Goodman and Gilman Manual of Pharmacology and Therapeutics

Section IV
Inflammation, Immunomodulation, and Hematopoiesis

Chapter 37
Hematopoietic Agents: Growth Factors, Minerals, and Vitamins

The finite life span of most mature blood cells requires their continuous replacement, a process termed hematopoiesis. New cell production must respond to basal needs and states of increased demand. Red blood cell production can increase >20-fold in response to anemia or hypoxemia, white blood cell production increases dramatically in response to a systemic infection, and platelet production can increase 10- to 20-fold when platelet consumption results in thrombocytopenia.

The regulation of blood cell production is complex. Hematopoietic stem cells are rare bone marrow cells that manifest self-renewal and lineage commitment, resulting in cells destined to differentiate into the 9 distinct blood-cell lineages. For the most part, this process occurs in the marrow cavities of the skull, vertebral bodies, pelvis, and proximal long bones; it involves interactions among hematopoietic stem and progenitor cells and the cells and complex macromolecules of the marrow stroma, and is influenced by a number of soluble and membrane-bound hematopoietic growth factors. Some of these hormones and cytokines have been identified and cloned, permitting their production in quantities sufficient for therapeutic use. Clinical applications range from the treatment of primary hematologic diseases to use as adjuncts in the treatment of severe infections and in the management of patients who are undergoing cancer chemotherapy or marrow transplantation.

Hematopoiesis also requires an adequate supply of minerals (e.g., iron, cobalt, and copper) and vitamins (e.g., folic acid, vitamin B12, pyridoxine, ascorbic acid, and riboflavin); deficiencies generally result in characteristic anemias or, less frequently, a general failure of hematopoiesis. Therapeutic correction of a specific deficiency state depends on the accurate diagnosis of the anemic state, and on knowledge about the correct dose, the use of these agents in appropriate combinations, and the expected response.

Hematopoietic Growth Factors

GROWTH FACTOR PHYSIOLOGY. Steady-state hematopoiesis encompasses the production of >400 billion blood cells each day. This production is tightly regulated and can be increased several-fold with increased demand. The hematopoietic organ also is unique in adult physiology in that several mature cell types are derived from a much smaller number of multipotent progenitors, which develop from a more limited number of pluripotent hematopoietic stem cells. Such cells are capable of maintaining their own number and differentiating under the influence of cellular and humoral factors to produce the large and diverse number of mature blood cells.

Stem cell differentiation can be described as a series of steps that produce so-called burst-forming units (BFUs) and colony-forming units (CFUs) for each of the major cell lines. These early progenitors (BFU and CFU) are capable of further proliferation and differentiation, increasing their number by some 30-fold. Subsequently, colonies of morphologically distinct cells form under the control of an overlapping set of additional growth factors (granulocyte colony-stimulating factor [G-CSF], macrophage colony-stimulating factor [M-CSF], erythropoietin, and thrombopoietin). Proliferation and maturation of the CFU for each cell line can amplify the resulting mature cell product by another 30-fold or more, generating >1000 mature cells from each committed stem cell.

Hematopoietic and lymphopoietic growth factors are glycoproteins produced by a number of marrow cells and peripheral tissues. They are active at very low concentrations and typically affect more than 1 committed cell lineage. Most interact synergistically with other factors and also stimulate production of additional growth factors, a process called networking. Growth factors generally exert actions at several points in the processes of cell proliferation and differentiation and in mature cell function. However, the network of growth factors that contributes to any given cell lineage depends absolutely on a nonredundant, lineage-specific factor, such that absence of factors that stimulate developmentally early progenitors is compensated for by redundant cytokines, but loss of the lineage-specific factor leads to a specific cytopenia.

Some of the overlapping and nonredundant effects of the more important hematopoietic growth factors are illustrated in Figure 37–1 and Table 37–1.

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Figure 37–1 Sites of action of hematopoietic growth factors in the differentiation and maturation of marrow cell lines. A self-sustaining pool of marrow stem cells differentiates under the influence of specific hematopoietic growth factors to form a variety of hematopoietic and lymphopoietic cells. Stem cell factor (SCF), ligand (FL), interleukin-3 (IL-3), and granulocyte-macrophage colony-stimulating factor (GM-CSF), together with cell–cell interactions in the marrow, stimulate stem cells to form a series of burst-forming units (BFU) and colony-forming units (CFU): CFU-GEMM (granulocyte, erythrocyte, monocyte and megakaryocyte), CFU-GM (granulocyte and macrophage), CFU-Meg (megakaryocyte), BFU-E (erythrocyte), and CFU-E (erythrocyte). After considerable proliferation, further differentiation is stimulated by synergistic interactions with growth factors for each of the major cell lines—granulocyte colony-stimulating factor (G-CSF), monocyte/macrophage-stimulating factor (M-CSF), thrombo-poietin, and erythropoietin. Each of these factors also influences the proliferation, maturation, and in some cases the function of the derivative cell line (Table 37–1).

Table 37–1

Hematopoietic Growth Factors

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ERYTHROPOIESIS-STIMULATING AGENTS

Erythropoiesis-stimulating agent (ESA) is the term given to a pharmacological substance that stimulates red blood cell production.

Erythropoietin is the most important regulator of the proliferation of committed erythroid progenitors (CFU-E) and their immediate progeny. In its absence, severe anemia is invariably present, commonly seen in patients with renal failure. Erythropoiesis is controlled by a feedback system in which a sensor in the kidney detects changes in oxygen delivery to modulate the erythropoietin secretion. The sensor mechanism is now understood at the molecular level.

Hypoxia-inducible factor (HIF-1), a heterodimeric (HIF-1α and HIF-1β) transcription factor, enhances expression of multiple hypoxia-inducible genes, such as vascular endothelial growth factor and erythropoietin. HIF-1α is labile due to its prolyl hydroxylation and subsequent polyubiquitination and degradation, aided by the von Hippel-Lindau (VHL)protein. During states of hypoxia, the prolyl hydroxylase is inactive, allowing the accumulation of HIF-1α and activating erythropoietin expression, which in turn stimulates a rapid expansion of erythroid progenitors. Specific alteration of VHL leads to an oxygen-sensing defect, characterized by constitutively elevated levels of HIF-1α and erythropoietin, with a resultant polycythemia.

Erythropoietin is expressed primarily in peritubular interstitial cells of the kidney. Erythropoietin contains 193 amino acids, of which the first 27 are cleaved during secretion. The final hormone is heavily glycosylated and has a molecular mass of ~30 kDa. After secretion, erythropoietin binds to a receptor on the surface of committed erythroid progenitors in the marrow and is internalized. With anemia or hypoxemia, synthesis rapidly increases by 100-fold or more, serum erythropoietin levels rise, and marrow progenitor cell survival, proliferation, and maturation are dramatically stimulated. This finely tuned feedback loop can be disrupted by kidney disease, marrow damage, or a deficiency in iron or an essential vitamin. With an infection or an inflammatory state, erythropoietin secretion, iron delivery, and progenitor proliferation all are suppressed by inflammatory cytokines, but this accounts for only part of the resultant anemia; interference with iron metabolism also is an effect of inflammatory mediator effects on the hepatic protein hepcidin.

PREPARATIONS. Available preparations of recombinant human erythropoietin (epoetin alfa) include EPOGEN, PROCRIT, and EPREX, supplied in single-use vials containing 2000-40,000 units/mL for intravenous or subcutaneous administration. When injected intravenously, epoetin alfa is cleared from plasma with a t1/2 of 4-8 h. However, the effect on marrow progenitors lasts much longer, and once-weekly dosing can be sufficient to achieve an adequate response. A novel erythropoiesis-stimulating protein, darbepoetin alfa (ARANESP), has been approved for clinical use in patients with indications similar to those for epoetin alfa. It is a genetically modified form of erythropoietin in which 4 amino acids have been mutated such that additional carbohydrate side chains are added during its synthesis, prolonging the circulatory survival of the drug to 24-26 h. Another erythropoiesis-stimulating peptide, Peginesatide (OMONTYS), was approved in 2012 for the treatment of anemia due to chronic kidney disease. Postmarketing reports of serious hypersensitivity reactions and anaphylaxis have necessitated a recall.

Recombinant human erythropoietin (epoetin alfa) is nearly identical to the endogenous hormone. The carbohydrate modification pattern of epoetin alfa differs slightly from the native protein, but this difference apparently does not alter kinetics, potency, or immunoreactivity of the drug. Modern assays can detect these differences and thereby identify athletes who use the recombinant product for “blood doping.”

THERAPEUTIC USES, MONITORING, AND ADVERSE EFFECTS. Recombinant erythropoietin therapy, in conjunction with adequate iron intake, can be highly effective in a number of anemias, especially those associated with a poor erythropoietic response. Epoetin alfa is effective in the treatment of anemias associated with surgery, AIDS, cancer chemotherapy, prematurity, and certain chronic inflammatory conditions. Darbepoetin alfa also has been approved for use in patients with anemia associated with chronic kidney disease.

During erythropoietin therapy, absolute or functional iron deficiency may develop. Functional iron deficiency (i.e., normal ferritin levels but low transferrin saturation) presumably results from the inability to mobilize iron stores rapidly enough to support the increased erythropoiesis. Supplemental iron therapy is recommended for all patients whose serum ferritin is <100 μg/L or whose serum transferrin saturation is <20%. During initial therapy and after any dosage adjustment, the hematocrit is determined once a week (HIV-infected and cancer patients) or twice a week (renal failure patients) until it has stabilized in the target range and the maintenance dose has been established; the hematocrit then is monitored at regular intervals. If the hematocrit increases by >4 points in any 2-week period, the dose should be decreased. Due to the time required for erythropoiesis and the erythrocyte half-life, hematocrit changes lag behind dosage adjustments by 2-6 weeks. The dose of darbepoetin should be decreased if the hemoglobin increase exceeds 1 g/dL in any 2-week period because of the association of excessive rate of rise of hemoglobin with adverse cardiovascular events.

During hemodialysis, patients receiving epoetin alfa or darbepoetin may require increased anticoagulation. The risk of thrombotic events is higher in adults with ischemic heart disease or congestive heart failure receiving epoetin alfa therapy with the goal of reaching a normal hematocrit (42%) than in those with a lower target hematocrit of 30%. ESA use is associated with increased rates of cancer recurrence and decreased on-study survival in patients in whom the drugs are administered for cancer-induced or for chemotherapy-induced anemia. The most common side effect of epoetin alfa therapy is aggravation of hypertension, which occurs in 20-30% of patients and most often is associated with a rapid rise in hematocrit. ESAs should not be used in patients with preexisting uncontrolled hypertension. Patients may require initiation of, or increases in, antihypertensive therapy. Hypertensive encephalopathy and seizures have occurred in chronic renal failure patients treated with epoetin alfa. Headache, tachycardia, edema, shortness of breath, nausea, vomiting, diarrhea, injection site stinging, and flu-like symptoms (e.g., arthralgias and myalgias) also have been reported in conjunction with epoetin alfa therapy.

Anemia of Chronic Renal Failure. Patients with anemia secondary to chronic kidney disease are ideal candidates for epoetin alfa therapy. The response in predialysis, peritoneal dialysis, and hemodialysis patients depends on the severity of the renal failure, the erythropoietin dose and route of administration, and iron availability. The subcutaneous route of administration is preferred over the intravenous route because absorption is slower and the amount of drug required is reduced by 20-40%. The dose of epoetin alfa should be adjusted to obtain a gradual rise in the hematocrit over a 2- to 4-month period to a final hematocrit of 33-36%. Treatment to hematocrit levels >36% is not recommended.

Patients are started on doses of 80-120 units/kg of epoetin alfa, given subcutaneously, 3 times a week. The final maintenance dose of epoetin alfa can vary from 10 units/kg to >300 units/kg, with an average dose of 75 units/kg, 3 times a week. Children <5 years of age generally require a higher dose. Resistance to therapy is common in patients who develop an inflammatory illness or become iron deficient, so close monitoring of general health and iron status is essential. Less common causes of resistance include occult blood loss, folic acid deficiency, carnitine deficiency, inadequate dialysis, aluminum toxicity, and osteitis fibrosa cystica secondary to hyperparathyroidism. Darbepoetin alfa is approved for use in patients who are anemic secondary to chronic kidney disease. The recommended starting dose is 0.45 µg/kg administered intravenously or subcutaneously once weekly, with dose adjustments depending on the response. Like epoetin alfa, side effects tend to occur when patients experience a rapid rise in hemoglobin concentration; a rise of <1 g/dL every 2 weeks generally is considered safe.

Anemia in AIDS Patients. Epoetin alfa therapy has been approved for the treatment of HIV-infected patients, especially those on zidovudine therapy. Excellent responses to doses of 100-300 units/kg, given subcutaneously 3 times a week, generally are seen in patients with zidovudine-induced anemia.

Cancer-Related Anemias. Epoetin alfa therapy, 150 units/kg 3 times a week or 450-600 units/kg once a week, can reduce the transfusion requirement in cancer patients undergoing chemotherapy. Therapeutic guidelines recommend the use of epoetin alfa in patients with chemotherapy-associated anemia when hemoglobin levels fall below 10 g/dL, basing the decision to treat less severe anemia (Hb, 10-12 g/dL) on clinical circumstances. For anemia associated with hematologic malignancies, the guidelines support the use of recombinant erythropoietin in patients with low-grade myelodysplastic syndrome. A baseline serum erythropoietin level may help to predict the response; most patients with blood levels >500 IU/L are unlikely to respond to any dose of the drug. Most patients treated with epoetin alfa experience an improvement in their anemia and their sense of well-being.

Recent case reports suggest a direct effect of both epoetin alfa and darbepoetin alfa in stimulation of tumor cells. A meta-analysis of a large number of patients and clinical trials estimates the risk at ~10% higher than nontreated cancer patients. This finding is being evaluated by the FDA and warrants serious attention.

Surgery and Autologous Blood Donation. Epoetin alfa has been used perioperatively to treat anemia (hematocrit 30-36%) and reduce the need for erythrocyte transfusion. Patients undergoing elective orthopedic and cardiac procedures have been treated with 150-300 units/kg of epoetin alfa once daily for the 10 days preceding surgery, on the day of surgery, and for 4 days after surgery. As an alternative, 600 units/kg can be given on days 21, 14, and 7 before surgery, with an additional dose on the day of surgery. Epoetin alfa also has been used to improve autologous blood donation.

Other Uses. Epoetin alfa has received orphan drug status from the FDA for the treatment of the anemia of prematurity, HIV infection, and myelodysplasia. In the latter case, even very high doses >1000 units/kg 2 to 3 times a week have had limited success. Highly competitive athletes have used epoetin alfa to increase their hemoglobin levels (“blood doping”) and improve performance. Unfortunately, this misuse of the drug has been implicated in the deaths of several athletes and is strongly discouraged.

MYELOID GROWTH FACTORS

The myeloid growth factors are glycoproteins that stimulate the proliferation and differentiation of one or more myeloid cell lines. Recombinant forms of several growth factors have been produced, including granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF, IL-3, M-CSF or CSF-1, and stem cell factor (SCF) (see Table 37–1).

Myeloid growth factors are produced naturally by a number of different cells, including fibroblasts, endothelial cells, macrophages, and T cells (Figure 37–2). These factors are active at extremely low concentrations and act via membrane receptors of the cytokine receptor superfamily to activate the JAK/STAT signal transduction pathway. GM-CSF can stimulate proliferation, differentiation, and function of a number of the myeloid cell lineages (see Figure 37–1). It acts synergistically with other growth factors, including erythropoietin, at the level of the BFU. GM-CSF stimulates CFU-granulocyte(G)/erythrocyte(E)/monocyte(M)/megakaryocyte(Meg) [CFU-GEMM], CFU-GM, CFU-M, CFU-E, and CFU-Meg to increase cell production. GM-CSF also enhances the migration, phagocytosis, superoxide production, and antibody-dependent cell-mediated toxicity of neutrophils, monocytes, and eosinophils.

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Figure 37–2 Cytokine-cell interactions. Macrophages, T cells, B cells, and marrow stem cells interact via several cytokines (IL-1, IL-2, IL-3, IL-4, IFN [interferon]-γ, GM-CSF, and G-CSF) in response to a bacterial or a foreign antigen challenge. See Table 37–1 for the functional activities of these various cytokines.

The G-CSF activity is restricted to neutrophils and their progenitors, stimulating their proliferation, differentiation, and function. It acts primarily on the CFU-G, although it also can play a synergistic role with IL-3 and GM-CSF in stimulating other cell lines. G-CSF enhances phagocytic and cytotoxic activities of neutrophils. G-CSF reduces inflammation by inhibiting IL-1, tumor necrosis factor, and interferon gamma. G-CSF also mobilizes primitive hematopoietic cells, including hematopoietic stem cells, from the marrow into the peripheral blood. This observation has virtually transformed the practice of stem cell transplantation, such that >90% of all such procedures today use G-CSF–mobilized peripheral blood stem cells as the donor product.

GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR. Recombinant human GM-CSF (sargramostim) is a 127–amino acid glycoprotein. The primary therapeutic effect of sargramostim is to stimulate myelopoiesis.

The initial clinical application of sargramostim was in patients undergoing autologous bone marrow transplantation. By shortening the duration of neutropenia, transplant morbidity was significantly reduced without a change in long-term survival or risk of inducing an early relapse of the malignant process. The role of GM-CSF therapy in allogeneic transplantation is less clear. Its effect on neutrophil recovery is less pronounced in patients receiving prophylactic treatment for graft-versus-host disease (GVHD). However, it may improve survival in transplant patients who exhibit early graft failure. It also has been used to mobilize CD34-positive progenitor cells for peripheral blood stem cell collection for transplantation after myeloablative chemotherapy. Sargramostim has been used to shorten the period of neutropenia and reduce morbidity in patients receiving intensive cancer chemotherapy. It also stimulates myelopoiesis in some patients with cyclic neutropenia, myelodysplasia, aplastic anemia, or AIDS-associated neutropenia.

Sargramostim (LEUKINE) is administered by subcutaneous injection or slow intravenous infusion at doses of 125-500 µg/m2/day. Plasma levels of GM-CSF rise rapidly after subcutaneous injection and then decline with a t1/2 of 2-3 h. When given intravenously, infusions should be maintained over 3-6 h. With the initiation of therapy, there is a transient decrease in the absolute leukocyte count secondary to margination and sequestration in the lungs. This is followed by a dose-dependent, biphasic increase in leukocyte counts over the next 7-10 days. Once the drug is discontinued, the leukocyte count returns to baseline within 2-10 days. When GM-CSF is given in lower doses, the response is primarily neutrophilic, whereas monocytosis and eosinophilia are observed at larger doses. After hematopoietic stem cell transplantation or intensive chemotherapy, sargramostim is given daily during the period of maximum neutropenia until a sustained rise in the granulocyte count is observed. Frequent blood counts are essential to avoid an excessive rise in the granulocyte count. Higher doses are associated with more pronounced side effects, including bone pain, malaise, flu-like symptoms, fever, diarrhea, dyspnea, and rash. An acute reaction to the first dose, characterized by flushing, hypotension, nausea, vomiting, and dyspnea, with a fall in arterial oxygen saturation due to granulocyte sequestration in the pulmonary circulation occurs in sensitive patients. With prolonged administration, a few patients may develop a capillary leak syndrome, with peripheral edema and pleural and pericardial effusions. Other serious side effects include transient supraventricular arrhythmia, dyspnea, and elevation of serum creatinine, bilirubin, and hepatic enzymes.

GRANULOCYTE COLONY-STIMULATING FACTOR. Recombinant human G-CSF, filgrastim (NEUPOGEN), is a 175–amino acid glycoprotein. The principal action of filgrastim is the stimulation of CFU-G to increase neutrophil production (see Figure 37–1).

Filgrastim is effective in the treatment of severe neutropenia after autologous hematopoietic stem cell transplantation and high-dose cancer chemotherapy. Like GM-CSF, filgrastim shortens the period of severe neutropenia and reduces morbidity secondary to bacterial and fungal infections. G-CSF also is effective in the treatment of severe congenital neutropenias. Filgrastim therapy can improve neutrophil counts in some patients with myelodysplasia or marrow damage (moderately severe aplastic anemia or tumor infiltration of the marrow). The neutropenia of AIDS patients receiving zidovudine also can be partially or completely reversed. Filgrastim is routinely used in patients undergoing peripheral blood stem cell (PBSC) collection for stem cell transplantation. It promotes the release of CD34+ progenitor cells from the marrow, reducing the number of collections necessary for transplant. G-CSF–induced mobilization of stem cells into the circulation has been promoted as a way to enhance repair of other damaged organs in which PBSC might play a role.

Filgrastim is administered by subcutaneous injection or intravenous infusion over at least 30 min at doses of 1-20 ′g/kg/day. The usual starting dose in a patient receiving myelosuppressive chemotherapy is 5 µg/kg/day. The distribution and clearance rate from plasma (t1/2 of 3.5 h) are similar for both routes of administration. As with GM-CSF therapy, filgrastim given daily after hematopoietic stem cell transplantation or intensive cancer chemotherapy will increase granulocyte production and shorten the period of severe neutropenia. Frequent blood counts should be obtained to determine the effectiveness of the treatment and guide dosage adjustment. In patients who received intensive myelosuppressive cancer chemotherapy, daily administration of G-CSF for ≥14-21 days may be necessary to correct the neutropenia. Adverse reactions to filgrastim include mild to moderate bone pain in patients receiving high doses over a protracted period, local skin reactions following subcutaneous injection, and rare cutaneous necrotizing vasculitis. Patients with a history of hypersensitivity to proteins produced by Escherichia coli should not receive the drug. Mild to moderate splenomegaly has been observed in patients on long-term therapy.

Pegylated recombinant human G-CSF pegfilgrastim (NEULASTA) is available. The clearance of pegfilgrastim by glomerular filtration is minimized, thus making neutrophil-mediated clearance the primary route of elimination. Consequently the circulating t1/2 of pegfilgrastim is longer than that of filgrastim, allowing for more sustained duration of action and less frequent dosing. The recommended dose for pegfilgrastim is fixed at 6 mg administered subcutaneously.

THROMBOPOIETIC GROWTH FACTORS

INTERLEUKIN-11. Interleukin-11 is a cytokine that stimulates hematopoiesis, intestinal epithelial cell growth, and osteoclasto-genesis and inhibits adipogenesis. Interleukin-11 also enhances megakaryocyte maturation in vitro. Recombinant human IL-11 oprelvekin (NEUMEGA) leads to a thrombopoietic response in 5-9 days when administered daily to normal subjects.

The drug is administered to patients at 25-50 μg/kg per day subcutaneously with a t1/2 ~7 h. Oprelvekin is approved for use in patients undergoing chemotherapy for nonmyeloid malignancies with severe thrombocytopenia (platelet count <20,000/μL), and it is administered until the platelet count returns to >100,000/μL. The major complications of therapy are fluid retention and associated cardiac symptoms, such as tachycardia, palpitation, edema, and shortness of breath; this is a significant concern in elderly patients and often requires concomitant therapy with diuretics. Also reported are blurred vision, injection-site rash or erythema, and paresthesias.

THROMBOPOIETIN. Thrombopoietin, a glycoprotein produced by the liver, marrow stromal cells, and other organs, is the primary regulator of platelet production. Two forms of recombinant thrombopoietin have been developed for clinical use. One is a truncated version of the native protein, termed recombinant human megakaryocyte growth and development factor (rHuMGDF) that is covalently modified with polyethylene glycol to increase the circulatory t1/2. The second is the full-length polypeptide termed recombinant human thrombopoietin (rHuTPO).

In clinical trials, both drugs are safe but data on efficacy are mixed. Due to several concerns including the immunogenicity of these agents, efforts are under way to develop small molecular mimics of recombinant thrombopoietin. Two of these agents are FDA-approved for use in patients with immune thrombocytopenic purpura (ITP) who have failed to respond to more conventional treatments.Romiplostim (NPLATE) contains 4 copies of a small peptide that binds with high affinity to the thrombopoietin receptor, grafted onto an immunoglobulin scaffold. Romiplostim is safe and efficacious in patients with ITP. The drug is administered weekly by subcutaneous injection, starting with a dose of 1 μg/kg, titrated to a maximum of 10 μg/kg, until platelet count increases above 50,000/μL.Eltrombopag (PROMACTA) is a small molecule that is a thrombopoietin receptor agonist. Eltrombopag is administered orally; the recommended starting dose is 50 mg/day, titrated to 75 mg depending on platelet response.

Drugs Effective in Iron Deficiency and Other Hypochromic Anemias

IRON AND IRON SALTS

Iron deficiency is the most common nutritional cause of anemia in humans. It can result from inadequate iron intake, malabsorption, blood loss, or an increased requirement, as with pregnancy. When severe, it results in a characteristic microcytic, hypochromic anemia. In addition to its role in hemoglobin, iron also is an essential component of myoglobin, heme enzymes (e.g., cytochromes, catalase, and peroxidase), and the metalloflavoprotein enzymes (e.g., xanthine oxidase and the α-glycerophosphate oxidase). Iron deficiency can affect metabolism in muscle independently of the effect of anemia on O2 delivery. This may reflect a reduction in the activity of iron-dependent mitochondrial enzymes. Iron deficiency also has been associated with behavioral and learning problems in children, abnormalities in catecholamine metabolism, and possibly, impaired heat production.

METABOLISM OF IRON. The body store of iron is divided between essential iron-containing compounds and excess iron, which is held in storage (Table 37–2).

Table 37–2

The Body Content of Iron

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Hemoglobin (Hb) dominates the essential fraction. Each Hb molecule contains 4 atoms of iron, amounting to 1.1 mg (20 μmol) of iron/mL of red blood cells. Other forms of essential iron include myoglobinand a variety of heme and nonheme iron-dependent enzymes. Ferritin is a protein-iron storage complex that exists as individual molecules or as aggregates. Apoferritin (MW ~450 kDa) is composed of 24 polypeptide subunits that form an outer shell around a storage cavity for polynuclear hydrous ferric oxide phosphate. More than 30% of the weight of ferritin may be iron (4000 atoms of iron per ferritin molecule). Ferritin aggregates, referred to as hemosiderin and visible by light microscopy, constitute about one-third of normal stores. The 2 predominant sites of iron storage are the reticuloendothelial system and the hepatocytes.

Internal exchange of iron is accomplished by the plasma protein transferrin, a 76-kDa glycoprotein that has 2 binding sites for ferric iron. Iron is delivered from transferrin to intracellular sites by means of specific transferrin receptors in the plasma membrane. The iron-transferrin complex binds to the receptor, and the ternary complex is internalized through clathrin-coated pits by receptor-mediated endocytosis. A proton-pumping ATPase lowers the pH of the intracellular vesicular compartment (the endosomes) to ~5.5. Iron subsequently dissociates and the receptor returns the apotransferrin to the cell surface, where it is released into the extracellular environment. Cells regulate their expression of transferrin receptors and intracellular ferritin in response to the iron supply. The synthesis of apoferritin and transferrin receptors is regulated post-transcriptionally by 2 iron-regulating proteins 1 and 2 (IRP1 and IRP2). These IRPs are cytosolic RNA-binding proteins that bind to iron-regulating elements (IREs) present in the 5′ or 3′ untranslated regions of mRNA encoding apoferritin or the transferrin receptors, respectively. Binding of these IRPs to the 5′ IRE of apoferritin mRNA represses translation, whereas binding to the 3′ IRE of mRNA encoding the transferrin receptors enhances transcript stability, thereby increasing protein production.

The flow of iron through the plasma amounts to a total of 30-40 mg/day in the adult (~0.46 mg/kg of body weight). The major internal circulation of iron involves the erythron and reticuloendothelial cells (Figure 37–3). About 80% of the iron in plasma goes to the erythroid marrow to be packaged into new erythrocytes; these normally circulate for ~120 days before being catabolized by the reticuloendothelial system. At that time, a portion of the iron is immediately returned to the plasma bound to transferrin, while another portion is incorporated into the ferritin stores of reticuloendothelial cells and returned to the circulation more gradually. With abnormalities in erythrocyte maturation, the predominant portion of iron assimilated by the erythroid marrow may be rapidly localized in the reticuloendothelial cells as defective red-cell precursors are broken down; this is termed ineffective erythropoiesis. The rate of iron turnover in plasma may be reduced by half or more with red-cell aplasia, with all the iron directed to the hepatocytes for storage.

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Figure 37–3 Iron metabolism in humans (excretion omitted).

The human body conserves its iron stores to a remarkable degree. Only 10% of the total is lost per year by normal men (i.e., ~1 mg/day). Two-thirds of this iron is excreted from the GI tract as extravasated red cells, iron in bile, and iron in exfoliated mucosal cells. The other third is accounted for by small amounts of iron in desquamated skin and in the urine. Additional losses of iron occur in women due to menstruation. Although the average loss in menstruating women is ~0.5 mg per day, 10% of menstruating women lose >2 mg per day. Pregnancy and lactation impose an even greater requirement for iron (Table 37–3). Other causes of iron loss include blood donation, the use of anti-inflammatory drugs that cause bleeding from the gastric mucosa, and GI disease with associated bleeding.

Table 37–3

Iron Requirements for Pregnancy

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The limited physiological losses of iron point to the primary importance of absorption in determining the body’s iron content. After acidification and partial digestion of food in the stomach, iron is presented to the intestinal mucosa as either inorganic iron or heme iron. A ferrireductase, duodenal cytochrome B (Dcytb), located on luminal surface of absorptive cells of the duodenum and upper small intestine, reduces the iron to the ferrous state, which is the substrate for the divalent metal (ion) transporter 1 (DMT1). DMT1 transports the iron to basolateral membrane, where it is taken up by another transporter, ferroportin (Fpn; SLC40A1), and subsequently reoxidized to Fe3+, primarily by hephaestin (Hp; HEPH), a transmembrane copper-dependent ferroxidase. Apo-transferrin (Tf) binds the resultant oxidized Fe3+.

IRON REQUIREMENTS AND THE AVAILABILITY OF DIETARY IRON. Adult men must absorb only 13 μg of iron/kg of body weight/day (~1 mg/day), whereas menstruating women require ~21 μg/kg (~1.4 mg) per day. In the last 2 trimesters of pregnancy, requirements increase to ~80 μg/kg (5-6 mg) per day; infants have similar requirements due to their rapid growth (Table 37–4).

Table 37–4

Daily Iron Absorption Requirement

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The difference between dietary supply and requirements is reflected in the size of iron stores, which are low or absent when iron balance is precarious and high when iron balance is favorable. In infants after the third month of life and in pregnant women after the first trimester, stores of iron are negligible. Menstruating women have approximately one-third the stored iron found in adult men (see Table 37–2).

Although the iron content of the diet obviously is important, of greater nutritional significance is the bioavailability of iron in food. Heme iron, which constitutes only 6% of dietary iron, is far more available and is absorbed independent of the diet composition; it therefore represents 30% of iron absorbed. The nonheme fraction represents the larger amount of dietary iron ingested by the economically underprivileged. In a vegetarian diet, nonheme iron is absorbed very poorly because of the inhibitory action of a variety of dietary components, particularly phosphates. Ascorbic acid and meat facilitate the absorption of nonheme iron. In developed countries, the normal adult diet contains ~6 mg of iron per 1000 calories, providing an average daily intake for adult men of between 12 and 20 mg and for adult women of between 8 and 15 mg. Foods high in iron (>5 mg/100 g) include organ meats such as liver and heart, brewer’s yeast, wheat germ, egg yolks, oysters, and certain dried beans and fruits; foods low in iron (<1 mg/100 g) include milk and milk products and most nongreen vegetables. Iron also may be added from cooking in iron pots. In assessing dietary iron intake, it is important to consider not just the amount of iron ingested but its bioavailability.

IRON DEFICIENCY. The prevalence of iron-deficiency anemia in the U.S. is on the order of 1-4% and depends on the economic status of the population. In developing countries, up to 20-40% of infants and pregnant women may be affected. Better iron balance has resulted from the practice of fortifying flour, the use of iron-fortified formulas for infants, and the prescription of medicinal iron supplements during pregnancy.

Iron-deficiency anemia results from dietary intake of iron that is inadequate to meet normal requirements (nutritional iron deficiency), blood loss, or interference with iron absorption. More severe iron deficiency is usually the result of blood loss, either from the GI tract, or in women, from the uterus. Finally, treatment of patients with erythropoietin can result in a functional iron deficiency. Iron deficiency in infants and young children can lead to behavioral disturbances and can impair development, which may not be fully reversible. Iron deficiency in children also can lead to an increased risk of lead toxicity secondary to pica and an increased absorption of heavy metals. Premature and low-birthweight infants are at greatest risk for developing iron deficiency, especially if they are not breast-fed and/or do not receive iron-fortified formula. After the age of 2-3 years, the requirement for iron declines until adolescence when rapid growth combined with irregular dietary habits again increases the risk of iron deficiency. Adolescent girls are at greatest risk; the dietary iron intake of most girls ages 11-18 is insufficient to meet their requirements.

TREATMENT OF IRON DEFICIENCY

GENERAL THERAPEUTIC PRINCIPLES. The response of iron-deficiency anemia to iron therapy is influenced by several factors, including the severity of anemia, the ability of the patient to tolerate and absorb medicinal iron, and the presence of other complicating illnesses. Therapeutic effectiveness is best measured by the resulting increase in the rate of production of red cells. The magnitude of the marrow response to iron therapy is proportional to the severity of the anemia (level of erythropoietin stimulation) and the amount of iron delivered to marrow precursors.

THERAPY WITH ORAL IRON. Orally administered ferrous sulfate is the treatment of choice for iron deficiency. Ferrous salts are absorbed about 3 times as well as ferric salts. Variations in the particular ferrous salt have relatively little effect on bioavailability; the sulfate (FEOSOL, others), fumarate (HEMOCYTE, FEOSTAT, others), succinate, gluconate (FERGON, others), aspartate, other ferrous salts, and polysaccharide-ferrihydrite complex (NIFEREX, others) are absorbed to approximately the same extent.

Other iron compounds have utility in fortification of foods. Reduced iron (metallic iron, elemental iron) is as effective as ferrous sulfate, provided that the material employed has a small particle size. Large-particle ferrum reductum and iron phosphate salts have a much lower bioavailability. Ferric edetate has been shown to have good bioavailability and to have advantages for maintenance of the normal appearance and taste of food. The amount of iron in iron tablets is important. It also is essential that the coating of the tablet dissolve rapidly in the stomach. Delayed-release preparations are available, but absorption from such preparations varies. Ascorbic acid (≥200 mg) increases the absorption of medicinal iron by at least 30%. However, the increased uptake is associated with an increase in the incidence of side effects. Preparations that contain other compounds with therapeutic action, such as vitamin B12, folate, or cobalt, are not recommended because the patient’s response to the combination cannot easily be interpreted.

The average dose for the treatment of iron-deficiency anemia is ~200 mg of iron per day (2-3 mg/kg), given in 3 equal doses of 65 mg. Children weighing 15-30 kg can take half the average adult dose; small children and infants can tolerate relatively large doses of iron, e.g., 5 mg/kg. When the object is the prevention of iron deficiency in pregnant women, e.g., doses of 15 to 30 mg of iron per day are adequate. Bioavailability of iron is reduced with food and by concurrent antacids. For a rapid response or to counteract continued bleeding, as much as 120 mg of iron may be administered 4 times a day. The duration of treatment is governed by the rate of recovery of hemoglobin (Table 37–5) and the desire to create iron stores.

Table 37–5

Average Response to Oral Iron

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UNTOWARD EFFECTS OF ORAL PREPARATIONS OF IRON. Side effects include heartburn, nausea, upper gastric discomfort, and diarrhea or constipation. A good policy is to initiate therapy at a small dosage, and then gradually to increase the dosage to that desired. Only individuals with underlying disorders that augment the absorption of iron run the hazard of developing iron overload (hemochromatosis).

IRON POISONING. Large amounts of ferrous salts are toxic, but fatalities are rare in adults. Most deaths occur in children, particularly between the ages of 12 and 24 months. As little as 1-2 g of iron may cause death, but 2-10 g usually is ingested in fatal cases. All iron preparations should be kept in childproof bottles. Signs and symptoms of severe poisoning may occur within 30 min after ingestion or may be delayed for several hours. They include abdominal pain, diarrhea, or vomiting of brown or bloody stomach contents containing pills. Of particular concern are pallor or cyanosis, lassitude, drowsiness, hyperventilation due to acidosis, and cardiovascular collapse. If death does not occur within 6 h, there may be a transient period of apparent recovery, followed by death in 12-24 h. The corrosive injury to the stomach may result in pyloric stenosis or gastric scarring. In the evaluation of a child thought to have ingested iron, a color test for iron in the gastric contents and determination of the concentration of iron in plasma can be performed. If the latter is >63 μmol (3.5 mg/L), the child is not in immediate danger. However, vomiting should be induced when there is iron in the stomach, and an X-ray should be taken to evaluate the number of pills remaining in the small bowel (iron tablets are radiopaque). When the plasma concentration of iron is greater than the total iron-binding capacity (63 μmol; 3.5 mg/L),deferoxamine should be administered (see Chapter 67). The speed of diagnosis and therapy is very important. With early treatment, the mortality from iron poisoning can be reduced from 45% to ~1%.Deferiprone is an oral iron chelator approved to treat iron overload due to blood transfusions in patients with thalassemia.

THERAPY WITH PARENTERAL IRON. When oral iron therapy fails, parenteral iron administration may be an effective alternative. Common indications are iron malabsorption (e.g., sprue, short bowel syndrome), severe oral iron intolerance, as a routine supplement to total parenteral nutrition, and in patients who are receiving erythropoietin. Parenteral iron can be given to iron-deficient patients and pregnant women to create iron stores, something that would take months to achieve by the oral route.

The rate of hemoglobin response is determined by the balance between the severity of the anemia (the level of erythropoietin stimulus) and the delivery of iron to the marrow from iron absorption and iron stores. When a large intravenous dose of iron dextran is given to a severely anemic patient, the hematologic response can exceed that seen with oral iron for 1-3 weeks. Subsequently, however, the response is no better than that seen with oral iron.

Parenteral iron therapy should be used only when clearly indicated because acute hypersensitivity, including anaphylactic and anaphylactoid reactions, can occur. Other reactions to intravenous iron include headache, malaise, fever, generalized lymphadenopathy, arthralgias, urticaria, and in some patients with rheumatoid arthritis, exacerbation of the disease. Four iron formulations are available in the U.S. These are iron dextran (DEXFERRUM or INFED), sodium ferric gluconate (FERRLECIT), ferumoxytol (FERAHEME), and iron sucrose (VENOFER). Ferumoxytol is a semisynthetic carbohydrate-coated superparamagnetic iron oxide nanoparticle that is approved for treatment of iron-deficiency anemia in patients with chronic kidney disease. The indications for the iron dextran preparations include treatment of any patient with documented iron deficiency and intolerance or irresponsiveness to oral iron. Indications for the ferric gluconate and iron sucrose are limited to patients with chronic kidney disease.

Iron Dextran. Iron dextran injection (INFED or DEXFERRUM) is a colloidal solution of ferric oxyhydroxide complexed with polymerized dextran (molecular weight, ~180,000 Da) that contains 50 mg/mL of elemental iron. The use of low-molecular-weight iron dextran has reduced the incidence of toxicity relative to that observed with high-molecular-weight preparations (IMFERON). Iron dextran can be administered by either intravenous (preferred) or intramuscular injection. Given intravenously in a dose <500 mg, the iron dextran complex is cleared exponentially with a plasma t1/2 of 6 h. When ≥1 g is administered intravenously as total dose therapy, reticuloendothelial cell clearance is constant at 10-20 mg/h.

Intramuscular injection of iron dextran should be initiated only after a test dose of 0.5 mL (25 mg of iron). If no adverse reactions are observed, the injections can proceed. The daily dose ordinarily should not exceed 0.5 mL (25 mg of iron) for infants weighing <4.5 kg (10 lb), 1 mL (50 mg of iron) for children weighing <9 kg (20 lb), and 2 mL (100 mg of iron) for other patients. However, local reactions and the concern about malignant change at the site of injection make intramuscular administration inappropriate except when the intravenous route is inaccessible. The patient should be observed for signs of immediate anaphylaxis and for an hour after injection for any signs of vascular instability or hypersensitivity, including respiratory distress, hypotension, tachycardia, or back or chest pain. Delayed hypersensitivity reactions also are observed, especially in patients with rheumatoid arthritis or a history of allergies. Fever, malaise, lymphadenopathy, arthralgias, and urticaria can develop days or weeks following injection and last for prolonged periods of time. Use iron dextran with extreme caution in patients with rheumatoid arthritis or other connective tissue diseases, and during the acute phase of an inflammatory illness. Once hypersensitivity is documented, iron dextran therapy must be abandoned. With multiple total-dose infusions such as those sometimes used in the treatment of chronic GI blood loss—accumulations of slowly metabolized iron dextran stores in reticuloendothelial cells can be impressive. The plasma ferritin level also can rise to levels associated with iron overload. It seems prudent, however, to withhold the drug whenever the plasma ferritin rises above 800 μg/L.

Sodium Ferric Gluconate. Sodium ferric gluconate is an intravenous iron preparation with a molecular size of ~295 kDa and an osmolality of 990 mOsm/kg-1. Administration of ferric gluconate at doses ranging from 62.5-125 mg during hemodialysis is associated with transferrin saturation exceeding 100%. Unlike iron dextran, which requires processing by macrophages that may require several weeks, ~80% of sodium ferric gluconate is delivered to transferrin with in 24 h. Sodium ferric gluconate also has a lower risk of inducing serious anaphylactic reactions than iron dextran.

Iron Sucrose. Iron sucrose is complex of polynuclear iron (III)-hydroxide in sucrose. Following intravenous injection, the complex is taken up by the reticuloendothelial system, where it dissociates into iron and sucrose. Iron sucrose is generally administered in daily amounts of 100-200 mg within a 14-day period to a total cumulative dose of 1000 mg. Like sodium ferric gluconate, iron sucrose appears to be better tolerated and to cause fewer adverse events than iron dextran. This agent is FDA-approved for the treatment of iron deficiency in patients with chronic kidney disease.

COPPER

Copper has redox properties similar to that of iron and is simultaneously essential and potentially toxic to the cell. Cells have virtually no free copper; rather, copper is stored by metallothioneins and distributed by specialized chaperones to sites that make use of copper’s redox properties.

Copper deficiency is extremely rare. Even in clinical states associated with hypocupremia (sprue, celiac disease, and nephrotic syndrome), effects of copper deficiency usually are not demonstrable. Anemia due to copper deficiency has been described in individuals who have undergone intestinal bypass surgery, in those who are receiving parenteral nutrition, in malnourished infants, and in patients ingesting excessive amounts of zinc. Copper deficiency interferes with the absorption of iron and its release from reticuloendothelial cells. In humans, the prominent findings have been leukopenia, particularly granulocytopenia, and anemia. Concentrations of iron in plasma are variable, and the anemia is not always microcytic. When a low plasma copper concentration is determined in the presence of leukopenia and anemia, a therapeutic trial with copper is appropriate. Daily doses up to 0.1 mg/kg of cupric sulfate have been given by mouth, or 1-2 mg per day may be added to the solution of nutrients for parenteral administration.

PYRIDOXINE. Patients with either hereditary or acquired sideroblastic anemia characteristically have impaired hemoglobin synthesis and accumulate iron in the perinuclear mitochondria of erythroid precursor cells, so-called ringed sideroblasts. Oral therapy with pyridoxine is of proven benefit in correcting the sideroblastic anemias associated with the antituberculosis drugs isoniazid and pyrazinamide, which act as vitamin B6 antagonists. A daily dose of 50 mg of pyridoxine completely corrects the defect without interfering with treatment, and routine supplementation of pyridoxine often is recommended (see Chapter 56). In contrast, if pyridoxine is given to counteract the sideroblastic abnormality associated with administration of levodopa, the effectiveness of levodopa in controlling Parkinson disease is decreased. Pyridoxine therapy does not correct the sideroblastic abnormalities produced by chloramphenicol or lead. Patients with idiopathic acquired sideroblastic anemia generally fail to respond to oral pyridoxine, and those individuals who appear to have a pyridoxine-responsive anemia require prolonged therapy with large doses of the vitamin, 50-500 mg/day.

RIBOFLAVIN. The spontaneous appearance in humans of red-cell aplasia due to riboflavin deficiency undoubtedly is rare, if it occurs at all. However, it seems reasonable to include riboflavin in the nutritional management of patients with gross, generalized malnutrition.

B12, Folic Acid, and the Treatment of Megaloblastic Anemias

Vitamin B12 and folic acid are dietary essentials. A deficiency of either vitamin impairs DNA synthesis in any cell in which chromosomal replication and division are taking place. Because tissues with the greatest rate of cell turnover show the most dramatic changes, the hematopoietic system is especially sensitive to deficiencies of these vitamins.

METABOLIC ROLES OF VITAMIN B12 AND FOLIC ACID. The major roles of vitamin B12 and folic acid in intracellular metabolism are summarized in Figure 37–4. Intracellular vitamin B12 is maintained as 2 active coenzymes: methylcobalamin and deoxyadenosylcobalamin.

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Figure 37–4 Interrelationships and metabolic roles of vitamin B12 and folic acid. See text for explanation and Figure 37–5 for structures of the various folate coenzymes. FIGLU, formiminoglutamic acid, which arises from the catabolism of histidine; TcII, transcobalamin II; CH3H4PteGlu1, methyltetrahydrofolate.

Methylcobalamin (CH3B12) supports the methionine synthetase reaction, which is essential for normal metabolism of folate. Methyl groups contributed by methyltetrahydrofolate (CH3H4PteGlu1) are used to form methylcobalamin, which then acts as a methyl group donor for the conversion of homocysteine to methionine. This folate–cobalamin interaction is pivotal for normal synthesis of purines and pyrimidines, and therefore of DNA. The methionine synthetase reaction is largely responsible for the control of the recycling of folate cofactors; the maintenance of intracellular concentrations of folylpolyglutamates; and, through the synthesis of methionine and its product, S-adenosylmethionine (SAM), the maintenance of a number of methylation reactions.

Deoxyadenosylcobalamin (deoxyadenosyl B12) is a cofactor for the mitochondrial mutase enzyme that catalyzes the isomerization of l-methylmalonyl CoA to succinyl CoA, an important reaction in carbohydrate and lipid metabolism. This reaction has no direct relationship to the metabolic pathways that involve folate.

Because methyltetrahydrofolate is the principal folate congener supplied to cells, the transfer of the methyl group to cobalamin is essential for the adequate supply of tetrahydrofolate (H4PteGlu1). Tetrahydrofolate is a precursor for the formation of intracellular folylpolyglutamates; it also acts as the acceptor of a 1-carbon unit in the conversion of serine to glycine, with the resultant formation of 5,10-methylenetetrahydrofolate (5,10-CH2H4PteGlu). The latter derivative donates the methylene group to deoxyuridylate (dUMP) for the synthesis of thymidylate (dTMP)—an extremely important reaction in DNA synthesis. In the process, the 5,10-CH2H4PteGlu is converted to dihydrofolate (H2PteGlu). The cycle then is completed by the reduction of the H2PteGlu to H4PteGlu by dihydrofolate reductase, the step that is blocked by folate antagonists such as methotrexate (see Chapter 61). As shown in Figure 37–4, other pathways also lead to the synthesis of 5,10-methylenetetrahydrofolate. These pathways are important in the metabolism of formiminoglutamic acid (FIGLU) and purines and pyrimidines.

Deficiency of either vitamin B12 or folate decreases the synthesis of methionine and SAM, which interferes with protein biosynthesis, a number of methylation reactions, and the synthesis of polyamines. In addition, the cell responds to the deficiency by redirecting folate metabolic pathways to supply increasing amounts of methyltetrahydrofolate; this tends to preserve essential methylation reactions at the expense of nucleic acid synthesis. With vitamin B12 deficiency, methylenetetrahydrofolate reductase activity increases, directing available intracellular folates into the methyltetrahydrofolate pool (not shown in Figure 37–4). The methyltetrahydrofolate then is trapped by the lack of sufficient vitamin B12 to accept and transfer methyl groups, and subsequent steps in folate metabolism that require tetrahydrofolate are deprived of substrate. This process provides a common basis for the development of megaloblastic anemia with deficiency of either vitamin B12 or folic acid.

The mechanisms responsible for the neurological lesions of vitamin B12 deficiency are less well understood. Damage to the myelin sheath is the most obvious lesion in this neuropathy. This observation led to the early suggestion that the deoxyadenosyl B12–dependent methylmalonyl CoA mutase reaction, a step in propionate metabolism, is related to the abnormality. However, other evidence suggests that the deficiency of methionine synthetase and the block of the conversion of methionine to SAM are more likely to be responsible.

VITAMIN B12

Humans depend on exogenous sources of vitamin B12 (see structure in Figure 37–5). In nature, the primary sources are certain microorganisms that grow in soil, or the intestinal lumen of animals that synthesize the vitamin. The daily nutritional requirement of 3-5 μg must generally be obtained from animal by-products in the diet. However, some vitamin B12is available from legumes, which are contaminated with bacteria that can synthesize vitamin B12, and vegetarians often fortify their diets with a wide range of vitamins and minerals; thus, strict vegetarians rarely develop vitamin B12 deficiency. The terms vitamin B12 and cyanocobalamin are used interchangeably as generic terms for all of the cobamides active in humans. Preparations of vitamin B12 for therapeutic use contain either cyanocobalamin or hydroxocobalamin because only these derivatives remain active after storage.

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Figure 37–5 The structures and nomenclature of vitamin B12 congeners. The vitamin B12 molecule has the 3 major portions:

1. A planar group porphyrin-like ring structure with 4 reduced pyrrole rings (A-D) linked to a central cobalt atom and extensively substituted with methyl, acetamide, and propionamide residues.

2. A 5,6-dimethylbenzimidazolyl nucleotide, which links almost at right angles to the planar nucleus with bonds to the cobalt atom and to the propionate side chain of the C pyrrole ring.

3. A variable R group—the most important of which are found in the stable compounds cyanocobalamin and hydroxocobalamin and the active coenzymes methylcobalamin and 5-deoxyadenosylcobalamin.

METABOLIC FUNCTIONS. The active coenzymes methylcobalamin and 5-deoxyadenosylcobalamin are essential for cell growth and replication. Methylcobalamin is required for the conversion of homocysteine to methionine and its derivative S-adenosylmethionine. In addition, when concentrations of vitamin B12 are inadequate, folate becomes “trapped” as methyltetrahydrofolate to cause a functional deficiency of other required intracellular forms of folic acid (see Figure 37–4). The hematologic abnormalities in vitamin B12–deficient patients result from this process. Deoxyadenosylcobalamin is required for the rearrangement of methylmalonyl CoA to succinyl CoA (see Figure 37–4).

ABSORPTION, DISTRIBUTION, ELIMINATION, AND DAILY REQUIREMENTS. In the presence of gastric acid and pancreatic proteases, dietary vitamin B12 is released from food and salivary binding protein and bound to gastric intrinsic factor. When the vitamin B12–intrinsic factor complex reaches the ileum, it interacts with a receptor on the mucosal cell surface and is actively transported into circulation. Vitamin B12 deficiency in adults is rarely the result of a deficient diet per se; rather, it usually reflects a defect in one or another aspect of this complex sequence of absorption (Figure 37–6). Antibodies to parietal cells or intrinsic factor complex also can play a prominent role in producing a deficiency. Several intestinal conditions can interfere with absorption, including pancreatic disorders (loss of pancreatic protease secretion), bacterial overgrowth, intestinal parasites, sprue, and localized damage to ileal mucosal cells by disease or as a result of surgery.

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Figure 37–6 Absorption and distribution of vitamin B12. Deficiency of vitamin B12 can result from a congenital or acquired defect in: (1) inadequate dietary supply; (2) inadequate secretion of intrinsic factor (classical pernicious anemia); (3) ileal disease; (4) congenital absence of transcobalamin II (TcII); or (5) rapid depletion of hepatic stores by interference with reabsorption of vitamin B12 excreted in bile. The utility of measurements of the concentration of vitamin B12 in plasma to estimate supply available to tissues can be compromised by liver disease and (6) the appearance of abnormal amounts of transcobalamins I and III (TcI and III) in plasma. The formation of methylco-balamin requires (7) normal transport into cells and an adequate supply of folic acid as CH3 H4 PteGlu1.

Absorbed vitamin B12 binds to transcobalamin II, a plasma β-globulin, for transport to tissues. The supply of vitamin B12 available for tissues is directly related to the size of the hepatic storage pool and the amount of vitamin B12 bound to transcobalamin II (see Figure 37–6). Vitamin B12 bound to transcobalamin II is rapidly cleared from plasma and preferentially distributed to hepatic parenchymal cells. As much as 90% of the body’s stores of vitamin B12, from 1-10 mg, is in the liver. Vitamin B12 is stored as the active coenzyme with a turnover rate of 0.5-8 μg per day. The recommended daily intake of the vitamin in adults is 2.4 μg. Approximately 3 μg of cobalamins are secreted into bile each day, 50-60% of which is not destined for reabsorption. Interference with reabsorption by intestinal disease can progressively deplete hepatic stores of the vitamin.

VITAMIN B12 DEFICIENCY. Vitamin B12 deficiency is recognized clinically by its impact on the hematopoietic and nervous systems. The sensitivity of the hematopoietic system relates to its high rate of cell turnover. Other tissues with high rates of cell turnover (e.g., mucosa and cervical epithelium) also have high requirements for the vitamin. As a result of an inadequate supply of vitamin B12, DNA replication becomes highly abnormal. Once a hematopoietic stem cell is committed to enter a programmed series of cell divisions, the defect in chromosomal replication results in an inability of maturing cells to complete nuclear divisions while cytoplasmic maturation continues at a relatively normal rate. This results in the production of morphologically abnormal cells and death of cells during maturation, a phenomenon referred to as ineffective hematopoiesis. Severe deficiency affects all cell lines, and a pronounced pancytopenia results.

The diagnosis of a vitamin B12 deficiency usually can be made using measurements of the serum vitamin B12 and/or serum methylmalonate (which is somewhat more sensitive and useful in identifying metabolic deficiency in patients with normal serum vitamin B12 levels). In managing a patient with severe megaloblastic anemia, a therapeutic trial using very small doses of the vitamin can be used to confirm the diagnosis. Serial measurements of the reticulocyte count, serum iron, and hematocrit are performed to define the characteristic recovery of normal red-cell production. The Schilling test can be used to measure the absorption of the vitamin and delineate the mechanism of the disease. By performing the Schilling test with and without added intrinsic factor, it is possible to discriminate between intrinsic factor deficiency by itself and primary ileal cell disease. Vitamin B12 deficiency can irreversibly damage the nervous system. Because the neurological damage can be dissociated from the changes in the hematopoietic system, vitamin B12 deficiency must be considered in elderly patients with dementia or psychiatric disorders, even if they are not anemic.

VITAMIN B12 THERAPY. Vitamin B12 is available for injection or oral administration; combinations with other vitamins and minerals also can be given orally or parenterally. The choice of a preparation always depends on the cause of the deficiency. Oral administration cannot be relied on for effective therapy in the patient with a marked deficiency of vitamin B12 and abnormal hematopoiesis or neurological deficits. The treatment of choice for vitamin B12 deficiency is cyanocobalamin administered by intramuscular or subcutaneous injection. Effective use of the vitamin B12 depends on accurate diagnosis and an understanding of the following general principles of therapy:

• Vitamin B12 should be given prophylactically only when there is a reasonable probability that a deficiency exists or will exist (i.e., dietary deficiency in the strict vegetarian, the predictable malabsorption of vitamin B12 in patients who have had a gastrectomy, and certain diseases of the small intestine). When GI function is normal, an oral prophylactic supplement of vitamins and minerals, including vitamin B12, may be indicated. Otherwise, the patient should receive monthly injections of cyanocobalamin.

• The relative ease of treatment with vitamin B12 should not prevent a full investigation of the etiology of the deficiency. The initial diagnosis usually is suggested by a macrocytic anemia or an unexplained neuropsychiatric disorder.

• Therapy always should be as specific as possible. Although a large number of multivitamin preparations are available, the use of shotgun vitamin therapy in the treatment of vitamin B12 deficiency can be dangerous: sufficient folic acid may be given to result in a hematologic recovery that can mask continued vitamin B12 deficiency and permit neurological damage to develop or progress.

• Although a classical therapeutic trial with small amounts of vitamin B12 can help confirm the diagnosis, acutely ill elderly patients may not be able to tolerate the delay in the correction of a severeanemia. Such patients require supplemental blood transfusions and immediate therapy with folic acid and vitamin B12 to guarantee rapid recovery.

• Long-term therapy with vitamin B12 must be evaluated at intervals of 6-12 months in patients who are otherwise well. If there is an additional illness or a condition that may increase the requirement for the vitamin (e.g., pregnancy), reassessment should be performed more frequently.

FOLIC ACID

Pteroylglutamic acid (PteGlu) (Figure 37–7) is the common pharmaceutical form of folic acid. It is not the principal folate congener in food or the active coenzyme for intracellular metabolism. After absorption, PteGlu is rapidly reduced at the 5, 6, 7, and 8 positions to tetrahydrofolic acid (H4PteGlu), which then acts as an acceptor of a number of one-carbon units. These are attached at either the 5 or the 10 position of the pteridine ring or may bridge these atoms to form a new five-membered ring. The most important forms of the coenzyme that are synthesized by these reactions are listed in Figure 37–4, and each plays a specific role in intracellular metabolism:

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Figure 37–7 The structures and nomenclature of pteroylglutamic acid (folic acid) and its congeners. X represents additional residues of glutamate; polyglutamates are the storage and active forms of the vitamin. The number of residues of glutamate is variable.

• Conversion of Homocysteine to Methionine. This reaction requires CH3H4PteGlu as a methyl donor and uses vitamin B12 as a cofactor.

• Conversion of Serine to Glycine. This reaction requires tetrahydrofolate as an acceptor of a methylene group from serine and uses pyridoxal phosphate as a cofactor. It results in the formation of 5,10-CH2H4PteGlu, an essential coenzyme for the synthesis of thymidylate.

• Synthesis of Thymidylate. 5,10-CH2H4PteGlu donates a methylene group and reducing equivalents to deoxyuridylate for the synthesis of thymidylate—a rate-limiting step in DNA synthesis.

• Histidine Metabolism. H4PteGlu also acts as an acceptor of a formimino group in the conversion of formiminoglutamic acid to glutamic acid.

• Synthesis of Purines. Two steps in the synthesis of purine nucleotides require the participation of 10-CHOH4PteGlu as a formyl donor in reactions catalyzed by ribotide transformylases: the formylation of glycinamide ribonucleotide and the formylation of 5-aminoimidazole-4-carboxamide ribonucleotide. By these reactions, carbon atoms at positions 8 and 2, respectively, are incorporated into the growing purine ring.

• Utilization or Generation of Formate. This reversible reaction uses H4PteGlu and 10-CHOH4PteGlu.

DAILY REQUIREMENTS. Many food sources are rich in folates, especially fresh green vegetables, liver, yeast, and some fruits. However, lengthy cooking can destroy up to 90% of the folate content of such food. Generally, a standard U.S. diet provides 50-500 μ;g of absorbable folate per day, although individuals with high intakes of fresh vegetables and meats will ingest as much as 2 mg per day. In the normal adult, the recommended daily intake is 400 μg; pregnant or lactating women and patients with high rates of cell turnover (such as patients with a hemolytic anemia) may require 500-600 μg or more per day. For the prevention of neural tube defects, a daily intake of at least 400 μg of folate in food or in supplements beginning a month before pregnancy and continued for at least the first trimester is recommended. Folate supplementation also is being considered in patients with elevated levels of plasma homocysteine.

ADME. As with vitamin B12, the diagnosis and management of deficiencies of folic acid depend on an understanding of the transport pathways and intracellular metabolism of the vitamin (Figure 37–8). Folates present in food are largely in the form of reduced polyglutamates, and absorption requires transport and the action of a pteroylglutamyl carboxypeptidase associated with mucosal cell membranes. The mucosae of the duodenum and upper part of the jejunum are rich in dihydrofolate reductase and can methylate most or all of the reduced folate that is absorbed. Because most absorption occurs in the proximal portion of the small intestine, it is not unusual for folate deficiency to occur when the jejunum is diseased. Both nontropical and tropical sprues are common causes of folate deficiency and megaloblastic anemia.

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Figure 37–8 Absorption and distribution of folate derivatives. Dietary sources of folate polyglutamates are hydro-lyzed to the monoglutamate, reduced, and methylated to CH3 H4 PteGlu1 during gastrointestinal transport. Folate deficiency commonly results from (1) inadequate dietary supply and (2) small intestinal disease. In patients with uremia, alcoholism, or hepatic disease there may be defects in (3) the concentration of folate-binding proteins in plasma and (4) the flow of CH3H4PteGlu1 into bile for reabsorption and transport to tissue (the folate enterohepatic cycle). Finally, vitamin B12deficiency will (5) “trap” folate as CH3H4PteGlu, thereby reducing the availability of H4PteGlu1 for its essential roles in purine and pyrimidine synthesis.

Once absorbed, folate is transported rapidly to tissues as CH3H4PteGlu. do bind folate derivatives, they have a greater affinity for nonmethylated analogs. The role of such binding proteins in folate homeostasis is not well understood. An increase in binding capacity is detectable in folate deficiency and in certain disease states, such as uremia, cancer, and alcoholism. A constant supply of CH3H4PteGlu is maintained by food and by an enterohepatic cycle of the vitamin. The liver actively reduces and methylates PteGlu (and H2 or H4PteGlu) and then transports the CH3H4PteGlu into bile for reabsorption by the gut and subsequent delivery to tissues. This pathway may provide ≥200 μg of folate each day for recirculation to tissues. The importance of the enterohepatic cycle is suggested by animal studies that show a rapid reduction of the plasma folate concentration after either drainage of bile or ingestion of alcohol, which apparently blocks the release of CH3H4PteGlu from hepatic parenchymal cells.

FOLATE DEFICIENCY. Folate deficiency is a common complication of diseases of the small intestine that interferes with the absorption of folate from food and the recirculation of folate through the enterohepatic cycle. In acute or chronic alcoholism, daily intake of folate in food may be severely restricted, and the enterohepatic cycle of the vitamin may be impaired by toxic effects of alcohol on hepatic parenchymal cells; this is the most common cause of folate-deficient megaloblastic erythropoiesis and the most amenable to therapy, via reinstitution of a normal diet. Disease states characterized by a high rate of cell turnover, such as hemolytic anemias, also may be complicated by folate deficiency. Additionally, drugs that inhibit dihydrofolate reductase (e.g., methotrexate and trimethoprim) or that interfere with the absorption and storage of folate in tissues (e.g., certain anticonvulsants and oral contraceptives) can lower the concentration of folate in plasma and may cause a megaloblastic anemia.

Folate deficiency is recognized by its impact on the hematopoietic system. As with vitamin B12, this fact reflects the increased requirement associated with high rates of cell turnover. The megaloblastic anemia that results from folate deficiency cannot be distinguished from that caused by vitamin B12 deficiency. In contrast to vitamin B12 deficiency, folate deficiency is rarely if ever associated with neurological abnormalities. After deprivation of folate, megaloblastic anemia develops much more rapidly than it does following interruption of vitamin B12 absorption (e.g., gastric surgery). This observation reflects the fact that body stores of folate are limited. Although the rate of induction of megaloblastic erythropoiesis may vary, a folate-deficiency state may appear in 1-4 weeks, depending on the individual’s dietary habits and stores of the vitamin. Folate deficiency is implicated in the incidence of neural tube defects. An inadequate intake of folate also can result in elevations in plasma homocysteine. Because even moderate hyperhomocysteinemia is considered an independent risk factor for coronary artery and peripheral vascular disease and for venous thrombosis, the role of folate as a methyl donor in the homocysteine-to-methionine conversion is getting increased attention.

Folic acid is marketed as oral tablets containing pteroylglutamic acid or L-methylfolate, as an aqueous solution for injection (5 mg/mL), and in combination with other vitamins and minerals. Folinic acid (leucovorin calcium, citrovorum factor) is the 5-formyl derivative of tetrahydrofolic acid. The principal therapeutic uses of folinic acid are to circumvent the inhibition of dihydrofolate reductase as a part of high-dose methotrexate therapy and to potentiate fluorouracil in the treatment of colorectal cancer (see Chapter 61). It also has been used as an antidote to counteract the toxicity of folate antagonists such as pyrimethamine or trimethoprim. Folinic acid provides no advantage over folic acid, is more expensive, and therefore is not recommended. A single exception is the megaloblastic anemia associated with congenital dihydrofolate reductase deficiency.

UNTOWARD EFFECTS. There have been rare reports of reactions to parenteral injections of folic acid and leucovorin. Oral folic acid usually is not toxic. Folic acid in large amounts may counteract the antiepileptic effect of phenobarbital, phenytoin, and primidone, and increase the frequency of seizures in susceptible children. The FDA recommends that oral tablets of folic acid be limited to strengths of ≤1 mg.

GENERAL PRINCIPLES OF THERAPY. The therapeutic use of folic acid is limited to the prevention and treatment of deficiencies of the vitamin. As with vitamin B12 therapy, effective use of the vitamin depends on accurate diagnosis and an understanding of the mechanisms that are operative in a specific disease state. The following general principles of therapy should be respected:

• Dietary supplementation is necessary when there is a requirement that may not be met by a “normal” diet. The daily ingestion of a multivitamin preparation containing 400-500 μg of folic acid has become standard practice before and during pregnancy to reduce the incidence of neural tube defects and for as long as a woman is breast-feeding. In women with a history of a pregnancy complicated by a neural tube defect, an even larger dose of 4 mg/day has been recommended. Patients on total parenteral nutrition should receive folic acid supplements as part of their fluid regimen because liver folate stores are limited. Adult patients with a disease state characterized by high cell turnover (e.g., hemolytic anemia) generally require 1 mg of folic acid given once or twice a day. The 1-mg dose also has been used in the treatment of patients with elevated levels of homocysteine.

• As with vitamin B12 deficiency, any patient with folate deficiency and a megaloblastic anemia should be evaluated carefully to determine the underlying cause of the deficiency state. This should include evaluation of the effects of medications, the amount of alcohol intake, the patient’s history of travel, and the function of the GI tract.

• Therapy always should be as specific as possible. Multivitamin preparations should be avoided unless there is good reason to suspect deficiency of several vitamins.

• The potential danger of mistreating a patient who has vitamin B12 deficiency with folic acid must be kept in mind. The administration of large doses of folic acid can result in an apparent improvement of the megaloblastic anemia, inasmuch as PteGlu is converted by dihydrofolate reductase to H4PteGlu; this circumvents the methylfolate “trap.” However, folate therapy does not prevent or alleviate the neurological defects of vitamin B12 deficiency, and these may progress and become irreversible.

See the 12th edition of the parent text for details on treating the patient who is acutely ill with megaloblastic anemia.