a number of proteins to which cobalamin is bound as it makes its way through the body (Table 25-2). Intrinsic factor is needed for the absorption of cobalamins given orally at physiologic dosage levels. Human intrinsic factor is a glycoprotein (Mr approximately 44,000) encoded by a gene on chromosome 11.132 It has binding sites for cobalamin and for a specific ileal receptor, the former situated near the carboxy terminus and the latter near the amino terminus of the intrinsic factor molecule.133 Binding to cobalamin is very tight.134,135 and 136 Its properties are summarized in Table 25-3. Bound vitamin alters the conformation of intrinsic factor, producing a more compact form that is resistant to proteolytic digestion. In humans, intrinsic factor is synthesized and secreted by the parietal cells of the cardiac and fundic mucosa.137 Secretion of intrinsic factor usually parallels that of HCl. It is enhanced by the presence of food in the stomach,138 by vagal stimulation,139 and by histamine and gastrin.140,141
TABLE 25-2 COBALAMIN-BINDING PROTEINS
TABLE 25-3 PROPERTIES OF HUMAN INTRINSIC FACTOR
Gastric juice also contains other cobalamin-binding proteins.142,143 These are known as the R binders because of their rapid electrophoretic mobility compared with intrinsic factor. The R binders are a group of immunologically related proteins of apparent Mr approximately 60,000 consisting of a single polypeptide species variably substituted with oligosaccharides that terminate with different quantities of sialic acid.144,145 These proteins are found in milk, plasma, saliva, gastric juice, and numerous other body fluids. They appear to be synthesized by mucosal cells of the organs that secrete them146 and by phagocytes.147 Though they bind cobalamin, they lack intrinsic factor activity, i.e., they are unable to promote the intestinal absorption of the vitamin.
Cobalamins in foods are liberated in the stomach by peptic digestion.148 They are then bound not to intrinsic factor but to R binders, because, at the acid pH of the stomach, cobalamin binds much more tightly to R binders than to intrinsic factor.149 On entering the duodenum, cobalamin is released from the cobalamin–R binder complex by digestion with pancreatic proteases, which in normal subjects act by selectively degrading R binders and the cobalamin–R binder complex while sparing intrinsic factor.149 It is at this point that the cobalamin finally reaches the intrinsic factor to form the intrinsic factor–cobalamin complex.
The intrinsic factor–cobalamin complex, which is very resistant to digestion,150 then journeys down the intestine until it reaches the intrinsic factor receptor cubulin,151,152 a 460-kDa peripheral membrane protein that is located in the microvillus pits of the ileal mucosa.153 (The same receptor is found in the brush border of renal proximal tubule cells.154,155,156 and 157 Its purpose there is unknown.) The ileal mucosa occupies the distal half of the small intestine, and the cubulin is found all along this portion of the intestine, its concentration rising progressively until a maximum is reached near the terminal ileum.158 A specific site on the intrinsic factor molecule avidly attaches to the receptor in a binding reaction that requires a pH of 5.4 or greater and Ca2+ (or other divalent cations) but no energy.159,160 and 161
Following attachment of intrinsic factor–cobalamin to receptor, the vitamin is taken into the ileal mucosal cells over 30 to 60 min by endocytosis,161,162 and 163 then over many hours is passed by the mucosal cells into the portal blood, while the receptors recycle to the surfaces of the microvilli for another load of intrinsic factor–cobalamin.163 During its sojourn in the ileal enterocyte the vitamin first appears in the lysosomes, but by 4 h most of it is located in the cytosol.164 During absorption the entire intrinsic factor–cobalamin complex appears to be taken into the cell, where the cobalamin is released while the intrinsic factor is degraded.161,165,166 and 167
It requires 3 to 4 h for the cobalamin from a small oral dose (10 to 20 µg) to start to appear in the blood and 8 to 12 h for the vitamin to reach a peak level. In the portal blood, the cobalamin is complexed with a cobalamin-transporting protein known as transcobalamin II (TC II).168 The cobalamin–TC II complex is probably formed in the ileal enterocyte, one of a variety of cells that have been shown to synthesize the transcobalamin.169,170,171,172 and 173 Large oral doses (1 mg) of cobalamin are absorbed by simple diffusion that is not mediated by intrinsic factor. In these instances, vitamin appears in blood within minutes, again as the cobalamin–TC II complex.
Like the folates, the cobalamins participate in an enterohepatic cycle. In humans, between 0.5 and 9 µg/day of cobalamins is secreted into the bile, where the cobalamins bind to an R binder and enter the intestine.174 In the intestine, the cobalamin–R binder complexes of biliary origin are treated exactly like those delivered from the stomach: The cobalamin is released by digestion of the R binder by pancreatic proteases then is taken up by intrinsic factor and reabsorbed. It has been estimated that 65 to 75 percent of the biliary cobalamin is reabsorbed by this mechanism.175 Because of the size of the cobalamin storage pool and the existence of this enterohepatic circulation, it takes a very long time—sometimes 20 years—to develop clinically significant cobalamin deficiency from a diet providing insufficient cobalamin (e.g., a strictly vegetarian diet). Patients who fail to absorb the vitamin, however, become clinically deficient in only 3 to 6 years, because biliary as well as dietary cobalamin is lost.176
COBALAMIN IN THE CELL: TRANSCOBALAMIN II177
The Uptake of Cobalamin by Cells Transcobalamin II (TC II) is the plasma protein that mediates the transport of cobalamin into the tissues. A simple protein of Mr=43,000,178,179 it binds cobalamin with exceedingly high affinity (Ka=1011/M).180 Unlike intrinsic factor, whose binding is relatively specific for cobalamins, TC II can also bind certain corrins that are chemically related to the cobalamins but are without function in mammalian systems and have come to be known as cobalamin “analogs.”181 TC II is synthesized by many types of cells, including enterocytes, hepatocytes, mononuclear phagocytes, fibroblasts, hematopoietic precursors in the marrow, and probably others.161,169,170,171,172 and 173
Although circulating TC II carries only a minor fraction of the cobalamin in the plasma, it is the protein to which newly acquired cobalamin is first bound. Cobalamin given parenterally associates almost immediately with unsaturated TC II,182 while cobalamin absorbed through the intestine is probably carried into the portal blood as the preformed cobalamin–TC II complex. Within minutes after their appearance in the bloodstream, these cobalamin–TC II complexes are transported into the tissues.182,183 The transport process begins with the binding of the cobalamin–TC II complex to a membrane receptor that is present on a wide variety of cells.184,185 The receptor-bound complex is then internalized by pinocytosis and delivered to a lysosome, where the TC II is digested and the cobalamin is freed.185,186,187 and 188 The cobalamin is then actively exported from the lysosome into the cytosol by a specific Mg2+-dependent carrier (Km for CNCbl=3.5 µM) that uses a proton gradient as the energy source.189,190
Formation of AdoCbl and MeCbl To be useful to the cell, CNCbl and OHCbl have to be converted to AdoCbl and MeCbl, the coenzymatically active cobalamins. This is accomplished by reduction and alkylation. CNCbl and OHCbl are first reduced to the Co2+ form [cob(II)alamin] by NADPH- and NADH-dependent reductases that are present in both mitochondria and microsomes.191,192 (NADPH–cobalamin reductase activity may be identical to that of NADPH–cytochrome c reductase193 and NADH–cobalamin reductase to the cytochrome b5/cytochrome b5 reductase system.)194 The CN– and OH– are displaced from the metal during reduction. Some of the cob(II) alamin in the mitochondria is reduced further to the intensely nucleophilic Co+ form [cob(I)alamin]. This is then alkylated by ATP to form AdoCbl in a reaction in which the 5′-deoxyadenosyl moiety of ATP is transferred to the cobalamin and the three phosphates of ATP are released as inorganic triphosphate.195,196 and 197 The rest of the cobalamin binds to cytosolic N5-methyltetrahydrofolate–homocysteine methyltransferase, where it is converted to MeCbl.198,199 The fate of cobalamin in the cell is summarized in Fig. 25-15.
FIGURE 25-15 The N5-methyl FH4:homocysteine methyltransferase reaction.
THE PLASMA COBALOPHILINS: TRANSCOBALAMINS I AND III
Transcobalamin I (TC I) is the principal R binder of plasma and carries most of the circulating cobalamin. It is a glycoprotein of Mr=approximately 50,000 containing nine potential glycosylation sites200 and is encoded by a gene on chromosome 11, the same chromosome that carries the intrinsic factor gene.201 In contrast to TC II, its clearance from the plasma is very slow (T½ 9 to 10 days).202 The cobalamin–TC I complexes are eliminated chiefly by the hepatocytes, into which they are carried by the asialoglycoprotein receptor, there to be degraded and their load of cobalamins excreted in the bile.202,203 and 204 TC I binds its ligands more tightly than does either intrinsic factor or TC II and is less restrictive than either with respect to ligand specificity, avidly taking up corrinoids of widely varying structure.181,205,206 The ligand-binding properties of TC I, together with its mode of clearance by the liver, have led to the suggestion that TC I helps clear the system of nonphysiologic cobalamin analogs that may be accidentally acquired in the normal course of events.207 As the liver metabolizes analog–TC I complexes, it will secrete the analogs into the bile. Since these analogs are bound poorly by intrinsic factor,180,208 they will be poorly reabsorbed from the intestine and instead will be eliminated in the feces. An alternative proposal is that TC I is a storage protein for cobalamins.209 In reality, however, the physiologic role of TC I is unknown.
A second circulating R binder is known as transcobalamin III (TC III).210 This protein is found in the plasma as well as in granulocytes,202,207,211 where it constitutes the cobalamin-binding protein of the specific granules and from which it is released into the serum when blood clots.212 Structurally, TC I is richer in sialic acid than is TC III. It is likely that the plasma R binders actually consist of half a dozen or more species whose pI values range from 2.9 to 4.0, with “TC I” and “TC III” representing the arbitrary division of these R binders into a low-pI group and a high-pI group.
ASSAY OF SERUM COBALAMIN AND THE TRANSCOBALAMINS
Cobalamin As with folate, cobalamin is measured using a radioisotope assay employing a cobalamin-binding protein.213 The misleading results formerly given by this assay were explained by the discovery in serum and tissue of a class of cobalamin analogs that are detected by the radioisotope assay when the binder is R binder but not when intrinsic factor is used as the binder.208 Current assays use intrinsic factor as binder and give accurate values for serum cobalamin. The chemical nature and biological significance of the analogs are unknown.214 The transcobalamins TC I and TC II are present in plasma in trace quantities (about 7 and 20 µg/liter, respectively), while TC III is often undetectable. In fasting plasma, 70 percent or more of the circulating cobalamin is bound to TC I.215,216 Nevertheless, TC I has substantial unsaturated binding capacity.217
TC II binds only 10 to 25 percent of the total plasma cobalamin202,218 but provides the majority (about 75 percent) of the total unsaturated cobalamin-binding capacity of plasma.217 Less than 2 percent of the TC II in plasma is saturated at any given moment.
Alterations in unsaturated cobalamin-binding capacity and in TC I and TC II levels in various disease states are listed in Table 25-4.
TABLE 25-4 LEVELS AND BINDING CAPACITY OF COBALAMIN-BINDING PROTEINS IN DISEASE213
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