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The erythron can be considered to be a single yet dispersed tissue, defined as the entire mass of erythroid cells from the first committed progenitor cell to the mature circulating erythrocyte. This functional definition emphasizes the integrated regulation of the erythron, both in health and disease. The primary function of the erythron is to transport molecular oxygen throughout the organism. To accomplish this, adequate numbers of circulating erythrocytes (nearly half of the blood by volume) must be maintained. These erythrocytes must be able to preserve their structure and flexibility to circuit repeatedly through the microcirculation and to resist oxidant stress accumulated during their life span.81 The erythrocyte also plays a key role in modulating vascular tone. Interactions between oxyhemoglobin and nitric oxide help match vasomotor tone to local tissue oxygen demands.30, 38, 42, 64, 88
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Homeostasis of erythron proliferation is primarily maintained by the equilibrium between stimulation via the hormone erythropoietin and apoptosis controlled by two receptors, Fas and FasL, expressed on the membranes of erythroid precursors. At the other extreme, erythrocytes are culled from the circulation at the end of their life span primarily by the action of the spleen. With age, erythrocytes become less able to negotiate the narrow red pulp passages in the spleen and are phagocytosed by macrophages. By filtering out these senescent cells, the spleen minimizes entrapment in the microvasculature of other organs and prevents spillage of intracellular contents including hemoglobin into the intravascular circulation.
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Erythropoietin (EPO) is a glycoprotein hormone of molecular weight 34,000 Da that is produced in the epithelial cells lining the peritubular capillaries of the kidney. Anemia and hypoxemia stimulate its synthesis. EPO receptors are found in human erythroid cells, megakaryocytes, and fetal liver. EPO promotes erythroid differentiation, the mobilization of marrow progenitor cells, and the premature release of marrow reticulocytes. The cell most sensitive to EPO is a cell between the erythroid colony-forming unit (CFU-E) and the proerythroblast. The absence of EPO results in DNA cleavage and erythroid cell death.
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The Mature Erythrocyte
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The mature erythrocyte (red blood cell) is a highly specialized cell, designed primarily for oxygen transport. Accordingly, it is densely packed with hemoglobin, which constitutes approximately 90% of the dry weight of the erythrocyte. During maturation, the erythrocyte loses its nucleus, mitochondria, and other organelles, rendering it incapable of synthesizing new protein, replicating, or using the oxygen being transported for oxidative phosphorylation. Its metabolic repertoire is severely limited and largely restricted to a few pathways (described below under Metabolism). In general, the enzymatic pathways are those required for optimizing oxygen and carbon dioxide exchange, transiting the microcirculation while maintaining cellular integrity and flexibility, and resisting oxidant stress on the iron and protein of the cell. The characteristic biconcave discocyte shape is dynamically maintained, increasing membrane surface-to-cell volume. This shape decreases intracellular diffusion distances to the cell membrane and allows plastic deformation when squeezing through the microcirculation. The shape is the net sum of elastic and electrostatic forces within the membrane, surface tension, and osmotic and hydrostatic pressures. The cell membrane contains globular proteins floating within the phospholipid bilayer. The major blood group antigens are carried on membrane ceramide glycolipids and proteins, particularly glycophorin A and the Rh proteins. Membrane proteins generally serve to maintain the structure of the cell, to transport ions and other substances across the membrane, or to catalyze a limited number of specific chemical reactions for the cell.
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The cell membrane is coupled to, and interacts dynamically with, the cytoskeleton, allowing changes in cell shape such as tank treading or rotation of the membrane relative to the cytoplasm. This cytoskeleton consists of a hexagonal lattice of proteins, especially spectrin, actin, and protein 4.1, which interact with ankyrin and band 3 in the membrane to provide a strong but flexible structure to the membrane. Other essential structural proteins include tropomyosin, tropomodulin, and adducin. Absence or abnormalities of these proteins can result in abnormal erythrocyte shapes such as spherocytes and elliptocytes.
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Many specialized transport proteins are embedded in the erythrocyte membrane. These include anion and cation transporters, glucose and urea transporters, and water channels. The erythrocyte membrane is relatively impermeable to ion flux. Band 3 anion-exchange protein plays an important role in the chloride-bicarbonate exchanges that occur as the erythrocyte moves between the lung and tissues. Glucose, the sole source of energy of the erythrocyte, crosses the membrane by facilitated diffusion mediated by a transmembrane glucose transporter. Sodium-potassium adenosine triphosphatase (Na+-K+-ATPase) maintains the primary cation gradient by pumping sodium out of the erythrocyte in exchange for potassium.
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Membrane-Associated Enzymes.
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At least 50 membrane-bound or membrane-associated enzymes are known to exist in the human erythrocyte. Acetylcholinesterase is an externally oriented enzyme whose role in the function of the erythrocyte remains obscure. Its function is inhibited by certain xenobiotics, most notably the organic phosphorus insecticides, and it can be conveniently assayed as a marker for such exposures (Chap. 113).
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Lacking mitochondria and the ability to generate adenosine triphosphate (ATP) using molecular oxygen, the mature erythrocyte has a severely limited repertoire of intermediary metabolism compared to most mammalian cells. Having lost its nucleus, ribosomes, and translational apparatus, new enzymes cannot be synthesized and existing enzymes decline in function over the lifetime of the cell. Fortunately, the metabolic demands of the erythrocyte are usually modest, but under conditions of stress the capacity can be overwhelmed, especially among senescent cells. The greatest expenditure of energy under physiologic conditions is for the maintenance of transmembrane gradients and for the contraction of cytoskeletal elements. However, oxidant stress can put severe strain on the metabolic reserves of the erythrocyte, and lead ultimately to the premature destruction of the cell, a process termed hemolysis.
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Figure 22–2 illustrates the main metabolic pathways and their purpose. Embden-Meyerhof glycolysis is the only source of ATP for the erythrocyte and consumes approximately 90% of the glucose imported by the cell. The reduced nicotinamide adenine dinucleotide (NADH) generated during glycolysis, which would ordinarily be used for oxidative phosphorylation in cells containing mitochondria, is directed toward the reduction of either methemoglobin to hemoglobin by cytochrome b5 reductase, or pyruvate to lactate. Both pyruvate and lactate are exported from the cell. During glycolysis, metabolism can be diverted into the Rapoport-Luebering shunt, generating 2,3-bisphosphoglycerate (2,3-BPG, formerly known as 2,3-diphosphoglycerate or 2,3-DPG) in lieu of ATP. 2,3-BPG binds to deoxyhemoglobin to modulate oxygen affinity and allow unloading of oxygen at the capillaries. In response to anemia, altitude, or changes in cellular pH, the activity of the shunt increases, thereby favoring synthesis of 2,3-BPG and increasing oxygen delivery considerably. Reduced [levels] of 2,3-BPG in stored blood result in impaired oxygen delivery following massive transfusion.98
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As an alternative to glycolysis, glucose can be directed toward the hexose monophosphate shunt during times of oxidant stress. This pathway results in the generation of reduced nicotinamide adenine dinucleotide phosphate (NADPH), which the erythrocyte uses to maintain reduced glutathione which, in turn, inactivates oxidants and protects the sulfhydryl groups of hemoglobin and other proteins. The initial, rate-limiting step of this pathway is controlled by glucose-6-phosphate dehydrogenase (G6PD). Accordingly, cells deficient in this enzyme are less able to maintain glutathione in a reduced state, and are vulnerable to irreversible damage under oxidant stress. The consequences of this deficiency are discussed in greater detail below under Glucose-6-Phosphate Dehydrogenase Deficiencies.
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The erythrocyte also contains enzymes to synthesize glutathione (γ-glutamyl-cysteine synthetase and glutathione synthetase), to convert CO2 to bicarbonate ion (carbonic anhydrase I), to remove pyrimidines resulting from the degradation of RNA (pyrimidine 5′-nucleotidase), to protect against free radicals (catalase, superoxide dismutase, glutathione peroxidase), and to conjugate glutathione to electrophiles (ρ-glutathione-S-transferase).
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Hemoglobin, the major constituent of the cytoplasm of the erythrocyte, is a conjugated protein with a molecular weight of 64,500 Da. To put things in perspective, the typical adult man has approximately 75 mL/kg of blood containing 15 g/100 mL of hemoglobin, or nearly 1 kg of hemoglobin. His 0.3-kg heart must pump this entire mass of hemoglobin every minute at rest, which is a substantial work expenditure. One molecule is composed of four protein (globin) chains, each attached to a prosthetic group called heme. Heme contains an iron molecule complexed at the center of a porphyrin ring. The globin chains are held together by noncovalent electrostatic attraction into a tetrahedral array. Hemoglobin is so efficient at binding and carrying oxygen that it enables blood to transport 100 times as much oxygen as could be carried by plasma alone (Chap. 29). In addition, the capacity of hemoglobin to modulate oxygen binding under different conditions allows adaptation to a wide variety of environments and demands. Three complex and integrated pathways are required for the formation of hemoglobin: globin synthesis, protoporphyrin synthesis, and iron metabolism.
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The protein chains of hemoglobin are produced with information from two different genetic loci. The α-globin gene cluster spans 30 kb on the short arm of chromosome 16, and codes for two identical adult α-chain genes, as well as the ζ-chain, an embryonic globulin. The β cluster is 50 kb on chromosome 11, and codes for the two adult globins β and α, as well as two nearly identical γ chains expressed in the fetus and an embryonic globulin ε. The expression of genes in each family changes during embryonic, fetal, neonatal, and adult development. Until 8 weeks of intrauterine life, ε, ζ, γ, and α chains are produced and assembled in various combinations in yolk sac–derived erythrocytes. With the shift in erythropoiesis from yolk sac to fetal liver and spleen, embryonic hemoglobin disappears, and the α and γ globin chains are paired into fetal hemoglobin (HbF = α2γ2). Erythrocytes containing HbF have a higher O2 affinity than those containing adult hemoglobin, which is important for oxygen transfer across the placenta into the relatively hypoxic uterine environment. Beginning shortly before birth, expression shifts to the α and β globins, which constitute the predominant adult hemoglobin termed hemoglobin A (α2β2). Approximately 2.5% of normal adult hemoglobin is in the form of hemoglobin A2(α2δ2). The thalassemias, a group of inherited disorders, result from defective synthesis of one or more of the globin chains. Clinically this results in a hypochromic, microcytic anemia.
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Heme is the iron complex of protoporphyrin IX. Protoporphyrin IX is a tetramer composed of four porphyrin rings joined in a closed, flat-ring structure. The IX designation refers to the order in which it was first synthesized in Hans Fischer’s laboratory. Of the 15 possible isomers, only protoporphyrin IX occurs in living organisms. Technically, only iron complexes with the iron in the Fe2+ state can be called heme, but the term is commonly used to refer to the prosthetic group of metalloproteins such as peroxidase (ferric) and cytochrome c (both ferric and ferrous), whether the iron is in the Fe2+ or Fe3+ state. The terms “hemiglobin” and “ferrihemoglobin” are synonymous with methemoglobin but rarely used. All animal cells can synthesize heme, with the notable exception of mature erythrocytes. Hemoproteins are involved in a multitude of biologic functions, including oxygen binding (hemoglobin, myoglobin), oxygen metabolism (oxidases, peroxidases, catalases, and hydroxylases), and electron transport (cytochromes), as well as metabolism of xenobiotics (cytochrome P450 family). Erythroid cells synthesize 85% of total body heme, with the liver synthesizing most of the balance. Hemoglobin is the most abundant hemoprotein, containing 70% of total body iron.
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The first step in the synthesis of heme takes place in the mitochondrion and is the condensation of glycine and succinyl-coenzyme A (CoA) to form 5-aminolevulinic acid (ALA) (Fig. 22–3). The formation of 5-ALA is catalyzed by aminolevulinic acid synthase (ALAS), the rate-limiting step of the pathway. The rate of heme synthesis is closely controlled, given that free intracellular heme is toxic and that this first step is essentially irreversible.
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Of the two isoforms of ALAS known to exist in mammals, erythroid cells express the ALAS2 isoform, which resides on the X chromosome. Comparatively more is known about ALAS1 (chromosome 3), a housekeeping gene with a short half-life expressed ubiquitously, allowing the synthesis of cellular and mitochondrial hemoproteins. ALAS1 activity is induced by many factors, which can increase its expression by two orders of magnitude. Moreover, it is strongly inhibited by heme in a classical negative feedback fashion. ALAS2 is constitutively expressed at very high concentrations in erythroid precursors, allowing sustained synthesis of heme during erythropoiesis. Pyridoxal phosphate (active vitamin B6) serves as a cofactor to both isoforms of ALAS. The clinical consequences of pyridoxine deficiency include a hypochromic, microcytic anemia, iron overload, and neurologic impairment.
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The next step in the synthesis of hemoglobin is the formation of the monopyrrole porphobilinogen via the condensation of two molecules of ALA. The subsequent steps in heme synthesis involve the condensation of four molecules of porphobilinogen into a flat ring, which is transported back into the mitochondrion by an unknown mechanism. The final step is the insertion of iron into protoporphyrin IX, a reaction that is catalyzed by ferrochelatase (also known as heme synthase) to form heme.
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Understanding this carefully regulated synthetic pathway is relevant to understanding the laboratory evidence of lead toxicity (Chap. 96), and predicting the response of porphyric patients to a range of xenobiotics. Most steps in the heme biosynthetic pathway are inhibited by lead. ALA dehydratase is the most sensitive, followed by ferrochelatase, coproporphyrinogen oxidase, and porphobilinogen deaminase. As a consequence, ALA is increased in plasma and especially urine. With increasing exposure, ferrochelatase inhibition coupled with iron deficiency causes zinc protoporphyrin to accumulate in erythrocytes, which can easily be detected by fluorescence. Coproporphyrinogen III also appears in the urine. Historically, these effects have served as the basis for a number of tests of lead exposure.
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The porphyrias are a group of disorders resulting from an inherited deficiency of any given enzyme that follows ALAS on the heme biosynthetic pathway. As such, when ALAS activity outpaces the activity of the deficient enzyme, the rate-limiting step shifts downstream, and intermediate metabolites accumulate. These metabolites can cause characteristic neuropsychiatric symptoms and palsies (caused by ALA and porphobilinogen), and cutaneous reactions, including photosensitivity (due to the fluorescence of the porphyrins). For example, porphobilinogen is excreted in large quantities by patients with acute intermittent porphyria (porphobilinogen deaminase deficiency), and the urine darkens with exposure to air and light due to oxidation to porphobilin and to nonenzymatic assembly into porphyrin rings. A variety of xenobiotics can precipitate a crisis in susceptible individuals, by inducing ALAS1 and overloading the deficient enzyme.97 The molecular mechanisms that allow xenobiotics to induce the ALAS1 gene closely resemble those accounting for induction of the cytochrome P450 (CYP) genes, which also require heme synthesis. The xenobiotic typically interacts with either the constitutive androstane receptor (CAR) or the pregnane X receptor (PXR), the two main so-called orphan nuclear receptors.104 These transcriptional factors are DNA-binding proteins that induce the expression of a range of drug metabolizing and transporting genes in response to the presence of a xenobiotic and are termed “xenosensors.” When activated, they associate with the 9-cis retinoic acid xenobiotic receptor (RXR), and then attach to enhancer sequences near the apoCYP or ALAS1 genes to enhance transcription.78 The multifunctional inducers capable of activating a wide range of hepatic enzymes are therefore extremely porphyrogenic and include phenobarbital, phenytoin, carbamazepine, and primidone. Furthermore, because CYP 3A4 and 2C9 represent nearly half of the hepatic CYP pool, inducers of these isoforms also stimulate heme synthesis and can induce a porphyric crisis. Examples include the anticonvulsants; nifedipine; sulfamethoxazole; rifampicin; ketoconazole; and the reproductive steroids progesterone, medroxyprogesterone, and testosterone. Glucocorticoids, on the other hand, despite binding to PXR, suppress ALAS1 induction and translation, and they are not porphyrogenic.97
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At equilibrium, approximately 1 to 2 mg of iron is absorbed from the diet, and a similar amount is shed from the intestinal epithelium each day. Unless appropriately chelated, free iron not bound by transport or storage proteins can generate harmful oxygen free radicals that damage cellular structures and metabolism (Chaps. 12 and 46). For this reason, serum iron circulates bound to a transfer protein, transferrin, and is stored in the tissues using ferritin (Fig. 22–4). Whereas each molecule of transferrin can bind two iron atoms, ferritin has a large internal cavity, approximately 80 Å in diameter, that can hold up to 4500 iron atoms per molecule. The amount of iron transported through plasma depends on total-body iron stores and the rate of erythropoiesis. Only about one-third of the iron-binding sites of circulating transferrin are normally saturated, as demonstrated by the usual serum iron content of 60 to 170 µg/dL (10–30 µmol/L) as compared to the total iron-binding capacity of 280 to 390 µg/dL (50–70 µmol/L).
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Only transferrin can directly supply iron for hemoglobin synthesis. The iron–transferrin complex binds to transferrin receptors on the surface of developing erythroid cells in bone marrow. Iron in the erythroid cell is used for hemoglobin synthesis or is stored in ferritin.
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The absorption of nonheme, free ferrous iron from the diet occurs via the divalent metal transporter one of the duodenal enterocyte, which then passes it into the circulation via ferroportin. Iron then circulates in the ferric form bound to transferrin.45 Dietary iron complexed with heme can be absorbed via the recently discovered heme carrier protein 1, which transports either iron or zinc protoporphyrin into the enterocyte.91 The iron may then be freed by microsomal heme oxygenase and follow the transport of atomic iron, or perhaps heme itself can be transferred to the circulation via specific export proteins and circulate bound to its carrier protein, hemopexin.5
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When the erythrocyte is removed from the circulation by splenic macrophages, heme is degraded by heme oxygenase to carbon monoxide and biliverdin, and the iron extracted. Some iron may remain in macrophages in the form of ferritin or hemosiderin. Most is delivered again by ferroportin back to the plasma and bound to transferrin.
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Iron homeostasis is largely regulated at the level of absorption, with little physiologic control over its rate of loss. Excess absorption relative to body stores is the hallmark of hereditary hemochromatosis. The iron regulatory hormone hepcidin produced by the liver is now believed to play a central role in iron control, including the anemia of chronic disease caused by inflammatory signals.45 Hepcidin is a 25-amino acid peptide that senses iron stores and controls the ferroportin-mediated release of iron from enterocytes, macrophages, and hepatocytes. The liver is an important reservoir of iron because it can store considerable amounts of iron taken from portal blood and release it when needed.
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Oxygen-Carbon Dioxide Exchange
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The evolutionary transition of organisms from anaerobic to aerobic life allowed the liberation of 18 times more energy from glucose. Vertebrates have developed two important systems to overcome the relatively small quantities of oxygen dissolved in aqueous solutions under atmospheric conditions: the circulatory system and hemoglobin. The circulatory system allows delivery of oxygen and removal of carbon dioxide throughout the organism. Hemoglobin plays an essential role in the transport and exchange of both gases. Moreover, the interactions between these gases and hemoglobin are directly linked in a remarkable story of molecular evolution. Understanding this interplay has allowed fundamental insight into protein conformation and the importance of allosteric interactions between molecules.
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The binding of one oxygen molecule to each of the four iron atoms in heme results in conformational changes that affect binding of oxygen at the remaining sites. This phenomenon is known as cooperativity, and it is necessary to both the transport of relatively large quantities of oxygen and the unloading of most of this oxygen at tissue sites. Cooperativity results from the intramolecular interactions of the tetrameric hemoglobin, and is expressed in the sigmoidal shape of the oxyhemoglobin dissociation curve (Fig. 29–2). Conversely, the monomeric myoglobin has a hyperbolic oxygen binding curve. The partial pressure of oxygen at which 50% of the oxygen bindings sites of hemoglobin are occupied is about 26 mm Hg, in contrast with about 1 mm Hg for myoglobin. Moreover, hemoglobin is nearly 100% saturated at partial oxygen pressures of about 100 mm Hg in the pulmonary capillaries, transporting 1.34 mL of oxygen per gram of hemoglobin A. About one-third of this oxygen can be unloaded under normal conditions at tissue capillaries with partial oxygen pressures around 35 mm Hg. The proportion unloaded rises during exercise and sepsis, as well as with xenobiotics that uncouple oxidative phosphorylation. Elite athletes can extract up to 80% of the available oxygen under conditions of maximal aerobic effort.
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The oxygen reserve, however, is only one of the benefits of the large quantity of hemoglobin in circulation. The ability of hemoglobin to buffer the acid equivalent of CO2 in solution is equally vital to respiratory physiology, because it allows the removal of large quantities of CO2 from metabolically active tissues with minimal changes in blood pH. Hemoglobin is by far the largest buffer in circulation, accounting for seven times the buffer capacity of the serum proteins combined (28 vs. 4 mEq H+/L of whole blood). For every 1 mole of oxygen unloaded in the tissue, about 0.5 mole of H+ is loaded onto hemoglobin.
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The linked interaction between oxygen and carbon dioxide transport can be first considered from the perspective of oxygen binding to hemoglobin. The affinity of oxygen for hemoglobin is directly affected by pH, which is a function of the CO2 content of the blood. The oxyhemoglobin dissociation curve shifts to the left in lungs, where the level of carbon dioxide, and thus carbonic acid, are kept relatively low as a result of ventilation, an effect that promotes oxygen binding. The curve shifts to the right in tissues where cellular respiration increases CO2 concentrations. This phenomenon, known as the Bohr effect, promotes the uptake of oxygen in the lungs and the release of oxygen at tissue sites.
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From the perspective of carbon dioxide transport, hemoglobin also plays an essential albeit indirect role. Carbon dioxide dissolves into plasma, and is slowly hydrated to carbonic acid which dissociates to H+ and HCO3– (pKa6.35). The speed of the hydration reaction is accelerated from about 40 seconds to 10 msec by the abundant enzyme carbonic anhydrase located within the erythrocyte. Most carbon dioxide collected at the tissues diffuses into erythrocytes, where it becomes H+ and HCO3–. This HCO3– is then rapidly transported back to the serum in exchange for chloride ion via the band 3 anion exchange transporter located in the erythrocyte membrane, thereby shifting serum Cl– into the erythrocyte (the chloride shift). The hydrogen ion is accepted by hemoglobin, largely at the imidazole ring of histidine residues, which have a pKa of about 7.0. A small amount of CO2 reacts directly with the amino terminal of the globin chains to form carbamino residues (HbNHCOO–). Thus, most of the transported carbon dioxide is transformed by the erythrocyte into bicarbonate ion that is returned to the serum and hydrogen ion that is buffered by hemoglobin. Each liter of venous blood typically carries 0.8 mEq dissolved CO2+ 16 mEq HCO3– in the plasma, 0.4 mEq dissolved CO2+ 4.6 mEq HCO3–, + 1.2 mEq HbNHCOO– in the erythrocyte (a total of 23 mEq CO2, equivalent to 510 mL CO2/L blood). Although two-thirds of the total CO2 content is ultimately carried in the plasma, nearly all of the bicarbonate is generated within erythrocytes. In the capillaries of the lungs, the reverse reactions occur to eliminate CO2. Because deoxyhemoglobin is better able to buffer hydrogen ions, the release of oxygen from hemoglobin at the tissues facilitates the uptake of carbon dioxide into venous blood. This effect is known as the Haldane effect. In fact, 1 L of venous blood at 70% oxygen saturation can transport an additional 20 mL of CO2 compared to arterial blood, which is nearly 100% saturated. Both the Bohr and Haldane effects can have important consequences at the extremes of acid–base perturbations, as can occur in a number of poisonings that interfere with oxygen metabolism.
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Finally, in addition to inactivating nitric oxide, hemoglobin can also reversibly bind it as S-nitrosohemoglobin, thereby playing an important role in the regulation of microvascular circulation and oxygen delivery. The ability of hemoglobin to vasodilate the surrounding microvasculature in response to oxygen desaturation using nitric oxide provides new insight into oxygen delivery and may be pivotal in such disorders as septic shock, pulmonary hypertension, and senescence of stored red blood cells.92
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Several alterations of the hemoglobin molecule are encountered in clinical toxicology. A detailed understanding of their molecular basis, clinical manifestations, and effects on gas exchange is essential. Unfortunately, the nomenclature can be ambiguous and overlaps with distinct clinical entities such as oxidant injury and hemolysis. Therefore, although a detailed discussion of these abnormal hemoglobins appears elsewhere (Chaps. 125 and 127), an overview of the subject is presented here. It is helpful to recall that the iron atom has six binding positions. Four of these positions are attached in a single plane to the protoporphyrin ring to form heme. The remaining two binding positions lie on opposite sides of this plane. One site is ordinarily bonded to the F8 proximal histidine residue of the globin chain. The remaining site is available for binding molecular oxygen, but it can also bind carbon monoxide, nitric oxide, cyanide, hydroxide ion, or water. The E7 distal histidine residue facilitates the binding of oxygen while stearically hindering carbon monoxide binding.
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Methemoglobin (ferrihemoglobin or hemiglobin) is the oxidized form of deoxyhemoglobin in which at least one heme iron is in the oxidized (Fe3+) valence state. A number of valency hybrids can occur, depending on the number of ferric versus ferrous heme units within the tetramer. Methemoglobin therefore represents oxidation (loss of electrons) of the hemoglobin molecule at the iron atom. It occurs spontaneously as a consequence of interactions between the iron and oxygen. Normally, in deoxygenated hemoglobin, the heme iron is in the ferrous (Fe2+) valence state. In this state, there are six electrons in the outer shell, four of which are unpaired. When oxygen is bound, one of these electrons is partially transferred to it and the iron is reversibly oxidized. When O2 is released, the electron is usually transferred back to heme iron, yielding the normal reduced state. Sometimes, the electron remains with the O2 yielding a superoxide anion radical O2– rather than molecular oxygen. In this case, heme iron is left in the Fe3+, or oxidized, state and is unable to release another electron to bind oxygen. This oxidation is primarily reversed via the action of cytochrome b5 reductase, also known as NADH methemoglobin reductase, which uses the electron carrier NADH generated by glycolysis (Metabolism discussion above and Chap. 13). Minor pathways are also involved in methemoglobin reduction, including NADPH methemoglobin reductase, which normally reduces only approximately 5% of the methemoglobin, and vitamin C, a nonenzymatic reducing agent. The activity of NADPH methemoglobin reductase may be significantly accelerated by the presence of the electron donor methylene blue (Antidotes in Depth: A42 and Chap. 127) or riboflavin. Equilibrium is maintained with methemoglobin concentrations of 1% of total hemoglobin. Many xenobiotics increase the rate of methemoglobin formation by as much as 1000-fold. Nitrites, nitrates, chlorates, and quinones are capable of directly oxidizing hemoglobin.19 Certain individuals may be especially vulnerable resulting from deficient methemoglobin reduction.28 The fetus and neonate are more susceptible to methemoglobinemia than the adult, because HbF is more susceptible to oxidation of the heme iron than adult hemoglobin. The newborn also has a limited capacity to reduce methemoglobin, because adult concentrations of cytochrome b5 reductase are only achieved at about 6 months of age.
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Carbon monoxide (CO) can reversibly bind to heme iron in lieu of molecular oxygen. The affinity of CO for hemoglobin is 200–300 times that of oxygen, despite the stearic hindrance of the E7 distal histidine. The presence of CO thereby precludes the binding of oxygen. In addition, CO binding within any one heme subunit degrades the cooperative binding of oxygen at the remaining heme groups of the same hemoglobin molecule. The oxyhemoglobin dissociation curve is therefore shifted to the left, reflecting the fact that oxygen is more tightly bound by hemoglobin and less able to be unloaded to the tissues. In addition, CO binds to the heme group of myoglobin and the cytochromes, interfering with cellular respiration and exacerbating the hypoxia (Chap. 125).41
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Sulfhemoglobin is a bright green molecule in which the hydrosulfide anion HS– irreversibly binds to ferrous hemoglobin. The sulfur atom is probably attached to a β carbon in the porphyrin ring and not at the normal oxygen-binding site.67 It has a spectrophotometric absorption band at approximately 618 nm,16 is ineffective in oxygen transport, and clinically produces a condition that resembles cyanosis. The oxygen affinity of sulfhemoglobin is approximately 100 times less than that of oxyhemoglobin, shifting the oxyhemoglobin dissociation curve to the right, in favor of O2 unbinding. Thus, the symptoms of hypoxia are not as severe with sulfhemoglobinemia as with carboxy- or methemoglobinemia.74
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Oxidation of the Globin Chain.
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Oxidation can also occur at the amino acid side chains of the globin protein. In particular, sulfhydryl groups can oxidize to form disulfide links between cysteine residues, which leads to the unfolding of the protein chain, exposure of other side chains, and further oxidation. When these disulfide links join adjacent hemoglobin molecules, they cause the precipitation of the concentrated hemoglobin molecules out of solution. Covalent links can also form between hemoglobin and other cytoskeletal and membrane proteins.25 Eventually, aggregates of denatured and insoluble protein are visible on light microscopy as Heinz bodies. The distortion of the cellular architecture and the loss of fluidity in particular signal reticuloendothelial macrophages to excise sections of erythrocyte membrane (“bite cells”) or to remove the entire erythrocyte from the circulation (see below). To guard against these oxidation reactions, the erythrocyte maintains a pool of reduced glutathione via the actions of the NADPH generated in the hexose monophosphate shunt (assuming adequate G6PD activity to initiate this pathway). This glutathione transfers electrons to break open disulfide links and to preserve sulfhydryl groups in their reduced state.
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Hemolysis is merely the acceleration of the normal process by which senescent or compromised erythrocytes are removed from the circulation.93 The normal life span of a circulating erythrocyte is approximately 120 days, and any reduction in this life span represents some degree of hemolysis. If sufficiently rapid, hemolysis can overwhelm the regenerative capacity of the erythron, resulting in anemia. Intravascular hemolysis occurs when the rate of hemolysis exceeds the capacity of the reticuloendothelial macrophages to remove damaged erythrocytes, and free hemoglobin and other intracellular contents of the erythrocyte appear in the circulation.
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Reticulocytosis, polychromasia, unconjugated hyperbilirubinemia, increased serum lactate dehydrogenase, and decreased serum haptoglobin are characteristic of hemolysis. A normal or elevated RBC distribution width and thrombocytosis are usually present. The presence of spherocytes on peripheral blood smear suggests an autoimmune or hereditary process and can be pursued with a Coombs’ test. Schistocytes suggest thrombotic thrombocytopenic purpura (TTP) or hemolytic uremic syndrome, disseminated intravascular coagulation, or valvular hemolysis. TTP and hemolytic uremic syndrome are characterized by a microangiopathic anemia, thrombocytopenia, and normal coagulation parameters (unlike disseminated intravascular coagulation). TTP is discussed under platelet disorders below. Hemoglobinemia, hemoglobinuria, and hemosiderinuria can occur with intravascular hemolysis. Specialized tests to measure hemolysis detect shortened erythrocyte survival, increased endogenous carbon monoxide generation from heme oxygenase, and increased fecal urobilinogen.
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Table 22–2 presents a brief classification of acquired causes of hemolysis relevant to toxicology. Oxidant injury following xenobiotic exposure is one of the triggers of hemolysis, as it may cause irreversible changes in the erythrocyte. Erythrocytes deficient in G6PD by virtue of cell age or enzyme mutations are particularly vulnerable to hemolysis following oxidant stress due to limited capacity to generate NADPH and reduced glutathione.93 The immune-mediated hemolytic anemias occur when ingested xenobiotics trigger an antigen antibody reaction. In general, these molecules are too small to be sensitizing agents, but antigenicity can be acquired after binding to carrier proteins in blood. The particulars of the xenobiotic-carrier immune activation sequence form the basis for the classification of this group of hemolytic anemias.7
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Nonimmune-Mediated Causes of Xenobiotic-Induced Hemolysis
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A number of xenobiotics or their reactive metabolites can cause hemolysis via oxidant injury. A Heinz-body hemolytic anemia can result, which typically resolves within a few weeks of drug discontinuation. Some xenobiotics cause hemolysis in the absence of overt oxidant injury (Table 22–2). Copper sulfate hemolysis is described in Chap. 95, while the delayed hemolysis following exposure to arsine or stibine is described here.
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Arsine (AsH3) is a colorless, odorless, nonirritating gas that is 2.5 times denser than air (Chap. 89). Clinical signs and symptoms appear after a latent period of up to 24 hours after exposure to concentrations above 30 ppm and may include headache, malaise, dyspnea, abdominal pain with nausea and vomiting, hepatomegaly, hemolysis with hemoglobinuric acute kidney injury, and death.23, 34, 53, 57, 77 The mechanism of hemolysis is believed to involve the fixation of arsine by sulfhydryl groups of hemoglobin and other essential proteins.39,103 Interestingly, hemolysis is prevented in vitro by conversion to carboxy- or methemoglobin.44 Impairment of membrane proteins, including Na+-K+-ATPase, is another potential mechanism for arsine-induced hemolysis.80 Chronic exposure to low levels of arsine can produce clinically significant disease.23 Stibine (SbH3), an antimony derivative, likely causes hemolysis via similar mechanisms (Chap. 88).
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Glucose-6-Phosphate Dehydrogenase Deficiencies.
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G6PD is the enzyme that catalyzes the first step of the hexose monophosphate shunt: the conversion of glucose-6-phosphate to phosphogluconolactone (Fig. 13–7). In the process, NADP+ is reduced to NADPH, which the erythrocyte uses to maintain a supply of reduced glutathione and to defend against oxidation. It follows that erythrocytes deficient in G6PD activity are less able to resist oxidant attack and, in particular, to maintain sulfhydryl groups of hemoglobin in their reduced state, resulting in hemolysis. It is important to recognize that the term G6PD deficiency encompasses a wide range of differences in enzyme activities among individuals. These differences may result from decreased enzyme synthesis, altered catalytic activity, or reduced stability of the enzyme. Approximately 7.5% of the world population is affected to some degree, with more than 400 variants having been identified. Most cases involve relatively mild deficiency and minimal morbidity. Ethnic populations from tropical and subtropical countries (the so-called malaria belt) have a much higher prevalence of G6PD deficiency, possibly because that phenotype protects against malaria.69
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The gene that encodes for G6PD resides near the end of the long arm of the X-chromosome. Most mutations consist of a single amino acid substitution, as complete absence of this enzyme is lethal. Although men hemizygous for a deficient gene are more severely affected, women randomly inactivate one X-chromosome during cellular differentiation according to the Lyon hypothesis. Thus, women carriers heterozygous for a deficient G6PD gene have a mosaic of erythrocytes, some proportion of which expresses the deficient gene during maturation. Accordingly, approximately 10% of carrier women may be nearly as severely affected as males hemizygous for the same deficient gene. Because of the high gene frequency in certain ethnic groups, another approximately 10% of women may be homozygous for the deficient gene.
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Normal G6PD has a half-life of about 60 days. Because the erythrocyte cannot synthesize new protein, the activity of G6PD normally declines by approximately 75% over its 120-day life span. Consequently, even in unaffected individuals, susceptibility to oxidant stress varies based on the age mix of circulating erythrocytes. In all cases, older erythrocytes are less likely to recover following exposure to an oxidant and will hemolyze first. Moreover, after an episode of hemolysis following acute exposure to an oxidant stress, the higher G6PD activity of surviving erythrocytes will confer some resistance against further hemolysis in most individuals with relatively mild deficiency, even if the offending xenobiotic is continued. For this reason, phenotypic testing for G6PD deficiency is best done 2 to 3 months after a hemolytic crisis, which is when the reticulocyte count has usually normalized.
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The World Health Organization classification of G6PD is based on the degree of enzyme deficiency and severity of hemolysis.18 Both class I and class II patients are severely deficient, with less than 10% of normal G6PD activity. Class I individuals are prone to chronic hemolytic anemia, whereas class II patients experience intermittent hemolytic crises. Class III patients have only moderate (10%–60%) enzyme deficiency, and experience self-limiting hemolysis in response to certain xenobiotics and infections. Approximately 11% of African Americans have a class III deficiency, traditionally termed type A-, and experience a decline of no more than 30% of the red blood cell mass during any single hemolytic episode. Another 20% of African Americans have type A+ G6PD enzyme, which is functionally normal, and therefore of no consequence despite a one-base substitution compared to wild-type B. The Mediterranean type found in Sardinia, Corsica, Greece, the Middle East, and India is a class II deficiency, and hemolysis can occur spontaneously or in response to ingestion of oxidants, such as the β-glycosides found in fava (Vicia fava) beans.
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The most common clinical presentation of previously unrecognized G6PD deficiency is the acute hemolytic crisis. Typically, hemolysis begins 1 to 4 days following the exposure to an offending xenobiotic or infection (Table 22–3). Jaundice, pallor, and dark urine may occur with abdominal and back pain. A decrease in the concentration of hemoglobin occurs. The peripheral smear demonstrates cell fragments and cells that have had Heinz bodies excised. Bone marrow stimulation results in a reticulocytosis by day 5 and an increased erythrocyte mass. In general, a normal bone marrow can compensate for ongoing hemolysis and can return the hemoglobin concentration to normal. Most crises are self-limiting because of the higher G6PD activity of younger erythrocytes. Historically, the anemia observed when primaquine was administered to type A– military recruits for malaria prophylaxis resolved within 3 to 6 weeks in most cases.17 Some xenobiotics, including APAP, vitamin C, and sulfisoxazole, are safe at therapeutic doses but can cause hemolysis in G6PD-deficient patients following overdose.
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Other presentations of more severe variants of G6PD include neonatal jaundice and kernicterus, chronic hemolysis with splenomegaly and black pigment gallstones, megaloblastic crisis caused by folate deficiency, and aplastic crisis after parvovirus B19 infection.
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Vitamin B12 and folate are essential for one-carbon metabolism in mammals. One-carbon fragments are necessary for the biosynthesis of thymidine, purines, serotonin, and methionine; the methylation of DNA, histones, and other proteins; and the complete catabolism of branched chain fatty acids and histidine. Unable to synthesize vitamin B12 or folate, mammals are dependent on dietary sources and microorganisms for these cofactors. The hematologic manifestation of vitamin B12 or folate deficiency is a characteristic panmyelosis termed megaloblastic anemia. The hallmark nuclear-cytoplasmic asynchrony is due to disrupted DNA synthesis, halted interphase and ineffective erythropoiesis.79 The hyperplastic bone marrow contains precursor cells with abnormal nuclei filled with incompletely condensed chromatin. Among circulating blood cells, macrocytic anemia (macro-ovalocytes) without reticulocytosis is followed by the appearance of granulocytes with an abnormally large, distorted nucleus (hypersegmented neutrophils with six or more lobes). Lymphocytes and platelets may appear normal but are also functionally impaired.
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In addition to dietary deficiencies, which have become less common, macrocytic anemia with or without megaloblastosis in adults is usually caused by chronic ethanol abuse, chemotherapeutics, or antiretrovirals, especially when mean corpuscular volumes are only moderately elevated (100–120 fL).87 The folate antagonists aminopterin, methotrexate, hydantoins, pyrimethamine, proguanil, sulfasalazine, trimethoprim sulfamethoxazole, and valproate can interfere with DNA synthesis. Ethanol affects folate metabolism and transport. Vitamin B12 deficiency can be induced by chronic exposure to nitrous oxide, biguanides, colchicine, neomycin, and the proton pump inhibitors. Purine analogs (eg, azathioprine, 6-mercaptopurine, 6-thioguanine, acyclovir) and pyrimidine analogs (eg, 5-fluorouracil, floxuridine, 5-azacitidine, and zidovudine) also disrupt nucleic acid synthesis. Hydroxyurea and cytarabine, which inhibit ribonucleotide reductase, also delay nuclear maturation and function and frequently cause megaloblastosis.
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Pure Red Cell Aplasia
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Pure red cell aplasia is an uncommon condition in which erythrocyte precursors are absent from an otherwise normal bone marrow. It results in a normocytic anemia with inappropriately low reticulocyte count. The other blood cell lines are unaffected, unlike aplastic anemia. Pharmaceuticals cause fewer than 5% of cases of this uncommon condition, having been implicated in fewer than 100 human reports.96 Only phenytoin, azathioprine, and isoniazid meet criteria for definite causality; chlorpropamide and valproic acid can be considered only as possible causes.96 Most other xenobiotics are cited only in single case reports, and drug rechallenge was not used, making the association uncertain.
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In part because of its rarity, a cluster of 13 cases in France of pure red cell aplasia in hemodialysis patients receiving subcutaneous recombinant EPO ultimately led to an international effort by researchers, regulatory authorities, and industry to identify the etiology.14,26 To reduce theoretical concerns regarding transmission of variant Creutzfeldt-Jakob disease, human serum albumin was replaced with polysorbate 80 as the stabilizer in a formulation used in Europe and Canada. It is suspected that this change allowed rubber to leach from the uncoated stopper of prefilled syringes, triggering an immune response against both recombinant and endogenous EPO in some patients.63 This episode not only serves as a recent example of successful pharmacovigilance for rare adverse drug effects but has also influenced safety assessments for an emerging class of biological therapies that include simple peptides, monoclonal antibodies, and recombinant DNA proteins.13,63
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Erythrocytosis denotes an increase in the red cell mass, either in absolute terms or relative to a reduced plasma volume. An increasingly recognized cause of drug-induced absolute erythrocytosis is the abuse of recombinant human EPO by athletes to enhance aerobic capacity (Chap. 40). Autologous blood transfusions (doping) are also used in this population, and both can cause dangerous increases in blood viscosity. Cobalt was once considered for the treatment of chronic anemia32 due to its ability to cause a secondary erythrocytosis (Chap. 94). The mechanism may involve impaired degradation of the transcription factor hypoxia-inducible factor 1α, thereby prolonging EPO transcription. This effect is more pronounced in high altitude dwellers, in whom elevated serum EPO concentrations persist despite hematocrits in excess of 75% and chronic mountain sickness is associated with increased serum cobalt concentrations.52