Folate Polyglutamates

Most intracellular folates exist as polyglutamylated derivatives, containing up to six or seven glutamate residues attached by γ-peptide bonds. Synthesis of these folate polyglutamates depends on activity of the enzyme folylpoly-γ-glutamate synthetase (EC 6.3.2.17), which adds glutamate residues one at a time to reduced folates, using adenosine triphosphate (ATP) as an energy source. Folate polyglutamates have higher affinity for most folate-dependent enzymes than the corresponding monoglutamates and are efficiently channeled between active sites in multifunctional enzymes. They do not readily cross the cell or mitochondrial membranes, and polyglutamylation of folates within cells thus contributes to the ability of cells to retain reduced folates in the presence of low extracellular concentrations. Separation of cytosolic and mitochondrial folate polyglutamate pools has important consequences for cellular folate metabolism.

Cellular Folate Uptake

Three distinct systems for cellular uptake of folates have been identified in mammals. The reduced folate carrier (RFC) supports a low-affinity, high-capacity system for uptake of reduced folates and methotrexate (an antifolate drug) at high concentrations (in the micromolar range) with a neutral optimum pH; it is less effective in uptake of folic acid. It appears to be an important folate transporter in many types of cells, including hematopoietic cells. RFC is encoded by the SLC19A1 gene on chromosome 21q22.3. Inactivation of Slc19a1 in mice results in neonatal lethality owing to failure of erythropoiesis.

A second, high-affinity low-capacity system for folate uptake, active at nanomolar folate concentrations, is mediated by members of the folate receptor family. Several different receptors have been identified, encoded by genes on the long arm of chromosome 11 ( FOLR1, FOLR2, FOLR3, and FOLR4 ), with different forms expressed at different times in different tissues. Folate receptor-α and folate receptor-β are attached to the cell surface by a glycosylphosphatidylinositol anchor. The circulating folate-binding protein that carries some of the circulating folate is derived from folate receptor-α by cleavage from this anchor. Binding of folates to folate receptors initiates carrier-mediated endocytosis. The level of cell surface folate receptors increases greatly in cultured mouse cells grown in very low folate concentrations, suggesting that these receptors play an important role in cellular folate uptake under these conditions.

The third folate transport protein, the proton-coupled folate transporter (PCFT), has an acidic optimal pH, and transports reduced and oxidized folates with similar efficiency. It is expressed at high levels in liver, kidney, and small intestine, with lower expression in other tissues. It was originally identified as a low-affinity heme transport protein but was subsequently shown to have higher affinity for folates. PCFT is encoded by the SLC46A1 gene on chromosome 17q11.2.

Absorption of Folates from the Gut

Dietary folates are principally folate polyglutamates. These are hydrolyzed within the gut lumen or brush border, or in the lysosomes of enterocytes, by the enzyme γ-glutamylhydrolase (EC 3.4.22.12), to generate folate monoglutamates that cross cell membranes much more easily than the corresponding polyglutamates. Adequate absorption is impossible without this hydrolysis. Uptake of folate monoglutamates occurs primarily in the proximal small intestine and is mediated by PCFT. RFC is also expressed in enterocytes, but its role in this tissue remains unknown. The efficiency of absorption of folic acid, folate polyglutamates, and food folate varies in different subjects. In general, absorption of synthetic folic acid is about twice as efficient as that of folate in food whereas folic acid from folic acid–fortified cereal grain food is absorbed about 1.7 times as efficiently as food folate. The carrier-mediated process in the proximal small intestine for reduced folates is saturable, with an upper limit approximating 200 µg of food folate. The absorption of unreduced folic acid, when taken in large doses, occurs more distally by nonsaturable high-capacity mechanisms, which helps explain both the higher bioavailability of folic acid than food folates and the frequent response to oral folic acid by patients with malabsorptive disease.

Folate appears in the portal venous blood within 15 minutes of entering the stomach and reaches a maximum level in portal venous plasma about 1 hour later. Reduced folates, such as 5-formylTHF, are rapidly converted to 5-methylTHF during absorption. A second potential source of folate is that synthesized by bacteria in the colon, some of which exists as monoglutamates. There is evidence for a folate transport system, with the same properties as the small intestine system, in the colon. This may contribute to whole-body folate homeostasis, or at least support folate requirements of colon cells.

It has been estimated that up to 100 µg of folates, including folate delivered to the liver and some hepatic folate, is excreted into the bile and subsequently reabsorbed. This enterohepatic recirculation mediates a slower and more even absorption than would occur in its absence. There is evidence that enterohepatic recirculation of folates is impaired with alcohol ingestion, contributing to folate deficiency.

Transport of Folates in Plasma

Plasma folate typically consists almost entirely of 5-methylTHF, but significant amounts of folic acid may be present in individuals who have consumed folic acid supplements or food supplemented with folic acid. Most folate circulates unbound, although some may be bound to albumin and a small proportion is bound to a folate-binding protein derived from the cell surface pro­tein folate receptor-α. Plasma folate-binding protein levels are increased in pregnancy, in folate deficiency, in renal failure, with breast and ovarian cancers, and with some types of inflammation. Its function in plasma is unclear. It is present in milk, where it may function to improve the absorption of folate by the newborn.

Folate Transport Across the Blood-Brain Barrier

The concentration of folate (primarily 5-methylTHF) in the cerebrospinal fluid (CSF) is twofold to threefold higher than in blood, indicating active transport occurs across the blood-brain barrier. Both PCFT and folate receptor-α are expressed at the choroid plexus, and mutations at either SLC46A1 or FOLR1 are associated with decreased cerebral 5-methylTHF levels. It has been suggested that folate receptor-α mediates endocytosis of folates and that PCFT is required for export of endocytosed folates from acidified endosomes.

Folate in the Kidney

Folate, free and bound to folate-binding protein or albumin, is filtered at the glomerulus and reabsorbed in the proximal tubule, with the result that little folate appears in the urine. Both folate receptor-α and folate receptor-β are expressed in the brush border of the proximal tubule and are responsible for reabsorption of free folate. Reabsorption of folate bound to albumin is carried out by the large multiligand endocytic receptors megalin and cubilin, whereas folate-binding protein is reabsorbed by megalin.

Folate Transport in the Placenta

Levels of plasma folate are higher in cord than maternal blood. Perfusion of the human placental cotyledon revealed a two-step maternal-to-fetal transfer of methylTHF, with binding of folate to placental folate-binding proteins on the maternally facing chorionic surface and gradual release generating an intervillous blood folate level three times that in the maternal circulation, followed by passive transfer to the fetal circulation along a downhill concentration gradient. There is expression of folate receptor-α, PCFT, and RFC in placenta, with folate receptor-α and PCFT localizing together to the syncytiotrophoblast microvillous plasma membrane, where they may function together in the same way that they do in the choroid plexus.

Folates and Arteriosclerotic Vascular Disease

Homocysteine was first implicated as an atherogenic factor by the observation of premature arteriosclerotic disease in children with severe hyperhomocysteinemia due to inborn errors of metabolism. It was subsequently suggested that moderate elevations of tHcy were associated with development of atherosclerotic disease in adults. A common thermolabile variant of MTHFR, c.677C→T (p.A222V), results in an approximately 50% decrease in enzyme activity when present in the homozygous state and in elevated tHcy in the presence of low folate concentration. Many observational studies showed an association between elevated tHcy and cardiovascular disease and a modest, but statistically significant, increased risk for cardiovascular disease in patients with the T allele. However, suggestions that lowering tHcy might improve cardiovascular outcomes in patients have not been supported in several large epidemiologic studies.

Folates and Neural Tube Defects and Other Birth Defects

As noted previously, folate supplementation during the periconceptional period (from initiation of pregnancy to closure of the neural tube at about 1 month of gestation) reduces the occurrence and recurrence of NTDs, leading to fortification of flour and other enriched grain products with folic acid in the United States and other countries. This has been associated with a decrease in prevalence of NTDs. Studies have also suggested a role for folate supplementation in preventing nonsyndromic cleft lip with or without cleft palate and congenital heart defects, but the evidence for these associations is not as strong.

Folates and Cancer

Folate status appears to have an inverse relationship with the risk for cancers of various types. However, epidemiologic studies have also suggested an association between higher folate status and progression of premalignant lesions in several tissues, and increased folate con­centrations have been associated with growth of some malignant cells. The effects of folate have been most thoroughly investigated in colorectal cancer, where results of meta-analysis were consistent with a small protective effect of folate against risk for cancer. There is less convincing evidence for a protective role for folate against cancers of the oropharynx, esophagus, stomach, pancreas, lungs, cervix, ovary, and breast as well as for neuroblastoma and leukemia. A transient increase in the rate of colorectal cancer in the United States and Canada following the start of folic acid fortification of grains may reflect the effect of increased folate concentration on proliferation of existing adenomas, however. Meta-analyses of studies of folic acid supplementation (at levels tenfold higher than those used in folic acid fortification of grain) showed no effect on the rate of cancer.

Folate Antimetabolites

Antifolates are analogues of folic acid that block folate metabolism, interrupting DNA synthesis and causing cell death. Methotrexate, a potent inhibitor of mammalian DHF reductase, is effective in combination chemotherapy of carcinomas of the breast, lung, bone, ovary, head and neck, and elsewhere and contributes to cure of choriocarcinoma and childhood acute lymphoblastic leukemia. It is also used for its immunosuppressive and antiproliferative properties in the treatment of psoriasis, rheumatoid arthritis, and graft-versus-host disease after bone marrow transplantation. Newer antifolates have been synthesized and tested over the past 40 years. Some are discussed in Chapter 46 , “Chemotherapy in the Pediatric Cancer Patient.”

Pyrimethamine is an antimalarial drug with greater affinity for parasitic than human DHF reductase. Trimethoprim is an antifolate with antibacterial activity and a higher affinity for bacterial than human DHF reductase. In combination with sulfisoxazole, which interferes with bacterial synthesis of folic acid, there is a double synergistic antifolate attack on bacteria. Such combinations are often used to treat urinary tract and other infections and as prophylaxis against pneumonia resulting from infection with Pneumocystis jirovecii (formerly Pneumocystis carinii ) in immunocompromised patients.

Nutritional Sources and Requirements

Although prokaryotes, animals, and many algae require cobalamin for metabolism, only prokaryotes synthesize it. Humans depend on microbial cobalamin, usually obtained second-hand through consumed animal-derived products, for their cobalamin requirement. Multicellular plants or fungi do not synthesize or accumulate cobalamin and thus do not contribute it to the diet. Many prokaryotes synthesize and utilize corrinoids other than cobalamin, containing a lower axial ligand other than dimethylbenzimidazole or lacking a lower ligand entirely that cannot be utilized by humans. The human cobalamin transport system largely excludes these analogues from internalization by cells.

The estimated daily cobalamin loss is 1 µg in adults, with wide individual variations. Much of the loss is biliary. Estimates of daily intake requirement have varied, in part because of different end points for sufficiency. The Institute of Medicine reviewed available dietary data to set requirements for children of all ages, adults, and pregnant and lactating women ( Table 10-2 ). The recommended daily intake of cobalamin for adults is set at 2.4 µg/day, on the assumption that the expected 50% absorption rate of such an amount from food will replace the estimated daily losses while leaving a cushion for individual variability. A subsequent Scandinavian study of adults found that metabolic status leveled off with ascending daily intakes that approximated 4.2 µg or even 6 to 7 µg, but interpretations should be tempered by the limitations of the short-term dietary assessments for cobalamin, the biomarker end points, and the study populations (e.g., a striking overrepresentation of vegetarians in one study).

Analysis of NHANES data from 2003 through 2006 showed that median daily cobalamin intake was 6.4 µg (95% confidence interval [CI], 4.7 to 8.6 µg) in males and 5.0 µg (95% CI, 3.7 to 6.9 µg) in females. It is useful to note that the daily intakes reported by NHANES exceeded the Institute of Medicine’s recommendations in children (see Table 10-2 ). The intakes were 5.1 µg at ages 1 to 3 years, 6.1 µg at 4 to 8 years, 5.6 µg at 9 to 13 years, and 5.9 µg at 13 to 19 years. The 95% confidence intervals for these intakes exceeded the Institute’s estimated requirements, and the children’s 95% confidence levels for methylmalonic acid (MMA) levels were within the expected reference ranges. Data were unavailable for infants.

Foods vary widely in cobalamin content. Liver and some seafood are among the richest in cobalamin but are not eaten often, beef has relatively rich cobalamin content, and cobalamin content of eggs and milk is smaller. How­ever, bioavailability can vary as much as tenfold among different food sources, and food cobalamin content, whose measurement can be imprecise, does not always predict effectiveness. Thus cow’s milk and fortified cereal, as well as fish, proved to be much more efficient providers of cobalamin than meat, despite the latter’s high cobalamin content. The specific receptor-mediated absorption of cobalamin via IF allows efficient absorption of more than 50% of this micronutrient while also excluding unutilizable corrinoid analogues. However, this mechanism is also easily saturable, and when more than 1 to 2 µg is ingested in a meal, much of the excess is relegated to nonspecific diffusion in which absorptive bioavailability falls to approximately 1%. The implications of such a small margin between the desirable level of intake and the total capacity of receptor-specific absorption have not been explored sufficiently. However, the existence of haptocorrin, a ubiquitous, relatively abundant, high-affinity cobalamin-binding protein that does not mediate specific delivery to intestinal cells or other tissue, raises the obvious question whether such an upper limit on cobalamin internalization serves a positive but still undefined biologic purpose, perhaps tied to corrin analogue exclusion from cells.

Cobalamin Stores

Estimates of stores vary with assessment methods and subject selection. The current consensus is that cobalamin contents in adults average 2 to 3 mg, with wide spreads. The liver contains approximately 1.5 mg of those stores, with concentration ranges of 0.7 to 1.5 µg/g. By comparison, liver concentration is 0.02 to 0.14 µg/g in patients with megaloblastic anemia caused by pernicious anemia, which is a reduction of more than 80%. Store estimates are scarce in children. Maternal-fetal cobalamin transfer favors the fetus even when maternal stores are limited, and cord blood cobalamin levels exceed maternal serum levels, 370 versus 240 ng/L, although maternal serum cobalamin levels tend to be spuriously low during pregnancy. Full-term newborns have a mean liver cobalamin content of 27.3 µg ; the total liver content is only 10.7 µg in premature newborns and 4.1 µg in the fetus. The total body cobalamin store in newborns is approximately 50 µg, which illustrates how greatly stores need to expand between infancy and adulthood. As will be discussed, the approximately 1000 : 1 stores-to-daily intake ratio in adults and adolescents may be only 100 : 1 in newborns.

Cobalamin-Binding Proteins

Cobalamin does not exist free in the body outside the gastrointestinal tract, where its carrier-free existence is transient. Cobalamin traffic, transport, and access to cells depend on three cobalamin-binding proteins: IF, transcobalamin, and haptocorrin. Only IF and transcobalamin interact with specific cell receptors, allowing efficient cellular internalization of cobalamin at low concentrations, but these systems have relatively low capacity. These binding proteins have similar primary sequences, and their genes have similar intron/exon structure, suggesting that they arose by duplications of an ancestral gene. They show 33% overall homology with each other, and specific regions demonstrate as much as 60% to 80% identity.

IF is a 45-kD glycoprotein synthesized by gastric parietal cells in humans; under some circumstances, ectopic IF synthesis may also occur in chief cells, which are the normal synthetic site in many other mammals. IF mediates efficient uptake of cobalamin in the intestine. It binds cobalamins less tightly than does transcobalamin or haptocorrin but has far greater binding affinity for cobalamins than for nonfunctional corrin analogues, which helps limit the analogues’ intestinal absorption. IF is encoded by the GIF gene on chromosome 11q13.

Transcobalamin (also called transcobalamin II) is synthesized by endothelial and other cells, and mediates entry of cobalamin into most types of peripheral cells. It is a nonglycosylated protein with a molecular weight of 43 kD that, like IF, binds cobalamin tightly but has low affinity for cobalamin analogues. Transcobalamin content is small in many secretions. The TCN2 gene is on chromosome 22q12-13.

Haptocorrin (also called transcobalamin I) differs from IF and transcobalamin in having a high affinity for nonfunctional corrinoid analogues as well as for cobalamin and in having no specific cellular receptors for its internalization. These characteristics suggest an important role in excluding analogues, and perhaps excess cobalamin, from cells. The protein, encoded by the TCN1 gene on chromosome 11q12.1, is approximately 46 kD, but its heavy glycosylation creates glycoproteins of different sizes, isoelectric points, and plasma clearance rates. The glycosylation variability, which is sometimes secretion specific, led to artificial distinctions and names for haptocorrin variants (e.g., transcobalamin III and R binder), which are no longer used. Haptocorrin is synthesized in myeloid precursors at early stages when specific granules form ; this appears to be the chief source of plasma haptocorrin. Epithelial or ductal cells of exocrine glands also synthesize haptocorrin and are its source in secretions, where haptocorrin achieves concentrations much higher than in plasma. The secretions include bile, saliva, tears, breastmilk, bronchial secretions, amniotic fluid, and seminal fluid ; and their high haptocorrin content suggests a still unspecified role in relation to the gut microbiome and its abundant content of cobalamin and corrinoid analogues.

Typically, more than 70% of the cobalamin in plasma at any moment exists bound to haptocorrin, but this seeming preponderance is misleading because haptocorrin is cleared slowly from plasma ( , 9.5 to 12 days), whereas transcobalamin ( , only 90 minutes) carries 40 times as much cobalamin daily in plasma as haptocorrin does. Study of haptocorrin interactions with each of the many corrinoid analogs has been difficult, but levels of plasma analogues, whose sources are unknown, approach or exceed cobalamin levels.

Absorption of Cobalamin

Cobalamin in food is almost entirely protein bound. It is released by the action of acid and pepsin in the stomach, where it binds to salivary or gastric haptocorrin in preference to IF when gastric pH is low ( Fig. 10-4 , 1 ). In the duodenum, the haptocorrin-cobalamin complex from the stomach, and from bile, is broken down by pancreatic proteases, releasing cobalamin, which subsequently binds to IF ( Fig. 10-4 , 2 ). The IF-cobalamin complex binds to receptors in the apical brush border of enterocytes; the receptors are most abundant in the distal ileum ( Fig. 10-4 , 3 ). The absorbed fraction varies inversely with the amount of ingested cobalamin. Absorption efficiency is more than 50% when the amount is 1 to 2 µg, which approximates some meal contents. High intake in a meal or supplement (e.g., 5 to 10 µg) exceeds the capacity of IF receptors and the excess free cobalamin is internalized only by nonspecific diffusion, a process with only approximately 1% efficiency.

The events of cobalamin absorption, transport, and internalization.

The events at four key sites of cobalamin transport are shown: the stomach, the duodenum, the ileum, and cellular sites, such as the bone marrow. ado-cbl, 5′-Deoxyadenosylcobalamin; HC, haptocorrin; IF, intrinsic factor; methyl-cbl, methylcobalamin; TC, transcobalamin; TC I, transcobalamin I; TC II, transcobalamin II.

(Modified from Carmel R: Folate deficiency. In Carmel R, Jacobsen DW, editors: Homocysteine in health and disease . Cambridge UK, 2001, Cambridge University Press, p. 271–288.) Used with permission.

The ileal receptor consists of two proteins, cubilin (the product of the CUBN gene on chromosome 10p12.1) and amnionless (the product of the AMN gene on chromosome 14q32), and has been named “cubam.” Cubilin, a 460-kD protein that binds many ligands other than IF, such as transferrin and vitamin D–binding protein, is anchored externally to the enterocyte membrane by amnionless, a 48-kD membrane-spanning protein. Both components must be synthesized for cubam to be expressed at the cell surface.

The IF-cobalamin complex is rapidly internalized by endocytosis after binding to cubam. Cobalamin bound to transcobalamin (holotranscobalamin [holo-TC]) appears in the portal circulation 4 hours after ingestion. Although some studies have suggested that attachment of cobalamin to transcobalamin occurs within enterocytes after its release from IF, studies in transgenic mice were consistent with export of free cobalamin from enterocytes by the ABCC1 (MRP1) membrane transporter with subsequent attachment to transcobalamin outside the enterocyte.

Enterohepatic Recirculation

Approximately 1.5 µg of cobalamin and varying amounts of cobalamin analogues bound to haptocorrin are excreted in the bile daily. Cross-species experiments in rabbits suggest that plasma haptocorrin uptake by asialoglycoprotein receptors on hepatocytes delivers cobalamin and analogues to bile, but reservations exist and direct data are unavailable. Pancreatic proteases degrade the biliary haptocorrin (see Fig 10-4 , 2 ). Released cobalamin binds to IF and is reabsorbed in the ileum, whereas released analogues, unavailable to IF, are excreted in the feces. This process salvages about two thirds of the cobalamin secreted in bile.

Renal Cobalamin Reabsorption

The amount of cobalamin filtered daily is approximately 1.5 µg. Little cobalamin is detected in urine under normal physiologic conditions because more than 99.8% is reabsorbed in the proximal tubules. Cubilin and a second large multiligand receptor, megalin, are expressed in the proximal renal tubule, where they reabsorb a variety of filtered proteins. Filtered holo-TC is reabsorbed by megalin. Unbound cobalamin does not normally exist in plasma, but if it is introduced by parenteral injection, the filtered free cobalamin cannot be reabsorbed in the proximal tubule.

Cobalamin in Pregnancy

Cobalamin is efficiently transported across the placenta during pregnancy, and cobalamin concentration is higher in cord blood than in the maternal circulation, even in cases of maternal cobalamin insufficiency. The mechanism of cobalamin transport remains poorly understood. Both cubilin and amnionless are expressed in the visceral yolk sac of rodents, where they support uptake of nutrients (although not cobalamin). It is not clear that the visceral yolk sac plays any role in nutrition of the postimplantation embryo in humans. The transcobalamin receptor is present in human placenta, but its role in transplacental cobalamin uptake has not been studied.

Cellular Cobalamin Metabolism

Cobalamin functions as a coenzyme in two reactions in humans ( Fig. 10-5 ). MeCbl is required for methylation of homocysteine to form methionine, catalyzed by the cytoplasmic enzyme methionine synthase (EC 2.1.1.13). This reaction utilizes 5-methyltetrahydrofolate as methyl group donor and represents the point in cellular metabolism where cobalamin and folate metabolism intersect. AdoCbl is required in the conversion of methylmalonyl-coenzyme A (CoA) to succinyl-CoA, which is catalyzed by the mitochondrial enzyme methylmalonyl-CoA mutase (EC 5.4.99.2). In microorganisms, a variety of other enzymes utilize cobalamin for reactions that appear not to occur in mammalian cells.

Scheme of cobalamin metabolism.

The sites affected by methylmalonyl-CoA mutase deficiency (mut) and the inborn errors of cobalamin metabolism ( cblA-cblG, cblJ, and cblX ) are shown in red. The cblA disorder is caused by defects in the MMAA protein; the cblB disorder is caused by defects in the cob(I)alamin adenosyltransferase (MMAB) protein; the cblC disorder is caused by defects in the MMACHC protein; the cblD, cblD variant 1 (cblDv1), and cblD variant 2 (cblDv2) disorders are caused by defects in the MMADHC protein; cblE disorder is caused by defects in the methionine synthase reductase (MSR) protein; cblG disorder is caused by defects in the methionine synthase (MS) protein; cblJ disorder is caused by mutations in the ABCD4 protein; cblX disorder is caused by mutations in the HCFC1 protein, and the mut disorder is caused by defects in methylmalonyl-CoA mutase (MCM) . The protein affected in the cblF disorder is unknown. MCM-AdoCbl, Holomethylmalonyl-CoA mutase (mutase with bound adenosylcobalamin); MS-cob(II)alamin, methionine synthase with bound cob(II)alamin; MS-MeCbl, holomethionine synthase (synthase with bound methylcobalamin; TC, transcobalamin.

Intracellular cobalamin concentrations are low, and many of the forms that are involved in metabolism are not stable in aqueous solution. This has led to the suggestion that once cobalamin enters cellular metabolism it must be bound to protein at all times. Most cellular cobalamin exists bound to proteins, primarily methionine synthase in the cytoplasm and methylmalonyl-CoA mutase in the mitochondria. It appears that cobalamin is not retained in cells unless it is bound to protein.

Uptake of circulating holo-TC is mediated by the transcobalamin receptor, which initiates endocytosis (see Figs. 10-4 and 10-5 ). Transcobalamin is degraded in lysosomes, releasing cobalamin that is transferred across the lysosomal membrane by a process involving the lysosomal membrane proteins LMBD1 and ABCD4. Once in the cytoplasm, cobalamin becomes associated with the molecular chaperone protein MMACHC, which catalyzes reduction of the central cobalt to the Co ²⁺ state and removal of the upper axial ligand, if one is present. Subsequently cobalamin may enter the mitochondria, where cobalamin is converted to AdoCbl by cobalamin adenosyltransferase and becomes associated with methylmalonyl-CoA mutase; a third protein, MMAA, is required for transfer of AdoCbl from adenosyltransferase to methylmalonyl-CoA mutase. Alternatively it can remain in the cytoplasm, where it becomes associated with methionine synthase and is converted to MeCbl.

Biochemical Effects of Cobalamin Deficiency

The biochemical consequences of cobalamin deficiency result from decreased activity of the cobalamin-dependent enzymes methionine synthase and methylmalonyl-CoA mutase. Decreased activity of methionine synthase results in elevated levels of its substrate, homocysteine, in the blood and excretion of homocystine in the urine. Decreased activity of methylmalonyl-CoA mutase results in accumulation of MMA in the blood and urine.

Methionine Metabolism

Methionine is an essential amino acid in humans. Methionine synthase remethylates the homocysteine generated from methionine during cellular metabolism, helping to maintain adequate methionine levels as well as preventing accumulation of potentially toxic homocysteine. A cobalamin-independent enzyme, betaine : homocysteine methyltransferase, catalyzes homocysteine remethylation in liver, but its activity is insufficient to prevent homocysteine accumulation when cobalamin-dependent methionine synthase activity is impaired. Methionine levels, however, often remain normal in cobalamin deficiency.

Cellular methionine may be utilized in protein synthesis or adenosylated to form S-adenosylmethionine, which acts as a methyl group donor in several dozen different transmethylation reactions, including methylation of DNA, which plays a role in regulation of gene expression, as well as methylation of RNA, lipids, proteins (including histones), neurotransmitters, and thiols. These reactions generate the homocysteine that is acted on by methionine synthase. Homocysteine may also react with serine to form cystathionine, in a reaction catalyzed by cystathionine β-synthase.

Interaction of Folate and Cobalamin Metabolism

Deficiency of either folate or cobalamin, when substantial, produces megaloblastic anemia. The mechanisms leading to the anemia are complex. Folate deficiency directly affects the one-carbon substituted THF derivatives required for de novo thymidine and purine biosynthesis, resulting in abnormal DNA synthesis in bone marrow cells. The explanation for the hematologic similarity in cobalamin deficiency is believed to be the result of a secondary deficiency of these THF derivatives, best explained by the “methyl trap” hypothesis. Because methionine synthase is the only reaction in human cells that can convert 5-methylTHF to THF, which can be utilized to form one-carbon substituted THF derivatives, and because the reaction that converts 5,10-methyleneTHF to 5-methyTHF is irreversible under physiologic conditions, cobalamin deficiency results in “trapping” of folate in the form of now unusable 5-methylTHF.

Incidence and Evolution of Megaloblastic Anemia

Although the megaloblastic anemia is identical in folate and cobalamin deficiencies, its appearance after the dietary or malabsorptive onset of vitamin depletion in adults is measured in 2 to 3 months for folate deficiency but in years for cobalamin deficiency because of their disparate stores-to-daily intake ratios. The time course has not been documented as well in children, and onset may occur sooner in infants because of their smaller vitamin stores.

The earliest RBC changes are a rise in mean corpuscular hemoglobin followed shortly by a rise in mean corpuscular volume (MCV). These cell content and size changes precede the overt evidence of anemia, in part because the early phases of the anemia involve declining RBC count but little change in blood hemoglobin and packed cell volume levels, which are compensated by the increasing RBC sizes and contents. Clinicians must pay attention to changes in RBC indices regardless of hemoglobin and packed cell values. Although macrocytosis has many causes ( Table 10-3 ), early megaloblastic anemia must be considered whenever a patient’s MCV, even if still within the normal reference interval, has changed as little as 3 to 5 fL compared with a previous value. The onset of macrocytosis can be documented even sooner by reticulocyte-specific indices of macrocytosis. However, macrocytosis is sometimes masked if a microcytic disorder, most often iron deficiency or thalassemia trait, coexists. These combinations can produce normal, low, or high MCV, but the MCV is always higher than normal for that particular patient and it always decreases after the megaloblastic anemia is treated. The frequency of nonelevated MCV was 7% in one study of adults with pernicious anemia. The macrocytosis typically features macroovalocytes, which suggest but are not specific to megaloblastic anemia. Anisocytosis and admixture of macrocytes with normocytes elevate the relative distribution of RBC width. As megaloblastic anemia progresses, poikilocytosis, including teardrop cells, becomes prominent. With severe anemia, circulating nucleated erythroid precursors, Cabot rings, and Howell Jolly bodies can appear.

The other early manifestation preceding anemia is nuclear hypersegmentation in neutrophils (see Fig. 10-6 ). It is also a much more specific feature of megaloblastic change than is macrocytosis. Unexplained hypersegmentation and other dysplastic granulocytic changes may also follow high-dose corticosteroid treatment of idiopathic thrombocytopenic purpura, however. The mechanism of hypersegmentation is unknown.

Neutropenia and thrombocytopenia are late features, appearing only infrequently before the hemoglobin level decreases below 10 g/dL; cytopenias that accompany mild anemia usually have independent explanations. The pancytopenia can ultimately become severe enough to mimic severe aplastic anemia. The two can be differentiated by the serum lactate dehydrogenase elevation (often exceeding 1000 units) and indirect hyperbilirubinemia in megaloblastic anemia.

Macrocytic anemia, the most common clinical feature of cobalamin or folate deficiency in adults, can be mild but it affects more than 70% of patients with clinically expressed deficiency, such as in pernicious anemia ; comparable data are not available in children. The reason why some patients with pernicious anemia do not develop anemia, even if they may have mild megaloblastic changes in the bone marrow that often escape detection, is unknown. Interestingly, a roughly inverse relationship between the severity of the hematologic and neurologic expressions has been described and the dominant expression tends to dominate again with relapses. A comparison of predominantly hematologically versus neurologically affected patients showed the predominantly anemic patients to have lower plasma glutathione, S-adenosylmethionine, cysteine, and folate levels, whereas a higher cysteine level was the most significant predictor of neurologic dysfunction.

Biochemistry of Megaloblastic Anemia

In deficiency of either folate or cobalamin, all dividing cells, especially bone marrow precursors and epithelial cells, show megaloblastic changes. MethyleneTHF, which mediates the methylation of deoxyuridylate to deoxythymidylate (see Fig. 10-2 ), is directly depleted in folate deficiency but can also be secondarily depleted in cobalamin deficiency, as best explained by the methylTHF trap hypothesis. Nevertheless, de novo thymidylate synthesis disruption, although necessary, may not by itself suffice to produce megaloblastic changes.

The misincorporation of excess uracil into DNA may exceed the capacity of excision repair, leading to single-strand breaks as well as occasional double-strand breaks. The latter may help explain the nuclear defects. Arrest at various stages of interphase occurs in megaloblastic precursors even while maturation proceeds, but the details are still unclear.

Mistaken folic acid therapy for clinically expressed cobalamin deficiency can bypass the methylTHF trap and improve the megaloblastic anemia. However, the response is often only partial and is usually impermanent. Concerns exist that neurologic manifestations may progress in the meantime and become irreversible as the cobalamin deficiency remains unrecognized and untreated. Systematic evidence is unavailable, and mistaken folic acid therapy may occasionally even improve neurologic status. However, ethical constraints pre­vent prospective studies and it is agreed that folate treatment must be avoided in cobalamin deficiency. These concerns prompted restriction of folic acid doses, as well as folic acid–fortified food intake, to 1 mg/day or less.

Ancillary Features of Megaloblastic Anemia

The ineffective hematopoiesis of megaloblastic anemia disrupts iron metabolism, raising serum iron, transferrin receptor, and ferritin levels, along with sideroblast counts and the ferritin content in erythroid precursors and macrophages. These changes can occur even in the presence of a coexisting iron deficiency, which may remain hidden until the ineffective erythropoiesis is corrected. Severe megaloblastic anemia also has a still unexplained element of intravascular hemolysis. Membrane protein changes, including spectrin, have been described in megaloblastic RBCs, but their impact is unknown.

Neurologic Manifestations

Neurologic dysfunction is common, but its frequency is unclear because neurologic disease is not easily quantified, unlike the probably more common hematologic abnormalities. Whereas 13% to 27% of adults with pernicious anemia have had neurologic symptoms without anemia, the proportion with anemia without neurologic abnormalities is higher.

The classic neurologic manifestations feature a symmetric myelopathy, largely limited to the posterior and lateral columns (“subacute combined degeneration”) and neuropathy. The myelopathy typically begins with sensory and proprioceptive changes in the feet and ascends at variable rates to involve hands and arms as deficiency progresses. If not caught early, the changes may become partially or completely irreversible and can evolve to spasticity, ataxia, and incontinence. Other manifestations include mental changes of varying severity (mild electroencephalographic abnormalities can occur even without overt symptoms), autonomic dysfunction, and, rarely, optic neuritis.

In newborns and infants, deficiency and its manifestations appear sooner than in adults because of the smaller storage reserves, because hereditary defects of cellular metabolism often act more quickly and directly on tissues than dietary or malabsorptive disorders, and most especially because the developing nervous system is at greater risk than the mature nervous system. Often, the manifestations include impaired growth and development. Thus the neurologic changes common to older children and adults are overshadowed in infants by failure to thrive, poor brain growth, developmental delay, seizures, motor retardation, and movement disorders ; the movement disorders, which sometimes increase transiently after cobalamin therapy, can be mistaken for seizures. The diagnostic connection to cobalamin deficiency is often diverted or delayed for months and, even in nonhereditary disorders, developmental and neurologic residua may persist long after therapy.

The mechanisms responsible for the demyelination in the central nervous system, followed by axonal degeneration and gliosis, in clinical cobalamin deficiency, or for the nondemyelinative peripheral neuropathy, are unknown. Still unproven etiologic hypotheses for neurologic dysfunction have included abnormal myelin lipid synthesis because of abnormal propionate metabolism, defective neurotransmitter metabolism, corrinoid analogue accumulation, and cytokine abnormalities. Evidence from the clinical patterns in diverse cbl mutations and other findings favors a central, but perhaps not sufficient, role for methionine synthase inactivation, which disrupts methionine metabolism and S-adenosylmethionine–dependent methylation reactions. However, folate deficiency, which also inactivates methionine synthase, rarely produces neurologic abnormalities of comparable severity to cobalamin deficiency in adults, although hereditary disorders of folate metabolism in children often do.

Other Clinical Manifestations

Cobalamin deficiency can cause atrophic glossitis and stomatitis, which can be painful. Somewhat similar changes occur in folate deficiency, but the oral changes respond only to the specific vitamin whose deficiency caused them. Unexplained weight loss, sometimes substantial, can occur with severe cobalamin deficiency in adults and children, and it reverses after cobalamin therapy. Infants sometimes feature prominent gastrointestinal dysfunction, such as diarrhea. Severely deficient infants can also present with recurrent infections that mimic immune deficiency states.

Young patients with severe deficiency, especially nonwhites, sometimes display pigmentary changes that reverse with cobalamin therapy: skin hyperpigmentation that overlies joints, dark hair that turns a light reddish hue, or blue discoloration of fingernails and toenails. The pigmentary changes should not be confused with the unrelated, irreversible autoimmune vitiligo that occasionally coexists with pernicious anemia. Adults with pernicious anemia display reduced levels of osteoblastic proteins, such as osteocalcin and bone alkaline phosphatase, which respond to cobalamin. Reports of clinical bone complications in adults await systematic proof. Bone effects have not been studied in children.

Subclinical Cobalamin Deficiency

Unexplained low serum cobalamin levels that were unaccompanied by clinical manifestations, did not progress to clinical manifestations, and displayed no clinical benefits after cobalamin treatment were originally regarded as spurious. However, biochemical studies established that some of these represented mild metabolic cobalamin insufficiency and, unlike more than 90% of cases with megaloblastic anemia and other clinical dysfunction, they were rarely associated with the loss of IF or IF-mediated absorption by ileal cells. This often purely biochemical abnormality became known as “subclinical cobalamin deficiency.” As accurate, sensitive, automated tests of cobalamin metabolism, such as MMA and tHcy, became widely available, epidemiologic studies established that subclinical deficiency affected many adults, especially the elderly, and indeed was several times as common as pernicious anemia and other clinical deficiency states. The causes of subclinical deficiency are often unknown, but a plurality of 30% to 50% of patients have malabsorption of food-bound cobalamin, which is usually associated with chronic gastritis, achlorhydria, or gastric surgery. Because poor dietary intake alone cannot usually deplete cobalamin stores sufficiently in adults and older children to cause clinical deficiency, most vegetarians, especially adults, display only subclinical deficiency.

The health consequences of subclinical cobalamin deficiency are unknown, and progression to clinically expressed deficiency seems infrequent. In the absence of compelling data from randomized clinical trials, the benefits of intervention to reverse isolated biochemical changes of still unproven likelihood of progression to clinical deficiency remain unsettled.

Subclinical deficiency has not been studied in children and was believed rare. However, a transient, purely metabolic cobalamin deficiency affecting many normal infants in the first year of life has been documented in Scandinavia and is discussed in the section on dietary cobalamin insufficiency. The metabolic phenomenon must also be borne in mind when using current cobalamin status–related reference intervals in infants, and hematologists and geneticists must consider this transient phenomenon when evaluating MMA elevations in infants.

Neurologic Manifestations

Folate deficiency in adults does not cause the severe demyelinative myelopathy characteristic of cobalamin deficiency. Forgetfulness and mood changes, such as irritability, have been described, as has neuropathy. However, their attribution and specificity remain unsettled because folate deficiency is often not an isolated deficiency. Nevertheless, the neurologic consequences can be severe in some folate deficiency disorders in newborns and infants. Children with hereditary folate malabsorption, cerebral folate deficiency, and severe MTHFR deficiency (the last two of which do not display megaloblastic anemia) often have seizures, microcephaly, developmental delay, mental retardation, and myelopathy.

Other Clinical Manifestations

Mucosal changes of the tongue and mouth can sometimes resemble those of cobalamin and other deficiencies. However, because folate deficiency is often part of a broader nutritional inadequacy, responsiveness specifically to folic acid provides the best proof of causation.

Subclinical Folate Deficiency

Subclinical deficiency, definable as isolated abnormalities of folate and tHcy levels without megaloblastic changes or other clinical expressions of folate deficiency, has not been studied or defined as formally as subclinical cobalamin deficiency. Certain subpopulations are at special risk because of marginal dietary folate intake, increased folate requirements, or both, such as children (including adolescents), pregnant women, people with homozygosity for common polymorphisms of the MTHFR gene, and alcoholics. Although not studied formally, subclinical folate deficiency is almost certainly more common than clinically expressed deficiency.

Countries such as the United States and Canada have mandated fortification of cereal and grain products with folic acid to reduce NTD risks. This policy has nearly eliminated clinical folate deficiency in the general populations, sharply reduced the prevalence of isolated low folate levels, and lowered tHcy levels. The influence of folate status on hyperhomocysteinemia and on NTDs is clear, but the record on vascular disease, cancer, and cognitive decline in the elderly is mixed or ambiguous, as discussed elsewhere in the chapter. It merits emphasis that many of the public health risks, including NTDs, have generally been associated with subclinical folate deficiency and possible genetic or acquired alterations of folate demand or utilization, rather than with clinically overt maternal folate deficiency.

Cobalamin Assay

Serum cobalamin measurement, although problematic, is the traditional test of choice for diagnosing cobalamin deficiency. Microbiologic assay, based on growth of cobalamin-dependent microbes on exposure to the test serum, is unavailable outside select reference and research laboratories. Clinical laboratories, which adopted isotopic, competitive-binding assays in the 1970s, have come to rely almost exclusively on automated, competitive-binding, chemiluminescence-based immunoassays since the late 1990s. Most laboratories define subnormal levels as below 200 or 250 ng/L (150 or 200 pmol/L). However, some authors raised cutpoints to 300 to 350 ng/L to capture nearly all elderly patients with otherwise isolated, mild MMA and tHcy elevations. This redefined reference interval led to claims that 40% or more of the elderly have cobalamin deficiency, but most of the redefined cases lacked clinical or metabolic evidence of deficiency, in part because abnormal MMA and tHcy themselves lack specificity. The cutpoint expansion worsened the already poor diagnostic specificity of a low cobalamin level. Most laboratories continue to use the 150-ng/L cutpoint in children, but levels, as shown in Table 10-4 , fluctuate during childhood, especially in early infancy, when cobalamin stores are small relative to intake and when maternal influences dominate.

Adults have many causes of falsely low cobalamin levels, including levels less than 100 ng/L sometimes. A major cause is pregnancy, with levels decreasing below 150 ng/L in almost half of women by the last trimester. The explanation is uncertain but may reflect maternofetal cobalamin transfer in part. Metabolic or clinical evidence of deficiency is rare, and cobalamin treatment is unnecessary. The levels become normal spontaneously a few weeks after delivery.

Folate deficiency often decreases serum cobalamin levels also, which become subnormal in 10% of patients. Folate therapy restores cobalamin levels to normal, but alertness is warranted because occasional patients, especially those with gastrointestinal disease, have concurrent deficiencies of both vitamins. Interestingly, hepatic cobalamin content is also somewhat low in folate deficiency. Other conditions associated with unexplained low cobalamin levels include oral contraceptive use, obesity, infection with human immunodeficiency virus, and multiple myeloma. Falsely low cobalamin levels also accompany hereditary or, less often, acquired deficiency of haptocorrin. Finally, the likelihood of falsely subnormal cobalamin levels rises sharply when overly liberal cutpoints, such as 300 ng/L, are applied.

Elevated cobalamin levels are more common than low levels in adults and can occasionally hide a coexisting cobalamin deficiency. Examples relevant to children include defects in cellular cobalamin metabolism, in which serum cobalamin (and holo-TC) levels remain normal or increase; these metabolic defects can be hereditary or acquired, as with nitrous oxide toxicity. A troubling malfunction peculiar to modern chemiluminescence-based cobalamin assays often causes spuriously normal (or elevated) levels in IF antibody–positive sera from cobalamin-deficient patients with pernicious anemia. Children with the juvenile form of pernicious anemia are often antibody positive and thus probably susceptible to the assay error. Renal failure can also cause spuriously elevated serum cobalamin. Race affects cobalamin levels; blacks have significantly higher levels at all ages than whites or Asians. Homozygosity for a common polymorphism of the FUT2 gene, affecting fucosylation, also causes slightly higher cobalamin levels. Finally, clinicians faced with unexpectedly normal or high cobalamin levels must consider recent cobalamin therapy as a possible cause.

Red cell cobalamin is rarely measured today because levels depend largely on the proportion of reticulocytes, which are rich in cobalamin, rendering results unreliable.

Serum Holotranscobalamin Assay

Holo-TC, the cobalamin fraction attached to transcobalamin, contains 10% to 30% of the total cobalamin in plasma at any moment in time (although the very high turnover of transcobalamin obscures the fact that transcobalamin carries much more cobalamin daily than haptocorrin does). Because holo-TC is the only cobalamin fraction that is available for delivery to cells, it is regarded by some authors to be a more reliable indicator of cobalamin status than serum (total) cobalamin. However, head-to-head receiver operator characteristics-based comparisons have shown holo-TC to be only marginally superior to total cobalamin (areas under the curves of 0.66 to 0.90 vs. 0.60 to 0.86 in six studies) in recognizing deficiency defined by MMA levels ; its specificity and positive predictive value are as poor as that of serum total cobalamin, but the causes await careful study. A recent study reported low holo-TC levels in folate-deficient patients also ; as noted earlier in this chapter, serum total cobalamin and hepatic cobalamin levels are also low in folate deficiency. Holo-TC testing also suffers from considerable cutpoint variability.

It is unclear whether the 90-minute half-life of plasma holo-TC leaves its levels too susceptible to transient changes in cobalamin flow from the gut to reliably reflect cobalamin stores and status, which change slowly. Evidence of relatively decreased holo-TC levels in patients with pernicious anemia in clinical and metabolic remission suggests that daily absorption flux, not just cobalamin adequacy, influence holo-TC levels, whose turnover is so great. More studies are needed to identify relevant influences that explain the poor specificity of low holo-TC levels.

Serum Folate Assay

As with cobalamin, folate testing began with microbiologic assay and, after 25 years of isotopic competitive-binding assay, is currently done by automated chemiluminescent assay. Current cutpoints approximate 2.5 to 3.0 µg/L (5.7 to 6.8 nmol/L) in adults. Normal levels fluctuate during childhood, with higher levels in early infancy that decline thereafter, reaching low-normal values in adolescence (see Table 10-4 ).

Serum folate has high sensitivity for folate deficiency. However, specificity is poorer because the serum levels disproportionately reflect recently ingested and absorbed folate. Transiently poor intake lowers the serum levels within 1 to 3 weeks, long before actual deficiency of stores.

A relevant cause of serum folate elevation is clinically expressed cobalamin deficiency, such as pernicious anemia, which causes RBC methylTHF to increase; however, the poor retention of methylTHF in RBC rapidly produces low RBC folate, along with high serum folate levels in 20% of cases. It is unknown if mild or subclinical cobalamin deficiency affects folate levels similarly. Dietary fortification with folic acid in the United States raised serum folate levels, and the frequency of levels below 3 µg/L decreased from 18% to 0.2%. Plasma tHcy levels improved commensurately.

Red Blood Cell Folate Assay

RBC folate content does not change after the reticulocyte stage and is thus less susceptible to short-term fluctuation than serum folate. The RBC level is a 120-day reflection of folate status, not a transient 1- to 3-week snapshot. However, RBC folate is subnormal in 60% of patients with pernicious anemia because the methylTHF accumulation caused by the folate trap in cobalamin deficiency is poorly retained by the RBC. Measuring serum folate, which is low in folate deficiency but high in cobalamin deficiency, can resolve the confusion. Other distortions of RBC folate content include blood transfusion, iron deficiency (which elevates RBC folate), and hemolytic anemia (reticulocytes have increased folate content). The choice between RBC and serum folate testing has generated disagreement, and clinicians should base their choice on the diagnostic question being asked.

Cutpoint selection has varied considerably. In adults, RBC folate cutpoints defined by megaloblastic anemia were set at 140 or 150 µg/L but later cutpoints based on metabolic criteria approximated 190 to 200 µg/L. A study of normal Scandinavian children identified age-related variation, with highest RBC folate in newborns (breastmilk has high folate content) and a steady decline from the first 6 months to the lowest levels in adolescents.

Methylmalonic Acid Assay

Current gas chromatography/high-performance liquid chromatography/mass spectrometry methods are accurate and, with automation, serum MMA assay has become increasingly available; urine assays are done less often. Serum MMA elevation has 98% sensitivity for clinically expressed cobalamin deficiency, although its sensitivity in subclinical deficiency is unknown because no diagnostic gold standard exists for comparison. Being the only test unaffected by folate deficiency, MMA assay is widely considered the best available metabolic test for cobalamin deficiency, but its specificity is limited ( Table 10-5 ). A survey in adults found that cobalamin status, renal status (MMA values correlate directly with serum creatinine, even within the normal range), age, and sex explained only 16% of the MMA level. Creatinine is the major known confounder and should be measured when MMA is elevated.

Most laboratories use a cutpoint approximating 270 nmol/L (0.27 µmol/L) in adults, but cutpoint selection has varied between 221 to more than 400 nmol/L, with predictably inverse impact on the frequency of cobalamin “deficiency.” MMA reference intervals have been lower in children than in adults, but they fluctuate in early infancy (see Table 10-4 ). Many infants display transient MMA elevations, now believed to reflect a mild subclinical cobalamin deficiency among exclusively breastfed infants but which had distorted pediatric MMA reference intervals for years.

MMA is also an effective, albeit more expensive, biochemical tool for monitoring response in cobalamin deficiency. Levels begin to decrease several days after cobalamin therapy (but not after folate therapy) and become normal within 2 to 3 weeks.

Plasma Total Homocysteine Assay

Plasma tHcy elevation is highly sensitive for folate deficiency and nearly as sensitive as MMA elevation for cobalamin deficiency. The degree of tHcy elevation in severe cobalamin or folate deficiency can mimic that seen in severe cystathionine β-synthase deficiency. However, inability of tHcy to distinguish between folate and cobalamin deficiencies limits its diagnostic utility. The many influences that elevate tHcy include serum creatinine, age, gender, and, uniquely among tests of cobalamin and folate status, preanalytic influences, especially using serum instead of plasma and failing to separate plasma from cells quickly (see Table 10-5 ). Cutpoint selection also affects diagnostic reliability. Reference intervals for tHcy fluctuate during childhood, peaking during the transient subclinical cobalamin deficiency affecting many healthy infants (see Table 10-4 ). A second, mild tHcy elevation occurs during adolescence, presumably reflecting a downward trend in folate status.

The high sensitivity of tHcy assay for cobalamin and folate deficiency and its relative inexpensiveness provide two valuable diagnostic roles: a normal tHcy level largely rules out the presence of clinically relevant folate deficiency, and the tHcy assay is the most cost-effective metabolic monitor of response of either cobalamin or folate deficiency to specific therapy. tHcy begins to decrease within a week and becomes normal within 1 to 3 weeks.

Miscellaneous Tests

Plasma cysteine increases along with tHcy in cobalamin and folate deficiencies, but methionine levels show little change. Measuring 2-methylcitric acid provides little diagnostic advantage over MMA measurement. The deoxyuridine suppression test, an older, indirect means of evaluating thymidylate adequacy, allows diagnosis of folate and cobalamin deficiencies at the cellular level when specific vitamins are added to cells in vitro but requires bone marrow cells and is labor intensive. Formiminoglutamic acid excretion lacks specificity and is no longer used diagnostically.

Dietary Insufficiency in Adults and Older Children

A narrowly focused cobalamin inadequacy, ranging from strict veganism to mild ovolactovegetarianism, in which dairy products and eggs are consumed, can produce cobalamin deficiency. However, the process of depleting 80% of the very high storage cushion found in adults and older children to induce clinical deficiency with megaloblastic anemia or myeloneuropathy takes years and requires uninterrupted dietary restriction. Indeed, depletion takes longer than in pernicious anemia and other malabsorptive diseases because vegetarians continue to reabsorb biliary cobalamin normally. Subclinical deficiency limited to mild abnormalities of serum cobalamin and MMA levels can be seen, but clinical deficiency is surprisingly infrequent, even in strict vegans, unless other events, such as malabsorption or increased metabolic requirements, supervene.

Dietary Insufficiency in Infants

Intake adequacy poses greater problems in infants than in older children. The much reduced stores–to–daily intake ratio in newborns requires expansion during infancy and the demands of growth and development. Despite maternofetal transfer of cobalamin that favors the fetus even when mothers have borderline cobalamin status, the small cobalamin stores at birth must then be expanded to accommodate the demands of growth, neurologic maturation, and cognitive development. In early infancy, much (and often, all) of the diet consists of breastmilk. The severity and duration of any maternal restrictions, whether in cobalamin transfer to the fetus, breastfeeding, or both, can influence the degree of deficiency in the infant. Indeed, maternal cobalamin status at delivery correlates significantly with the infant’s status not just at birth but also 6 months later. Breastmilk cobalamin content is also influenced by maternal status.

Mild Dietary Insufficiency in Breastfed Infants.

A series of Norwegian studies beginning in 2001 documented unexplained elevations of serum MMA levels in 60% of normal, asymptomatic 4- to 6-month-old Norwegian infants whose mothers were not cobalamin deficient. The MMA elevation exceeded 1000 nmol/L in 30% of the infants, and median serum MMA was 780 nmol/L in infants aged 6 weeks to 6 months, compared with 290 nmol/L in newborns and 130 nmol/L in children aged 1 to 10 years. As Table 10-4 shows, plasma tHcy was also higher in the 6-week to 6-month age group, although not as strikingly abnormal as MMA. Cobalamin levels tended to be low-normal or, in only 8% of cases, slightly subnormal. These biochemical changes, which could also be easily reversed with cobalamin supplements, usually normalized spontaneously after several months. The absence of clinical signs of deficiency suggests that these normal infants had subclinical deficiency.

The explanation for the transient cobalamin status changes in early infancy has now become clearer. A recent longitudinal Danish study documented an unexplained, apparently spontaneous decline in breastmilk cobalamin levels in healthy, omnivore mothers 4 months after giving birth, often despite their taking cobalamin supplements. The authors associated this decline in breastmilk cobalamin with the transiently worse cobalamin status in exclusively breastfed infants compared with infants eating mixed diets ( Table 10-6 ). At month 9, the breastmilk cobalamin content had improved spontaneously, as had the cobalamin status of the infants (whose diet had also become mixed). The mothers’ cobalamin status remained unchanged throughout these fluctuations of breastmilk cobalamin. It remains possible that additional factors play a role because breastfed infants who also ingest a mixed diet showed somewhat similar cobalamin findings as exclusively breastfed infants at 4 and 9 months. The findings are compelling, but their surprisingly high frequency in seemingly normal mothers and infants requires clarification. More studies outside Scandinavia are needed as well. It is also important to note that this transient cobalamin deficiency of early infancy is subclinical. Convincing clinical accompaniments, including changes in hemoglobin and MCV levels, have not been reported. Finally, it is noteworthy that plasma folate levels remained unchanged throughout these transient cobalamin deficiencies but decreased sharply after the age of 1 year and continued to decline throughout childhood (see Table 10-4 ). The relatively high folate content of human breastmilk provides breastfed babies significantly better folate status than nonbreastfed infants.

TABLE 10-6

Relationships between Breastmilk Cobalamin Content and Maternal and Infant Cobalamin Status *

	Birth/2 wk Postpartum	At 4 mo	At 9 mo
Mean breast hindmilk	561 (760)	214 (290)	325 (440)
Cobalamin concentration (n = 25)
Paired mean plasma
Cobalamin levels:
Mothers (n = 60)	295 (400)	288 (390)	310 (420)
Infants (n = 60)	325 (440)	177 (240)	236 (320)
Mean serum MMA
Levels (nmol/L):
Mothers (n = 60)	180	170	160
Infants (n = 28)	270	440	240

Only gold members can continue reading. Log In or Register to continue

Share this:

• Click to share on Twitter (Opens in new window)
• Click to share on Facebook (Opens in new window)

Related

Related posts:

The Neonatal Erythrocyte and Its Disorders Diagnostic Approach to the Anemic Patient Acquired Disorders of Hemostasis Targeted Therapies in Oncology Pediatric Radiation Oncology Nonrhabdomyosarcoma Soft Tissue Sarcomas and Other Soft Tissue Tumors

Stay updated, free articles. Join our Telegram channel

Join