Chapter 41: Folate, Cobalamin, and Megaloblastic Anemias

Folate and cobalamin (vitamin B₁₂) play key roles in the metabolism of all cells, particularly proliferating cells.

CHEMISTRY

The group of compounds referred to as folates consists of folic acid and its various derivatives. Folic acid (pteroylglutamic acid) is composed of a pteridine moeity, a p-aminobenzoate residue, and an L-glutamic acid residue (Fig. 41–1A). The first two together are called pteroic acid.¹ In nature, folates occur largely as conjugates in which multiple glutamic acids are linked by peptide bonds involving their γ-carboxyl groups (Fig. 41–1B). Additionally, the naturally occurring polyglutamated folates are reduced in the 5, 6, 7, and 8 positions of the pteridine ring (as described below, Fig. 41–1B). Conjugates are named according to the length of the glutamate chain (e.g., pteroylmonoglutamate, pteroyldiglutamate, pteroylhexaglutamate). Therapeutic folic acid (pteroylglutamic acid, abbreviated PteGlu, or F) has one glutamic acid and the pteridine ring is not reduced.

To form a functional compound, folate must be reduced to tetrahydrofolate (FH₄; see Fig. 41–1B). In this reduction, dihydrofolate (FH₂) is an intermediate. A single enzyme, FH₂ 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 with regard to the identity of the one-carbon unit and the site of its attachment to FH₄. Figure 41–2 shows one-carbon substituents of biochemical significance and their major interconversions.

These substituents are attached to FH₄ through N⁵,N¹⁰, or both (see Fig. 41–2). Specific enzymes interconvert these various FH₄ derivatives through oxidations that require nicotinamide adenine dinucleotide phosphate (NADP) and reductions that utilize the reduced form of NADP, NADPH.²

Reduced derivatives of folic acid usually are sensitive to air oxidation. An important exception is N⁵-formyl FH₄, also called citrovorum factor, leucovorin, or folinic acid, which, because of its stability, is the form preferred for clinical use.

NUTRITION

Sources

Folic acid comes from many sources. The richest vegetable sources are asparagus, broccoli, endive, spinach, lettuce, and lima beans. Each vegetable contains more than 1 mg of folate per 100 g dry weight. The best fruit sources are oranges, lemons, bananas, strawberries, and melons. Folates also are abundant in liver, kidney, yeast, mushrooms, and peanuts. Since the advent of folic acid fortification of the food supply, the median daily intake of folate from an average American diet is estimated to be 350 mcg.³ Foods are readily depleted of folate by excessive cooking, especially with large amounts of water, discarded before ingestion.

Daily Requirements

In the normal adult, the minimum daily requirement for folic acid is approximately 50 mcg. The average diet contains many times this amount, but some of the folate may be unavailable. Accordingly, the officially recommended dietary allowance of food folate for an adult is 0.4 mg.³ This figure is derived through considerations of the nutrient requirements to satisfy the needs of 97 to 98 percent of healthy individuals and the relative differences in absorption and bioavailability between dietary folate and the more bioavailable synthetic folic acid. One microgram of food folate is the dietary equivalent of 0.6 mcg folic acid added to food. The body is thought to contain approximately 5 mg of folate.⁴ When folate intake is reduced to 5 mcg/day, megaloblastic anemia develops in approximately 4 months.⁵

Folic acid requirements increase in hemolytic anemia, leukemia, and other malignant diseases, in alcoholism⁶ and during growth; in pregnancy and during lactation requirements increase threefold to sixfold.⁷ Adequate folate supplies are particularly important in pregnant and lactating women, in whom the recommended daily allowance is increased to 600 and 500 mcg/day, respectively, to meet requirements.⁸

METABOLISM

Folate-Dependent Enzymes

FH₄ is an intermediate in reactions involving the transfer of one-carbon units from a donor to an acceptor. Table 41–1 summarizes the metabolic systems of animal tissues known to require folic acid coenzymes.

One-carbon units enter the folate pool principally via the serine hydroxymethyltransferase (SHMT) reaction which requires pyridoxal phosphate as a cofactor.⁹ There are both cytoplasmic (SHMT1) and mitochondrial (SHMT2) isoforms of SHMT which impart a state of functional redundancy for this important enzyme, which is the primary source of one-carbon units for purine and pyrimidine biosynthesis. The cytoplasmic form can undergo sumoylation during S-phase, allowing nuclear localization.¹⁰

Among the several one-carbon transfers mediated by folic acid, the transfer that appears to be the most important clinically is the methylation of deoxyuridylate to thymidylate, catalyzed by the enzyme thymidylate synthase.^2,10,11 This reaction is an essential step in the synthesis of DNA (Fig. 41–3). 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.¹² The reaction generates FH₂, which must be reduced again to FH₄ by FH₂ reductase and NADPH before it can again be utilized as a coenzyme:

where dUMP = deoxyuridine monophosphate; dTMP = deoxythymidine monophosphate; and NADP = nicotinamide adenine dinucleotide phosphate. Limitation of thymidylate synthesis in folic acid deficiency causes incorporation of uracil instead of thymine into DNA.¹³

A key enzyme, methylenetetrahydrofolate reductase (MTHFR) regulates the distribution of reduced folates by controlling the rate of NADPH-mediated conversion of N⁵,N¹⁰-methylene FH₄ to N-methyltetrahydrofolate. Because N⁵,N¹⁰-methylene FH₄ is the obligate one-carbon donor for thymidylate synthesis, its conversion to N-methyltetrahydrofolate serves as a brake on DNA synthesis and repair, while diverting more folate to the methionine synthase reaction (see Fig. 41–2). The activity of MTHFR therefore serves as a checkpoint for intracellular folate trafficking and distribution, and thus becomes more critical in states of folate depletion.

A polymorphic form of MTHFR, MTHFR 677C→T, is of some clinical importance. The mutation results in a thermolabile form of the enzyme with a higher K_m (Michaelis-Menten dissociation constant) for its methylene-FH₄ substrate. Retardation of the folate methylation cycle makes more methylene-FH₄ available for thymidylate synthesis (Fig. 41–4), and also affects the levels of homocysteine, an amino acid whose rate of production depends on both folate and cobalamin.

Folate deficiency 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 carboxy-amide ribotide, the reaction that places the C-2 in the purine ring.¹⁴ Additional reactions dependent on biopterin, a nonfolate pteridine derivative, that are of potential metabolic importance are hydroxylation of phenylalanine to tyrosine, oxidation of long-chain alkyl ethers of glycerol to fatty acid, hydroxylation of tryptophan to 6-hydroxytryptophan (a precursor of serotonin), 17α-hydroxylation of progesterone,¹⁵ and production of nitric oxide.¹⁶ Tetrahydrofolic acid is weakly active in some of these systems in vitro¹⁷; whether it plays any such role in vivo is unknown.

Significance of Folylpolyglutamates

Intracellular folates exist primarily as polyglutamate conjugates.¹⁸ Approximately 75 percent of the folate in human erythrocytes and leukocytes is polyglutamylated.¹⁹ Plasma folate consists largely of the monoglutamate N⁵-methyltetrahydrofolate and is transported into the cells in this form.²⁰ Inside the cells, the polyglutamate chain is sequentially built up by an ATP-dependent folylpoly-γ-glutamyl synthase (FPGS).²¹ The activity of human FPGS depends strongly on the form of the folate substrate, declining in the order FH₄ > N¹⁰-formyl FH₄ > N⁵-methyltetrahydrofolate, toward which the enzyme is almost inert.²² In humans, conjugated folates carry on average seven to eight glutamyl residues.²³ Intracellular folylmonoglutamates leak out of the cells at a fairly rapid rate whereas polyglutamates do not, presumably because of the highly charged polyglutamate tail.²⁴ Therefore, attachment of the polyglutamate chain is essential for retaining folates within cells. Folylpolyglutamates are superior to monoglutamates as substrates for folate-dependent enzyme reactions.¹⁹

PHYSIOLOGY

Intestinal Absorption

The duodenum and proximal jejunum is the principal site of folate absorption. Absorption of a dose of either monoglutamate or polyglutamate forms of folate begins within minutes. Peak plasma levels are reached in 1 to 2 hours. Because only folylmonoglutamate appears in plasma, all folylpolyglutamates must first be hydrolyzed by the enzyme glutamate carboxypeptidase II (folate hydrolase)²⁵ during absorption across the intestine.²⁶ This enzyme (previously referred to as “conjugase”) is located on the brush-border membrane and plays an important role in the intestinal absorption of folate.²⁷ Folylpolyglutamate is hydrolyzed at the brush-border of the intestinal cell (Fig. 41–5). Folate hydrolase purified from human jejunum catalyzes the Zn²⁺-dependent deconjugation of folate polyglutamates ranging from PteGlu₂ to at least PteGlu₇.²⁸ It is an exopeptidase that successively removes single glutamate residues from the end of the polyglutamate chain, ultimately yielding the folylmonoglutamate. The monoglutamate forms are then taken up by one of two folate-specific transporters located on the apical brush-border, the reduced folate carrier (RFC) or the proton-coupled folate transporter (PCFT).²⁵ Although RFC has a pH optimum of 7.4, PCFT is a high-affinity folate transporter that uses a proton-coupled system to facilitate folate absorption and shows maximum transport activity at a low pH.^25,29 This property is consistent with the observation that cancer cells retain a high affinity for the new-generation antifolate pemetrexed. Defects in the PCFT are the underlying cause of hereditary folate malabsorption.³⁰

Folate hydrolases also are found outside the intestine. For example, human plasma contains sufficient hydrolase activity to convert polyglutamates containing more than three glutamyl residues to monoglutamates. Other γ-glutamyl hydrolases appear to be lysosomal carboxypeptidases³¹ that are not involved in absorption of folates from the intestine but that play a role in the release of folate from storage sites in the liver and kidney.²⁵

Folate monoglutamates are actively transported across the intestinal epithelium by PCFT-mediated transport (K_m = 1 to 2 μM) that is independent of Na⁺, K⁺, and transmembrane potential.³² The 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.³³ Passive transport also may occur.³⁴ In the intestinal cell, the absorbed folate monoglutamates are reduced if necessary, and then converted to N⁵-methyltetrahydrofolate (some N¹⁰-formyl FH₄ also is made) and transported into the bloodstream without further change.³⁵

Folate undergoes an enterohepatic cycle in which it is first secreted against a concentration gradient into the bile, appearing there chiefly as N⁵-methyltetrahydrofolate monoglutamate, and then is reabsorbed from the small intestine.³⁶ Bile contains approximately two to 10 times the folate concentration of normal serum, with biliary excretion accounting for up to 0.1 mg of folate per day. This quantity is sufficiently large that interruption of the enterohepatic cycle by biliary diversion causes serum folate levels to fall by more than 50 percent in less than 1 day.³⁷ The enterohepatic cycle has been proposed to redistribute folate between hepatic stores and peripheral tissues according to the state of the exogenous folate supplies.³⁸

METABOLISM

Tritiated folylmonoglutamate (³H-F) administered intravenously is almost completely removed from the bloodstream in a few minutes.³⁹ Uptake involves two classes of folate-binding proteins⁴⁰: high-affinity folate receptors⁴¹ that concentrate folate in intracellular vesicles and a membrane folate transporter that transports folate from the vesicles into the cytosol. The high-affinity receptors, which are attached to the outer surface of the cell membrane by glycosyl-phosphatidylinositol linkages and lack an intracytoplasmic portion,⁴² bind very tightly (K_d [distribution coefficient] in the nanomolar range) to most physiologic folate monoglutamates,⁴³ particularly N⁵-methyltetrahydrofolate, the major circulating folate.⁴⁴ The folate receptors exist in various isoforms (of which α and β are the most important). Folate receptor-α, despite its missing cytoplasmic extension, is effective in mediating endocytosis.⁴⁵ Their very high affinity enables the receptors to take up N⁵-methyltetrahydrofolate from the plasma, even at its ambient concentration of approximately 10 nM. The membrane folate transporter is a probenecid-inhibitable organic anion carrier that, among other functions, carries reduced folates and methotrexate in and out of the cytoplasm.⁴⁰ Its K_m for folate is in the micromolar range. Once internalized, the folates are retained by the cells partly through polyglutamylation⁴⁶ as well as through tight association with a set of intracellular folate-binding proteins.⁴⁷ Three of these proteins are enzymes involved in methyl group metabolism: sarcosine dehydrogenase and dimethylglycine dehydrogenase (mitochondrial)⁴⁸ and glycine N-methyl transferase (cytosolic).⁴⁹ Why these enzymes bind folate so avidly or whether this binding affects overall methyl group metabolism is unknown, although glycine N-methyl transferase is speculated to regulate methyl group metabolism by controlling the tissue concentration of S-adenosylhomocysteine (SAH), 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 and in blood appears to be the N⁵-methyl form.⁵⁰ The total folate pool turns over very slowly.⁵¹ Degradation accounts for a portion of this turnover. 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 are released from cell membranes by proteolysis.⁵² These proteins can be detected in approximately 15 percent of normal individuals⁵³ and are found at increased levels in some pregnant women, women taking oral contraceptives, folate-deficient alcoholics (but not patients with cobalamin deficiency),⁵⁴ and patients with uremia, hepatic cirrhosis, and chronic myelogenous leukemia.⁵⁵ In normal subjects, the proteins are approximately two-thirds saturated and have a total folate-binding capacity of approximately 175 pg/mL of serum.⁵⁶ The proteins may not be detected in some subjects because of complete saturation of the proteins with endogenous unlabeled folate.⁵⁷ Serum folate-binding protein has an Mr of 40,000 and prefers oxidized to reduced folates.⁵⁵

Folate-binding proteins have been found in milk and in normal granulocytes.⁵⁸ Folate bound to the milk folate binder in suckling animals is absorbed chiefly in the ileum⁵⁹ 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.⁶⁰ The milk folate binder is speculated to protect an infant’s folate supply by preventing bacteria from sequestering the vitamin away from the intestinal absorptive surface. The folate-binding protein in granulocytes has been localized to the specific granules, from which it is released when the granulocytes are stimulated.

EXCRETION

Folates are both resorbed and secreted by the kidney. Resorption is accomplished by a membrane-bound high-affinity folate receptor (K_m for N⁵-methyltetrahydrofolate = 0.4 nM) located in the brush-borders of the proximal tubules.⁶¹ Filtered folate may thus be returned to the bloodstream. There is resorption of most, but not all, of the filtered folate.

In humans, intact folates and their cleavage products are excreted by the kidney at a rate of 2 to 5 mcg/day.⁶² A small percentage of parenterally administered labeled folate is recoverable in the feces and mainly represents unreabsorbed folate from the enterohepatic cycle.⁶³

ASSAY OF SERUM FOLATE

Folates are measured by chemiluminescent methods using various folate binders. These assays are identical in principle to the radioligand binding assays that they have replaced.

CHEMISTRY

Structure and Nomenclature

The cobalamin molecule has two major portions: a porphyrin-like near-planar macrocycle known as corrin, and a nucleotide that lies almost perpendicular to the corrin ring (Fig. 41–6). The corrin moiety contains four reduced pyrrole rings that bind a central cobalt atom whose two remaining coordination positions are occupied by a 5,6-dimethylbenzimidazolyl (5,6-DMB) group, below the ring and various ligands (in this case, —N in the form of CN) above the ring.⁶⁴

Compounds containing the corrin ring are known as corrinoids. The cobalamins are corrinoids whose nucleotide contains 5,6-DMB. Two connections exist 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. Figure 41–7 summarizes the numbering and ring designations of the corrin system.

The term vitamin B₁₂ is sometimes used as a generic term for the cobalamins. The term probably is best reserved, however, as an alternative name for cyanocobalamin, the usual therapeutic form of cobalamin.

Four cobalamins are important in animal cell metabolism. Two are cyanocobalamin (CnCbl; vitamin B₁₂) and hydroxocobalamin (OHCbl) or aquocobalamin (HOH Cbl). The other two cobalamins are alkyl derivatives that are synthesized from OHCbl and serve as coenzymes. In one, adenosylcobalamin (AdoCbl), a 5′-deoxyadenosyl replaces OH as the cobalt ligand above the ring (Fig. 41–8).⁶⁵ In the second, methylcobalamin (MeCbl), the upper ligand is a methyl group. MeCbl is the major form of cobalamin in human blood plasma.⁶⁶

NUTRITION

Sources

Cobalamin is synthesized only by certain microorganisms; animals ultimately depend on microbial synthesis for their cobalamin supply. Foods that contain cobalamin are of animal origin: meat, liver, seafood, and dairy products. Cobalamin has not been found in plants.

Daily Requirements

The average daily diet in Western countries contains 5 to 30 mcg of cobalamin, of which 1 to 5 mcg is absorbed.⁶⁷ 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,⁶⁸ with approximately 1 mg in the liver. The kidneys also are rich in cobalamin.⁶⁹ 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 does not develop for several years after cessation of cobalamin intake. The officially recommended dietary allowance (RDA) for adults is 2.4 mcg²; growth, hypermetabolic states, and pregnancy increase daily requirements. The RDA for children ages 1 to 13 years is 0.9 to 1.8 mcg. Because of insufficient data, no RDA has been established for infants. Instead, adequate intakes of 0.4 mcg for age 0 to 6 months and 0.5 mcg for age 7 to 12 months have been estimated.

ROLE IN METABOLISM

The only two recognized cobalamin-dependent enzymes in human cells are AdoCbl-dependent methylmalonyl CoA (coenzyme A) mutase and MeCbl-dependent methyltetrahydrofolate-homocysteine methyltransferase.

Methylmalonyl Coenzyme A Mutase

Methylmalonyl CoA mutase is a mitochondrial enzyme that participates in the disposal of the propionate formed during breakdown of valine, isoleucine, and odd-carbon fatty acids. The enzyme is a homodimer of a 78-kDa subunit that is encoded by a gene on chromosome 6.⁷⁰ In the reaction catalyzed by methylmalonyl CoA mutase, methylmalonyl CoA, which is produced during catabolism of propionate,⁷¹ 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. 41–9).

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 migration of —COSCoA.

N⁵-Methyltetrahydrofolate-Homocysteine Methyltransferase

MeCbl participates in cobalamin-dependent synthesis of methionine from homocysteine by the enzyme N⁵-methyltetrahydrofolate–homocysteine methyltransferase. S-adenosylmethionine (SAMe) and a second enzyme, methionine synthase reductase are required for methyltransferase activity.⁷² The reductase converts the oxidized cobalt to the readily alkalizable Co⁺, which then accepts a methyl group from SAMe, a powerful biologic methylating agent, thereby restoring activity of the methyltransferase. In humans, this pathway also serves as a mechanism critical for converting N⁵-methyltetrahydrofolate to FH₄ required for synthesis of polyglutamates as well as other important one-carbon adducts of folate. The demethylation of N⁵-methyltetrahydrofolate is a prerequisite for attachment of the polyglutamate chain to newly acquired folate, which is largely taken up by the cell in the form of N⁵-methyltetrahydrofolate monoglutamate.²⁴ Nitrous oxide (N₂O) impairs the methyltransferase by oxidizing cob(I)alamin (a catalytic intermediate in the methyltransferase reaction) to cob(II)alamin. This reaction depletes MeCbl and produces a cobalamin deficiency-like state.

Nonenzymatic Metabolism

Because cobalamin has the capacity to bind cyanide, it may participate in detoxification of cyanide. Tobacco and certain foods (fruits, beans, tubers, and nuts) contain cyanide in the form of thiocyanate. Although the evidence is inconclusive, cobalamin is believed to play a role in neutralizing cyanide taken in via these substances.⁶⁴

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 also is variably corrected by folic acid supplementation even if no cobalamin is given, although the remission may be partial and only temporary. Conversely, the anemia of folate deficiency is generally not helped at all by cobalamin although partial responses to high doses of cobalamin have been reported in some patients with folate deficiency.⁷³ These clinical observations indicate that the megaloblastic anemia in cobalamin deficiency actually results from an abnormality in folate metabolism.²⁴ The observation that urinary excretion of formiminoglutamic acid (FIGlu) and AICAR, normally regarded as a sign of folate deficiency, is seen occasionally in pure cobalamin deficiency⁷⁴ provides further evidence that folate metabolism is deranged by cobalamin deficiency. Two explanations have been proposed to account for the folate responsiveness of cobalamin-deficient megaloblastic anemia: (1) the methylfolate trap hypothesis, which is accepted by the majority of authorities, and (2) the formate starvation hypothesis (Fig. 41–10).

METHYLFOLATE TRAP HYPOTHESIS

The methylfolate trap hypothesis⁷⁵ is based on the fact that the folate-requiring enzyme N⁵-methyltetrahydrofolate–homocysteine methyltransferase is also dependent on cobalamin. The hypothesis states that in cobalamin deficiency tissue folates are gradually diverted into the N⁵-methyltetrahydrofolate pool because of slowing of the methyltransferase reaction,⁷⁶ the only route out of that pool for folate. As N⁵-methyltetrahydrofolate levels increase, the levels of other forms of folate decline, with a consequent fall in the rates of reactions in which those forms participate. In particular, because the MTHFR reaction is irreversible, methylene-FH₄ becomes depleted, the synthesis of dTMP is slowed, and megaloblastic anemia ensues.

In its simplest form, the hypothesis predicts that in cobalamin deficiency tissue levels of N⁵-methyltetrahydrofolate are abnormally high and those of other forms of folate are abnormally low. Although serum N⁵-methyltetrahydrofolate levels are frequently elevated in cobalamin deficiency,⁷⁷ tissue folate levels, predominantly polyglutamates, decline.⁷⁸ The decreased level appears to be related to the substrate specificity of the folate-conjugating enzyme. This enzyme works very poorly with N⁵-methyltetrahydrofolate; therefore, it is unable to carry out normal γ-glutamylation of newly internalized N⁵-methyltetrahydrofolate 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, although sequestration of tissue folates in an expanded N⁵-methyltetrahydrofolate 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 is development of tissue folate deficiency as the unconjugated folate leaks out (see Fig. 41–10). The whole process is aggravated by a drop in tissue levels of SAMe as the methionine supply is curtailed because of the diminished activity of the methyltransferase.⁷⁹ SAMe, which is necessary for methyltransferase activity, is also a powerful inhibitor of N⁵,N¹⁰-methylene FH₄ reductase MTHFR,⁸⁰ the enzyme responsible for production of N⁵-methyltetrahydrofolate. The relief of this inhibition as SAMe levels fall accelerates the flow of folates toward N⁵-methyltetrahydrofolate, further aggravating the metabolic imbalance resulting from impairment in methyltransferase activity.

This problem could be overcome if N⁵-methyltetrahydrofolate were converted into a substrate for the conjugating enzyme by another route. In theory, this could be accomplished by reversal of the N⁵,N¹⁰-methylene FH₄ reductase reaction. For practical purposes, however, the N⁵,N¹⁰-methylene FH₄ reductase reaction is irreversible in vivo.⁸¹

FORMATE STARVATION HYPOTHESIS

This hypothesis holds that formate starvation is the basis for folate-responsive megaloblastic anemia of cobalamin deficiency.⁸² This theory is based on the diminished capacity of cobalamin-deficient lymphoblasts to incorporate formaldehyde into purine and methionine⁷⁹ 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.⁸³ The hypothesis states that with the decrease in methionine production in cobalamin-deficient conditions, the generation of formate is depressed (because normally the methyl group of excess methionine is rapidly oxidized to formate),⁸⁴ leading to a decline in the production of N⁵-formyl FH₄.

INTESTINAL ABSORPTION

Intrinsic Factor

Intrinsic factor is one of a number of binding proteins in which cobalamin is ensconced as it makes its way through the body (Table 41–2). Intrinsic factor is needed for the absorption of cobalamins taken orally at physiologic dosage levels. Human intrinsic factor is a glycoprotein (Mr approximately 44,000) encoded by a gene on chromosome 11.⁸⁵ It has binding sites for cobalamin and a specific ileal receptor, the former situated near the carboxy-terminus and the latter near the aminoterminus of the intrinsic factor molecule.⁸⁶ Binding to cobalamin is very tight, and involves the 5,6-DMB lower axial ligand of the molecule. This specificity allows for the exclusion of other noncobalamin corrinoids during the tightly regulated absorptive process.⁶⁴ The entrapment of the 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.⁸⁷ Secretion of intrinsic factor usually parallels that of hydrochloric acid (HCl). It is enhanced by the presence of food in the stomach, vagal stimulation, histamine, and gastrin. Gastric juice also contains other cobalamin-binding glycoproteins.⁸⁸ These proteins were known as the R proteins because of their rapid electrophoretic mobility compared with intrinsic factor. Elucidation of the primary protein structure of the R proteins reveals that they belong to the same family of isoproteins as the plasma haptocorrin (HC) binder (previously known as transcobalamins I and III). These HC-like proteins are produced mainly by the salivary glands.

Absorption of Cobalamin: Cubilin

Cobalamins in foods are liberated in the stomach by peptic digestion.⁸⁹ They are then bound not to intrinsic factor but to the HC-like protein because cobalamin binds much more tightly to HC than to intrinsic factor at the acid pH of the stomach.⁹⁰ Upon entering the duodenum, cobalamin is released from the cobalamin–HC protein complex through digestion by pancreatic proteases, which in normal subjects act by selectively degrading HC and the cobalamin–HC complex while sparing intrinsic factor.⁹⁰ Only at this point can cobalamin bind to intrinsic factor to form the intrinsic factor–cobalamin complex.

The intrinsic factor–cobalamin complex, which is very resistant to digestion,⁹¹ traverses the intestine until it reaches the intrinsic factor receptor, cubilin,⁹² a 460-kDa peripheral membrane glycoprotein located in the microvillus pits of the ileal mucosa brush-border. Cubilin forms part of a multifunctional epithelial receptor complex also found in the yolk sac and renal proximal tubule cells.⁹³ In the kidney, it appears to serve a role in the overall body economy through tubular reabsorption of cobalamin,⁹⁴ but the function of the cubilin receptor complex in the kidney and other polarized epithelial surfaces extends beyond cobalamin. The ileal cubilin receptor complex consists of two proteins, cubilin (CUB) and amnionless (AMN), the product of two distinct genes, CUB and AMN. Both proteins, which together have been designated the “CUBAM complex,” colocalize in the endocytic compartment and are required for the process of assimilation of cobalamin,⁹⁵ AMN serving as a chaperon for endosomal targeting. Mutations affecting either of the two proteins disrupt the normal process of the intestinal phase of cobalamin absorption. In addition to the tightly embracing components of the CUBAM complex, a distinct large multifunctional protein, megalin, which belongs to the low-density lipoprotein family,⁹⁶ also participates in the conformational changes that accompany internalization. The concentration of the CUBAM complex rises progressively to a maximum near the terminal ileum.⁹⁷ 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.⁹⁸

The intrinsic factor–cobalamin receptor complex is taken into the ileal mucosal cells over 30 to 60 minutes by endocytosis,⁹⁹ where the vitamin is processed and released into the portal blood over many hours. The receptors recycle to the microvillus surface to shuttle another load of intrinsic factor–cobalamin complex.⁹⁹ That this process has a limited capacity is evident from estimates of the maximum amount of cobalamin that can be absorbed from a single dose via this physiologic pathway.^64,100 Defects in the genes that regulate the complex mechanism of ileal absorption are implicated in autosomal recessive megaloblastic anemia (MGA1), caused by intestinal malabsorption of cobalamin (see “Selective Malabsorption of Cobalamin, Autosomal Recessive Megaloblastic Anemia, Imerslund-Gräsbeck Disease” below).

During its sojourn in the ileal enterocyte, the vitamin first appears in the lysosomes, but by 4 hours most of the vitamin is located in the cytosol.¹⁰¹ 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.¹⁰²

Cobalamin from a small oral dose (10 to 20 mcg) starts to appear in the blood after 3 to 4 hours, and the vitamin reaches a peak level in 6 to 12 hours. In the portal blood, the cobalamin is complexed with a cobalamin-transporting protein known as transcobalamin (TC) previously known as TC II.¹⁰³ There is evidence that cobalamin leaves the enterocyte through a portal that is part of the ABC drug transport system, ABCC1 (also known as the multidrug resistance protein1 [MRP1]) located on the basolateral surface of the intestinal epithelium, as well as other polar cells and nonpolar cells (macrophages).¹⁰⁴ The cobalamin–TC complex is now believed to be formed as it exits the ileal enterocyte, one of a variety of cells that synthesize TC, including the neighboring vascular endothelial cells in the submucosa.¹⁰⁵ Large oral doses (1 mg) of cobalamin are absorbed inefficiently (1 to 2 percent of an oral dose) by simple diffusion that is not mediated by intrinsic factor.¹⁰⁰ In these instances, the vitamin appears in blood within minutes, again as the cobalamin–TC complex.

Like the folates, the cobalamins undergo appreciable enterohepatic recycling.¹⁰⁶ In humans, between 0.5 and 9 mcg/day of cobalamin is secreted into the bile, where it is bound to HC.¹⁰⁷ After entering the intestine, the cobalamin–HC complexes of biliary origin are treated exactly like those delivered from the stomach. The cobalamin is released by digestion of the HC by pancreatic proteases, and then is taken up by intrinsic factor and reabsorbed. From 65 to 75 percent of biliary cobalamin is estimated to be reabsorbed by this mechanism.¹⁰⁸ Because of the size of the cobalamin storage pool and the existence of this enterohepatic circulation, a very long time—as long as 20 years—is required for a clinically significant cobalamin deficiency to develop from a diet providing insufficient cobalamin (e.g., a strictly vegetarian diet).¹⁰⁹ Patients who are unable to absorb the vitamin, however, become clinically deficient in only 3 to 6 years because the absorption of both biliary and dietary cobalamin are interdicted.¹¹⁰

COBALAMIN IN THE CELL: TRANSCOBALAMIN

Uptake of Cobalamin By Cells

TC is the plasma protein that mediates the transport of cobalamin into the tissues.¹¹¹ A β-globulin protein with a calculated molecular weight of 45,538 from the deduced amino acid sequence,^112,113 TC binds cobalamin with exceedingly high affinity (K_a = 10^–11 M).¹¹⁴ Unlike intrinsic factor, whose binding is highly specific for cobalamins, TC shows some promiscuity and also can bind certain corrins that are chemically related to the cobalamins but have no function in mammalian systems and are known as cobalamin “analogues.”¹¹⁵ TC is synthesized by many types of cells, including enterocytes, hepatocytes, endothelial cells, mononuclear phagocytes, fibroblasts, and hematopoietic precursors in the marrow.⁶⁴ Although circulating TC carries only a minor fraction of the cobalamin in the plasma, it is the protein on which cobalamin absorbed through the intestine and is transported into the portal blood as the preformed cobalamin–TC complex. It also is the protein with which cobalamin given parenterally associates almost immediately.¹¹⁶ These cobalamin–TC complexes are transported into the tissues within minutes of appearing in the bloodstream.¹¹⁷ The transport process begins with binding of the cobalamin–TC complex to a specific membrane receptor that is present on a wide variety of cells.¹¹⁸ The protein and gene encoding the TC receptor has been purified from placental membranes and characterized.¹¹⁹ Designated as CD320, the receptor belongs to the low-density lipoprotein receptor family and its internalization involves megalin. The receptor-bound complex is internalized by receptor-mediated endocytosis and delivered to a lysosome, where the TC is digested and the cobalamin is freed.^120,121

Formation of Adenosylcobalamin and Methylcobalamin

To become metabolically active, CnCbl and OHCbl must first be converted to AdoCbl and MeCbl, the coenzymatically active cobalamins. The conversion is accomplished by reduction and alkylation. CnCbl and OHCbl are first reduced to the Co⁺⁺ form [cob(II)alamin] by NADPH- and NADH (nicotinamide adenine dinucleotide phosphate)-dependent reductases that are present in mitochondria and microsomes.¹²² CN– and OH– are displaced from the metal during reduction. Some of the cob(II)alamin is reduced further in the mitochondria 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 (Fig. 41–11). The rest of the cobalamin binds to cytosolic N⁵-methyltetrahydrofolate–homocysteine methyltransferase, where it is converted to MeCbl. The several steps involved in the conversion of cobalamin to its coenzymatically active forms are regulated by genes that play a critical role in the processing of the vitamin. There are a number of inherited metabolic errors that correspond to one or more of these specific steps and that result in characteristic syndromes affecting aspects of cobalamin metabolism that are discussed later in this chapter.

PLASMA HAPTOCORRIN (TRANSCOBALAMINS I AND III; “R” PROTEINS)

The HCs (previously known as R proteins) 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.¹²³ They 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 them¹²⁴ and by phagocytes.¹²⁵ Although the HCs bind cobalamin, they lack intrinsic factor activity, that is, they are unable to promote the intestinal absorption of the vitamin.

Plasma HC carries most (70 to 90 percent) of the circulating cobalamin. It contains nine potential glycosylation sites¹²⁶ and is encoded by a gene on chromosome 11, the same chromosome that carries the intrinsic factor gene.¹²⁷ In contrast to TC, HC clearance from the plasma is very slow (half-life [T_1/2]: 9 to 10 days).¹²⁸ The asialoglycoprotein receptor carries the cobalamin–HC complexes into the hepatocytes, where they are chiefly eliminated. The complexes are degraded, and their load of cobalamins is excreted in the bile.^106,129 HC binds its ligands more tightly than does either intrinsic factor or TC. Furthermore, HC is less restrictive than either intrinsic factor or TC with respect to ligand specificity; it avidly takes up corrinoids of widely varying structure.¹³⁰ The ligand-binding properties of HC and its mode of clearance by the liver suggest that HC helps clear the system of nonphysiologic cobalamin analogues that may have been acquired or may have arisen through degradation of cobalamin.^131,132 As the liver metabolizes analogue–HC complexes, it secretes the analogues into the bile. Because these analogues are bound poorly by intrinsic factor,¹³⁰ they are poorly reabsorbed from the intestine and are eliminated in the feces. The precise role of HC is unknown, although it may play a role in the body economy of cobalamin by facilitating excretion of cobalamin analogues while conserving cobalamin through enterohepatic recycling. Additionally, it has been proposed that HC may serve an antimicrobial role.⁶⁴

ASSAY OF SERUM COBALAMIN AND THE TRANSCOBALAMINS

As with folate, cobalamin is usually measured with automated competitive displacement assays using intrinsic factor as a cobalamin-binding protein. The misleading results previously provided by competitive ligand displacement assays were explained by the discovery in serum and tissue of a class of cobalamin analogues that are detected by the radioisotope assay when HC-type proteins and not intrinsic factor were used as the binding protein.¹³³ Current assays use intrinsic factor as the binder and give more reliable values for serum cobalamin. The chemical nature and biologic significance of the analogues are unknown,¹³⁴ but recent evidence suggests that they may arise in the gastrointestinal tract.^131,132

TC and HC are present in plasma in trace quantities (approximately 7 and 20 mcg/L, respectively). In fasting plasma, at least 70 percent of the circulating cobalamin is bound to HC.¹³⁵ TC binds only 10 to 25 percent of the total plasma cobalamin,¹³⁶ but provides the majority (approximately 75 percent) of the total unsaturated cobalamin-binding capacity of plasma.¹³⁵ Table 41–3 lists alterations in unsaturated cobalamin-binding capacity and in HC and TC levels in various disease states. In recent years, assays have been developed that measure the fraction of the plasma cobalamin that is bound to TC. This component, known as holotranscobalamin (holoTC), shows improved specificity compared with the standard cobalamin assay for identifying true cobalamin deficiency, although the assays appear to be generally comparable with respect to sensitivity.^{137,138,139,140,141,142,143}

DEFINITION

Megaloblastic anemias are disorders caused by impaired DNA synthesis. The presence of megaloblastic cells is the morphologic hallmark of this group of anemias. Megaloblastic red cell precursors are larger than normal and have more cytoplasm relative to the size of the nucleus. Promegaloblasts show a blue granule-free cytoplasm and a fine “salt and pepper” granular chromatin that contrasts with the ground-glass texture of its normal counterpart. As the cell differentiates, the chromatin condenses more slowly than normal into darker aggregates that coalesce, but do not fuse homogeneously, giving the nucleus a characteristic fenestrated appearance. Continuing maturation of the cytoplasm as it acquires hemoglobin contrasts with the immature-looking nucleus—a feature termed nuclear-cytoplasmic asynchrony.

Megaloblastic granulocyte precursors are also larger than normal and show nuclear-cytoplasmic asynchrony. A characteristic cell is the giant metamyelocyte, which has a large horseshoe-shaped nucleus, sometimes irregularly shaped, containing ragged open chromatin.

Megaloblastic megakaryocytes may be abnormally large and polylobated, with deficient granulation of the cytoplasm. In severe megaloblastosis, the nucleus may show detached lobes. Further details are provided in “Laboratory Features” below and in Figs. 41–12 and 41–13.

ETIOLOGY AND PATHOGENESIS

Table 41–4 lists the causes of megaloblastic anemia. By far the most common causes worldwide are folate deficiency and cobalamin deficiency. There has, however, been a marked reduction in the prevalence of folate deficiency in North America and a growing number of other countries that have implemented folic acid fortification of the food supply.

Megaloblastic cells have much more cytoplasm and RNA than do their normal counterparts, but they have a relatively normal amount of DNA,¹⁴⁴ suggesting that cytoplasmic constituents (RNA and protein) are synthesized faster than is DNA. Evidence that maturation is retarded in megaloblastic precursors supports this conclusion.¹⁴⁵ DNA synthesis is impaired,¹⁴⁶ and migration of the DNA replication fork and the joining of DNA fragments synthesized from the lagging strand (Okazaki fragments) are delayed,¹⁴⁷ and the S-phase is prolonged.¹⁴⁶

Slowing of DNA replication in the megaloblastic anemias of folate and cobalamin deficiency appears to arise from failure of the folate-dependent conversion of dUMP to dTMP. Because of this failure, deoxyuridine triphosphate (dUTP) levels become abundant and because DNA polymerase is promiscuous with respect to its substrate specificity, allows dUTP to become incorporated into the DNA of folate-deficient cells in place of deoxythymidine triphosphate (dTTP).¹⁴⁸ DNA excision–repair mechanisms to repair the DNA by replacing uridine with thymidine fail for the same reason that uridine triphosphate was incorporated into the DNA in the first place. The result is a repetitive iteration of flawed DNA repair that ultimately leads to DNA strand breaks, fragmentation, and apoptotic cell death.¹⁴⁹

Addition of deoxyuridine (dU) to marrow cells in culture normally decreases the incorporation of tritiated thymidine into DNA, because the dU is converted via dUMP→dTMP to unlabeled dTTP, which competes with the tritiated thymidine. In megaloblastic cells, this effect of added dU is greatly diminished. This finding is consistent with impairment in the dUMP→dTMP reaction in the megaloblastic cells and was the basis for the now defunct dU suppression test.¹⁵⁰ The failed excision–repair model following dUTP misincorporation into DNA also explains the chromosome breaks and other abnormalities that occur in megaloblastic cells.¹⁵¹

CLINICAL FEATURES

All megaloblastic anemias share certain general clinical features. Because the anemia develops slowly, with opportunity for cardiopulmonary and intraerythrocytic compensatory changes,¹⁵² it produces few symptoms until the hematocrit is severely depressed. Symptoms, when they appear, are those of anemia: weakness, palpitation, fatigue, light-headedness, and shortness of breath. The blending of severe pallor and slight jaundice caused by a combination of intramedullary and extravascular hemolysis produce a characteristic lemon-yellow skin. Leukocyte and platelet counts may be low, but rarely cause clinical problems. Details of the clinical manifestations are given in the sections on the specific forms of megaloblastic anemia later in this chapter.

LABORATORY FEATURES

Blood Cells

All cell lines are affected. Erythrocytes vary markedly in size and shape, often are large and oval, and in severe cases can show basophilic stippling and nuclear remnants (Cabot rings and Howell-Jolly bodies). Erythroid activity in the marrow is enhanced, although the megaloblastic cells usually die before they are released, accounting for the reduced reticulocyte count. The more severe the anemia, the more pronounced the morphologic changes in the red cells. When the hematocrit is less than 20 percent, erythroblasts with megaloblastic nuclei, including an occasional promegaloblast, may appear in the blood. The anemia is macrocytic (mean corpuscular volume [MCV] is 100 to 150 fL or more), although coexisting iron deficiency, thalassemia trait,¹⁵³ or inflammation can prevent macrocytosis.¹⁵⁴ Slight macrocytosis may be the earliest sign of megaloblastic anemia. Because of the progressive nature of gradual replacement of normocytic red cells with the macrocytic progeny of a megaloblastic marrow, the earliest observable change in red cell indices is an increase in the red cell distribution width (RDW), reflecting an increase in anisocytosis.

Neutrophil nuclei often have more than the usual three to five lobes (see Fig. 41–12).¹⁵⁵ Typically, more than 5 percent of the neutrophils have five lobes. Cells may contain six or more lobes, a morphology rarely seen in normal neutrophils but not pathognomonic of megaloblastic hematopoiesis. In nutritional megaloblastic anemias caused by folate deficiency, hypersegmented neutrophils are an early sign of megaloblastosis⁵ and persist in the blood for many days after treatment.¹⁵⁵ Neutrophil hypersegmentation was not found to be a sensitive test for mild cobalamin deficiency.¹⁵⁶ Cytogenetic studies are nonspecific and show chromosomes that are elongated and broken. Specific therapy corrects these abnormalities, usually within 2 days, although some abnormalities do not disappear for months.^151,157 Platelets are often reduced in number and slightly smaller than normal with a wider variation in size (increased platelet distribution width [PDW]).¹⁵⁸ The morphologic features of megaloblastic anemia may be grossly exaggerated in patients who have been splenectomized or lack a functional spleen as occurs in celiac disease or sickle cell anemia. Numerous circulating megaloblasts and bizarre red cell morphology may be present.¹⁵⁹

Marrow

Aspirated marrow is cellular and shows striking megaloblastic changes, especially in the erythroid series with well-hemoglobinized erythroblasts containing nuclei that possess less-mature, more-open nuclear chromatin than their normal counterparts. There is a preponderance of earlier basophilic erythroblasts over more mature forms which gives the overall impression of a maturation arrest (see Fig. 41–13). Sideroblasts are increased in number and contain increased numbers of iron granules. The ratio of myeloid to erythroid precursors falls to 1:1 or lower, and granulocyte reserves may be decreased. In severe cases, promegaloblasts containing an unusually large number of mitotic figures are plentiful. Macrophage iron content often is increased. Megaloblastic features in the granulocytic series is also usually present with giant forms and large horseshoe-shaped nuclei. Occasionally megakaryocytes with hyperlobated nuclei are present.

Coexisting Microcytic Anemia

Many features of megaloblastic anemia may be masked when megaloblastic anemia is combined with a microcytic anemia.¹⁵⁴ The anemia can be normocytic or even microcytic, whereas the blood film may show both microcytes and macroovalocytes (a “dimorphic anemia”). The marrow may contain “intermediate” megaloblasts¹⁶⁰ that are smaller and look less “megaloblastic” than usual. In this kind of mixed anemia, the microcytic component usually is iron-deficiency anemia,¹⁵⁴ but it may be thalassemia minor¹⁵³ or the anemia of chronic disease. Even megaloblastic anemia masked by a severe microcytic anemia usually shows hypersegmented neutrophils in the blood and giant metamyelocytes and bands in the marrow. Neutrophil myeloperoxidase levels are high.¹⁶¹

Less commonly, the megaloblastic component of a mixed iron-deficiency anemia can be overlooked, and the patient may be treated only with iron. In this situation, the anemia may respond only partly to therapy, and megaloblastic features become more conspicuous as iron stores fill. The masking of macrocytosis in these situations may be responsible for delay or difficulty in diagnosis of pernicious anemia, particularly in certain geographic areas and ethnic groups where there is a high incidence of thalassemia and microcytic hemoglobinopathies.^153,162,163 There are several situations that favor the coexistence of a megaloblastic state with iron deficiency. Both folate and iron deficiency occur in celiac disease,¹⁶⁴ and cobalamin and iron deficiency both complicate gastric reduction surgery for morbid obesity.¹⁶⁵ Furthermore, Helicobacter pylori infection is associated with gastric atrophy that can result first in iron deficiency and later lead to cobalamin malabsorption and perhaps even predispose to pernicious anemia.^166,167

Incomplete Megaloblastic Anemia

If a patient with a full-blown megaloblastic anemia receives cobalamin or folate before marrow aspiration, the anemia persists but the megaloblastic changes may be obscured. Attenuated megaloblastic changes also are seen in patients with early megaloblastic anemia, in patients with coexisting infection,¹⁵⁴ or in patients after transfusion.

Megaloblastic Anemia Misdiagnosed as Acute Leukemia

Occasionally, very severe megaloblastic anemia produces marrow morphology so bizarre as to be mistaken for acute leukemia. In some cases, the erythroid series does not mature, and the megaloblastic pronormoblast dominates the marrow with prominent mitotic figures and dysmorphic forms, raising the possibility of erythroid leukemia.

Megaloblastic Changes in Other Cells

In most forms of megaloblastic anemia, cytologic abnormalities resembling megaloblastosis may appear in other proliferating cells. Epithelial cells from the mouth, stomach, small intestine, and cervix uteri may look megaloblastic, appearing larger than their normal counterparts and containing atypical immature-looking nuclei. Distinguishing these “megaloblastic” changes from the changes of malignancy may be difficult.¹⁶⁸

Chemical Changes in Body Fluids

Plasma bilirubin, iron, and ferritin levels are increased.¹⁶⁹ Serum lactate dehydrogenase-1 (LDH-1) and LDH-2, both found in red cells, are markedly elevated as a result of rapid intramedullary erythroblast turnover and increase with the severity of the anemia.¹⁷⁰ In megaloblastic anemia LDH-1 is greater than LDH-2, whereas in other anemias LDH-2 is greater than LDH-1.¹⁷¹ Serum muramidase (lysozyme) levels are high,¹⁷² whereas serum glutamic oxaloacetic transaminase (aspartate transaminase [AST]) is normal.¹⁷³ Erythropoietin levels rise, but less than in other anemias of similar severity.¹⁷⁴ Surprisingly, the elevated erythropoietin levels fall sharply within 1 day of beginning treatment, an interval too short either to have been mediated by the hematocrit or to affect it.

Cytokinetics

Megaloblastic anemia is associated with two pathophysiologic abnormalities: ineffective erythropoiesis and hemolysis. Ineffective erythropoiesis increases the red cell precursor to reticulocyte ratio, plasma iron turnover,¹⁷⁵ LDH-1 and LDH-2 levels,¹⁷¹ and “early labeled” bilirubin.¹⁷⁶ Both intramedullary and extramedullary hemolysis occurs in megaloblastic anemia, with red cell life span decreased by 30 to 50 percent.¹⁷⁷

Increased serum muramidase in megaloblastic anemia can be caused by increased granulocyte turnover,¹⁷² possibly induced by disintegration of granulocyte precursors in the marrow (ineffective granulopoiesis). In cobalamin deficiency, platelet production is only 10 percent of that expected from the megakaryocyte mass,¹⁷⁸ perhaps reflecting ineffective thrombopoiesis. Platelets in severe cobalamin deficiency are functionally abnormal.¹⁷⁹

FOLIC ACID DEFICIENCY

Etiology and Pathogenesis

Folate deficiency is caused by (1) dietary deficiency, (2) impaired absorption, and (3) increased requirements or losses (see Table 41–4).

Decreased Intake Caused by Poor Nutrition

Prior to the mid-1990s, inadequate dietary intake was the major cause of folate deficiency. However, in the era of folic acid fortification, the prevalence of folate deficiency has fallen dramatically. In the United States, the prevalence of low plasma folate has dropped from 22 to 1.7 percent of the population.¹⁸⁰ Because folate reserves are limited, deficiency develops rapidly in malnourished persons, typically the old, the poor, and the alcoholic. Folate deficiency can also occur during hyperalimentation¹⁸¹ and subclinical folate deficiency has been reported in subtotal gastrectomy.¹⁸² Folate deficiency can occur in premature infants, especially with infection, diarrhea, or hemolytic anemia¹⁸³; in children on a synthetic diet because of inborn errors¹⁸⁴; and in infants raised on goat’s milk, which is poor in available folate.¹⁸⁵ Destruction of folate through excessive cooking can aggravate folate deficiency.

In alcoholic cirrhosis, megaloblastic anemia usually is caused by folate deficiency.¹⁸⁶ Alcohol may acutely depress serum folate, even if folate stores are replete,¹⁸⁷ and accelerates the development of megaloblastic anemia in persons with early folate deficiency.¹⁸⁸ Alcohol causes acute marrow suppression, decreases in reticulocyte, platelet, and granulocyte levels¹⁸⁹; reversible vacuolation of erythroid and myeloid precursors; and dysfunction of granulocytes.¹⁹⁰ These changes occur even if large doses of folate are given with the alcohol.¹⁹¹

Decreased Intake Caused by Impaired Absorption Nontropical Sprue

Nontropical sprue (celiac disease in children) is related to ingestion of wheat gluten.¹⁹² Pathologically, nontropical sprue shows atrophy and chronic inflammation of the small intestinal mucosa that is most severe proximally. Findings include weight loss; glossitis (typical of folate deficiency); other signs of a generalized vitamin deficiency; diarrhea; and passage of light-colored, bulky stools with a particularly foul odor caused by steatorrhea. Iron deficiency, hypocalcemia, osteoporosis, and osteomalacia may occur.

Folate malabsorption occurs in most patients with this disorder.¹⁹³ Serum folate levels are low,¹⁹⁴ and megaloblastic anemia occurs frequently.

Tropical Sprue

Tropical sprue is endemic in the West Indies, southern India, parts of Southern Africa, and Southeast Asia. It can be acquired by travelers to those regions and persists for many years after the travelers return.¹⁹⁵ Tropical sprue is rapidly corrected by folate therapy, even though folate deficiency does not cause the disease. The precise etiology of tropical sprue is unknown, although the response of the disease to antibiotics suggests infection.¹⁹⁶

Clinically and pathologically, tropical sprue is like nontropical sprue, except that tropical sprue is more severe in the distal small intestine.¹⁹⁷ Therefore, tropical sprue eventually also leads to cobalamin deficiency¹⁹⁸ and should be strongly considered as a cause of cobalamin deficiency in former residents of the tropics, even though they have been away from the tropics for 20 years or more. Folate malabsorption may occur,¹⁹⁹ possibly because the diseased intestine fails to deconjugate folate polyglutamates.²⁰⁰ Consequently, megaloblastic anemia is very common in patients with this disease,²⁰¹ and may result from both folate and cobalamin deficiency.

Other Intestinal Disorders

Malabsorption of folic acid commonly occurs in regional enteritis,²⁰¹ after extensive resections of the small intestine,²⁰² and in conditions such as lymphomatous or leukemic infiltration of the small intestine,²⁰³ Whipple disease,²⁰³ scleroderma and amyloidosis,²⁰⁴ and diabetes mellitus.²⁰⁵ Systemic bacterial infections impair folate absorption.²⁰⁶

Increased Folate Requirements in Pregnancy

During pregnancy (Chap. 8),²⁰⁷ folate requirements increase five- to 10-fold because of transfer of folate to the growing fetus,²⁰⁸ which draws down maternal folate stores even in the face of severe maternal folate deficiency.²⁰⁹ Further increases in requirements may result from the presence of multiple fetuses, a poor diet, infection, coexisting hemolytic anemia, or anticonvulsant medication. Lactation aggravates folate deficiency.²¹⁰ Consequently, folate deficiency is very common in pregnancy and is the major cause of the megaloblastic anemia of pregnancy,²¹¹ particularly in developing countries.²¹²

Folate deficiency is difficult to diagnose in pregnancy because the signs of deficiency are obscured by the normal hematologic changes of pregnancy. During pregnancy, a physiologic “anemia” develops because of increased plasma volume that is only partly offset by an accompanying increase in red cell mass. Hemoglobin levels may fall to 10 g/dL. The anemia is associated with a physiologic macrocytosis; MCV may increase to 120 fL, although the average at term is 104 fL.²¹³ Serum and red cell folate levels fall steadily during pregnancy, even in well-nourished women who are not taking a folic acid supplement.²¹⁴ Conversely, hypersegmented neutrophils, usually a reliable clue to early megaloblastic anemia, are inconspicuous in early megaloblastic anemia of pregnancy.²¹⁵

Increased Cell Turnover

Because of increased marrow cell turnover, the folate requirement rises sharply in chronic hemolytic anemia.²¹⁶ During bouts of acute hemolysis that can occur in these anemias, the marrow may become megaloblastic within days.

Folic acid deficiency may arise in chronic exfoliative dermatitis, in which folate losses of 5 to 20 mcg/day may occur.²¹⁷ Patients with psoriasis who are treated with methotrexate have an added reason for developing signs of folate deficiency. Pretreating such patients with folate may prevent these signs without impairing the therapeutic effect of methotrexate.²¹⁷ During hemodialysis, folate is lost in the dialysis fluid.²¹⁸

Clinical Features

The clinical picture of folate deficiency includes all the general manifestations of megaloblastic anemia plus the following specific features: (1) a history and laboratory studies indicating folate deficiency, (2) absence of the neurologic signs of cobalamin deficiency (see “Cobalamin Deficiency” below), and (3) a full response to physiologic doses of folate.

Laboratory Features

The earliest specific indicator of folate deficiency is a low serum or plasma folate. Raised plasma levels of homocysteine may precede the lowering of plasma folate. However, elevated homocysteine has poor specificity as there are several causes of a raised plasma homocysteine.²¹⁹ Plasma folate follows folate intake closely, so an isolated low serum folate (less than approximately 3 ng/mL) may simply indicate a drop in folate intake over the preceding few days.⁵ Similarly, a low plasma folate, except in malabsorption, rises quickly on refeeding.

A better indicator of the tissue folate status is the red cell folate,²²⁰ which remains relatively unchanged while a red cell is circulating and thus reflects folate status over the preceding 2 to 3 months. Red cell folate usually is quite low in folate-deficient megaloblastic anemia. However, red cell folate also is low in more than 50 percent of patients with cobalamin-deficient megaloblastic anemia¹⁰⁰ owing to the poor retention of methyltetrahydrofolate monoglutamate within the cells; consequently, red cell folate cannot be used to distinguish between folate and cobalamin deficiencies. Conversely, red cell folate may be normal in the megaloblastic state that occurs, often with little accompanying anemia, in rapidly developing folate deficiency (see “Acute Megaloblastic Anemia” below).²²¹

The dU suppression test has been used in research on pathogenetic mechanisms in megaloblastic states. It adds little to the clinical evaluation of a megaloblastic anemia. The test is further discussed in “Deoxyuridine Suppression” below.

Differential Diagnosis

Macrocytosis without megaloblastic anemia occurs in alcoholism, liver disease, hypothyroidism, aplastic anemia, certain forms of myelodysplasia, pregnancy, and any condition associated with reticulocytosis (e.g., autoimmune hemolytic anemia). Macrocytosis has also been reported among smokers.²²² However, MCV rarely exceeds 110 fL in these conditions, whereas in folate deficiency, uncomplicated by causes of microcytosis, the MCV is usually over 110 fL.

A full hematologic response to physiologic doses of folate (i.e., 200 mcg daily) distinguishes folate deficiency from cobalamin deficiency, in which a response occurs only at pharmacologic doses of folate (e.g., 5 mg daily). This is not recommended as a diagnostic test because neurologic problems may develop in cobalamin-deficient patients treated with folate alone. High doses of cobalamin may produce a partial response in folate deficiency.⁷³

The diagnosis of nontropical sprue rests on (1) the demonstration of malabsorption, (2) noninvasive serologic testing including detection of antibodies to gliadin, endomysium, tissue transglutaminase, and deamidated gliadin,²²³ (3) a jejunal biopsy showing villus atrophy, and (4) the response to a gluten-free diet. In 80 percent of patients, a gluten-free diet gradually reverses the functional disorder by correcting folate malabsorption.²²⁴

Nonhematologic Effects of Folate Deficiency

The hematologic problems associated with folate deficiency have been recognized for decades. However, folate deficiency has been associated with a number of serious disorders not involving the hematopoietic system. Moreover, these disorders occur at folate levels usually regarded as low normal. They include developmental, neurologic, cardiovascular, and neoplasic diseases.²²⁵

Abnormalities of Neural Tube Closure

A close association exists between mild folate deficiency and congenital anomalies of the fetus, most notably defects in neural tube closure, but also abnormalities involving the heart, urinary tract, limbs, and other sites.²²⁶ A portion of the neural tube closure defects appear to be associated with antibodies against folate receptors that may be overcome by higher folate intake.²²⁷ Mutations and polymorphisms affecting enzymes of folate metabolism, especially the common 677C→T polymorphism of the MTHFR gene (also designated as MTHFR 677C→T),²²⁸ also predispose to congenital anomalies. As noted above, this polymorphism results in diminished conversion of its substrate methylene FH₄ to methyltetrahydrofolate, supporting the view that it is the role of folate in methylation through methionine synthesis (see Table 41–1) that is critical in embryonic development. Folic acid fortification programs, which were mandated in the United States and Canada in the mid-1990s, have been highly successful as a public health measure in reducing the incidence of neural tube defect births by between 20 and 50 percent.^229,230

Cobalamin also plays a significant role as a risk factor for neural tube defects. Levels of TC in normal pregnant women correlate with their likelihood of bearing an infant with a defect in neural tube closure. Patients in the lowest quintile of TC concentration are five times more likely to give birth to a defective infant as patients in the highest quintile.²³¹ Evidence indicates that in populations exposed to folic acid fortification, there is an approximately threefold increase in the risk of neural tube defects in offspring of mothers in the lowest quartile of TC.²³²

Several poorly defined neuropsychiatric abnormalities that respond to folate therapy have been reported in patients with folate deficiency. The most convincing associations are with depressive illness.^225,233

Vascular Disease

Even a mildly elevated homocysteine level is a major independent risk factor for atherosclerosis and venous thrombosis, possibly because of an effect on the vascular endothelium.²³⁴ In folate deficiency, homocysteine levels may rise considerably, resulting in varying degrees of hyperhomocysteinemia. This is true also in cobalamin and pyridoxine deficiencies; consequently, the notion of seeking to ameliorate hyperhomocysteinemia and thus diminish the risk of cardiovascular disease has seemed appealing. However, the effect of lowering homocysteine levels by the use of folate, cobalamin, and pyridoxine supplements, on the risk of recurrent vascular disease is unclear. Although there is some evidence that such supplements reduce risk,²³⁵ contradictory evidence suggests that supplement use may actually increase the risk of in-stent coronary restenosis²³⁶ or other adverse cardiovascular outcome.²³⁷ On the other hand, an accelerated rate of decrease in stroke mortality has been observed in the United States and Canada that coincided with the introduction of folic acid fortification in these countries.²³⁸ The disparate designs of these studies makes it difficult to draw firm conclusions regarding the question of whether lowering of plasma homocysteine in subjects at risk for cardiovascular disease has any ameliorative or deleterious effect on outcome. In a meta-analysis of eight randomized trials involving over 37,000 individuals, the authors concluded that supplementation with folic acid in various combinations with vitamin B₁₂ and vitamin B₆ for periods of up to 7.3 years, despite an overall reduction of plasma homocysteine of 22 percent and 25 percent in folic acid fortified and non-fortified populations, respectively, there were no significant effects on cardiovascular events, overall cancer, or mortality.²³⁹ Critical factors might relate to several considerations including the preexisting degree of vascular damage in relation to the time of the intervention and the form and dosage of administered vitamins.

Another potential link between folate deficiency and morbidity risk that relates to hyperhomocysteinemia is the association between elevated homocysteine levels and incident dementia or cognitive impairment without dementia, independent of the vascular complications of hyperhomocysteinemia.^240,241 The MTHFR polymorphism MTHFR 677C→T leads to increased homocysteine levels in subjects with low folate or cobalamin levels,²⁴² although controversy exists as to whether MTHFR 677C→T contributes to an increased incidence of vascular disease. Like folate, cobalamin seems to be important in decreasing the risk of vascular disease.²⁴³ A 1561C→T polymorphism in the gene for glutamate carboxypeptidase-II increases serum folate and decreases serum homocysteine in the homozygote, possibly protecting against vascular disease.²⁴⁴

HELLP Syndrome

Severe folate deficiency reportedly mimics the hemolysis, elevated liver enzymes, low platelets (HELLP) syndrome (preeclampsia with liver swelling and abnormal liver function studies in pregnant women; Chap. 8).²⁴⁵ In these patients, the diagnosis of severe folate deficiency can be made based on the presence of anemia and a megaloblastic blood film and marrow. Serum and red cell folate, serum cobalamin, homocysteine, and methylmalonic acid levels all should be assayed before treatment is started.²⁴⁶ The patient should immediately be given high doses of folate plus cobalamin, the latter in case the megaloblastic anemia actually results from cobalamin deficiency, a possibility rendered more likely in folic acid-fortified populations. A major goal of treatment is preventing preterm delivery of the fetus.

Colon Cancer

A large study of nurses in the United States indicated that supplementation with more than 400 mcg of folic acid per day reduces the incidence of colon cancer by 31 percent.²⁴⁷ Furthermore, individuals who are homozygous for the 677C→T MTHFR mutation also have a decreased incidence for colon cancer compared with 677C→T heterozygotes and normal controls.²⁴⁸ Other evidence points to possible deleterious effect of folic acid on colon cancer incidence. Although only circumstantial, a recent epidemiologic study reported that after several successive years of a declining incidence of colorectal cancer in the United States and Canada, there was a significant increase in the rate in both countries that coincided with and followed the introduction of folic acid fortification.²⁴⁹ These apparently contradictory observations may be reconcilable because of the several roles of folate on cellular proliferation and repair as well as on the stage of tumorigenesis.²⁵⁰ Because folate is critical for de novo thymidine synthesis, it plays an important part in DNA repair, thus correcting mutations and DNA strand breaks that could potentially initiate cancer. On the other hand, the growth of established neoplastic clones might be accelerated by additional folate, allowing more rapid tumor progression. The situation is rendered even more complex if the potential role of folate in epigenetic regulation of gene expression is considered. Folate is necessary for synthesis of the universal methyl donor, SAMe, which is required for both cytosine and histone methylation. In this pathway, too, the role of folate theoretically may be cancer promoting or cancer protective, depending on whether oncogenes or tumor-suppressor genes are silenced by methylation of CpG islands in DNA or by conformational changes in chromatin resulting from histone methylation. The question of a possible effect of increased folate intake through the use of folic acid supplements on overall and site-specific cancer incidence was recently examined in a meta-analysis of 50,000 individuals. The authors concluded that there was no substantial increase or decrease in incidence over a 5-year period of folic acid supplement use.²⁵¹

Therapy, Course, and Prognosis

Folate, usually in the form of folic acid, 1 to 5 mg/day, is given orally, although 1 mg usually is sufficient. At this dose, anemia usually is corrected even in patients with malabsorption. A parenteral preparation containing 5 mg/mL of folate also is available.

Treatment for tropical sprue consists of the usual doses of folate, plus cobalamin if indicated. To prevent relapse, treatment should be maintained for at least 2 years. Broad-spectrum antibiotics are helpful adjuncts, although antibiotics alone fail to correct the condition.

Pregnant women must be given at least 400 mcg of folate per day.²⁵² As to the possibility of overlooking cobalamin deficiency resulting from folate administration, although pernicious anemia (PA) in women of childbearing age is rare in whites, this is not the case among persons of African and Hispanic descent.^253,254 In pregnant women at risk for cobalamin deficiency (e.g., vegans or patients with malabsorption), the risk of an associated cobalamin deficiency is easily prevented with vitamin B₁₂, 1 mg given parenterally every 3 months during the pregnancy.

Therapeutic doses of folate partially and temporarily correct the hematologic abnormalities in cobalamin deficiency, but the neurologic manifestations can progress, with disastrous results.²⁵⁵ Therefore, both folate status and cobalamin status must be evaluated early in the workup of a megaloblastic anemia. If treatment is urgent and the nature of the deficiency is unclear, both folate and cobalamin can be given after suitable specimens have been obtained for assay.

Patients who receive low-dose methotrexate therapy as an immunosuppressant may develop side effects, the worst of which is hepatotoxicity. The incidence of side effects, including hepatotoxicity, has been correlated with reduced folate levels.²⁵⁶ Administration of folic or folinic acid can prevent or greatly diminish the major side effects without reducing the therapeutic effect of low-dose methotrexate. Coadministration of folic acid together with vitamin B₁₂ also reduces side-effects without adversely affecting the therapeutic efficacy of the newer multitargeted antifolate drug, pemetrexed.²⁵⁷

COBALAMIN DEFICIENCY

Etiology and Pathogenesis

There are several causes and varying degrees of severity of cobalamin depletion and deficiency. From the hematologic standpoint, it is convenient to divide the causes of B₁₂ deficiency into those that frequently lead to megaloblastic anemia and those that usually do not.^64,258

Table 41–4 lists disorders that lead to cobalamin deficiency.

Decreased Uptake Caused by Impaired Absorption

Cobalamin deficiency most often results from defective absorption, most commonly PA, a condition characterized by failure of gastric intrinsic factor production. Many other causes of defective cobalamin absorption involve mainly the stomach, or small intestine and to lesser extent, the pancreas.

Gastric Disorders in Pernicious Anemia

PA is a disease of insidious onset that generally begins in middle age or later (usually after age 40 years).²⁵⁹ In this condition, intrinsic factor secretion fails because of gastric mucosal atrophy. PA is an autoimmune disease. The gastric atrophy of PA probably results from immune destruction of the acid- and pepsin-secreting portion of the gastric mucosa. The term pernicious anemia sometimes is used as a synonym for cobalamin deficiency, but it should be reserved for the condition resulting from defective secretion of intrinsic factor by an atrophic gastric mucosa caused by an autoimmune process primarily directed against the parietal cells and their products.

In patients with PA, antibodies occur that recognize the H⁺/K⁺-adenosine triphosphatase (ATPase), which resides in the secretory membrane of the parietal cell and is responsible for acidifying the stomach contents. These antiparietal cell antibodies occur in approximately 60 percent of patients with simple atrophic gastritis and in 90 percent of patients with PA, but in only 5 percent of a random 30- to 60-year-old population.²⁶⁰ Antiparietal cell antibodies also occur in a significant percentage of patients with thyroid disease.²⁶¹ Conversely, patients with PA have a higher than expected incidence of antibodies against thyroid epithelium, lymphocytes, and renal collecting duct cells.²⁶²

Antiparietal cell antibodies are not thought to be responsible for the pathogenesis of PA. Rather, studies in mice suggest the gastric atrophy in PA is caused by CD4+ T cells whose receptors recognize the H⁺/K⁺-ATPase. Thus, thymectomized BALB/c mice develop an autoimmune atrophic gastritis similar to that seen in PA patients. CD4+ T cells from these mice produce atrophic gastritis when injected into nude mice.²⁶³

Antibodies to intrinsic factor (“type I,” or “blocking,” antibodies) or the intrinsic factor–cobalamin (Cbl) complex (“type II,” or “binding,” antibodies) are highly specific to PA patients.²⁶⁴ Blocking antibodies, which prevent formation of the intrinsic factor–Cbl complex, are found in up to 70 percent of PA sera.²⁶⁴ Binding antibodies, which prevent the intrinsic factor–Cbl complex from binding to its ileal receptors, are found in about half the sera that contain blocking antibody. Some findings in humans support the idea that T cells are responsible for the gastric atrophy in PA. First, lymphocytes from patients with PA are hyperresponsive to gastric antigens.²⁶⁵ Second, the incidence of PA is higher than expected in patients with agammaglobulinemia, even though their sera contain none of the antibodies typical of PA.²⁶⁶

Other Autoimmune Diseases

The coexistence of several other autoimmune diseases and PA is further evidence that PA is an autoimmune disease. Antiparietal cell antibodies and PA are unexpectedly frequent in patients with other autoimmune diseases,²⁶⁷ including autoimmune thyroid disorders (thyrotoxicosis, hypothyroidism, and Hashimoto thyroiditis),²⁶⁸ type I diabetes mellitus, hypoparathyroidism,²⁶⁹ Addison disease, postpartum hypophysitis,²⁷⁰ vitiligo,²⁷¹ acquired agammaglobulinemia,²⁶⁶ infertile female patients younger than age 40 years,²⁷² and hypospermia and infertility in males.^273,274 Infertility may, however, relate to impairment of DNA synthesis in gonadal cells rather than to an autoimmune mechanism.

Inherited Predisposition to Pernicious Anemia

Predisposition to PA can be inherited. The disease is associated with human leukocyte antigen types A2, A3, B7, and B12,²⁷⁵ and with blood group A.²⁷⁶ PA and antiparietal cell antibodies occur more frequently than expected in the families of PA patients.²⁷⁷ In one study, gastric atrophy was found in more than 30 percent of the relatives of patients with PA; of these relatives, 65 percent had antiparietal cell antibodies and 22 percent had antiintrinsic factor antibodies.²⁷⁸ PA occurs relatively frequently in northern Europeans (especially Scandinavians)²⁷⁹ as well as Africans,^163,280 but is uncommon in Asians. In Americans of African descent, the disease tends to begin early, occurs with high frequency in women, and often is severe.^163,254

Stomach and Intestine in Pernicious Anemia

Gastric manifestations of PA include achlorhydria, acquired intrinsic factor deficiency previously demonstrable by the Schilling test, and an increased incidence of certain malignancies. There is an approximately twofold increase in the incidence of gastric cancer, similar increases in the incidence of certain hematologic malignancies, and an increase in the incidence of gastric carcinoid.²⁷⁹ Achlorhydria may precede by many years the loss of intrinsic factor secretion and the development of PA.²⁸¹ The absence of achlorhydria excludes the diagnosis of PA. H. pylori, a microorganism that infects the gastric mucosa, is a major cause of gastritis and peptic ulcers. Evidence is conflicting regarding the role of H. pylori in PA. In two studies, cultures of gastric biopsies showed a very low incidence of H. pylori infection in PA patients.²⁸² One study reported that anti–H. pylori antibodies were found in only a small fraction of the sera from these patients. The other study reported that these antibodies were present in most of the PA sera, indicating that most of the patients described in the study had been infected previously. Whether H. pylori participates in the pathogenesis of PA is an open question. An intriguing hypothesis has been advanced that chronic infection with H. pylori may be responsible for triggering an autoimmune reaction directed against the host H⁺/K⁺-ATPase protein as a result of molecular mimicry.^167,283

Fasting plasma gastrin levels are high in most patients with PA, whereas somatostatin levels are low.²⁸⁴ In biopsies from PA stomachs, however, fundal gastrin and somatostatin levels were high, correlating with increases in argyrophilic cells in the basal crypts; antral gastrin and somatostatin were normal. Gastrin levels are high in simple achlorhydria without PA.²⁸⁵

The stomach shows characteristic histologic abnormalities in PA (Fig. 41–14). The mucosa of the cardia and fundus is atrophic, containing few chief (i.e., pepsin-secreting) or parietal cells. The withered mucosa is infiltrated with lymphocytes²⁸⁶ and plasma cells. In contrast, the antral and pyloric mucosa are normal. Gastric atrophy is partly reversible by glucocorticoid treatment, with some regeneration and return of intrinsic factor secretion, further evidence for the autoimmune nature of PA.²⁸⁷ Clinical response to administration of glucocorticoids or adrenocorticotropic hormone in patients with neurologic disease may reflect temporary amelioration of underlying and undiagnosed PA.²⁸⁸

Megaloblastic changes reversible by cobalamin are seen in the gastrointestinal epithelium. Cells recovered by lavage are large¹⁶⁸ and show atypical nuclei resembling early malignant change.²⁸⁹ Small intestinal biopsy shows decreased mitoses in crypts, shortening of villi, megaloblastic changes in epithelial cells, and infiltration in the lamina propria.²⁹⁰ These changes may account for the malabsorption of D-xylose and carotene observed in PA.²⁹¹

Recognizing PA may be difficult. PA combines the general features of megaloblastic anemia and features specific for cobalamin deficiency with unique clinical features related to its (probable) autoimmune etiology and gastric pathology. The disease is easily missed because of its (1) insidious onset, (2) tendency to be masked by the use of multivitamin preparations containing folic acid,²⁹² and (3) many atypical presentations,²⁹³ including its presentation as a neurologic disease without hematologic findings,^77,294 and its tendency to be overlooked in patients with another autoimmune disease.

Antiparietal cell and antiintrinsic factor antibodies are rarely measured, even though antiintrinsic factor antibodies in particular could be of considerable diagnostic value.²²¹ In the absence of a reliable method to assess vitamin B₁₂ absorption, following the Schilling test becoming obsolete, measurement of antiintrinsic factor antibodies in serum represents the only available method to confirm a diagnosis of PA. Antiintrinsic factor antibody is highly specific for PA (although its sensitivity is only modest); its presence in a megaloblastic anemia makes the diagnosis of PA almost certain.

Gastrectomy Syndromes

Gastric surgery often leads to anemia. Iron-deficiency anemia is most common, but cobalamin deficiency with megaloblastic anemia can occur. After total gastrectomy, cobalamin deficiency develops within 5 or 6 years because the operation removes the source of intrinsic factor.²⁹⁵ The delay between surgery and the onset of cobalamin deficiency reflects the time needed to exhaust cobalamin stores after cobalamin absorption ceases. This may occur more rapidly because of abrogation of the enterohepatic reabsorption of biliary cobalamin.

After partial gastrectomy, few patients show frank cobalamin deficiency, but approximately 5 percent have intermediate megaloblastosis, approximately 25 to 50 percent have low serum cobalamin levels, and many have varying degrees of decreased cobalamin absorption.²⁹⁶ Achlorhydria not present before surgery often develops some years after gastrectomy. Postgastrectomy patients with low serum cobalamin levels usually have low serum iron levels,²⁹⁷ in contrast to the high iron levels otherwise typical of cobalamin deficiency.

Cobalamin deficiency after partial gastrectomy can be caused by mucosal atrophy in the unresected remnant of the stomach²⁹⁸ or, if a gastrojejunostomy was performed, by bacterial overgrowth in the afferent loop (see “Competing Intestinal Flora and Fauna: ‘Blind Loop Syndrome’” below). A surgical procedure that has gained popularity for the treatment of morbid obesity is gastric reduction surgery. This procedure results in multiple deficiencies of micronutrients including cobalamin.²⁹⁹

Of the various causes of cobalamin malabsorption described, those that most often lead to megaloblastic anemia include PA, total or partial gastrectomy, intestinal blind loop syndrome, fish tapeworm, ileal resection, regional enteritis (Crohn disease) and tropical sprue.⁶⁴ In addition, several of the inherited disorders affecting cobalamin absorption and metabolism, such as congenital intrinsic factor deficiency, selective cobalamin malabsorption and congenital TC deficiency can also result in megaloblastic anemia.

Zollinger-Ellison Syndrome

In Zollinger-Ellison syndrome, a gastrin-producing tumor, usually in the pancreas, stimulates the gastric mucosa to secrete immense amounts of HCl. The major clinical problem is a severe ulcer diathesis. Malabsorption of cobalamin occurs when the vast quantities of HCl secreted by the overactive gastric mucosa cannot be completely neutralized by the pancreatic secretions. The resulting acidification of the duodenal contents prevents transfer of Cbl from HC binder to intrinsic factor and also inactivates pancreatic proteases.³⁰⁰

Intestinal Diseases

Because the terminal ileum is the site for physiologic cobalamin absorption, a number of intestinal disorders can lead to cobalamin deficiency, including (1) extensive resection of the ileum³⁰¹; (2) inflammatory bowel disease or regional ileitis or other disease affecting the ileum (e.g., lymphoma, radiation damage³⁰²); (3) cobalamin malabsorption associated with hypothyroidism,³⁰³ or certain drugs³⁰⁴; (4) the effects of cobalamin deficiency itself³⁰⁵; and (5) sprue, either tropical or, less often, nontropical.¹⁹⁸ In each of these disorders, administration of exogenous intrinsic factor, as was carried out in the Schilling test, would fail to correct subnormal cobalamin absorption.

Competing Intestinal Flora and Fauna: “Blind Loop Syndrome”

The blind loop syndrome is a state of cobalamin malabsorption with megaloblastic anemia caused by intestinal stasis from anatomic lesions (strictures, diverticula, anastomoses, surgical blind loops) or impaired motility (scleroderma, amyloid).³⁰⁶ Serum cobalamin is low, but intrinsic factor secretion is normal. Cobalamin malabsorption is not corrected by exogenous intrinsic factor but may be corrected by antibiotic treatment. The defect in cobalamin absorption is caused by colonization of the diseased small intestine by bacteria that take up ingested cobalamin before it can be absorbed from the intestine.³⁰⁷ Steatorrhea is also seen in the blind loop syndrome.

Another cause of cobalamin deficiency is infestation with the fish tapeworm Diphyllobothrium latum. Prevalence is highest near the Baltic Sea, Canada, and Alaska, where raw or undercooked fish is consumed. Cobalamin deficiency results from competition between the worm and the host for ingested cobalamin.³⁰⁸ The clinical picture of D. latum infestation ranges from no symptoms to a full-blown megaloblastic anemia with neurologic changes. The infestation is diagnosed by finding tapeworm ova in the feces.

Acquired Immunodeficiency Syndrome

A substantial number of patients with AIDS have low serum cobalamin levels with associated evidence of cobalamin malabsorption.³⁰⁹ In addition, individuals testing seropositive for HIV infection may also have low serum cobalamin and evidence of cobalamin malabsorption.³⁰⁹ The cause of the malabsorption may be intestinal or gastric or a combination of both.^310,311

Pancreatic Disease

Some degree of cobalamin malabsorption has been demonstrated in 50 to 70 percent of patients with exocrine pancreatic insufficiency.³¹² Cobalamin malabsorption in pancreatic insufficiency is caused by a deficiency in pancreatic proteases, resulting in a partial failure to destroy HC–Cbl complexes whose destruction is a prerequisite for the transfer of cobalamin to intrinsic factor.³¹³ Pancreatic insufficiency rarely causes clinically significant cobalamin deficiency.³¹⁴

Dietary Cobalamin Deficiency

Dietary cobalamin deficiency was previously considered very unusual and restricted largely to complete vegetarians who also do not consume dairy products and eggs (vegans).³¹⁵ Low serum cobalamin levels occur in 50 to 60 percent of individuals in this group. The onset of cobalamin deficiency in vegans is slower than in conditions associated with cobalamin malabsorption. Thus it may take 10 to 20 years for an individual consuming a vegan diet to manifest features of cobalamin deficiency.³¹⁶ This is because the enterohepatic pathway for biliary cobalamin absorption remains intact, thus conserving body cobalamin stores.⁶⁴ Breastfed infants of vegan mothers also may develop cobalamin deficiency.³¹⁷ Cobalamin deficiency in vegans presents with mild megaloblastic anemia, glossitis, and neurologic disturbances. In addition to vegans, however, there is mounting evidence of cobalamin inadequacy in children and young adults in developing countries that cannot be explained on the basis of cobalamin malabsorption, and has therefore been attributed to inadequate dietary intake.³¹⁸

Cobalamin deficiency may occur in severe general malnutrition. A megaloblastic anemia not related to cobalamin deficiency may accompany kwashiorkor or marasmus.³¹⁹

Neurologic Effects of Cobalamin Deficiency

Previously, the neurologic abnormalities of cobalamin deficiency were attributed to disordered metabolism of myelin lipids caused by an impaired methylmalonyl CoA mutase reaction.³²⁰ Similar neurologic abnormalities do not, however, occur in patients with inherited methylmalonyl CoA mutase deficiency.^260,321 Authentic combined system disease has occurred in a patient with nutritional folate deficiency³²² and in a patient with MTHFR deficiency.³²³ The latter reports suggest the neurologic lesions of cobalamin deficiency result from deranged methyl group metabolism. Animal studies support this hypothesis. Neurologic disorders closely resembling combined system disease develop in cobalamin-deficient fruit bats,³²⁴ pigs, and monkeys.³²⁵ The development of these disorders is prevented by methionine, which is produced in a cobalamin-dependent reaction and is the precursor of the biologic methylating reagent SAMe. A finding that further supports a methylation defect is that brains from cobalamin-deficient pigs contain increased levels of SAH,³²⁶ a powerful methylation inhibitor produced in SAMe-dependent methylation reactions:

where RH is any unmethylated compound and RCH₃ is its methylated form. Against the methylation defect hypothesis is the finding that cobalamin deficiency had no effect on SAMe, SAH, or methylation of phospholipids or myelin basic protein³²⁷ in the brains of fruit bats.

CLINICAL FEATURES

The more typical clinical picture of cobalamin deficiency includes the nonspecific manifestations of megaloblastosis, such as anemia, thrombocytopenia, neutropenia, smooth tongue, cardiomyopathy, pale yellow skin and/or weight loss, plus specific features caused by the lack of cobalamin, chiefly neurologic abnormalities. Disturbances in either or both cellular and hormonal immune functions have been reported in cobalamin deficiency.^328,329 Cobalamin deficiency may also contribute to the risk of vascular disease through elevation of homocysteine levels. Other disease associations with cobalamin deficiency have been described. These include a possible increase in breast cancer risk in premenopausal women³³⁰ and in osteoporosis.^331,332 Because cobalamin reserves are large, years may pass between the cessation of cobalamin absorption and the appearance of deficiency symptoms.

Neurologic Abnormalities

Cobalamin deficiency causes a neurologic syndrome that is particularly dangerous because the syndrome can develop in isolation,³³³ with no megaloblastic anemia to suggest a lack of cobalamin,^294,334 and because the syndrome cannot be reversed by treatment when it is sufficiently far advanced. The syndrome usually begins with paresthesias in feet and fingers as a result of early peripheral neuropathy and disturbances of vibratory sense and proprioception. The earliest signs, which precede other neurologic findings by months, are loss of position sense in the second toe and loss of vibration sense for a 256-Hz but not a 128-Hz tuning fork.³³⁵ Left untreated, the neurologic disorder progresses to spastic ataxia resulting from demyelination of the dorsal and lateral columns of the spinal cord, so-called combined system disease (Fig. 41–15).³³⁶

The peripheral nerves, the spinal cord, and the brain are affected by cobalamin deficiency. Somnolence and perversion of taste, smell, and vision with occasional optic atrophy are accompanied by slow waves on the electroencephalogram. A dementia mimicking Alzheimer disease can develop.³³⁷ There is recent evidence linking low cobalamin status with brain volume loss and cerebral white matter lesions.^338,339 Psychological derangements, including psychotic depression and paranoid schizophrenia, can occur.³⁴⁰ Frank psychosis in cobalamin deficiency has been given the sobriquet megaloblastic madness.³⁴¹

The neurologic lesions of cobalamin deficiency can be detected by magnetic resonance imaging (MRI). Demyelination appears as T2-weighted hyperintensity of the white matter.³⁴² MRI is particularly useful for confirming the diagnosis of a neurologic disorder resulting from cobalamin deficiency. MRI also has been used to follow the progress of neurologic abnormalities during treatment of cobalamin-deficient patients.³⁴²

Subtle Cobalamin Deficiency

Some observations suggest the existence of a large group of patients who are hematologically normal, with a normal hematocrit and MCV, but who have cobalamin-responsive neuropsychiatric disease.²⁹⁴ Neuropsychiatric findings include peripheral neuropathy, gait disturbance, memory loss, and psychiatric symptoms, often with abnormal evoked potentials. Serum cobalamin may be normal, borderline, or low, but tissue cobalamin deficiency is suggested by consistently high levels of serum methylmalonic acid and/or homocysteine. Most of the neuropsychiatric abnormalities appear to respond to cobalamin therapy.

LABORATORY FEATURES

Plasma or Serum Cobalamin Levels

Plasma or serum cobalamin is low in most but not all patients with cobalamin deficiency.²¹⁹ Cobalamin levels are usually normal in cobalamin deficiency resulting from exposure to nitrous oxide, TC deficiency, and inborn errors of cobalamin metabolism. Levels also may be normal in cobalamin-deficient patients with high HC levels resulting from myeloproliferative diseases. Conversely, plasma cobalamin levels may be low in the presence of normal tissue cobalamins in vegetarians, in subjects taking megadoses of ascorbic acid,³⁴³ in pregnancy (25 percent), in the presence of HC deficiency,^344,345 and in megaloblastic anemia resulting from folate deficiency (30 percent).²¹⁹ Plasma folate may be high in cobalamin deficiency because of retardation in conversion of methyltetrahydrofolate, which is the predominant form in plasma. Patients deficient in both cobalamin and folate may show normal serum folate levels.

Plasma or Serum Holotranscobalamin

The fraction of the cobalamin in plasma that is bound to TC constitutes only 10 to 30 percent of the total plasma cobalamin. Even so, it is this fraction that is functionally important and also better reflects the integrity of the cobalamin absorptive status of an individual.^142,346,347 The major fraction of plasma cobalamin bound to HC is considered functionally inert and is therefore less relevant for the consideration of cobalamin status. Consequently, and with the development of assays to measure the TC-bound fraction of the plasma cobalamin, an increasing body of evidence has accumulated to support the usefulness of TC-associated cobalamin (holoTC).^{64,137,140,346}

Methylmalonic Acid

Except when caused by an inborn error, methylmalonic aciduria is a reliable indicator of cobalamin deficiency.³⁴⁸ Normal subjects excrete only traces of methylmalonate (0 to 3.4 mg/day). In cobalamin deficiency, urine methylmalonate usually is elevated.³⁴⁹ Cobalamin therapy restores excretion to normal in a few days. Another possible advantage of measurement of urine rather than plasma methylmalonic acid is that in conditions of impaired renal function, when plasma methylmalonic acid may give misleadingly elevated levels, measurement of the metabolite in urine when correlated with creatinine obviates this problem.³⁵⁰

Serum or Plasma Methylmalonic Acid and Homocysteine

Elevated plasma or serum methylmalonic acid and homocysteine levels are indicators of tissue cobalamin deficiency. Their levels are high in more than 90 percent of cobalamin-deficient patients and rise before plasma cobalamin falls to subnormal levels.^219,351 Elevated plasma methylmalonic acid and/or elevated homocysteine are both indicators of cobalamin deficiency in patients without a congenital disorder in their metabolism. Of the two, methylmalonic acid measurement is both more sensitive and more specific, and elevated methylmalonic acid will persist for several days, even after cobalamin treatment is instituted. Unlike homocysteine levels that rise in folate and pyridoxine deficiencies, as well as in hypothyroidism, methylmalonic acid elevation is seen only in cobalamin deficiency.²¹⁹ In renal diseases however, both homocysteine and methylmalonate, acid levels are frequently elevated. Additionally, intestinal bacteria synthesize propionate, a precursor of methylmalonate, and in conditions of bacterial overgrowth, microbially derived methylmalonic acid may contribute to elevations in plasma methylmalonic acid.^351,352 Although measurement of these metabolites may be used for population screening for evidence of cobalamin deficiency, the finding of an isolated elevation of plasma methylmalonate cannot be taken as a priori evidence of clinically attributable cobalamin deficiency, absent any demonstration of a therapeutic response to the administration of cobalamin.^352,353

Spinal fluid methylmalonic acid levels are markedly elevated in cobalamin deficiency.³⁵⁴

Assays of Cobalamin Absorption and Intrinsic Factor

Despite its numerous shortcomings the previous “gold standard” for assessment of cobalamin absorption was the Schilling test. The Schilling test assessed cobalamin absorption by measuring urinary radioactivity after an oral dose of radioactive cobalamin. The test could be performed even after cobalamin deficiency had been corrected. The test consisted of administering a physiologic dose of radiolabeled Co-CnCbl by mouth followed 2 hours later by injection of a large “flushing” dose of unlabeled CnCbl and determination and radioactivity in a 24-hour collection of urine. Subjects with normal absorption excreted 7 percent or more of the radioactivity in the urine. Subjects with subnormal urinary excretion would have the test repeated with addition of an animal-derived intrinsic factor to determine whether the malabsorption could be corrected.³⁵⁵ The use of the Schilling test has dropped to a point of obsolescence as a consequence of reduced availability of the test components, cost, radioactive waste disposal, and concern about the use of animal-derived tissues for human use, which were required for the intrinsic factor administered in the second part of the test.⁶⁴ Replacements for the Schilling test are currently under development. One approach uses measurement of the change in holoTC following oral administration of non–radiolabeled cobalamin.^346,347,356 A different approach involves the use of accelerator mass spectrometry and microbially produced ¹⁴C at attomolar concentrations.³⁵⁷ In this approach, ¹⁴C is measured in blood at the time of peak appearance 6 to 8 hours following the dose. Both methods show promise but have not been approved or validated for routine clinical use.

Deoxyuridine Suppression Test

The dU suppression test is based on the finding that unlabeled dU can suppress the uptake of [³H]thymidine ([³H]Thd) into the DNA of cultured lymphocytes or marrow cells through dilution of the label in the thymidine pool.³⁵⁸ This occurs when the thymidylate synthase reaction is functionally intact, which requires adequate quantities of both folate and cobalamin.

The dU suppression test is chiefly a research tool. It can help diagnose certain special clinical problems,³⁵⁸ but these problems also can be diagnosed using other laboratory tests, therapeutic trials with vitamins or iron, or watchful waiting. Furthermore, in more than 40 years of use, the test has not moved from the research laboratory into the clinic. The dU suppression test seems unlikely to enjoy more widespread clinical use in the future.

THERAPY, COURSE, AND PROGNOSIS

Treatment of cobalamin deficiency consists of parenteral CnCbl (vitamin B₁₂) or OHCbl to replace daily losses and refill storage pools, which normally contain 2 to 5 mg of cobalamin.³⁵⁹ Toxicity is highly unusual, and there is no defined upper limit.² Doses exceeding 100 mcg saturate the cobalamin-binding proteins (TC and HC), and the excess is lost in the urine. A typical treatment schedule consists of 1000 mcg cobalamin intramuscular (IM) daily for 2 weeks, then weekly until the hematocrit is normal, and then monthly for life. For neurologic manifestations, 1000 mcg every 2 weeks for 6 months is recommended. Higher doses are given for certain inherited disorders (e.g., TC deficiency). Transfusion occasionally is required when the hematocrit is less than 15 percent or the patient is debilitated, infected, or in heart failure. In such instances, packed cells should be given slowly to avoid pulmonary edema. Infections can impair the response to cobalamin and must be treated vigorously.

Response to Treatment and Therapeutic Trial

Following parenteral administration of cobalamin to deficient patients, elevated plasma bilirubin, iron, and LDH levels fall rapidly (Fig. 41–16).³⁶⁰ Decreasing plasma iron turnover and fecal urobilinogen reflect cessation of ineffective erythropoiesis. Within 12 hours, the marrow begins to change from megaloblastic to normoblastic, a process that is complete in 2 to 3 days. Consequently, morphologic diagnosis may be difficult after treatment is initiated. Reticulocytosis begins on days 3 to 5 and peaks on days 4 to 10.³⁶¹ The new red cells come from new normoblasts, not from the old megaloblasts, most of which die before leaving the marrow.¹⁴⁹ Blood hemoglobin concentration becomes normal within 1 to 2 months. If normal values are not achieved by 2 months, another cause of anemia should be sought.

Other changes include the following: (1) prompt and dramatic improvement in the sense of well-being; (2) normalization of leukocyte and platelet counts, although neutrophil hypersegmentation may persist for 10 to 14 days; (3) rise in serum cobalamin and folate. Cobalamin deficiency does not respond to a physiologic dose of folate (100 to 400 mcg/day), although this dose produces a maximal response in folate deficiency. Larger doses of folate (5 to 15 mg/day) can produce a reticulocytosis and partially or temporarily correct the anemia in cobalamin deficiency. To avoid the risk of masking an underlying cobalamin deficiency by inducing a hematologic remission in response to folate, doses in excess of 1 mg folic acid daily should be shunned until an underlying cobalamin deficiency has been ruled out.³

Special Circumstances

After Gastrectomy

Cobalamin should always be given after total gastrectomy. Cobalamin administration is not necessary after partial gastrectomy, but patients need to be watched for megaloblastic anemia, bearing in mind that this anemia can be masked by postgastrectomy iron deficiency.^352,362

Blind Loop Syndrome

The anemia of the blind loop syndrome can be treated by parenteral cobalamin therapy. It also responds after approximately 1 week to oral broad-spectrum antibiotics (cephalexin monohydrate [Keflex] 250 mg QID plus metronidazole 250 mg TID for 10 days),³⁶³ and cobalamin absorption is restored. Successful surgical correction of an anatomic lesion also cures the syndrome.

Fish Tapeworm

Treatment consists of a single oral dose of a 50 mg/kg of niclosamide or a dose of 5 to 10 mg/kg of praziquantel.

Rekindled Use of Oral Cobalamin

Much interest has been kindled³⁶⁴ regarding the possibility of treating cobalamin deficiency with oral cobalamin as had been proposed previously.³⁶⁵ Oral cobalamin can be used not only for treatment of dietary cobalamin deficiency that occurs in vegans and in patients with very severe general malnutrition, but also for patients with food cobalamin malabsorption³⁶⁶ and for patients with PA, provided the patients are followed carefully.³⁶⁷ In patients lacking intrinsic factor, approximately 1 percent of an oral dose of the vitamin crosses the intestinal epithelium by mass action. Therefore, 1000 to 2000 mcg/day of oral cobalamin supplies most PA patients with their daily cobalamin requirement without the need for injections and their accompanying pain and expense. Cobalamin should be given by mouth to patients with dietary cobalamin deficiency and patients (e.g., hemophiliacs, the frail elderly) who cannot take IM injections.

ACUTE MEGALOBLASTIC ANEMIA

Megaloblastic anemia usually is a chronic condition that requires weeks or months to develop, but a potentially fatal megaloblastic state resulting from acute tissue folate or cobalamin deficiency can arise over the course of only a few days. Patients with acute megaloblastic anemia present with rapidly developing thrombocytopenia and/or leukopenia and counts that sometimes fall to very low levels, but little change in red cell levels unless another cause of anemia is present. The discrepancy between platelet and leukocyte counts on the one hand and red cells on the other hand is a reflection of the much longer red cell life span. The clinical picture can suggest an immune cytopenia. The diagnosis is made from the marrow aspirate, which is floridly megaloblastic, and confirmed by the rapid response to appropriate replacement therapy.

The most common cause of acute megaloblastic anemia is nitrous oxide (N₂O) anesthesia.³⁶⁸ N₂O rapidly destroys MeCbl,³⁶⁹ leading to a megaloblastic state. AdoCbl eventually is lost, SAMe and total folate levels decline, and the proportion of folate in the form of N⁵-methyltetrahydrofolate increases.³⁷⁰ Clinical findings develop quickly. Grossly megaloblastic changes are seen in the marrow after 12 to 24 hours.³⁷¹ Hypersegmented neutrophils do not appear until 5 days after exposure but then persist for several days.³⁷² The effects of N₂O disappear spontaneously after a few days; disappearance can be hastened by folinic acid or cobalamin.³⁷³ Fatalities resulting from N₂O-induced megaloblastosis have occurred in tetanus patients given N₂O for weeks.³⁶⁸ Long-term recreational use of N₂O has led to a neurologic disorder similar to combined system disease.³⁷⁴

Acute megaloblastic anemia occurs in other clinical settings. A rapidly developing megaloblastic state with acute thrombocytopenia has occurred in seriously ill patients, often in intensive care units.³⁷⁵ Especially at risk are patients who are transfused extensively at surgery,³⁷⁶ those on dialysis or total parenteral nutrition, and those receiving weak folate antagonists such as trimethoprim. Morphologic clues to the diagnosis (e.g., hypersegmented neutrophils) often are absent from the blood film. Both red cell folate and serum cobalamin levels may be normal, but the marrow is always megaloblastic. A rapid response to therapeutic doses of parenteral folate (5 mg/day) and cobalamin (1 mg) is the rule.

MEGALOBLASTIC ANEMIA CAUSED BY DRUGS

Table 41–5 lists the drugs that cause megaloblastic anemia. Aminopterin and methotrexate are almost structurally identical to folic acid. After they enter cells via the folate carrier³⁷⁷ and acquire a polyglutamate chain,³⁷⁸ they act as very powerful inhibitors of FH₂ reductase.³⁷⁹ By blocking the FH₂→FH₄ reaction and perhaps inhibiting other enzymes of folate metabolism, they effect the rapid withdrawal of folates from the one-carbon fragment carrier pool, causing a fall in nucleotide (especially thymidine) biosynthesis that leads to a major derangement in DNA replication (Chaps. 10 and 22).³⁸⁰

Toxic effects include necrotic mouth lesions; ulcerations of the esophagus, small intestine, and colon, with abdominal pain, vomiting, and diarrhea; megaloblastic anemia; alopecia; and hyperpigmentation. The drug is excreted by the kidney, so effects and toxicity are prolonged and enhanced if renal function is impaired. Toxicity caused by these folate antagonists is treated with folinic acid (N⁵-formyl FH₄). Folic acid itself is useless in this setting because the blocked reductase cannot convert folate to the active tetrahydro form. Folinic acid is already in the tetrahydro form, so folinic acid is effective despite reductase blockade. The usual dose of folinic acid is 3 to 6 mg/day IM. Larger doses are given in chemotherapy protocols that use folinic acid to rescue patients deliberately treated with otherwise fatal doses of methotrexate. Folinic acid was used intrathecally in a patient in whom a large overdose of methotrexate was accidentally delivered into the subarachnoid space.³⁸¹

Zidovudine (azidothymidine [AZT]) is used for HIV infections (AIDS; Chap. 81).³⁸² Its principal toxic effect is severe megaloblastic anemia. Anemia or neutropenia produced by zidovudine limits use of this drug.³⁸³

HIV infection itself suppresses hematopoiesis, leading to pancytopenia with myelodysplastic features (Chaps. 81 and 87). The blood film shows vacuolated monocytes. Megaloblastosis in HIV infection may result from folate or cobalamin deficiency³⁸⁴ or AZT or trimethoprim toxicity.

Hydroxyurea is used at high doses to treat chronic myelogenous leukemia, polycythemia vera, and essential thrombocythemia, and at lower doses to treat psoriasis, rheumatoid arthritis, and sickle cell disease (Chap. 22). It inhibits conversion of ribonucleotides to deoxyribonucleotides.³⁸⁵ Marked megaloblastic changes are routinely found in the marrow 1 to 2 days after initiating hydroxyurea therapy. These changes are rapidly reversed after the drug is withdrawn. Megaloblastosis as a result of N₂O is discussed in “Acute Megaloblastic Anemia” above.

Long-term use of omeprazole and presumably other H⁺/K⁺-ATPase inhibitors is associated with reduced serum cobalamin levels, presumably because of the ability of these drugs to inhibit parietal cell function.³⁸⁶ Reduced serum cobalamin levels are not a problem when these drugs are used for short intervals.³⁸⁷

Pemetrexed is an antifolate approved for use in mesothelioma. It also has been used for treatment of non–small cell lung cancer. Like other antifolate agents, pemetrexed can result in a megaloblastic anemia that is treated with cobalamin and folate. Coadministration of the drug with cobalamin and folate also reduces toxicity. Trimethoprim is a FH₂ reductase inhibitor that is designed to act on microbial rather than the mammalian enzyme. Still, in patients with borderline folate status, trimethoprim can precipitate a state of folate deficiency.

MEGALOBLASTIC ANEMIA IN CHILDHOOD

Megaloblastic anemia in childhood is usually the result of genetic disorders affecting either the cobalamin binding proteins or the enzymes concerned with intracellular trafficking of cobalamin or its conversion to coenzymatically active forms. Several recent reviews have dealt comprehensively with this topic.^321,388,389

Defects Involving Cobalamin-Binding Proteins

Several genetic mutations and polymorphisms exist that affect the key binding proteins for cobalamin. Their effects range from being clinically benign to causing severe cobalamin deficiency with megaloblastic anemia and neurologic complications usually manifesting in infancy or early childhood, occasionally in adolescence or early adulthood. In general, the mutations and deletions affecting the encoded proteins cause serious health consequences whereas the polymorphic variants may be totally inconspicuous or result only in a modified likelihood of disease risk.

Cobalamin malabsorption occurs in four childhood conditions associated with a genetic component: (1) cobalamin malabsorption in the presence of normal intrinsic factor secretion, (2) congenital abnormality of intrinsic factor, (3) TC deficiency, and (4) true PA of childhood. The management of cobalamin deficiency in childhood has been comprehensively reviewed.³⁸⁹

Selective Malabsorption of Cobalamin, Autosomal Recessive Megaloblastic Anemia, Imerslund-Gräsbeck Disease

Imerslund-Gräsbeck disease³⁹⁰ is an inherited failure of transport of the intrinsic factor–Cbl complex by the ileum, usually accompanied by proteinuria, mostly of albumin.³⁸⁹ It may be the most common cause of cobalamin deficiency in infancy in some populations.³⁹¹ Cobalamin deficiency usually is seen before age 2 years, but may appear earlier or later. Cobalamin malabsorption is not corrected by addition of intrinsic factor. Endogenous intrinsic factor and HCl secretion, TC and HC levels, and gastric and intestinal histology are all normal. Intrinsic factor antibodies are absent. Intrinsic factor–Cbl receptors are present in some but not all patients. The molecular defect responsible for this disease has been elucidated. For the ileal phase of cobalamin absorption, two genes code distinct proteins that form part of the cobalamin–IF receptor (cobalamin-intrinsic factor receptor) complex. The first, which codes for the protein CUBN, is affected by several mutations described in Finnish patients with MGA1.^95,392 The second, affecting the protein AMN results in a milder MGA1 phenotype and is found in Norwegian patients.^95,393 Again, several mutations in the gene coding for the AMN protein have been described.⁹⁵ Patients are treated with IM cobalamin. The anemia is corrected, but proteinuria persists.

Congenital Intrinsic Factor Deficiency

Congenital intrinsic factor deficiency is an autosomal recessive disease in which parietal cells fail to produce functionally normal intrinsic factor.³⁹⁴ Patients present with irritability and megaloblastic anemia when cobalamin stores (<25 mcg at birth) are exhausted. The disease usually presents at age 6 to 24 months. HCl secretion and gastric histology are normal, proteinuria is not present, and antiintrinsic factor antibodies are absent.³⁹⁵ Abnormal cobalamin absorption is corrected by oral intrinsic factor.³⁹⁶ Treatment consists of standard doses of IM cobalamin.

Transcobalamin Deficiency

TC deficiency is an autosomal recessive disorder causing a flagrant megaloblastic anemia that generally presents in early infancy.³⁹⁵ The disease is dangerously deceptive because it results from a very severe deficiency of tissue cobalamin, usually with serum cobalamin levels in the normal range because most of the plasma cobalamin is bound to HC resulting in a misleading test result if reliance is placed simply on serum cobalamin measurement. Undiagnosed TC deficiency causes irreversible CNS damage.³⁹⁷ Patients are healthy at birth but over the next few weeks develop signs and symptoms of cobalamin deficiency, such as rapidly progressive pancytopenia, mouth ulcers, vomiting, and diarrhea. Recurrent bacterial infections may occur.³⁹⁵ Neurologic findings are not prominent in the early stages of the disease.³⁹⁷

Serum folate and cobalamin are normal (the latter because most cobalamin is carried by HC). Homocysteine and/or methylmalonic acid levels are elevated in the plasma.³⁹⁸ The marrow is megaloblastic and the cobalamin absorption is usually but not always abnormal and is not corrected by intrinsic factor.³⁹⁹ The diagnosis is made by measuring plasma TC.³⁸⁹ Prenatal diagnosis is possible.⁴⁰⁰ Serum should be obtained prior to treatment because TC levels in normal individuals drop sharply after cobalamin is given. TC deficiency is treated with cobalamin doses sufficiently large to force enough vitamin into the cells to allow normal function. Initial therapy can consist of oral CnCbl or OHCbl 500 to 1000 mcg twice a week, or IM OHCbl 1000 mcg/week. Blood counts and symptoms should be monitored and doses adjusted upward if necessary.

Several single nucleotide polymorphisms in the TC gene have been described and the allele frequency of the most common form (776 C>G) is high in certain populations.^401,402 HoloTC levels are lower and methylmalonate levels are higher in individuals homozygous for the G allele,^401,402 suggesting that this genotype may be associated with less-favorable cobalamin status.⁶⁴

Haptocorrin Deficiency

Congenital deficiency is not associated with clinically manifested cobalamin deficiency, although the plasma or serum cobalamin levels are well below normal,³⁴⁵ and this is how the condition is recognized. The absence of morbidity in these patients indicates that HCs are not essential for health.

True Juvenile Pernicious Anemia

True PA, with gastric atrophy and a defect in intrinsic factor secretion, is exceedingly rare in childhood.⁴⁰³ Patients usually present in their teens with cobalamin deficiency. Serum antiintrinsic factor antibodies usually are present.²⁶⁵ The diagnosis and treatment are the same as for PA in adults.

INBORN ERRORS OF COBALAMIN METABOLISM

Cobalamin is converted to AdoCbl and MeCbl by a complex series of transformations involving several steps.^388,398,404 Eight disorders affecting this cobalamin transformation pathway have been described, one for each of the steps. Because the molecular causes of these disorders have not yet been fully characterized, the disorders themselves are not named for a defective protein but instead are designated by sequential capital letters preceded by a cbl prefix. The disorders can be grouped into three broad clinical syndromes based on the abnormal metabolites in the patient’s urine (Table 41–6). These disorders are usually discovered during investigation of infants with unexplained developmental delay, acidosis, anemia, or unexplained neurologic difficulties. Typically they have normal plasma cobalamin levels.

Methylmalonic Aciduria Only (cblA, cblB, and cblH)

In cblA and cblB, AdoCbl production is impaired but MeCbl production is normal. This may result either from an abnormal methylmalonyl CoA mutase (designated muto or mut–) or from a defect in activation or production of its cofactor, AdoCbl. The cblH variant appears to represent an interallelic variant of cblA.⁴⁰⁵ Patients present in infancy with acidosis because they cannot catabolize methylmalonic acid. Symptoms include lethargy and failure to thrive, vomiting, and neurologic problems. Mental retardation is not prominent, and megaloblastic anemia is absent. Most patients respond to 1000 mcg/day of OHCbl or CnCbl, although muto and mut– patients are unresponsive.

Homocystinuria Only (Cble and Cblg)

In these disorders, N⁵-methyltetrahydrofolate-homocysteine methyltransferase is defective and lacks the capacity to produce MeCbl.⁴⁰⁶ In patients with cblG, methionine synthase is missing or defective.⁴⁰⁷ cblE results from failure to reactivate methionine synthase that was inactivated by oxidation of its bound cobalamin.⁴⁰⁸ Patients present in infancy with vomiting, mental retardation, and megaloblastic anemia. They have marked homocystinuria and hyperhomocysteinemia without methylmalonic aciduria or methylmalonic acidemia. They respond well to CnCbl 1000 mcg/day or 1000 mcg/week. Infants diagnosed prenatally and treated from birth usually show normal development. On rare occasions, this disorder may first become apparent in adult life.

Methylmalonic Aciduria and Homocystinuria (Cblc, Cbld, and Cblf)

In these disorders, the defect in Cbl transformation affects AdoCbl and MeCbl, probably because reduction of cobalt from Co⁺⁺ to Co⁺ is defective. These patients have both hyperhomocysteinemia and methylmalonic acidemia. The age at initial presentation ranges from early infancy to adolescence. In addition to lethargy and failure to thrive, affected infants present with serious neurologic difficulties. Older patients present with psychological problems, progressive dementia, and motor signs and symptoms. cblC disease is the most common of the cobalamin inborn errors. In cblF the defect lies in an inability to release cobalamin from lysosomes.⁴⁰⁹ Megaloblastic anemia occurs in about half the cases. Patients respond partially to 1000 mcg/day of OHCbl or CnCbl.

A tentative diagnosis of a cobalamin mutation can be made by demonstrating methylmalonic aciduria and/or homocystinuria in a patient with the clinical findings described above in “Methylmalonic Aciduria Only” or “Homocystinuria Only,” respectively. Establishing a diagnosis requires a specialized laboratory equipped to do cultured fibroblast complementation studies.³⁸⁸ In a patient suspected of having a cobalamin mutation, treatment should be started pending the test results because early high-dose cobalamin treatment is risk-free and may reduce the chance of damage to the CNS. Fetuses with these diseases have been successfully treated in utero with very large doses of CnCbl given parenterally to the mother.⁴¹⁰

INBORN ERRORS OF FOLATE METABOLISM

Megaloblastic anemia in infancy has been described in three inherited disorders of folate metabolism.^30,389,411

Hereditary Folate Malabsorption

Hereditary folate malabsorption is a rare inherited disorder in which patients cannot absorb folate from the gastrointestinal tract or transport it across the choroid plexus and into the cerebrospinal fluid.^29,30 The molecular basis for this disorder is caused by abnormalities in the PCFT.²⁹ Patients present with severe megaloblastic anemia, seizures, mental retardation, and other CNS findings.⁴¹² Folate levels are low in the serum and nil in the cerebrospinal fluid. Folate given parenterally has corrected the anemia and seizures in some patients but has had no effect on other CNS symptoms or on the cerebrospinal fluid folate level. Treatment with daily folinic acid by injection maintains the spinal fluid level and can lead to normal development.³⁸⁹

Dihydrofolate Reductase Deficiency

Dihydrofolate reductase deficiency may present isolated megaloblastic anemia within days or weeks after birth. The anemia responds to folinic acid but not to folic acid.⁴¹³

N⁵-Methyltetrahydrofolate–Homocysteine Methyltransferase Deficiency

Decreased methyltransferase activity was described in a liver biopsy from a child with megaloblastic anemia and mental retardation. The anemia failed to respond to folate, cobalamin, or pyridoxal phosphate.⁴¹⁴ The phenotype of this disorder resembles the inborn errors of cobalamin metabolism affecting the methionine synthesis reaction and has not been well characterized as a distinct entity at the molecular level.

Methylene Tetrahydrofolate Reductase Deficiency

In this rare autosomal recessive disorder there is a severe hyperhomocysteinemia and homocystinuria with low plasma methionine. Patients have neurologic and vascular complications but no megaloblastic anemia or methylmalonic aciduria.³⁸⁹ The polymorphic variations in MTHFR have been discussed earlier as well as their influence on disease susceptibility and the influence of the enzyme on the distribution of major folate species toward either methylation or DNA synthetic pathways.

OTHER INBORN ERRORS

Hereditary Orotic Aciduria

Hereditary orotic aciduria is an autosomal recessive disorder of pyrimidine metabolism⁴¹⁵ characterized by megaloblastic anemia, growth impairment, and excretion of orotic acid in the urine. Cobalamin and folate levels are normal.

Lesch-Nyhan Syndrome

The Lesch-Nyhan syndrome is an X-linked disorder of purine metabolism characterized by hyperuricemia, hyperuricosuria, and a neurologic disease with self-mutilation. It is caused by a hypoxanthine–guanine phosphoribosyltransferase deficiency. One patient described had megaloblastic anemia.⁴¹⁶

Thiamine-Responsive Megaloblastic Anemia

Seven children with severe megaloblastic anemia, sensorineural deafness, and diabetes mellitus, all beginning in infancy, have been reported. The anemia responded to thiamine (25 to 100 mg/day). The marrow was reported as myelodysplastic in two patients with the disorder.⁴¹⁷ The gene for this puzzling disorder has been mapped to the long arm of chromosome 1, and the underlying biochemical defect is caused by reduced nucleic acid production through impairment of the thiamine dependent pentose cycle enzyme transketolase that results in cell-cycle arrest and the megaloblastic phenotype.⁴¹⁸ This condition is also discussed in Chap. 44.

OTHER CAUSES OF MEGALOBLASTIC ANEMIA

Congenital Dyserythropoietic Anemia

The congenital dyserythropoietic anemias are lifelong anemias. They often are mild, showing dysplastic changes affecting the red cell line only, most typically multinuclearity of the normoblasts. They appear to result from defects in glycosylation of polylactosaminoglycans linked to membrane proteins and ceramides.⁴¹⁹ Of the three types, two (type I usually⁴²⁰ and type III occasionally⁴²¹) show megaloblastic red cell precursors (Chap. 39).

Refractory Megaloblastic Anemia

Refractory megaloblastic anemia is regarded as a manifestation of some sideroblastic anemias (Chap. 59) and myelodysplastic disorders (Chap. 87).⁴²² The megaloblastic changes are atypical. Dysplastic features are confined to the erythroid series. Giant metamyelocytes and bands are absent from the marrow. A few patients with refractory megaloblastic anemia respond to pharmacologic doses of pyridoxine (200 mg/day),⁴²³ perhaps because of an effect on serine transformylase, which requires both pyridoxine and folate.

Acute Erythroid Leukemia

In acute erythroid leukemia, a variety of acute myelogenous leukemia (Chap. 89)⁴²⁴ nucleated red cells appear on the blood film, there is usually marked anisocytosis and anisochromia, and macrocytes are usually present. The marrow shows pronounced erythroid hyperplasia involving very bizarre looking megaloblast-like red cell precursors, often containing multiple nuclei or nuclear fragments (see Chap. 88, Fig. 88–1) together with increased numbers of blasts. The megaloblastoid erythroid precursors frequently appear vacuolated.

Consideration of the rarer causes of megaloblastic anemia is important when the common and correctable causes resulting from folate or cobalamin deficiencies have been excluded. This is particularly important in the pediatric age group, but also in patients who are refractory to treatment with either folate or cobalamin.