Megaloblastic Anemias:Disorders of Impaired Dna Synthesis
Ralph Carmel
HISTORICAL BACKGROUND
The story of megaloblastic anemia, its causes, and how they were decoded is a wonderfully instructive chapter of medicine. Clinical observations set the stage for a series of insightful clinical investigations that converted a dreaded “pernicious” condition into one that is now easily treated.
A puzzling illness with anemia, debilitation, languor, and, finally, torpor and death was described by Addison1 in 1849. Although possibly similar cases were reported earlier and Addison believed the anemia to be related to adrenal dysfunction, this is generally taken as the first description of pernicious anemia. Neuropathy was noted by Osler and Gardner in 1877, and Lichtheim associated it with myelopathy 10 years later. In 1880, Ehrlich identified megaloblasts and proposed them as the precursors of the “giant blood corpuscles” described in the peripheral blood by Hayem.
The clinical breakthrough occurred in 1926 when Minot and Murphy,2 using the then new technique of reticulocyte assessment, showed that the manifestations could be reversed and held in abeyance by eating prodigious amounts of liver; for this, they shared the Nobel Prize. Three years later, Castle,3 building on earlier descriptions of achylia gastrica, demonstrated that gastric juice contains an “intrinsic factor” (IF) that combines with an “extrinsic factor” in meat and allows it to be absorbed. Several decades later, the extrinsic factor, vitamin B12, was synthesized,4, 5 and its structure was demonstrated by Hodgkin,6 who was awarded a Nobel Prize for her crystallographic work.
Studies by Wills,7 who treated macrocytic anemia with yeast, and by many others defined the need for folate, which was isolated and characterized by 1948.8 The ability of nutritional folate deficiency to cause megaloblastic anemia was proven in a notable selfexperiment by Herbert.9 The stories of these and other discoveries are available in several highly readable books and articles.10, 11, 12, 13
Cure is so simple now that the once deadly diseases are considered domesticated. That gratifying development has been so successful that the potential for clinical neglect has emerged,14, 15, 16 even as major metabolic and molecular advances continue. The definitions of insufficiency have expanded,17 and accurate and accessible biochemical tools have facilitated exploration of potential impact on public health. This has not been free of controversy.
NORMAL PHYSIOLOGY AND PATHOPHYSIOLOGY
Megaloblastic anemia is, most often, a product of folate or cobalamin (vitamin B12) deficiency. These metabolically intertwined vitamins, whose disorders are sometimes difficult to differentiate, require very distinct diagnostic and therapeutic approaches. The two are discussed separately throughout this chapter, which signals the need to always differentiate between them.
Biochemistry and Metabolism
Folate
Folate, or pteroylglutamate, consists of a pteridine ring, paraminobenzoate, and one or more glutamate side chains (Fig. 36.1). Folates vary in their reduction states, one-carbon moieties, and glutamation states. Polyglutamated folates, with three or more γ-carboxyl-linked glutamate residues, predominate intracellularly; they are better retained in cells than monoglutamated forms, which traverse membranes more easily.18 Polyglutamated forms are also more effective participants in enzymatic reactions, although N5-methyltetrahydrofolate (methylTHF), the major folate in the body, tends to be a poor substrate for polyglutamation.
Folic acid is pharmacologically stable but must be reduced to be active. Folates reduced to tetrahydrofolate (THF) at positions N5 through N8 (Fig. 36.1) function as one-carbon group donors, acceptors, or modifiers. THFs with varying one-carbon moieties at the N5, N10, or both positions, attach to enzymes and serve as co-substrates in cytoplasmic or mitochondrial one-carbon group transfers involving amino acids (Fig. 36.2, reactions 4, 5, and 7),
purines (reactions 10 and 11), and pyrimidines (reaction 2). By indirectly contributing to S-adenosylmethionine (AdoMet) generation, folate is linked to important methylations, including methylation of DNA cytosine (Fig. 36.2, reaction 13). Cellular folate metabolism and its compartments are reviewed elsewhere.18, 19
purines (reactions 10 and 11), and pyrimidines (reaction 2). By indirectly contributing to S-adenosylmethionine (AdoMet) generation, folate is linked to important methylations, including methylation of DNA cytosine (Fig. 36.2, reaction 13). Cellular folate metabolism and its compartments are reviewed elsewhere.18, 19
 FIGURE 36.1. Folate structure. The constituents are, from left to right, pteridine and p-aminobenzoic acid (PABA), which constitute the pteroyl moiety, and one or more glutamates that are attached by γ-carboxyl linkage (in this diagram, three glutamates are linked). Metabolic activity requires reduction to tetrahydrofolate at positions 5, 6, 7, and 8. Various one-carbon moieties are attached to the nitrogen at position 5 (N5-methyl, N5-formyl, or N5-formimino) or 10 (N10-formyl), or bridging 5 and 10 (N5,10-methylene or N5,10-methenyl). Each folate participates in specific reactions by transferring, accepting, or transforming its one-carbon moiety. |
 FIGURE 36.2. Folate metabolism and its linkage with cobalamin (reaction 4) to homocysteine and methionine metabolism (right). Vitamin co-factors are highlighted in boxes. Not all bidirectional reactions are noted as such, but the unidirectional nature of reaction 3 is the basis for the methyltetrahydrofolate trap hypothesis. The enzymes for the reactions include the following: (1) dihydrofolate (DHF) reductase; (2) thymidylate synthase; (3) methylene tetrahydrofolate (THF) reductase; (4) methionine synthase (maintained by a reductive system mediated by methionine synthase reductase); (5) serine hydroxymethyltransferase; (6) two sequential reactions mediated by two enzymatic activities of a single protein (methyleneTHF dehydrogenase and methenylTHF cyclohydrolase); (7) glutamate formiminotransferase; (8) formiminoTHF cyclodeaminase; (9) methenylTHF synthase; (10) glycinamide ribonucleotide (GAR) transformylase; (11) 5-aminoimidazole-4carboxamide ribonucleotide (AICAR) transformylase; (12) methionine adenosyltransferase; (13) diverse S-adenosylmethionine (AdoMet)-dependent methyltransferases; (14) S-adenosylhomocysteine (AdoHcy) hydrolase (kinetics usually favor synthesis of AdoHcy); (15) betaine:homocysteine methyltransferase; (16) cystathionine β-synthase;(17) cystathionine γ-lyase. dTMP, deoxythymidine monophosphate; dUMP, deoxyuridine monophosphate; FAD, flavin adenine dinucleotide (riboflavin); FAICAR, formylAICAR; FGAR, formylGAR; FIGLU, formiminoglutamic acid; Glu, glutamic acid; MeCbl, methylcobalamin; PLP, pyridoxal 5′-phosphate. |
Cobalamin
Cobalamin, a 1,355-Da member of the corrin family, is an organometallic co-factor. Four nitrogens of its planar tetrapyrrole anchor a central cobalt atom linked with a nucleotide (5,6-dimethylbenzimidazole) below and a variable prosthetic moiety above the pyrrole plane (Fig. 36.3). Specific cobalamins are named for their prosthetic moiety: methylcobalamin, which predominates in plasma and cytoplasm, and 5′-deoxyadenosylcobalamin, which predominates in mitochondria. Vitamin B12, used popularly as a synonym for cobalamin, was the name given to cyanocobalamin,4 a stable pharmacologic preparation that must be converted to other forms for utilization. Noncobalamin corrinoids, often with major structural changes or deletions, are ubiquitous in microbial metabolism, but cannot be used by humans. Some noncobalamin analogs are nevertheless bound avidly by transcobalamin (TC) I (also called haptocorrin)20, 21 and are found in the gut, plasma, and, much less often, tissues.22
To serve as an active co-enzyme, cobalamin requires reduction of its cobalt to the monovalent cob(I)alamin or divalent cob(II) alamin.23 Reduced cobalamin has two known roles in humans. Attached to methionine synthase, it accepts the methyl group from methylTHF as methylcobalamin and transfers it to homocysteine (Fig. 36.2, reaction 4). Cobalamin activity for this reaction is maintained by methionine synthase reductase24 and requires AdoMet. In cobalamin’s other role, reduced mitochondrial cobalamin accepts 5′-deoxyadenosyl from adenosine triphosphate and binds to methylmalonyl coenzyme A (CoA) mutase. This mutase mediates an intramolecular rearrangement of L-methylmalonyl CoA to succinyl CoA in propionate metabolism.25
Cobalamin deficiency has a major impact on folate, homocysteine, and methionine metabolism by impairing the methionine synthase reaction. Most evidence supports the concept that cobalamin deficiency thereby “traps” methylTHF, which has no alternative metabolic role or route.26, 27 Feedback mechanisms activate methyleneTHF reductase (Fig. 36.2, reaction 3) in an effort to mobilize more methylTHF but only end up diverting even more methyleneTHF from its other roles and into the trap. A paralysis of folate metabolism spreads as THF and other active folates decrease. An alternative to the trap hypothesis emphasizes that the failure of methionine synthesis ultimately leads to formate “starvation,”28 with depletion of formylTHF, methenylTHF, and methyleneTHF.
Homocysteine and Methionine
Folate and cobalamin participate intimately in homocysteine and methionine metabolism. The eventual generation of AdoMet (Fig. 36.2, reaction 12) serves several major functions. AdoMet helps regulate the metabolism of folate and disposition of homocysteine
by inhibiting both methyleneTHF reductase (reaction 3) and betaine hydroxymethyltransferase (an alternate remethylator of homocysteine [reaction 15] confined to liver and perhaps kidney and lens), and by activating cystathionine β-synthase (reaction 16).29
by inhibiting both methyleneTHF reductase (reaction 3) and betaine hydroxymethyltransferase (an alternate remethylator of homocysteine [reaction 15] confined to liver and perhaps kidney and lens), and by activating cystathionine β-synthase (reaction 16).29
 FIGURE 36.3. Cobalamin structure. Attached to the central cobalt atom of the corrin tetrapyrrole and to one of the pyrrole rings is the α-ligand, the 5,6-dimethylbenzimidazole nucleotide, extending below the corrin plane. The β-ligand (marked as X in the diagram) above the plane can be any one of several moieties, such as methyl, 5′-deoxyadenosyl, hydroxyl, or cyanide. |
Even more importantly, AdoMet is the methyl donor for most cellular methylation reactions affecting proteins, nucleic acids, histones, phospholipids, and neurotransmitters, all mediated by various methyltransferases (Fig. 36.2, reaction 13).30 DNA hypomethylation, which contributes to neoplasia, can result from AdoMet deficiency. In the methylation reactions, S-adenosylhomocysteine (AdoHcy) is formed. AdoHcy, which in excess inhibits AdoMet activity, can be hydrolyzed to homocysteine; under normal conditions, however, the direction of this reaction favors AdoHcy synthesis (reaction 14).
An alternate fate of homocysteine is its transsulfuration to cystathionine and then to cysteine; both of these steps require pyridoxal 5′-phosphate (reactions 16 and 17). This is the major catabolic pathway for homocysteine, which is cytotoxic in concentrations normal for other amino acids. The pathway also influences redox activity by generating glutathione, a major intracellular reducing agent.
Normal Folate Physiology
Nutritional Characteristics
Folate, named for green leafy vegetables, exists in nearly all classes of foods. Organ meats (e.g., liver and kidney) and yeast are particularly rich, although not frequently eaten, sources. More common sources are other meats, nuts, beans, orange juice, dairy products, grains, and cereals. However, food folate, mostly methylTHF and formylTHF, is labile. Large losses occur with storage and some cooking processes,31 especially when the water is discarded.9
Fortification of the American food supply in 1998 with 140 µg folic acid/100 g in grain and cereal products was mandated to lower the risk of neural tube defect births. Many food producers exceeded that amount, increasing the average folate intake by 190 µg/day.32 Folate levels have risen sharply, reducing the frequency of subnormal red cell folate from 37.6% to 4.5% in young women.33 Supplement use has also grown in Western societies, especially among women and the aged.34 However, overall folate intake remains poor in other countries.
Dietary recommendations35 take into account folate bioavailability, which is highest for synthetic folic acid and lower for food folate, which tends to be polyglutamated and requires conversion to monoglutamate for optimal absorption. Recommended intake is therefore expressed in dietary folate equivalents, in which 0.5 µg of folic acid supplement or 0.85 µg folic-acid-fortified food delivers the dietary equivalent of 1 µg of food folate. The recommended dietary folate equivalent intake is 400 µg daily for adults, 600 µg for pregnant women, 500 µg for lactating women, and lesser amounts scaled for age in children. Women of reproductive age are advised to take 400 µg of folic acid as an additional supplement to prevent neural tube defect births.
Physiologic Cycle of Assimilation
Folate Absorption
The absorption combines specific saturable processes that tend to predominate in the upper small intestine and nonsaturable ones that predominate in the ileum.36 Luminal binding proteins appear not to be involved. The saturable process allows absorption of all folates, usually after the polyglutamated forms are hydrolyzed to monoglutamated ones by glutamate carboxypeptidase II in the jejunal brush borders. Two transmembrane, high-affinity members of the solute carrier superfamily mediate the saturable absorption of reduced folates in the upper small intestine. The main one, the proton-coupled folate transporter, has optimal affinity for folate at the duodenal pH of 5.5.36, 37, 38 The reduced folate carrier has a higher affinity at pH 7.4 and thus has a lesser duodenal role.36 Both these carriers exist in other tissues also. Conversion of absorbed folate to methylTHF appears to occur in the intestinal cell, which then exports the methylTHF into portal blood via carrier-mediated basolateral membrane transport, but some have proposed later hepatic conversion instead.39 The liver either polyglutamates and stores the folate, secretes it for biliary enterohepatic recycling, or exports it for delivery elsewhere. Folate achieves peak levels in the blood 1 hour after ingestion.
Nonsaturable ileal absorption of folate assumes a dominant role whenever more than 200 µg of folate, which exceeds the capacity of the jejunal process, is ingested.35 Such absorption of nearly unlimited amounts of unreduced monoglutamates may explain why oral folic acid is effective even when the more specific jejunal mechanisms fail in some malabsorptive disorders.
Folate Binders and Receptors
The various specific and nonspecific folate binders are incompletely understood and may not be mandatory for cellular uptake of folate. Specific binding proteins in the blood bind only a small fraction of circulating folate. Secretions, especially milk, also contain specific binding protein whose role is unclear but may withhold folate from bacteria.
The reduced folate carrier belongs to the SLC19 family of transporters, some members of which transport thiamine, and transports reduced folates (and methotrexate) into cells by a process linked to organic anions.36 With its high activity at neutral
pH and operating at higher concentrations of folate, reduced folate carrier assumes major responsibility for folate uptake from the blood by the liver, kidney, leukocytes, placenta, and parts of the brain.
pH and operating at higher concentrations of folate, reduced folate carrier assumes major responsibility for folate uptake from the blood by the liver, kidney, leukocytes, placenta, and parts of the brain.
Folate receptor-α (FR-α or FR1) and FR-β (or FR2), are specific, partially homologous receptors in epithelial cells, placenta, hematopoietic cells, renal tubular cells, and malignant cells.36, 40 Their related genes lie on chromosome 11q13. FR-α and FR-β are covalently linked to glycosylphosphatidyl inositol anchors on cell membranes, bind and internalize methylTHF by endocytosis via clathrin-coated pits, and are recycled.41 FR-γ is confined to hematopoietic cells but lacks glycosylphosphatidyl inositol anchoring and is therefore readily secreted.42 The roles of these FR isoforms are not known, but FRα is active in renal tubular reabsorption of excreted folates. Some FR is found intracellularly, and some circulating folate-binding proteins appear to derive from FR. FRs are being explored as enhancers of drug delivery and cellular imaging because of their high expression in malignant and inflammatory cells. FR-α is also a co-factor for entry of Marburg and Ebola viruses into cells.43
Folate Transport and Cellular Utilization
Dietary folate and reabsorbed biliary folate enter the bloodstream and are rapidly cleared to tissues.44 The rapid flux probably explains why plasma folate declines within 3 weeks when intake is reduced. Plasma folate, which is mostly methylTHF, is bound nonspecifically and with low affinity to proteins such as albumin, but approximately one third circulates unbound and a tiny fraction is bound specifically by the previously discussed FR-derived binding proteins. None of the circulating proteins plays any obvious role in cell uptake of folates.
Cellular uptake of monoglutamated folate occurs via either membrane FR or reduced folate carrier, the details varying among cell types and conditions.18, 36 Passive diffusion also occurs. In the cell, folates are rapidly polyglutamated by folylpoly-γ-glutamate synthetase, which enhances cellular retention and promotes attachment to enzymes. The folate is transported by saturable mechanisms into organelles; 35% of intracellular folate is in mitochondria.
Folate Stores and Turnover
A classic depletion study in one volunteer suggests that body stores of folate can be depleted in a few months to levels that do not support normal hematopoiesis,9 but precise data are scarce. Old data estimate that normal liver folate content exceeds 7.5 mg.45 The liver concentrates much of the body’s folate and is a major site of one-carbon metabolism. Several folate pools exist, with different turnover rates ranging up to 100 days.46 Enterohepatic recycling involves as much as 100 µg of folate daily.44
Approximately 200 µg of folate is excreted fecally every day. However, that amount includes not only the endogenous losses via sloughed intestinal cells and nonreabsorbed biliary folate but also external folates, such as unabsorbed food folate and folate synthesized by intestinal bacteria.46 Approximately 1 mg of folate is filtered daily in the glomerulus but is largely reabsorbed as its folate-binding protein attaches to megalin, the multiligand receptor in proximal tubules.47 Oxidatively degraded folate is lost, primarily in urine. Daily losses thus approximate 1% to 2% of body stores, which is compatible with the folate depletion rate reported in a normal person after 2 to 3 months of dietary restriction.9
Normal Cobalamin Physiology
Nutritional Characteristics
Humans obtain their cobalamin secondhand from animals and fish, which ingested the bacteria that synthesize cobalamin, or from animal products, such as milk.45, 48 The more prodigious bacteria ingestors, such as ruminants and oysters, are rich sources. Plants do not contain cobalamin. Bioavailability varies with different foods45, 48, 49, 50 and is at least as important as content. Cereals, fortified with cobalamin, dairy products, and fish provide better cobalamin bioavailability than meat.49, 50 Cobalamin supplement use, often as part of a multivitamin, has grown.49, 51
When absorption is normal, the recommended daily requirement of 2.4 µg in adults52 provides more than 1 µg cobalamin to compensate for the approximate daily loss. In children, recommended daily intake rises from 0.4 µg in infancy to 1.8 µg in preadolescence. A survey in 1995 indicated that typical daily intake of cobalamin in the United States is 4.0 to 6.2 µg.53 European surveys suggested that metabolic sufficiency plateaus at intakes of 4.2 µg or higher, depending on the population surveyed.50, 54, 55
Physiologic Cycle of Assimilation
Intestinal absorption and tissue uptake (Fig. 36.4) are designed to internalize and concentrate available cobalamin efficiently while excluding possibly harmful nonfunctional analogs. The process is tightly regulated by a system of binding proteins and receptors.56, 57, 58 Whether food-bound or as a free supplement, cobalamin that exceeds the capacity of IF-mediated uptake is poorly absorbed,59 unlike the ready absorption of excess, free folic acid. IF mediates the absorption of more than 75% of the typically small cobalamin load in a meal but can accommodate little more than a total of 1 to 2 µg at a time.45
Cobalamin-binding Proteins
Three proteins with nonoverlapping roles bind cobalamin with high affinity56, 57, 58, 60 (Table 36.1). IF and TC II (called TC by authors who eschew TC I and TC II terminology) share moderate structural homology but their genes are located on different chromosomes.61, 62 Crystallographic analysis of TC II has defined key functional regions63 that may be shared by IF. Each protein binds cobalamin specifically and ferries it into cells by endocytosis via its specific receptor; neither protein binds nonfunctional corrinoid analogs avidly.20 IF, which originates in parietal cells although chief cells may synthesize it ectopically in patients with gastritis,64 is confined to the gastrointestinal tract. TC II is synthesized by endothelial and other cells and is found in plasma and other fluids (Table 36.1). Holo-TC II (TC II with bound cobalamin) is cleared quickly by cellular uptake; its half-life in plasma is 90 minutes.65 Plasma levels of total TC II tend to rise in inflammatory and other disorders, suggesting an acute-phase reactant character unrelated to cobalamin status,56, 66, 67 but correlation with C-reactive protein levels is poor.68 A common missense polymorphism of the TCN2 gene, C776G, displays no consistent phenotypic impact,69, 70, 71 but the less common A67G mutation was associated with lower holo-TC II without affecting metabolic cobalamin status.71
TC I (also called haptocorrin, R binder, or cobalophilin) is heavily glycosylated.56 Its carbohydrate variability is prominent and affects major physicochemical characteristics.56, 72 Some carbohydrate variations led to ill-advised isoelectric point demarcation of a “TC III” subset of TC I, but others may substantively alter TC I clearance from plasma. TC I is synthesized in the specific granules of late myeloid precursors73 and in exocrine gland epithelium.56 TC I has only 33% to 36% homology to IF and TC II.62, 74 It also differs from them in two important ways: it binds nonfunctional corrinoid analogs as avidly as cobalamin, and it lacks specific receptors for cellular delivery. As a result, plasma TC I exists largely saturated with cobalamin (i.e., holo-TC I) and has a halflife of 9 to 10 days.65 Thus, TC I carries more than 70% of cobalamin75, 76 and virtually 100% of analogs in plasma,20, 77 whose concentrations are nearly equal. Only 10% to 30% of plasma cobalamin is holo-TC II at any moment, which greatly underestimates the heavy but rapidly turning over holo-TC II traffic.
Holoprotein proportions can vary widely in some disorders, and the reason is not always known.76, 78 Desialylated TC I is cleared nonspecifically by hepatic receptors for asialoglycoproteins,79 whereby some of its cobalamin is cleared in bile.
Holoprotein proportions can vary widely in some disorders, and the reason is not always known.76, 78 Desialylated TC I is cleared nonspecifically by hepatic receptors for asialoglycoproteins,79 whereby some of its cobalamin is cleared in bile.
 FIGURE 36.4. Assimilation and utilization of cobalamin. ado-cbl, 5′-deoxyadenosylcobalamin; IF, intrinsic factor; methyl-cbl, methylcobalamin; TC I (HC), transcobalamin I (haptocorrin); TC II, transcobalamin II. (Modified from Carmel R. Cobalamin deficiency. In Carmel R, Jacobsen DW, eds. Homocysteine in health and disease. Cambridge, MA: Cambridge University Press, 2001:289-305, with permission.) |
TC I concentrations in all secretions exceed plasma levels manyfold.56 TC I carries nearly all the cobalamin in breast milk, salivary TC I is the first protein to bind ingested cobalamin, and biliary TC I is involved in enterohepatic recycling of cobalamin. These facts suggest that much of the TC I role lies outside the bloodstream. It is plausible to suggest that TC I, whose role is considered unknown, is the primary “withholder”of excess cobalamin and, perhaps more important, all analogs that are used by
the vast human microbiome in the gut and elsewhere, as well as withholding unusable analogs from human cells.80 The subject requires careful study. Several minor cobalamin-binding proteins and complexes have also been described in plasma, but their roles are unknown.56, 79, 81, 82, 83
the vast human microbiome in the gut and elsewhere, as well as withholding unusable analogs from human cells.80 The subject requires careful study. Several minor cobalamin-binding proteins and complexes have also been described in plasma, but their roles are unknown.56, 79, 81, 82, 83
TABLE 36.1 COBALAMIN-BINDING PROTEINS | ||||||||||||||||||||||||||||||||||||||||||||
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Cobalamin Absorption
Cobalamin is released from food by pepsin at an acid pH in the stomach and binds preferentially to salivary TC I rather than IF at this pH (Fig. 36.4, panel 1). Pancreatic secretions entering the duodenum neutralize the pH and provide proteases to degrade TC I.84 The released cobalamin, including biliary cobalamin,21 thus becomes available to IF (Fig. 36.4, panel 2).
The IF-cobalamin complex eventually attaches to the cubilin receptor for IF in the ileum (Fig. 36.4, panel 3). Cubilin is a 460-kDa glycoprotein whose 27 CUB domains provide binding sites for many other ligands, such as vitamin-D-binding protein, transferrin, immunoglobulin light chain, albumin, and apolipoprotein A1;47 it also exists in yolk sac, renal proximal tubules, and elsewhere. Cubilin lacks a transmembrane domain but is anchored by amnionless, a 45-kDa transmembrane protein that provides cell signaling for the “cubam” receptor complex.85, 86, 87 After endocytosis, the cubilin-IF-cobalamin complex is split, and cobalamin exits the ileal cell into the bloodstream several hours after its oral ingestion. Whether the exit is mediated by TC II or multidrug resistance protein-188 is unclear.
Plasma Transport and Cellular Utilization
Some of the portal blood holo-TC II is internalized by hepatocytes via TC II receptors.21 The remainder finds its way to other tissues for calcium-dependent, TC II receptor-mediated cellular endocytosis89, 90 (Fig. 36.4, panel 4). After lysosomal degradation of the TC II, its cobalamin is released for attachment to cytoplasmic methionine synthase and conversion to methylcobalamin and to mitochondria for conversion to adenosylcobalamin.
Specific uptake of holo-TC II also occurs in yolk sac, gut, and especially in the proximal renal tubule by a second receptor, megalin, a 600-kDa glycoprotein member of the low-density lipoprotein receptor family.47 As is cubilin, with which it sometimes co-localizes, megalin is a multiligand receptor, but it has a considerably greater clientele of ligands; these include additional vitamin-binding proteins (such as folate-binding protein and retinol-binding protein), several lipoproteins, carrier proteins (such as albumin, lactoferrin, and transthyretin), hormones, enzymes, hemoglobin, and myoglobin. Most information on megalin derives from studies in the proximal renal tubule, which is rich in the protein, and in knockout mice. Megalin prevents the urinary loss of many filtered proteins, and virtually completely reabsorbs the 1.5-µg cobalamin load in the holo-TC II that is filtered by the glomerulus daily.
Body Stores and Turnover
Cobalamin stores exceed daily requirement more than 1,000-fold. This contrast with the much smaller ratio for folate explains why cobalamin depletion takes years whereas folate depletion takes weeks to months. The total body content of cobalamin approximates 2.5 mg (2,500 µg) in adults91 and only approximately 1 µg is lost daily. Some of the loss occurs by biliary excretion of 1.4 µg daily, but two thirds of that is reabsorbed via the IF mechanism.21 If the IF mechanism fails, as in pernicious anemia, biliary reabsorption ceases. Urinary loss of cobalamin is minor because of reabsorption by megalin (however, if plasma cobalamin exceeds TC II capacity, as happens after cobalamin injection, the free cobalamin escapes renal reabsorption).
CLINICAL AND LABORATORY FEATURES
The hematologic consequences are identical in folate and cobalamin deficiency. So are many nonhematologic manifestations, with the notable exception of neurologic dysfunction. Some clinical variations reflect the ways in which the two deficiencies arise. Folate deficiency typically evolves rapidly, and it is often
associated with broad malnutrition and with alcohol abuse. In contrast, the evolution of cobalamin deficiency is usually measured in years, and it tends to be a purer deficiency state because malabsorption is often restricted to cobalamin alone.
associated with broad malnutrition and with alcohol abuse. In contrast, the evolution of cobalamin deficiency is usually measured in years, and it tends to be a purer deficiency state because malabsorption is often restricted to cobalamin alone.
Megaloblastic Anemia in Cobalamin and Folate Deficiency
Biochemistry
Deficiency of folate compromises the methylation of deoxyuridylate to deoxythymidylate (Fig. 36.2, reaction 2), and deficiency of cobalamin does so indirectly. However, impaired de novo thymidylate synthesis only partially explains megaloblastic anemia. Observations in nonanemic patients with cobalamin deficiency93, 94, 95, 96 and animal models97 show that thymidylate synthase impairment alone need not lead to anemia. Other steps contribute.98 The excess uracil from deoxyuridylate is misincorporated into DNA in place of thymine and active excision repair produces many single-strand breaks.99 When excisions coincide at opposing DNA strand sites, double-stranded breaks result, which may explain the nuclear defects of megaloblastic anemia. The end result appears to be an arrest at various stages of interphase in hematopoietic precursors.100 Even so, the details are incomplete and megaloblastic anemia is not restricted to cobalamin or folate deficiency.
Hematopathology
Megaloblastic anemia is a panmyelosis, even though its name suggests a disorder limited to red cells and erythroid hyperplasia is a prominent feature. Indeed, the immature appearance of megaloblastic nuclei and the occasionally intense myeloid proliferation in the marrow have led to a misdiagnosis of leukemia in rare cases. The morphologic hallmark is nuclear-cytoplasmic dissociation, which is best appreciated in precursor cells in the bone marrow aspirate (Fig. 36.5). Megaloblastic nuclei are larger than normoblastic nuclei, and their chromatin appears abnormally dispersed due to its retarded condensation. Random chromosomal abnormalities are seen, including centromere spreading,101 but nonrandom changes may also occur.102, 103 Cytoplasmic maturation appears unremarkable.
Giant band cells and metamyelocytes with large and often misshapen nuclei are typical. Neutrophils with characteristic hypersegmented nuclei (Fig. 36.6) appear in the blood early in the course,9, 104 but they do not arise directly from the giant metamyelocytes.98 The mechanism of hypersegmentation and why it persists in the blood for more than a week after therapy105 are unknown. As megaloblastic anemia worsens, neutropenia and thrombocytopenia develop. These can be severe in advanced cases but are uncommon when anemia is mild.45 Platelets are often functionally impaired,106 although megakaryocytes do not show definable morphologic changes. The same may apply to lymphocytes.107
 FIGURE 36.5. Normal and megaloblastic precursor cells in the bone marrow. A: Pronormoblast. B: Megaloblastic equivalent of cell in plate A. C: Late normoblast. D: Megaloblastic equivalent of cell in plate C. (From Lee RG, Foerster J, Lukens J, et al., eds. Wintrobe’s clinical hematology, 10th ed. Philadelphia: Lippincott Williams & Wilkins, 1999:913, with permission.) |
Erythroid macrocytosis (Fig. 36.6) is an early change. Individual macrocytes appear first, as detectable by hemoglobin and cell size measurements confined to reticulocytes,108 followed by a gradual rise in overall mean corpuscular hemoglobin (MCH) and then mean corpuscular volume (MCV) that eventually crosses the line into abnormality (>97 fl) before the hemoglobin levels fall.9 In cobalamin deficiency, with its slow progression, macrocytosis precedes anemia by months.14, 15 Macro-ovalocytes are especially characteristic of megaloblastic anemia but are not specific.109 Early megaloblastic changes in the bone marrow precede the macrocytosis but are easily missed. Eventually, poikilocytosis becomes more pronounced with teardrop cells. Nucleated red cells, Howell-Jolly bodies, and even Cabot rings appear in the blood in severe megaloblastosis.
As anemia progresses, iron and transferrin receptor levels, sideroblast counts, and the ferritin content of erythroid precursors and macrophages increase.45, 110, 111 Erythropoietin levels correlate with the severity of anemia but can vary widely.112
Megaloblastic anemia is the chief exemplar of ineffective hematopoiesis in all three hematopoietic cell lines; bone marrow hyperplasia is intense but reticulocytosis does not occur.45, 98, 100 Precursor cells are arrested at various stages in interphase but continue to mature. As megaloblastosis advances, most precursors die within the hypercellular bone marrow and are phagocytosed. Whether early cell death is primarily apoptotic or not is controversial and may depend on the model studied.100, 113, 114 Advanced megaloblastic anemia has a poorly understood component of intravascular hemolysis also; survival of normal red cells transfused into cobalamin-deficient patients is short.115 Serum glutathione, an antioxidant, appears to be the most significant metabolic predictor of anemia in cobalamin deficiency.116 Abnormalities of red cell membrane proteins, including spectrin, have also been described.117
The evolution of hematologic changes has been detailed elsewhere45 and its laboratory characteristics are detailed further in the section “Hematologic Assessment.” With progressive anemia also come fatigue, hypervolemia, and cardiovascular symptoms, as well as the pallor combined with hyperbilirubinemia that gives a classic lemon yellow skin color, and even retinal hemorrhages and, on occasion, pseudotumor cerebri.
Neurologic Dysfunction
Cobalamin Deficiency
Pathophysiology
Demyelination with subsequent axonal disruption and gliosis can affect all parts of the central nervous system. Peripheral nerves, however, tend to show axonal degeneration without
demyelination.118, 119 The classic myelopathic syndrome is subacute combined degeneration, in which posterior and lateral column damage predominates; dorsal, pyramidal, and spinocerebellar tracts are affected. The earliest changes appear in the cervical or thoracic spine and can be detected by magnetic resonance imaging (MRI) as hyperintensity on T2-weighted images (Fig. 36.7). Larger, more heavily myelinated fibers tend to be affected most often.120
demyelination.118, 119 The classic myelopathic syndrome is subacute combined degeneration, in which posterior and lateral column damage predominates; dorsal, pyramidal, and spinocerebellar tracts are affected. The earliest changes appear in the cervical or thoracic spine and can be detected by magnetic resonance imaging (MRI) as hyperintensity on T2-weighted images (Fig. 36.7). Larger, more heavily myelinated fibers tend to be affected most often.120
 FIGURE 36.6. Blood smear from a patient with megaloblastic anemia due to cobalamin deficiency. Note characteristic large macro-ovalocytes (A) and hypersegmented neutrophils (B). (Courtesy of Irma Pereira MT [ASCP]SH.) |
The biochemical mechanisms are unknown. Because myeloneuropathy is much more prominent in cobalamin deficiency than in folate deficiency, suspicion fell on the folate-unrelated role of adenosylcobalamin in propionate metabolism and its possible consequences to fatty acids in myelin. Most evidence now points to methionine synthase impairment and its effects on methionine metabolism and methylations instead.121 Animal data suggested that methionine ameliorates cobalamin deficiency-induced neurologic dysfunction, preliminary cerebrospinal fluid studies in humans described low AdoMet levels, and high AdoHcy-to-AdoMet ratios suggested inhibition of AdoMet’s methylation activity by AdoHcy. Plasma homocysteine-related metabolite comparisons in neurologically impaired versus hematologically impaired patients with pernicious anemia suggest even greater complexity:116 cysteine levels were significantly higher in the neurologically affected patients and, surprisingly, AdoMet levels were actually higher in neurologically impaired patients. Neurologically impaired patients also had significantly higher serum folate levels than those without neurologic problems.116, 122, 123 It is unclear if these differences represent cause, concurrence, or consequence.
 FIGURE 36.7. Magnetic resonance imaging (T2 weighted) of a sagittal section of the cervical spine of a man with pernicious anemia and severe myelopathy. Note the posterior localization of the high signal intensity lesion (arrow). (From Larner AJ, Zeman AZ, Allen CMC, et al. MRI appearances in subacute combined degeneration of the spinal cord due to vitamin B12 deficiency. J Neurol Neurosurg Psychiatry 1997;62:99-101, with permission.) |
Clinical Features
The frequency of neurologic involvement in cobalamin deficiency is presumed lower than the frequency of anemia but is undefined (perhaps because it is not as explicit and quantifiable as anemia and because of patient selection biases). Often regarded as a late development, neurologic changes can precede anemia instead.124, 125, 126 Interestingly, the extents of neurologic and hematologic expressions of cobalamin deficiency tend to vary inversely in patients,116, 126 and the predilection tends to recur when deficiency relapses.126, 127, 128 Although genetic influences on clinical expression seem plausible, methyleneTHF reductase polymorphisms that increase folate availability for thymidylate synthase over methionine synthase did not predispose to neurologic manifestations in pernicious anemia.129
Neuromyelopathy is the most common neurologic feature of cobalamin deficiency,120, 126, 130, 131 but it is not specific to it; copper deficiency, for example, produces similar findings,132 as well as macrocytosis.133 Sensory changes in cobalamin deficiency include position sense disturbance and dysesthesia; pyramidal tract signs include spasticity and a Babinski reflex; neuropathy is exemplified by loss of tendon reflexes; and gait disturbances are common signs of advanced involvement. Manifestations tend to be symmetrical. Neuropathy, which is usually sensory but can be sensorimotor,134 can be hard to differentiate clinically from posterior column involvement.126, 130, 135, 136
The earliest manifestations are loss of vibratory sense in the feet and numbness, tingling, and loss of fine sensation. Others include loss of proprioception and, depending on the balance between myelopathy and neuropathy, hyperactive or diminished deep tendon reflexes. Involvement ascends up the legs, and, eventually, hands are affected as well. Muscle weakness and tenderness have been described sometimes. Ataxia, spasticity, gait disturbances, positive Babinski reflex, impotence, and bladder137 and bowel dysfunction appear in advanced cases.
Cerebral symptoms, notably cognitive and emotional changes, can be severe in some cases124, 126 or so mild as to be recognized
only in retrospect after treatment. MRI reveals focal and diffuse changes in the brain138 (Fig. 36.8). Tensor diffusion imaging also shows white matter changes in adjacent areas that seem normal on MRI.139 Nevertheless, despite low cobalamin levels in 10% to 20% of patients with chronic dementias,95, 140 the low levels usually reflect only subclinical cobalamin deficiency (SCCD) and appear unrelated to chronic dementias, which rarely improve with cobalamin therapy.141 In very young children, however, some cobalamin deficiencies can cause developmental delay, lethargy, cerebral atrophy, and seizures.142
only in retrospect after treatment. MRI reveals focal and diffuse changes in the brain138 (Fig. 36.8). Tensor diffusion imaging also shows white matter changes in adjacent areas that seem normal on MRI.139 Nevertheless, despite low cobalamin levels in 10% to 20% of patients with chronic dementias,95, 140 the low levels usually reflect only subclinical cobalamin deficiency (SCCD) and appear unrelated to chronic dementias, which rarely improve with cobalamin therapy.141 In very young children, however, some cobalamin deficiencies can cause developmental delay, lethargy, cerebral atrophy, and seizures.142
 FIGURE 36.8. Magnetic resonance imaging (T2 weighted) of the brain in a woman with pernicious anemia and cognitive dysfunction. Large confluent and focal areas of increased signal intensities are seen, predominating around the ventricles. The changes improved after therapy. (From Stojsavljevic N, Levic Z, Drulovic J, et al. A 44-month clinical-brain MRI follow-up in a patient with B12 deficiency. Neurology 1997;49:878-881, with permission.) |
Autonomic dysfunction is sometimes demonstrable with deficiency.118, 143, 144, 145 Other neurologic manifestations include visual changes, optic neuritis, which predominates in men, and disturbances of smell or taste.126, 146 Classic motor dysfunction is rare, other than that caused by spasticity and proprioceptive loss, but abnormal central motor conduction times have been described.134 At early or late stages, MRI (Figs. 36.7 and 36.8) and functional electrophysiologic tests, such as electroencephalography, evoked potentials, and nerve conduction, show abnormalities.134, 138, 147, 148, 149, 150, 151
Unlike anemia, neurologic dysfunction does not always reverse after cobalamin therapy. Residual deficits persist in 6% of patients;126 they are not always predictable but tend to accompany more extensive involvement and longer duration before treatment begins.45, 130 Mistaken folate therapy has also been tied to the risk of neurologic irreversibility in patients with clinical cobalamin deficiency. The delayed cobalamin treatment when the anemia responds to folate presumably allows neurologic abnormalities to appear, progress, and, in occasional reports,152 perhaps even accelerate. The events are more ambiguous than assumed. Hematologic improvement after folate is usually neither complete nor long lived in pernicious anemia,152, 153 and transient neurologic responses to folate may occur too.153 Nevertheless, the anecdotal reports of clinical acceleration cannot be completely dismissed and cannot be studied prospectively in pernicious anemia. Folate should never be given alone to cobalamin-deficient patients, even those whose deficiency is subclinical and lacks convincing evidence for adverse effects.154
Folate Deficiency
Neurologic defects occur in only occasional adults with folate deficiency155, 156, 157 and are rarely as severe as in cobalamin deficiency. Although myelopathy has been reported,158 it is exceptional. Mild mental changes, such as forgetfulness and irritability,9 and mild neuropathy have been better accepted. A causative association with depression is controversial.159, 160 Attributions of any neurologic changes to folate deficiency, which often occurs in a setting of malnutrition, require high standards of proof, including consideration of alternative explanations such as alcohol abuse.
In sharp contrast, however, children with inborn errors of folate metabolism often have severe myelopathy and brain dysfunction, including seizures and mental retardation.142, 161, 162 The explanations for the neurologic differences between folate and cobalamin deficiency or between acquired and hereditary folate disorders are unknown.
Other Manifestations
Miscellaneous Clinical Findings
Atrophy of tongue papillae is common with cobalamin deficiency and sometimes gives rise to a beefy red tongue that may or may not cause symptoms.45, 163 Aphthous stomatitis and oral soreness can be prominent in some patients, including some without anemia. However, glossitis occurs with folate deficiency and other deficiencies also. The glossitis of cobalamin deficiency does not respond to folate.164 Mucosal changes occur elsewhere too, including megaloblastic changes in buccal epithelium, cervical cells, and intestinal villi;45, 165, 166 the latter apparently caused temporary malabsorption of cobalamin and other nutrients.167, 168 Other reversible but unexplained clinical changes in pernicious anemia include: impaired osteoblastic activity,169 although reports of bone density changes and risk of fractures have been inconsistent; darkening of nails and skin and change of hair color in severely deficient non-Caucasians;170, 171, 172 and sometimes marked weight loss.173
Miscellaneous Laboratory Abnormalities
Platelet function is sometimes impaired,106 but neutrophil dysfunction has been variable. Hemoglobin A2 levels rise slightly.174 Occasional patients with sickle cell trait have shown striking changes in hemoglobin S and F levels when folate-deficient.175 Cobalamin deficiency also affects some nonhematologic serum analytes: bone alkaline phosphatase is often decreased169 and immunoglobulin levels sometimes decline.176 An adolescent with severe megaloblastic anemia had reduced ADAMTS13 activity.177
Subclinical Deficiency and Indirect Consequences of Altered Vitamin Status
Automated metabolic tests have simplified identification of asymptomatic, nonanemic vitamin deficiency. The high frequency of these early subclinical states has opened them to scrutiny as possible public health issues, with varying outcomes. The uncertainties apply especially to cobalamin, because SCCD is widespread, dominates epidemiologic surveys, and is often confused with clinical deficiency, yet has unclear health implications and, therefore, unknown need for intervention.17, 178, 179
Subclinical Cobalamin Deficiency
Many patients and healthy people—especially, but not exclusively, the elderly—have low cobalamin levels but are asymptomatic and have normal blood counts. These low serum levels were thought artifactual until sensitive deoxyuridine suppression tests
showed in 1985 that most of them reflected mild biochemical insufficiency that responded to cobalamin and, equally important, rarely involved IF-related malabsorption.94, 95, 96, 180 None of the subjects had cobalamin-related anemia although bone marrow cells displayed reversible metabolic defects. Similarly, none had clinical neurologic findings although some,96, 141, 181 but not all,182 displayed mild, reversible electrophysiologic changes.
showed in 1985 that most of them reflected mild biochemical insufficiency that responded to cobalamin and, equally important, rarely involved IF-related malabsorption.94, 95, 96, 180 None of the subjects had cobalamin-related anemia although bone marrow cells displayed reversible metabolic defects. Similarly, none had clinical neurologic findings although some,96, 141, 181 but not all,182 displayed mild, reversible electrophysiologic changes.
Widespread methylmalonic acid (MMA) and homocysteine testing183, 184, 185 next showed that asymptomatic nonanemic SCCD was much more common than clinical deficiency.17, 186, 187, 188 Thus, a survey of community-dwelling elderly subjects found that 12% had biochemical insufficiency188 but only 2% of them had IF antibody-positive pernicious anemia189 and, by extrapolation, 1% to 2% more may have had undetected antibody-negative disease. Readers must be aware that epidemiologic data apply to SCCD alone because too few subjects have clinical deficiency.190 Moreover, falsely low cobalamin levels (i.e., with normal MMA and homocysteine) also outnumber clinical deficiency in surveys.191 Nevertheless, the mild metabolic abnormalities in some asymptomatic subjects with low-normal cobalamin levels led many laboratories to raise the cutpoint for “suspicious” cobalamin levels from 200 to 300 or 350 ng/L to capture more cases.186 The change is open to criticism because nearly 70% of cases added thereby have normal MMA and homocysteine levels and are not cobalamin-deficient. The influx of suspected SCCD expanded the concept of cobalamin deficiency at the cost of overdiagnosis based on often nonspecific biochemical findings whose need for therapy was unclear.17, 179, 190, 192, 193
Assumptions that SCCD, especially when unaccompanied by malabsorption, progresses inevitably to clinical deficiency are untested.179, 190 Asymptomatic persons without malabsorption have been free of symptoms and anemia for 10 years despite persistently low cobalamin levels,194 and metabolic monitoring over 1 to 4 years showed infrequent progression of mildly elevated MMA levels.192 Only a small minority of people with SCCD have early pernicious anemia,189 whose progression to clinical deficiency is very predictable. Most persons with SCCD have no identifiable cause for it,17, 178 and the likelihood of progression in the 30% to 40% who have food-cobalamin malabsorption appears limited.195
TABLE 36.2 COMPARISON BETWEEN CLINICAL AND SUBCLINICAL COBALAMIN DEFICIENCY STATES | ||||||||||||||||||||||||||||||||||||||||||||||||
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SCCD is compared and contrasted with clinical cobalamin deficiency in Table 36.2.
Subclinical Folate Deficiency
Subclinical folate deficiency is less explicitly defined than SCCD for several reasons: folate status fluctuates more readily than cobalamin status because folate turnover is rapid;35 folate deficiency tends to be accompanied by other deficiencies; and attention is often diverted from the deficiency to its hyperhomocysteinemia. The latter, and growing pharmacogenetic and gene-nutrient data, suggest that subclinical variations of folate status below the level of deficiency may subtly influence many health issues, which are discussed next.
Vitamins and Public Health Risks
Subclinical Cobalamin Deficiency and Cognition
Many epidemiologic associations with SCCD have been pursued but clinical trials have been infrequent. Cognitive decline in the elderly has undergone the most active study because neurologic manifestations are frequent in clinical deficiency. The most consistent associations for cognitive dysfunction have been with homocysteine status, with variable links to folate and, to often lesser extent, cobalamin status. As reviewed elsewhere,179, 196, 197 early clinical trials were inadequate or inconclusive.198, 199, 200, 201, 202 However, two long-term randomized clinical trials in Europe, where dietary fortification is not mandatory, reported reduced progression of brain atrophy and cognitive decline. One found that 0.8 mg of folic acid daily was effective.203 The second demonstrated that three vitamins (0.8 mg folic acid, 500 µg cobalamin,
and 25 mg pyridoxine) slowed brain atrophy by 30%204 and reduced cognitive decline.205 Its important features were that the subjects were mildly cognitively impaired at baseline (which predicts likely progression, but avoids the irreversibility of dementia), the responders were hyperhomocysteinemic, and it is not clear which of the three vitamins was the beneficial one.
and 25 mg pyridoxine) slowed brain atrophy by 30%204 and reduced cognitive decline.205 Its important features were that the subjects were mildly cognitively impaired at baseline (which predicts likely progression, but avoids the irreversibility of dementia), the responders were hyperhomocysteinemic, and it is not clear which of the three vitamins was the beneficial one.
Folate and Neural Tube Defect Prevention
Supplementation with folic acid has also been scrutinized for prevention of conditions not directly attributable to folate deficiency. Of the many studied targets and associations, such as cancer, vascular and thrombotic disease, osteoporosis, cognitive dysfunction, and birth defects, the only unambiguous effect has been on neural tube defect (NTD). Folate supplementation reduces a woman’s risk of having a child with NTD,206, 207 but the benefit seems pharmacologic rather than amelioration of folate inadequacy. Genetic and other susceptibilities related to folate, cobalamin, or homocysteine probably underlie the NTD risk, more so than does maternal folate deficiency. For example, the MTHFR C677T polymorphism increases the risk of NTD.208 High rates of antibody to FR have also been reported in women with NTD pregnancies209 and deficiency of FR-α in mice is associated with neural tube and other birth defects.210
Folate prophylaxis must begin at conception because the neural tube closes at approximately 3 weeks. Daily doses of 400 µg are effective, with 4 mg recommended for women who use anticonvulsants or have a history of previous NTD. All grain and cereal foods have been fortified with folic acid since 1998 in the United States, Canada, and a few other countries. NTD rates have fallen but not disappeared, leading some experts to urge additional fortification.211 The benefits must be weighed against the consequences of chronic use of high folic acid doses, as discussed in the section “Preventive and Other Uses of Supplements.”
Considerable human and animal data associated lower folate status with increased risk of cancer, best demonstrated with colon cancer. However, the associations are complex, may include double-edged folate impact that depends on neoplastic status and details as much as on folate status, and are beyond the scope of this chapter. These issues are discussed in the section “Preventive and Other Uses of Supplements” at the end of the chapter.
Hyperhomocysteinemia
Extrapolation from inborn errors of homocysteine metabolism suggested that severe hyperhomocysteinemia may predispose to thrombotic manifestations.212 Extensive epidemiologic data associated even mild hyperhomocysteinemia with increased risks for coronary, cerebral, and peripheral vascular complications, although the associations were less firm in prospective, rigorously designed studies than in retrospective ones.213, 214 Genetic polymorphisms, primarily of methyleneTHF reductase, contribute to 9% of homocysteine level variation,215 but even in the absence of vitamin deficiency folate, cobalamin, and vitamin B6 often reverse the hyperhomocysteinemia. Indeed, folic acid fortification reduced the frequency of hyperhomocysteinemia from 20% to 32% to 5% to 14% in the elderly.216 Nevertheless, the outcomes of large interventional vitamin trials on coronary and cerebrovascular disease prevention have been disappointing.217 Indeed, some trials even suggested adverse effects.218, 219, 220, 221, 222
Laboratory Evaluation
Laboratory evaluation must have two separate targets: documenting hematologic, metabolic, and clinical chemistry changes that identify cobalamin or folate deficiency, and documenting the underlying condition or disease that caused the deficiency.
Hematologic Assessment
The mechanisms of megaloblastosis were discussed in the section “Hematopathology.” The classic blood count findings in cobalamin or folate deficiency are anemia, a high MCV and MCH, and, in more advanced cases, thrombocytopenia and neutropenia. Patients are often identifiable at an early stage in which they have MCV and MCH elevation alone, which precedes anemia by months in cobalamin deficiency15 but only by a few weeks in the more rapidly developing folate deficiency.9 At first, the MCV and MCH may simply be higher than the patient’s baseline, without being explicitly abnormal (e.g., an MCV of 90 fl replacing one of 85 fl). An early anisocytosis as new macrocytes begin to emerge can be detected by measuring the MCV or hemoglobin content of reticulocytes108 or by an elevated red cell distribution width, which, however, is not invariable in early deficiency.223 Red cell counts decline before hemoglobin and packed cell volume levels.
Megaloblastic anemia is not the only cause of macrocytic anemia, however, or even the most common (Table 36.3). A hospital survey found 64% of MCV values >100 fl to be due to chemotherapy, antiretroviral therapy, or alcohol abuse.109 Cobalamin and folate deficiencies caused only 6% of the high MCV values but accounted for most MCV values above 110 fl.
Absolute reticulocyte counts typically fall slightly. The laboratory signs of ineffective erythropoiesis, serum lactate dehydrogenase and indirect bilirubin levels, are initially inapparent but rise as the hemoglobin approaches 10 g/dl.45 As anemia worsens, lactate dehydrogenase levels may reach thousands of units per liter as intravascular hemolysis is added to ineffective erythropoiesis. Other markers include rising serum transferrin receptor, iron, ferritin, nontransferrin-bound iron, and methemalbumin levels, as well as low serum haptoglobin levels. Platelet and neutrophil counts usually decline only as the anemia progresses. The pancytopenia can ultimately mimic aplastic anemia, which too is usually macrocytic (but does not display the bilirubin and lactate dehydrogenase elevations).
Most of the hematologic variability is dictated by the stage at which the patient is discovered.45 However, not every patient expresses the same degree of anemia for the degree of cobalamin deficiency. Many severely deficient patients have surprisingly mild anemia or even lack it.125 The reasons are usually unknown but some may be linked to the unexplained tendency for inverse association between anemia and neurologic dysfunction.124, 125 Another influence on hematologic expression is coexisting iron deficiency or thalassemia that produces normal or low MCV in approximately 7% of patients with pernicious anemia;224 these MCVs are above baseline for the patients and fall after vitamin therapy.225 Iron deficiency sometimes also blunts the erythroid megaloblastic changes themselves, both morphologically and by deoxyuridine suppression testing.226, 227 Iron studies in untreated megaloblastic anemia often do not reveal the coexisting iron deficiency.45 Because all marrow and blood indicators of iron status fall within 24 to 48 hours of vitamin therapy, sometimes transiently to low levels before rebounding, it is advisable to wait several days for the tests to stabilize and reveal the patient’s true iron status.
Hypersegmentation of neutrophil nuclei is a constant feature but is variably defined45 and may be unreliable in inexpert hands. The most serviceable criteria are finding one or more neutrophils with six or more nuclear lobes or showing that at least 4% to 5% of neutrophils have five lobes. Calculating lobe averages is considered the gold standard but comparison against published reference ranges is unreliable because interobserver variation is great. Hypersegmentation often precedes anemia,9, 104 but it is not found in subclinical deficiency.228 Hypersegmented neutrophils are not specific for cobalamin or folate deficiency; they are found in patients receiving chemotherapeutic drugs such as 5-fluorouracil or hydroxyurea, in some patients receiving steroid therapy for immune thrombocytopenic purpura,229 and in rare
patients with myelofibrosis or chronic myelogenous leukemia. It is unclear whether iron deficiency can cause hypersegmentation.230 Neutrophil segmentation is normally greater in blacks than in whites.228
patients with myelofibrosis or chronic myelogenous leukemia. It is unclear whether iron deficiency can cause hypersegmentation.230 Neutrophil segmentation is normally greater in blacks than in whites.228
TABLE 36.3 CAUSES OF MACROCYTOSIS, DEFINED AS MEAN CORPUSCULAR VOLUME (MCV) GREATER THAN 97 FLa | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Laboratory Tests of Deficiency
Cobalamin and folate levels are measurable in serum and cells. The original microbiologic methods exploited the cobalamin or folate requirements of various micro-organisms, including differential sensitivities to specific forms of folate.45, 231, 232 Although rarely used today, microbiologic assays remain the gold standard. Radioisotope dilution competitive-binding assays for cobalamin and folate gave way to automated chemiluminescence-based competitive-binding assays a decade ago. Technical ease and demand have grown, but transparency, documentation, and validation of assay performance have diminished.233, 234 Long suspected of critical failures to identify low cobalamin levels, and sometimes producing results above 1,000 ng/L,234, 235 many automated assays have misidentified 22% to 35% of sera from untreated patients with pernicious anemia.236 The errors appear to involve failure to inactivate serum autoantibodies to IF, but at present the manufacturers’ corrections are awaited.
No single biochemical test is diagnostically definitive for either cobalamin or folate deficiency.190 In most clinical settings, the cobalamin test, when free of error, nevertheless suffices if the clinical picture is clear.193 Several metabolic tests are available to clarify the vitamin levels (Table 36.4), and can be clinically decisive in difficult cases. However, a clinical “picture” is unavailable in SCCD, and all tests lack sufficient specificity.190 Therefore, a panel evaluating cobalamin testing in epidemiologic research237 recommended that the diagnosis of SCCD should be based on finding at least two abnormalities, one metabolic (e.g., MMA or homocysteine) and one quantitating cobalamin content (e.g., serum cobalamin
or holo-TC II).190 Metabolic tests are essential in the diagnosis of inborn errors of metabolism, in which vitamin levels are often normal. Metabolites are also ideal for monitoring response of deficiency to therapy because their levels do not change unless the therapy was effective, whereas vitamin levels (serum cobalamin, holo-TC II, or folate) rise upon vitamin entry into the bloodstream regardless of efficacy. Metabolite improvement is also delayed for several days, allowing a post-therapy window of time for diagnostic reassessment if needed.
or holo-TC II).190 Metabolic tests are essential in the diagnosis of inborn errors of metabolism, in which vitamin levels are often normal. Metabolites are also ideal for monitoring response of deficiency to therapy because their levels do not change unless the therapy was effective, whereas vitamin levels (serum cobalamin, holo-TC II, or folate) rise upon vitamin entry into the bloodstream regardless of efficacy. Metabolite improvement is also delayed for several days, allowing a post-therapy window of time for diagnostic reassessment if needed.
TABLE 36.4 BIOCHEMICAL TESTS FOR THE DIAGNOSIS AND DIFFERENTIATION OF CLINICALLY RELEVANT COBALAMIN AND FOLATE DEFICIENCIES | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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