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either increased iron requirements (eg, growing children, pregnant women) or increased losses of iron (eg, menstruating women), iron requirements can exceed normal dietary supplies, and iron deficiency can develop.

A. Absorption

The average American diet contains 10–15 mg of elemental iron daily. A normal individual absorbs 5–10% of this iron, or about 0.5–1 mg daily. Iron is absorbed in the duodenum and proximal jejunum, although the more distal small intestine can absorb iron if necessary. Iron absorption increases in response to low iron stores or increased iron requirements. Total iron absorption increases to 1–2 mg/d in menstruating women and may be as high as 3–4 mg/d in pregnant women.

Iron is available in a wide variety of foods but is especially abundant in meat. The iron in meat protein can be efficiently absorbed, because heme iron in meat hemoglobin and myoglobin can be absorbed intact without first having to be dissociated into elemental iron (Figure 33–1). Iron in other foods, especially vegetables and grains, is often tightly bound to organic compounds and is much less available for absorption. Nonheme iron in foods and iron in inorganic iron salts and complexes must be reduced by a ferrireductase to ferrous iron (Fe2+) before it can be absorbed by intestinal mucosal cells.

Iron crosses the luminal membrane of the intestinal mucosal cell by two mechanisms: active transport of ferrous iron by the divalent metal transporter DMT1, and absorption of iron complexed with heme (Figure 33–1). Together with iron split

TABLE 33 1 Iron distribution in normal adults.1

1Values are based on data from various sources and assume that normal men weigh 80 kg and have a hemoglobin level of 16 g/dL and that normal women weigh 55 kg and have a hemoglobin level of 14 g/dL.

Adapted, with permission, from Kushner JP: Hypochromic anemias. In: Wyngaarden JB, Smith LH (editors). Cecil Textbook of Medicine, 18th ed. Saunders, 1988. Copyright Elsevier.

from absorbed heme, the newly absorbed iron can be actively transported into the blood across the basolateral membrane by a transporter known as ferroportin and oxidized to ferric iron (Fe3+) by the ferroxidase hephaestin. The liver-derived hepcidin inhibits intestinal cell iron release by binding to ferroportin and triggering its internalization and destruction. Excess iron is stored in intestinal epithelial cells as ferritin, a water-soluble complex consisting of a core of ferric hydroxide covered by a shell of a specialized storage protein called apoferritin.

B. Transport

Iron is transported in the plasma bound to transferrin, a β-globulin that can bind two molecules of ferric iron (Figure 33–1). The transferrin-iron complex enters maturing erythroid cells by a specific receptor mechanism. Transferrin receptors—integral membrane glycoproteins present in large numbers on proliferating erythroid cells—bind and internalize the transferrin-iron complex through the process of receptor-mediated endocytosis. In endosomes, the ferric iron is released, reduced to ferrous iron, and transported by DMT1 into the cytoplasm, where it is funneled into hemoglobin synthesis or stored as ferritin. The transferrintransferrin receptor complex is recycled to the cell membrane, where the transferrin dissociates and returns to the plasma. This process provides an efficient mechanism for supplying the iron required by developing red blood cells.

Increased erythropoiesis is associated with an increase in the number of transferrin receptors on developing erythroid cells and a reduction in hepatic hepcidin release. Iron store depletion and iron deficiency anemia are associated with an increased concentration of serum transferrin.

C. Storage

In addition to the storage of iron in intestinal mucosal cells, iron is also stored, primarily as ferritin, in macrophages in the liver, spleen, and bone, and in parenchymal liver cells (Figure 33–1). The mobilization of iron from macrophages and hepatocytes is primarily controlled by hepcidin regulation of ferroportin activity.

Low hepcidin concentrations result in iron release from these storage sites; high hepcidin concentrations inhibit iron release. Ferritin is detectable in serum. Since the ferritin present in serum is in equilibrium with storage ferritin in reticuloendothelial tissues, the serum ferritin level can be used to estimate total body iron stores.

D. Elimination

There is no mechanism for excretion of iron. Small amounts are lost in the feces by exfoliation of intestinal mucosal cells, and trace amounts are excreted in bile, urine, and sweat. These losses account for no more than 1 mg of iron per day. Because the body’s ability to excrete iron is so limited, regulation of iron balance must be achieved by changing intestinal absorption and storage of iron in response to the body’s needs. As noted below, impaired regulation of iron absorption leads to serious pathology.

Clinical Pharmacology

A. Indications for the Use of Iron

The only clinical indication for the use of iron preparations is the treatment or prevention of iron deficiency anemia. This manifests as a hypochromic, microcytic anemia in which the erythrocyte mean cell volume (MCV) and the mean cell hemoglobin concentration are low (Table 33–2). Iron deficiency is commonly seen in populations with increased iron requirements. These include infants, especially premature infants; children during rapid growth periods; pregnant and lactating women; and patients with chronic kidney disease who lose erythrocytes at a relatively high rate during hemodialysis and also form them at a high rate as a result of treatment with the erythrocyte growth factor erythropoietin (see below). Inadequate iron absorption also can cause iron deficiency. This is seen after gastrectomy and in patients with severe small bowel disease that results in generalized malabsorption.

TABLE 33 2 Distinguishing features of the nutritional anemias.

MCV, mean cell volume; MCHC, mean cell hemoglobin concentration; SI, serum iron; TIBC, transferrin iron-binding capacity.

The most common cause of iron deficiency in adults is blood loss. Menstruating women lose about 30 mg of iron with each menstrual period; women with heavy menstrual bleeding may lose much more. Thus, many premenopausal women have low iron stores or even iron deficiency. In men and postmenopausal women, the most common site of blood loss is the gastrointestinal tract. Patients with unexplained iron deficiency anemia should be evaluated for occult gastrointestinal bleeding.

B. Treatment

Iron deficiency anemia is treated with oral or parenteral iron preparations. Oral iron corrects the anemia just as rapidly and completely as parenteral iron in most cases if iron absorption from the gastrointestinal tract is normal. An exception is the high requirement for iron of patients with advanced chronic kidney disease who are undergoing hemodialysis and treatment with erythropoietin; for these patients, parenteral iron administration is preferred.

1.Oralirontherapy—A wide variety of oral iron preparations is available. Because ferrous iron is most efficiently absorbed, ferrous salts should be used. Ferrous sulfate, ferrous gluconate, and ferrous fumarate are all effective and inexpensive and are recommended for the treatment of most patients.

Different iron salts provide different amounts of elemental iron, as shown in Table 33–3. In an iron-deficient individual, about 50–100 mg of iron can be incorporated into hemoglobin daily, and about 25% of oral iron given as ferrous salt can be absorbed. Therefore, 200–400 mg of elemental iron should be given daily to correct iron deficiency most rapidly. Patients unable to tolerate such large doses of iron can be given lower daily doses of iron, which results in slower but still complete correction of iron deficiency. Treatment with oral iron should be continued for 3–6 months after correction of the cause of the iron loss. This corrects the anemia and replenishes iron stores.

Common adverse effects of oral iron therapy include nausea, epigastric discomfort, abdominal cramps, constipation, and diarrhea. These effects are usually dose-related and often can be overcome by lowering the daily dose of iron or by taking the tablets immediately after or with meals. Some patients have less severe

TABLE 33 3 Some commonly used oral iron preparations.

gastrointestinal adverse effects with one iron salt than another and benefit from changing preparations. Patients taking oral iron develop black stools; this has no clinical significance in itself but may obscure the diagnosis of continued gastrointestinal blood loss.

2. Parenteral iron therapy—Parenteral therapy should be reserved for patients with documented iron deficiency who are unable to tolerate or absorb oral iron and for patients with extensive chronic anemia who cannot be maintained with oral iron alone. This includes patients with advanced chronic renal disease requiring hemodialysis and treatment with erythropoietin, various postgastrectomy conditions and previous small bowel resection, inflammatory bowel disease involving the proximal small bowel, and malabsorption syndromes.

The challenge with parenteral iron therapy is that parenteral administration of inorganic free ferric iron produces serious dosedependent toxicity, which severely limits the dose that can be administered. However, when the ferric iron is formulated as a colloid containing particles with a core of iron oxyhydroxide surrounded by a core of carbohydrate, bioactive iron is released slowly from the stable colloid particles. In the United States, the three traditional forms of parenteral iron are iron dextran, sodium ferric gluconate complex, and iron sucrose. Two newer preparations are available (see below).

Iron dextran is a stable complex of ferric oxyhydroxide and dextran polymers containing 50 mg of elemental iron per milliliter of solution. It can be given by deep intramuscular injection or by intravenous infusion, although the intravenous route is used most commonly. Intravenous administration eliminates the local pain and tissue staining that often occur with the intramuscular route and allows delivery of the entire dose of iron necessary to correct the iron deficiency at one time. Adverse effects of intravenous iron dextran therapy include headache, light-headedness, fever, arthralgias, nausea and vomiting, back pain, flushing, urticaria, bronchospasm, and, rarely, anaphylaxis and death. Owing to the risk of a hypersensitivity reaction, a small test dose of iron dextran should always be given before full intramuscular or intravenous doses are given. Patients with a strong history of allergy and patients who have previously received parenteral iron dextran are more likely to have hypersensitivity reactions after treatment with parenteral iron dextran. The iron dextran formulations used clinically are distinguishable as high-molecular-weight and low-molecular-weight forms. In the United States, the INFeD preparation is a low-molecular-weight form while Dexferrum is a high-molecular-weight form. Clinical data—primarily from observational studies—indicate that the risk of anaphylaxis is largely associated with high-molecular-weight formulations.

Sodium ferric gluconate complex and iron-sucrose complex are alternative parenteral iron preparations. Ferric carboxymaltose is a colloidal iron preparation embedded within a carbohydrate polymer. Ferumoxytol is a superparamagnetic iron oxide nanoparticle coated with carbohydrate. The carbohydrate shell is removed in the reticuloendothelial system, allowing the iron to be stored as ferritin, or released to transferrin. Ferumoxytol may interfere with magnetic resonance imaging (MRI) studies. Thus if

imaging is needed, MRI should be performed prior to ferumoxytol therapy or alternative imaging modality used if needed soon after dosing. The U.S. Food and Drug Administration (FDA) has issued a black box warning about risk of potentially fatal allergic reactions associated with the use of ferumoxytol.

For patients treated chronically with parenteral iron, it is important to monitor iron storage levels to avoid the serious toxicity associated with iron overload. Unlike oral iron therapy, which is subject to the regulatory mechanism provided by the intestinal uptake system, parenteral administration—which bypasses this regulatory system—can deliver more iron than can be safely stored. Iron stores can be estimated on the basis of serum concentrations of ferritin and the transferrin saturation, which is the ratio of the total serum iron concentration to the total iron-binding capacity (TIBC).

sickle cell anemia, aplastic anemia, etc. Deferiprone rarely has been associated with agranulocytosis; thus weekly monitoring of the CBC is required for patients treated with this drug.

VITAMIN B12

Vitamin B12 (cobalamin) serves as a cofactor for several essential biochemical reactions in humans. Deficiency of vitamin B12 leads to megaloblastic anemia (Table 33–2), gastrointestinal symptoms, and neurologic abnormalities. Although deficiency of vitamin B12 due to an inadequate supply in the diet is unusual, deficiency of B12 in adults—especially older adults—due to inadequate absorption of dietary vitamin B12 is a relatively common and easily treated disorder.

Clinical Toxicity

A. Acute Iron Toxicity

Acute iron toxicity is seen almost exclusively in young children who accidentally ingest iron tablets. As few as 10 tablets of any of the commonly available oral iron preparations can be lethal in young children. Adult patients taking oral iron preparations should be instructed to store tablets in child-proof containers out of the reach of children. Children who are poisoned with oral iron experience necrotizing gastroenteritis with vomiting, abdominal pain, and bloody diarrhea followed by shock, lethargy, and dyspnea. Subsequently, improvement is often noted, but this may be followed by severe metabolic acidosis, coma, and death. Urgent treatment is necessary. Whole bowel irrigation (see Chapter 58) should be performed to flush out unabsorbed pills. Deferoxamine, a potent iron-chelating compound, can be given intravenously to bind iron that has already been absorbed and to promote its excretion in urine and feces. Activated charcoal, a highly effective adsorbent for most toxins, does not bind iron and thus is ineffective. Appropriate supportive therapy for gastrointestinal bleeding, metabolic acidosis, and shock must also be provided.

B. Chronic Iron Toxicity

Chronic iron toxicity (iron overload), also known as hemochromatosis, results when excess iron is deposited in the heart, liver, pancreas, and other organs. It can lead to organ failure and death. It most commonly occurs in patients with inherited hemochromatosis, a disorder characterized by excessive iron absorption, and in patients who receive many red cell transfusions over a long period of time (eg, individuals with β-thalassemia).

Chronic iron overload in the absence of anemia is most efficiently treated by intermittent phlebotomy. One unit of blood can be removed every week or so until all of the excess iron is removed. Iron chelation therapy using parenteral deferoxamine or the oral iron chelators deferasirox or deferiprone (see Chapter 57) is less efficient as well as more complicated, expensive, and hazardous, but it may be the only option for iron overload that cannot be managed by phlebotomy, as is the case for many individuals with inherited and acquired causes of refractory anemia such as thalassemia major,

Chemistry

Vitamin B12 consists of a porphyrin-like ring with a central cobalt atom attached to a nucleotide. Various organic groups may be covalently bound to the cobalt atom, forming different cobalamins. Deoxyadenosylcobalamin and methylcobalamin are the active forms of the vitamin in humans. Cyanocobalamin and hydroxocobalamin (both available for therapeutic use) and other cobalamins found in food sources are converted to the active forms. The ultimate source of vitamin B12 is from microbial synthesis; the vitamin is not synthesized by animals or plants. The chief dietary source of vitamin B12 is microbially derived vitamin B12 in meat (especially liver), eggs, and dairy products. Vitamin B12 is sometimes called extrinsic factor to differentiate it from intrinsic factor, a protein secreted by the stomach that is required for gastrointestinal uptake of dietary vitamin B12.

Pharmacokinetics

The average American diet contains 5–30 mcg of vitamin B12 daily, 1–5 mcg of which is usually absorbed. The vitamin is avidly stored, primarily in the liver, with an average adult having a total vitamin B12 storage pool of 3000–5000 mcg. Only trace amounts of vitamin B12 are normally lost in urine and stool. Because the normal daily requirements of vitamin B12 are only about 2 mcg, it would take about 5 years for all of the stored vitamin B12 to be exhausted and for megaloblastic anemia to develop if B12 absorption were stopped. Vitamin B12 is absorbed after it complexes with intrinsic factor, a glycoprotein secreted by the parietal cells of the gastric mucosa. Intrinsic factor combines with the vitamin B12 that is liberated from dietary sources in the stomach and duodenum, and the intrinsic factor–vitamin B12 complex is subsequently absorbed in the distal ileum by a highly selective receptor-mediated transport system. Vitamin B12 deficiency in humans most often results from malabsorption of vitamin B12 due either to lack of intrinsic factor or to loss or malfunction of the absorptive mechanism in the distal ileum. Nutritional deficiency is rare but may be seen in strict vegetarians after many years without meat, eggs, or dairy products.

Once absorbed, vitamin B12 is transported to the various cells of the body bound to a family of specialized glycoproteins, transcobalamin I, II, and III. Excess vitamin B12 is stored in the liver.

Pharmacodynamics

Two essential enzymatic reactions in humans require vitamin B12 (Figure 33–2). In one, methylcobalamin serves as an intermediate in the transfer of a methyl group from N5-methyltetrahydrofolate to homocysteine, forming methionine (Figure 33–2A; Figure 33–3, section 1). Without vitamin B12, conversion of the major dietary and storage folate—N5-methyltetrahydrofolate—to tetrahydrofolate, the precursor of folate cofactors, cannot occur. As a result, vitamin B12 deficiency leads to deficiency of folate cofactors necessary for several biochemical reactions involving the transfer of one-car- bon groups. In particular, the depletion of tetrahydrofolate prevents synthesis of adequate supplies of the deoxythymidylate (dTMP) and purines required for DNA synthesis in rapidly dividing cells, as shown in Figure 33–3, section 2. The accumulation of folate as N5-methyltetrahydrofolate and the associated depletion of tetrahydrofolate cofactors in vitamin B12 deficiency have been referred to as the “methylfolate trap.” This is the biochemical step whereby vitamin B12 and folic acid metabolism are linked, and it explains why the megaloblastic anemia of vitamin B12 deficiency can be partially corrected by ingestion of large amounts of folic acid. Folic acid can be reduced to dihydrofolate by the enzyme dihydrofolate

A. Methyl transfer

N5-Methyltetrahydrofolate Tetrahydrofolate

Cobalamin Methylcobalamin

Methionine Homocysteine

B. Isomerization of L-Methylmalonyl-CoA

Methylmalonyl-CoA

mutase

L-Methylmalonyl-CoA Succinyl-CoA Deoxyadenosylcobalamin

FIGURE 33 2 Enzymatic reactions that use vitamin B12.

Purines

reductase (Figure 33–3, section 3) and thereby serve as a source of the tetrahydrofolate required for synthesis of the purines and dTMP required for DNA synthesis.

Vitamin B12 deficiency causes the accumulation of homocysteine due to reduced formation of methylcobalamin, which is required for the conversion of homocysteine to methionine (Figure 33–3, section 1). The increase in serum homocysteine can be used to help establish a diagnosis of vitamin B12 deficiency (Table 33–2). There is evidence from observational studies that elevated serum homocysteine increases the risk of atherosclerotic cardiovascular disease. However, randomized clinical trials have not shown a definitive reduction in cardiovascular events (myocardial infarction, stroke) in patients receiving vitamin supplementation that lowers serum homocysteine.

The other reaction that requires vitamin B12 is isomerization of methylmalonyl-CoA to succinyl-CoA by the enzyme methylmalonyl-CoA mutase (Figure 33–2B). In vitamin B12 deficiency, this conversion cannot take place and the substrate, methylmalonyl-CoA, as well as methylmalonic acid accumulate. The increase in serum and urine concentrations of methylmalonic acid can be used to support a diagnosis of vitamin B12 deficiency (Table 33–2). In the past, it was thought that abnormal accumulation of methylmalonyl-CoA causes the neurologic manifestations of vitamin B12 deficiency. However, newer evidence implicates the disruption of the methionine synthesis pathway as the cause of neurologic problems. Whatever the biochemical explanation for neurologic damage, the important point is that administration of folic acid in the setting of vitamin B12 deficiency will not prevent neurologic manifestations even though it will largely correct the anemia caused by the vitamin B12 deficiency.

Clinical Pharmacology

Vitamin B12 is used to treat or prevent deficiency. The most characteristic clinical manifestation of vitamin B12 deficiency is megaloblastic, macrocytic anemia (Table 33–2), often with associated mild or moderate leukopenia or thrombocytopenia (or both), and a characteristic hypercellular bone marrow with an accumulation of megaloblastic erythroid and other precursor cells. The neurologic syndrome associated with vitamin B12 deficiency usually begins with paresthesias in peripheral nerves and weakness and progresses to spasticity, ataxia, and other central nervous system dysfunctions. Correction of vitamin B12 deficiency arrests the progression of neurologic disease, but it may not fully reverse neurologic symptoms that have been present for several months. Although most patients with neurologic abnormalities caused by vitamin B12 deficiency have megaloblastic anemia when first seen, occasional patients have few if any hematologic abnormalities.

Once a diagnosis of megaloblastic anemia is made, it must be determined whether vitamin B12 or folic acid deficiency is the cause. (Other causes of megaloblastic anemia are very rare.) This can usually be accomplished by measuring serum levels of the vitamins. The Schilling test, which measures absorption and urinary excretion of radioactively labeled vitamin B12, can be used to further define the mechanism of vitamin B12 malabsorption when this is found to be the cause of the megaloblastic anemia.

The most common causes of vitamin B12 deficiency are pernicious anemia, partial or total gastrectomy, and conditions that affect the distal ileum, such as malabsorption syndromes, inflammatory bowel disease, or small bowel resection. Strict vegans eating a diet free of meat and dairy products may become B12 deficient.

Pernicious anemia results from defective secretion of intrinsic factor by the gastric mucosal cells. Patients with pernicious anemia have gastric atrophy and fail to secrete intrinsic factor (as well as hydrochloric acid). These patients frequently have autoantibodies to intrinsic factor. Historically, the Schilling test demonstrated diminished absorption of radioactively labeled vitamin B12, which is corrected when intrinsic factor is administered with radioactive B12, since the vitamin can then be normally absorbed. This test is now rarely performed due to use of radioactivity in the assay.

Vitamin B12 deficiency also occurs when the region of the distal ileum that absorbs the vitamin B12–intrinsic factor complex is damaged, as when the ileum is involved with inflammatory bowel disease or when the ileum is surgically resected. In these situations, radioactively labeled vitamin B12 is not absorbed in the Schilling test, even when intrinsic factor is added. Rare cases of vitamin B12 deficiency in children have been found to be secondary to congenital deficiency of intrinsic factor or to defects of the receptor sites for vitamin B12–intrinsic factor complex located in the distal ileum. Alternatives to the Schilling test include testing for intrinsic factor antibodies and testing for elevated homocysteine and methylmalonic acid levels (Figure 33–2) to make a diagnosis of pernicious anemia with high sensitivity and specificity.

Almost all cases of vitamin B12 deficiency are caused by malabsorption of the vitamin; therefore, parenteral injections of vitamin B12 are required for therapy. For patients with potentially reversible diseases, the underlying disease should be treated after initial treatment with parenteral vitamin B12. Most patients, however, do not have curable deficiency syndromes and require lifelong treatment with vitamin B12.

Vitamin B12 for parenteral injection is available as cyanocobalamin or hydroxocobalamin. Hydroxocobalamin is preferred because it is more highly protein-bound and therefore remains longer in the circulation. Initial therapy should consist of 100–1000 mcg of vitamin B12 intramuscularly daily or every other day for 1–2 weeks to replenish body stores. Maintenance therapy consists of 100–1000 mcg intramuscularly once a month for life. If neurologic abnormalities are present, maintenance therapy injections should be given every 1–2 weeks for 6 months before switching to monthly injections. Oral vitamin B12–intrinsic factor mixtures and liver extracts should not be used to treat vitamin B12 deficiency; however, oral doses of 1000 mcg of vitamin B12 daily are usually sufficient to treat patients with pernicious anemia who refuse or cannot tolerate the injections. After pernicious anemia is in remission following parenteral vitamin B12 therapy, the vitamin can be administered intranasally as a spray or gel.

FOLIC ACID

Reduced forms of folic acid are required for essential biochemical reactions that provide precursors for the synthesis of amino acids, purines, and DNA. Folate deficiency is relatively common, even

Starting in January 1998, all products made from enriched grains in the United States and Canada were required to be supplemented with folic acid.These rulings were issued to reduce the incidence of congenital neural tube defects (NTDs). Epidemiologic studies show a strong correlation between maternal folic acid deficiency and the incidence of NTDs such as spina bifida and anencephaly. The requirement for folic acid supplementation is a public health measureaimedat thesignificantnumber ofwomenwhodonot receive prenatal care and are not aware of the importance of adequate folic acid ingestion for preventing birth defects in their infants. Observational studies from countries that supplement grains with folic acid have found that supplementation is associated with a significant (20–25%) reduction in NTD rates. Observational studies also suggest that rates of other types of congenital anomalies (heart and orofacial) have fallen since supplementation began.

There may be an added benefit for adults. N5-Methyl- tetrahydrofolate is required for the conversion of homocysteine to methionine (Figure 33–2; Figure 33–3, reaction 1). Impaired synthesis of N5-methyltetrahydrofolate results in elevated serum concentrations of homocysteine. Data from several sources suggest a positive correlation between elevated serum homocysteine and occlusive vascular diseases such as ischemic heart disease and stroke. Clinical data suggest that the folate supplementation program has improved the folate status and reduced the prevalence of hyperhomocysteinemia in a population of middle-aged and older adults who did not use vitamin supplements. There is also evidence that adequate folic acid protects against several cancers, including colorectal, breast, and cervical cancer.

though the deficiency is easily corrected by administration of folic acid. The consequences of folate deficiency go beyond the problem of anemia because folate deficiency is implicated as a cause of congenital malformations in newborns and may play a role in vascular disease (see Box: Folic Acid Supplementation: A Public Health Dilemma).

Chemistry

Folic acid (pteroylglutamic acid) is composed of a heterocycle (pteridine), p-aminobenzoic acid, and glutamic acid (Figure 33–4). Various numbers of glutamic acid moieties are attached to the pteroyl portion of the molecule, resulting in monoglutamates, triglutamates, or polyglutamates. Folic acid undergoes reduction, catalyzed by the enzyme dihydrofolate reductase (“folate reductase”), to give dihydrofolic acid (Figure 33–3, section 3). Tetrahydrofolate is subsequently transformed to folate cofactors possessing one-carbon units attached to the 5-nitrogen, to the 10-nitrogen, or to both positions (Figure 33–3). Folate cofactors are interconvertible by various enzymatic reactions and serve the important biochemical function of donating one-carbon units at various levels of oxidation. In most of these, tetrahydrofolate is regenerated and becomes available for reutilization.

Although the potential benefits of supplemental folic acid during pregnancy are compelling, the decision to require folic acid in grains was controversial. As described in the text, ingestion of folic acid can partially or totally correct the anemia caused by vitamin B12 deficiency. However, folic acid supplementation does not prevent the potentially irreversible neurologic damage caused by vitamin B12 deficiency. People with pernicious anemia and other forms of vitamin B12 deficiency are usually identified because of signs and symptoms of anemia, which typically occur before neurologic symptoms. Some opponents of folic acid supplementation were concerned that increased folic acid intake in the general population would mask vitamin B12 deficiency and increase the prevalence of neurologic disease in the elderly population. To put this in perspective, approximately 4000 pregnancies, including 2500 live births, in the United States each year are affected by NTDs. In contrast, it is estimated that more than 10% of the elderly population in the United States, or several million people, are at risk for the neuropsychiatric complications of vitamin B12 deficiency. In acknowledgment of this controversy, the FDA kept its requirements for folic acid supplementation at a somewhat low level. There is also concern based on observational and prospective clinical trials that high folic acid levels can increase the risk of some diseases, such as colorectal cancer, for which folic acid may exhibit a bell-shaped curve. Further research is needed to more accurately define the optimal level of folic acid fortification in food and recommendations for folic acid supplementation in different populations and age groups.

Pharmacokinetics

The average American diet contains 500–700 mcg of folates daily, 50–200 mcg of which is usually absorbed, depending on metabolic requirements. Pregnant women may absorb as much as 300–400 mcg of folic acid daily. Various forms of folic acid are

Folic acid

FIGURE 33 4 The structure of folic acid. (Reproduced, with permission, from Murray RK et al: Harper’s Biochemistry, 24th ed. McGraw-Hill, 1996. Copyright © The McGraw-Hill Companies, Inc.)

present in a wide variety of plant and animal tissues; the richest sources are yeast, liver, kidney, and green vegetables. Normally, 5–20 mg of folates is stored in the liver and other tissues. Folates are excreted in the urine and stool and are also destroyed by catabolism, so serum levels fall within a few days when intake is diminished. Because body stores of folates are relatively low and daily requirements high, folic acid deficiency and megaloblastic anemia can develop within 1–6 months after the intake of folic acid stops, depending on the patient’s nutritional status and the rate of folate utilization.

Unaltered folic acid is readily and completely absorbed in the proximal jejunum. Dietary folates, however, consist primarily of polyglutamate forms of N5-methyltetrahydrofolate. Before absorption, all but one of the glutamyl residues of the polyglutamates must be hydrolyzed by the enzyme α-1-glutamyl transferase (“conjugase”) within the brush border of the intestinal mucosa. The monoglutamate N5-methyltetrahydrofolate is subsequently transported into the bloodstream by both active and passive transport and is then widely distributed throughout the body. Inside cells, N5-methyltetrahydro-folate is converted to tetrahydrofolate by the demethylation reaction that requires vitamin B12 (Figure 33–3, section 1).

Pharmacodynamics

Tetrahydrofolate cofactors participate in one-carbon transfer reactions. As described earlier in the discussion of vitamin B12, one of these essential reactions produces the dTMP needed for DNA synthesis. In this reaction, the enzyme thymidylate synthase catalyzes the transfer of the one-carbon unit of N5, N10-methylenetetrahydrofolate to deoxyuridine monophosphate (dUMP) to form dTMP (Figure 33–3, section 2). Unlike all the other enzymatic reactions that use folate cofactors, in this reaction the cofactor is oxidized to dihydrofolate, and for each mole of dTMP produced, 1 mole of tetrahydrofolate is consumed. In rapidly proliferating tissues, considerable amounts of tetrahydrofolate are consumed in this reaction, and continued DNA synthesis requires continued regeneration of tetrahydrofolate by reduction of dihydrofolate, catalyzed by the enzyme dihydrofolate reductase. The tetrahydrofolate thus produced can then reform the cofactor N5, N10-methylenetetrahydrofolate by the action of serine transhydroxymethylase and thus allow for the continued synthesis of dTMP. The combined catalytic activities of dTMP synthase, dihydrofolate reductase, and serine transhydroxymethylase are referred to as the dTMP synthesis cycle. Enzymes in the dTMP cycle are the targets of two anti-cancer drugs: methotrexate inhibits dihydrofolate reductase, and a metabolite of 5-fluorouracil inhibits thymidylate synthase (see Chapter 54).

Cofactors of tetrahydrofolate participate in several other essential reactions. N5-Methylenetetrahydrofolate is required for the vitamin B12-dependent reaction that generates methionine from homocysteine (Figure 33–2A; Figure 33–3, section 1). In addition, tetrahydrofolate cofactors donate one-carbon units during the de novo synthesis of essential purines. In these reactions, tetrahydrofolate is regenerated and can reenter the tetrahydrofolate cofactor pool.

Clinical Pharmacology

Folate deficiency results in a megaloblastic anemia that is microscopically indistinguishable from the anemia caused by vitamin B12 deficiency (see above). However, folate deficiency does not cause the characteristic neurologic syndrome seen in vitamin B12 deficiency. In patients with megaloblastic anemia, folate status is assessed with assays for serum folate or for red blood cell folate. Red blood cell folate levels are often of greater diagnostic value than serum levels, because serum folate levels tend to be labile and do not necessarily reflect tissue levels.

Folic acid deficiency is often caused by inadequate dietary intake of folates. Patients with alcohol dependence and patients with liver disease can develop folic acid deficiency because of poor diet and diminished hepatic storage of folates. Pregnant women and patients with hemolytic anemia have increased folate requirements and may become folic acid-deficient, especially if their diets are marginal. Evidence implicates maternal folic acid deficiency in the occurrence of fetal neural tube defects. (See Box: Folic Acid Supplementation: A Public Health Dilemma.) Patients with malabsorption syndromes also frequently develop folic acid deficiency. Patients who require renal dialysis are at risk of folic acid deficiency because folates are removed from the plasma during the dialysis procedure.

Folic acid deficiency can be caused by drugs. Methotrexate and, to a lesser extent, trimethoprim and pyrimethamine, inhibit dihydrofolate reductase and may result in a deficiency of folate cofactors and ultimately in megaloblastic anemia. Long-term therapy with phenytoin also can cause folate deficiency, but it only rarely causes megaloblastic anemia.

Parenteral administration of folic acid is rarely necessary, since oral folic acid is well absorbed even in patients with malabsorption syndromes. A dose of 1 mg folic acid orally daily is sufficient to reverse megaloblastic anemia, restore normal serum folate levels, and replenish body stores of folates in almost all patients. Therapy should be continued until the underlying cause of the deficiency is removed or corrected. Therapy may be required indefinitely for patients with malabsorption or dietary inadequacy. Folic acid supplementation to prevent folic acid deficiency should be considered in high-risk patients, including pregnant women, patients with alcohol dependence, hemolytic anemia, liver disease, or certain skin diseases, and patients on renal dialysis.

■ HEMATOPOIETIC GROWTH FACTORS

The hematopoietic growth factors are glycoprotein hormones that regulate the proliferation and differentiation of hematopoietic progenitor cells in the bone marrow. The first growth factors to be identified were called colony-stimulating factors because they could stimulate the growth of colonies of various bone marrow progenitor cells in vitro. Many of these growth factors have been purified and cloned, and their effects on hematopoiesis have been extensively studied. Quantities of these growth factors sufficient for clinical use are produced by recombinant DNA technology.

Of the known hematopoietic growth factors, erythropoietin (epoetinalfa and epoetin beta), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 11 (IL-11), and thrombopoietin receptor agonists (romiplostim and eltrombopag) are currently in clinical use.

The hematopoietic growth factors and drugs that mimic their action have complex effects on the function of a wide variety of cell types, including nonhematologic cells. Their usefulness in other areas of medicine, particularly as potential anti-cancer and anti-inflammatory drugs, is being investigated.

ERYTHROPOIETIN

Chemistry & Pharmacokinetics

Erythropoietin, a 34to 39-kDa glycoprotein, was the first human hematopoietic growth factor to be isolated. It was originally purified from the urine of patients with severe anemia. Recombinant human erythropoietin (rHuEPO, epoetin alfa) is produced in a mammalian cell expression system. After intravenous administration, erythropoietin has a serum half-life of 4–13 hours in patients with chronic renal failure. It is not cleared by dialysis. It is measured in international units (IU). Darbepoetin alfa is a modified form of erythropoietin that is more heavily glycosylated as a result of changes in amino acids. Darbepoetin alfa has a twofold to threefold longer half-life than epoetin alfa. Methoxy polyethylene glycol–epoetin beta is an isoform of erythropoietin covalently attached to a long polyethylene glycol polymer. This long-lived recombinant product is administered as a single intravenous or subcutaneous dose at 2-week or monthly intervals, whereas epoetin alfa is generally administered three times a week and darbepoetin is administered weekly.

Pharmacodynamics

Erythropoietin stimulates erythroid proliferation and differentiation by interacting with erythropoietin receptors on red cell progenitors. The erythropoietin receptor is a member of the JAK/ STAT superfamily of cytokine receptors that use protein phosphorylation and transcription factor activation to regulate cellular function (see Chapter 2). Erythropoietin also induces release of reticulocytes from the bone marrow. Endogenous erythropoietin is produced primarily in the kidney. In response to tissue hypoxia, more erythropoietin is produced through an increased rate of transcription of the erythropoietin gene. This results in correction of the anemia, provided that the bone marrow response is not impaired by red cell nutritional deficiency (especially iron deficiency), primary bone marrow disorders (see below), or bone marrow suppression from drugs or chronic diseases.

Normally, an inverse relationship exists between the hematocrit or hemoglobin level and the serum erythropoietin level. Nonanemic individuals have serum erythropoietin levels of less than 20 IU/L. As the hematocrit and hemoglobin levels fall and anemia becomes more severe, the serum erythropoietin level rises exponentially. Patients with moderately severe anemia usually have

erythropoietin levels in the 100–500 IU/L range, and patients with severe anemia may have levels of thousands of IU/L. The most important exception to this inverse relationship is in the anemia of chronic renal failure. In patients with renal disease, erythropoietin levels are usually low because the kidneys cannot produce the growth factor. These are the patients most likely to respond to treatment with exogenous erythropoietin. In most primary bone marrow disorders (aplastic anemia, leukemias, myeloproliferative and myelodysplastic disorders, etc) and most nutritional and secondary anemias, endogenous erythropoietin levels are high, so there is less likelihood of a response to exogenous erythropoietin (but see below).

Clinical Pharmacology

The availability of erythropoiesis-stimulating agents (ESAs) has had a significant positive impact for patients with several types of anemia (Table 33–4). The ESAs consistently improve the hematocrit and hemoglobin level, often eliminate the need for transfusions, and reliably improve quality of life indices. The ESAs are used routinely in patients with anemia secondary to chronic kidney disease. In patients treated with an ESA, an increase in reticulocyte count is usually observed in about 10 days and an increase in hematocrit and hemoglobin levels in 2–6 weeks. Dosages of ESAs are adjusted to maintain a target hemoglobin up to, but not exceeding, 10–12 g/dL. To support the increased erythropoiesis, nearly all patients with chronic kidney disease require oral or parenteral iron supplementation. Folate supplementation may also be necessary in some patients.

In selected patients, erythropoietin is also used to reduce the need for red blood cell transfusion in patients undergoing myelosuppressive cancer chemotherapy who have a hemoglobin level of less than 10 g/dL, and for selected patients with low-risk myelodysplastic syndromes and anemia requiring red blood cell transfusion. Patients who have disproportionately low serum erythropoietin levels for their degree of anemia are most likely to respond to treatment. Patients with endogenous erythropoietin levels of less than 100 IU/L have the best chance of response, although patients with erythropoietin levels between 100 and 500 IU/L respond occasionally. Methoxy polyethylene glycol– epoetin beta should not be used for treatment of anemia caused by cancer chemotherapy because a clinical trial found significantly more deaths among patients receiving this form of erythropoietin.

Erythropoietin is one of the drugs commonly used illegally by endurance athletes to enhance performance. Other methods such as autologous transfusion of red cells or use of androgens also have been used to increase hemoglobin. “Blood doping” constitutes a serious health risk to athletes and as a form of cheating is universally banned and routinely tested for in athletic events.

Toxicity

The most common adverse effects of erythropoietin are hypertension and thrombotic complications. ESAs increase the risk of serious cardiovascular events, thromboembolic events, stroke, and mortality in clinical studies when given to support hemoglobin

levels greater than 11 g/dL. In addition, a meta-analysis of 51 placebo-controlled trials of ESAs in cancer patients reported an increased rate of all-cause mortality and venous thrombosis in those receiving an ESA. Based on the accumulated evidence, it is recommended that the hemoglobin level not exceed 11 g/dL in patients with chronic kidney disease receiving an ESA, and that ESAs be used conservatively in cancer patients (eg, when hemoglobin levels are <10 g/dL) and with the lowest dose needed to avoid transfusion. It is further recommended that ESAs not be used when a cancer therapy is being given with curative intent.

Allergic reactions to ESAs have been infrequent. There have been a small number of cases of pure red cell aplasia (PRCA) accompanied by neutralizing antibodies to erythropoietin. PRCA was most commonly seen in dialysis patients treated subcutaneously for a long period with a particular form of epoetin alfa (Eprex with a polysorbate 80 stabilizer rather than human serum albumin) that is not available in the United States. After regulatory agencies required that Eprex be administered intravenously rather than subcutaneously, the rate of ESA-associated PRCA diminished. However, rare cases have still been seen with all ESAs administered subcutaneously for long periods to patients with chronic kidney disease.

MYELOID GROWTH FACTORS

Chemistry & Pharmacokinetics

G-CSF and GM-CSF, the two myeloid growth factors currently available for clinical use, were originally purified from cultured human cell lines (Table 33–4). Recombinant human G-CSF (rHuG-CSF; filgrastim) is produced in a bacterial expression system. It is a nonglycosylated peptide of 175 amino acids, with a molecular weight of 18 kDa. Tbo-filgastrim is similar

to filgrastim, with minor structural differences and equivalent activity. Recombinant human GM-CSF (rHuGM-CSF; sargramostim) is produced in a yeast expression system. It is a partially glycosylated peptide of 127 amino acids, comprising three molecular species with molecular weights of 15,500, 15,800, and 19,500. These preparations have serum half-lives of 2–7 hours after intravenous or subcutaneous administration. Pegfilgrastim, a covalent conjugation product of filgrastim and a form of polyethylene glycol, has a much longer serum half-life than recombinant G-CSF, and it can be injected once per myelosuppressive chemotherapy cycle instead of daily for several days. Lenograstim, used widely in Europe, is a glycosylated form of recombinant G-CSF.

Pharmacodynamics

The myeloid growth factors stimulate proliferation and differentiation by interacting with specific receptors found on myeloid progenitor cells. Like the erythropoietin receptor, these receptors are members of the JAK/STAT superfamily (see Chapter 2). G-CSF stimulates proliferation and differentiation of progenitors already committed to the neutrophil lineage. It also activates the phagocytic activity of mature neutrophils and prolongs their survival in the circulation. G-CSF also has a remarkable ability to mobilize hematopoietic stem cells, ie, to increase their concentration in peripheral blood. This biologic effect underlies a major advance in transplantation—the use of peripheral blood stem cells (PBSCs) rather than bone marrow stem cells for autologous and allogeneic hematopoietic stem cell transplantation (see below).

GM-CSF has broader biologic actions than G-CSF. It is a multipotential hematopoietic growth factor that stimulates proliferation and differentiation of early and late granulocytic progenitor cells as well as erythroid and megakaryocyte progenitors. Like G-CSF, GM-CSF also stimulates the function of mature