Chapter 33: Erythrocyte Turnover

Normal human red blood cells have a life span of approximately 120 days, after which they are engulfed by macrophages. This is an extremely efficient process as macrophages phagocytose approximately 5 million erythrocytes every second without a significant release of hemoglobin into the circulation. The precise molecular mechanism by which macrophages recognize senescent red blood cells for phagocytosis remains largely unknown. As red blood cells age, several physiologic changes occur that may serve as signals for recognition by macrophages.^1,2 These include a decrease in the activity of enzymes,³ a progressive decrease of ATP content,⁴ a loss of lipid asymmetry with exposure of phosphatidylserine,⁵ an accumulation of lipid peroxidation products,⁶ a desialylation of membrane glycoprotein,⁷ an exposure of cryptic senescent antigens,⁸ aggregation of band 3 protein (Chap. 46),⁹ a decrease in deformability as the result of increased oxidative stress,¹⁰ and an increase in cell surface-bound immunoglobulins and complement components (Chap. 54).^11,12 All of these changes have been investigated as signals for recognition by the macrophages.

MEASUREMENT OF RED CELL DESTRUCTION

The original method for the measurement of the red cell life span consisted in the transfusion of cells that were compatible but identifiable immunologically—the Ashby technique; type O red cells were infused into individuals with type A or B cells. The differential agglutination technique used anti-A or anti-B antiserum to measure the life span of type O red cells that were transfused to type A or type B recipients and the recipients’ own cells were removed using anti-A or anti-B serum.¹³ During World War II and shortly after, this method was used extensively, but in recent years, because of the hazards associated with the administration of allogeneic erythrocytes, it has been completely replaced by techniques based on labeling of autologous blood. Furthermore, this method could not be applied to autologous red cells.

In 1946, Shemin and Rittenberg demonstrated that the incorporation of nitrogen (¹⁵N)-labeled glycine into heme could be used to measure the life span of the red cells.¹⁴ Since then a number of other isotopic methods have been developed. These can be divided into three groups: (1) those that label a cohort of cells, (2) those that label cells randomly, and (3) those that use indirect measurements such as the rate of production of red cells or the rate of heme breakdown. The first two methodologic approaches yield information about the nature of the shortening of the red cell life span, age-dependent or random. The methodology yields only mean life span.

COHORT METHODS

Cohort methods depend on the biosynthetic incorporation of the label into the developing red cells. In these methods, a group of cells of approximately the same age is labeled. The labels used are glycine-containing labeled ¹⁵N,¹⁴ radioactive carbon (¹⁴C),¹⁵ or radioactive iron (either ⁵⁵Fe or ⁵⁹Fe).^16,17,18 The main disadvantage of cohort labeling is the need for prolonged periods of sampling, especially if the life span is only moderately reduced (Fig. 33–1). In addition, radioiron from destroyed red cells may be reused, making it difficult to interpret results. Furthermore, the increasing restrictions on use of radiochemicals has drastically decreased availability of these two previously widely used nuclear medicine tests.

A simple double-labeling technique that allows nonradioactive cohort labeling was described using two distinct labeling steps separated by a defined time interval. Cells are subsequently evaluated by the relative proportions of these labels. The initial labeling step uses biotin that binds to all circulating cells (the red blood cells accounting for most of the label) the second administered labeling substance at later time digoxigenin then distinguishes erythrocyte subpopulation of known age.¹⁹

RANDOM-LABEL METHODS

The random-label methods are the Ashby differential agglutination technique,¹³ which uses an immunologic marker, and or the use of various red cell labels such as chromium-50 (⁵⁰Cr), chromium-51 (⁵¹Cr), or chromium-53 (⁵³Cr),^20,21,22 diisopropylfluorophosphate (DFP) labeled with phosphorus-32 (³²P),^{23 14}C,²⁴ or ¹⁴C cyanate,²⁵ a lipophilic dye,^26,27 or biotin.^28,29

Chromium-51 Method

By far the most commonly used radioactive isotope for the measurement of the red cell life span is ⁵¹Cr. As the chromate ion penetrates the red cell membrane it binds to the β and γ chains of globin. Unfortunately, these bonds are not covalent and there is a continuous elution of the isotope, varying from 0.5 to 2.9 percent per day.³⁰ DFP, on the other hand, is irreversibly bound to red cell cholinesterase. There is some elution of unbound DFP during the first 2 to 3 days of study, but after that, DFP disappearance closely matches red cell destruction.^23,31 Nevertheless, because sample preparation is somewhat complicated, this label is not commonly used.

The life span is estimated by measuring the survival of randomly labeled red cells. Immediately following transfusion, the labeled red cells equilibrate with unlabeled red blood cells (RBCs). This takes normally approximately 5 minutes,³² but may be longer in patients with splenomegaly. Following equilibration, the cells that have been damaged by the labeling process will be removed from the circulation during the next 24 hours. RBCs that survive this period will usually have their expected long-term survival.³³

To accurately calculate red cell life span using a random label method requires steady-state conditions or that correction can be made for concurrent blood loss or blood transfusion. Fortunately, it is usually possible to gain an accurate estimate of red cell half-life by sampling three times a week for 1 to 2 weeks.

In the normal human the red cell, life span is finite with an average of approximately 120 days, with very little random destruction, that is, loss irrespective of cell age (0.06 to 0.4 percent per day). In some mammalian species the amount of random destruction is much greater.³⁴ The survival curve of randomly labeled human red cells should consequently be nearly linear from day 0 to day 120, with a half-life of 60 days. When ⁵¹Cr is used as the label, approximately 1 percent of label elutes per day and the survival curve becomes exponential with a half-life of approximately 30 days (see Fig. 33–1). For clinical use, the red cell life span is usually expressed as chromium half-life (T_1/2) and compared to the normal value for the method of 30 days.

Because merely expressing the red cell life span measured by chromium as chromium T_1/2 will not give information as to the character of destruction, senescence versus random, it has been recommended that in addition a correction factor for chromium elution be used and the data recorded using linear coordinates.³⁵ If the data lie on a straight line, the destruction is by senescence and the life span can be calculated as twice the half-life. If the data indicate exponential disappearance and it is necessary to use a semilogarithmic paper in order to depict the data on a straight line, the destruction is random and the life span is 1.44 times the half-life. One objection to this method is that the degree of chromium elution is not a constant but varies from day to day and is influenced by various disease states.³⁰ Furthermore, the best fit of data is rarely linear or exponential, but somewhere between. Although computer-assisted methods can resolve ambiguities, the inherent biologic and technical variations in measuring red cell life span are such that it is better to rely on chromium T_1/2 with intuitive adjustments based on clinical findings.

Biotin Method

A nonradioactive label has also been developed to label the RBCs in humans by covalent attachment of biotin to red cell membrane proteins and enumerating the survival by flow cytometry.³⁶ Biotin labeling estimates RBC survival comparable to that obtained with a concurrent ⁵¹Cr label for both normal volunteers and patients with sickle-cell disease. In addition to being a nonradioactive probe, biotin labeling has other advantages. The transfused cells can be isolated from the patients on avidin substrates for further characterization. Biotin labeling has been used to demonstrate that sickle cells without fetal hemoglobin have a shorter in vivo survival compared to those with fetal hemoglobin,³⁷ and has been also instrumental in showing a role for phosphatidylserine exposure in the clearance of sickle cells.³⁸

INDIRECT METHODS

There are two approaches to the calculation of the red cell life span by indirect methods: from a measurement of the rate of production of red cells using radioactive iron and from a measurement of the rate of breakdown of heme to bilirubin,³⁹ that is, the release of carbon monoxide from catabolized heme.⁴⁰ Both of these compounds are derived almost exclusively from catabolized hemoglobin and measurements of their rate of production have provided useful information about the red cell life span. There are too many variables that affect the serum bilirubin level to make it a reliable, quantitative measurement of red cell destruction. The measurement of carbon monoxide (CO) production was formerly very tedious, requiring elaborate rebreathing apparatus. With the development of newer technologies,^41,42 measuring CO levels has become more practical. An advantage of the measurement of blood CO as an indication of the rate of red cell destruction is that it gives the rate of destruction at a single point in time. An instrument by CoSense (Capnia Inc., Palo Alto, CA) obtained FDA 510(k) clearance in December 2013 and provides a substitute for the previous Natus end-tidal breath analyzer (Natus Medical, San Carlos, CA) that had not been available for about a decade. CoSense is compact and portable, and uses a sterile one-time-use single nasal cannula for quantifying end-tidal breath for CO quantification and also samples ambient CO, which is subtracted from the value in exhaled breath and is suitable for use in neonates. This device also counts the breath rate, analyzes CO in individual breaths, and provides CO measurements in parts per million in a matter of minutes.

As part of routine erythrokinetic studies both radioactive iron and radioactive chromium may be used to localize red cell production and red cell destruction. This is accomplished by positioning probes for external counting over the sacrum, liver, spleen, and heart and measuring the distribution of radioactivity in the body.⁴³

In a normal subject, ⁵⁹Fe injected intravenously is cleared rapidly from the plasma, and within 24 hours approximately 85 percent of the radioactivity can be accounted for in the marrow. The liver and the spleen divide the remaining 15 percent. Over the next 10 days the marrow radioactivity decreases gradually as a result of the release into circulating blood of red cells labeled with radioactive hemoglobin. Patterns showing different uptake and distribution of the radioactive iron have been found for various hematologic disorders.⁴⁴ In hypersplenism, the trapping and destruction of iron-labeled cells in the spleen increases splenic radioactivity rapidly, and in patients with erythroid hypoplasia the distribution of radioactive iron between liver and marrow is reversed (Fig. 33–2).

IMAGING MACROPHAGES IN THE MARROW, LIVER, AND SPLEEN

More effective methods demonstrating in situ erythropoiesis involve imaging the macrophages in the marrow, liver, and spleen with a technetium-99m (^99mTc) sulfur colloid or indium-111 (¹¹¹In).⁴⁵ Although these isotopes label primarily the monocyte-macrophage system, their uptake is similar to that of ⁵⁹Fe and they can be used as surrogate markers to estimate the distribution of erythroid tissue.

SURFACE COUNTING FOR CHROMIUM-51

Surface counting for ⁵¹Cr-labeled red cells provides a characteristic organ distribution of radioactivity and has been used to demonstrate the degree of red cell sequestration and destruction in an enlarged spleen (see Fig. 33–2).⁴⁶ This approach has been used to predict the results of elective splenectomy, but the utility of this method has been challenged.⁴⁷ The in situ localization of red cell sequestration or destruction can also be determined by following the tissue distribution of ⁵⁹Fe-labeled red cells, especially if the red cell life span is very short.

SENESCENCE OF NORMAL ERYTHROCYTES

Methodologic Considerations

Labeling a cohort of human erythrocytes with ⁵⁹Fe and centrifuging the cells in a density gradient demonstrates that reticulocytes and young red cells are less dense than mature red cells.^48,49 However, at the end of the life span of the labeled cohort, radioactivity is fairly evenly distributed throughout red cells of all densities, with only a slight tendency of the radioactivity to be concentrated in the more dense cells. Unfortunately, many studies of the properties of senescent cells in the past have been based upon the characteristics of the most dense fraction of erythrocytes, using various fractionating techniques. In fact, the most dense fraction of red cells is only slightly enriched with old erythrocytes.^50,51 A combination of density separation and elutriation seemed to provide results superior to density separation alone using hemoglobin A_1C content as a marker, but the degree of enrichment with older cells has not been documented using actual old red cells as separated by biotinylation or by the mouse hypertransfusion technique.⁵²

There are two animal models and one human disease model that provide cells that are truly aged. In mice, in vivo aged cells have been produced by serially transfusing mice, maintaining polycythemia to suppress virtually all erythropoiesis.⁵³ In other species, particularly the rabbit, red cells have been labeled with traces of biotin, which allows them to be recovered from the circulation.⁵⁴ The human model is transient erythroblastopenia of childhood (Chaps. 36 and 55), a disorder in which there is cessation of all erythropoiesis for several months; however, the density and deformability of the aged cells in erythroblastopenia of childhood is normal.⁵⁰ The use of the latter model has been criticized because this disorder is not fully understood and the red cells in the circulation may not be entirely normal.⁵⁵ However, the results that have been obtained are consistent with those obtained in animal models and are probably reliable (see “Properties of Aged Cells” below).

PROPERTIES OF AGED CELLS

Although the activities of a large number of enzymes, including hexokinase, glucose-6-phosphate dehydrogenase (G6PD), and pyruvate kinase (PK), are higher in reticulocytes than in mature erythrocytes, the activities of these enzymes do not normally continue to decline during the aging of the erythrocyte.^54,56 Pyrimidine-5′-nucleotidase^57,58 and adenosine monophosphate (AMP)-deaminase^59,60,61 appear to be exceptions to this rule in that there is continuing decline of enzyme activity throughout the life span of the red cell. The decrease in the activity of these enzymes is not linear with age but exponential.³ This stability of many of the red cell enzymes during the aging of normal erythrocytes contrasts to the circumstances that are brought about by mutations in enzymes such as G6PD and PK, where instability of the abnormal enzyme leads to accelerated decay in the amount of enzyme protein, a factor that surely plays an important role in the ultimate demise of the cell (Chap. 47). Fluorescent sorting of blood type NN erythrocytes transfused into humans shows that the most dense fractions are only minimally enriched with old cells,⁶² and biotinylated aged cells of rabbits have been found to have only a modestly decreased surface area, volume, cell water, and density, and therefore slightly decreased deformability.^51,63

As they circulate red cells lose a substantial portion of their membrane and hemoglobin in the form of vesicles (reviewed in Ref. 64). The loss of membrane material in hemoglobin vesicles may play a role in the aging process.⁶⁵ In normal blood, a small number of red cell vesicles, approximately 190/μL blood, can be harvested. During storage of RBCs, the aggregates of vesicles are formed and may contribute to acute lung injury by interacting with neutrophils.⁶⁶

MECHANISM OF DESTRUCTION OF NORMAL, AGED CELLS

Several different mechanisms of senescent red cell destruction have been proposed. Determining the actual mechanism(s) is especially difficult because the cells that are marked for removal are bound to be present at very low concentrations or not at all in the circulating blood—they have been removed. Many of the earlier data are predicated upon the isolation of dense cells and the consideration that they are “old”; we now recognize that they are not (see “Methodologic Considerations” above). Moreover, it is likely that there is more than one mechanism that serves to remove effete red cells from the circulation; there is no known mutation that lengthens red cell life span.

Band 3 Clustering Models

It has been proposed that an altered membrane band 3 serves as a receptor for antibodies directed against a neoantigen, designated senescent-cell antigen, and that possibly after-binding complement marks the senescent cell for destruction. It is not known how clustering of band 3 occurs in vivo and recent work suggests peroxidation of cytoplasmic aspect of band 3 results in carbonylation. Methemoglobin binds to the cytoplasmic peroxidized domain of band 3 and induces cluster formation.⁶⁷ But much, if not all, of the evidence for these models depends upon the assumption that dense cells are old, and the uptake of cells by monocytes as a surrogate for their being marked for destruction.⁶⁸ However, immunoglobulin levels on aged, biotinylated rabbit cells are not increased,⁶⁹ and the fact that red cell life span has never been demonstrated to be prolonged in agammaglobulinemic patients casts serious doubt upon the concept that immunoglobulins mediate removal of senescent red cells.

Phosphatidylserine Exposure Models

In RBCs, as in most other cells, the anionic phospholipid phosphatidylserine is present exclusively in the inner cytoplasmic leaflet of the membrane bilayer.⁷⁰ The exposure of phosphatidylserine on the outer leaflet of the cell membrane is one of the signals that allows macrophages to recognize apoptotic cells. It is likely that this is, indeed, at least one of the signals by which macrophages recognize senescent erythrocytes.^5,71,72 Data from a biotinylated rabbit erythrocyte model suggests that the average time during which phosphatidylserine is exposed is only 0.3 to 0.5 days, so that few cells with increased exposure of the phospholipid are in the circulation at any time.⁷¹ An increase of phosphatidylserine exposure has also been documented in humans descending from high altitudes.⁷³ The exposure of phosphatidylserine on the outer leaflet of the cell membrane is one of the signals. A proposed model for the destruction of newly formed cells was that endothelial cells might respond to changes in circulating erythropoietin by influencing the interaction of phagocytes with young red cells, targeting the cells by surface adhesion molecules.⁷⁴ A study in mice, using somewhat different methods, suggested that phosphatidylserine exposure is greatest in young erythrocytes, and does not increase with aging.⁷⁵ It is not yet clear whether phosphatidylserine exposure is the only or even the primary signal that indicates that a cell has reached the end of its life span, but it is the only major difference between senescent and nonsenescent erythrocytes that has been documented clearly.⁷²

Several proteins were described that bind to phosphatidylserine-expressing apoptotic cells including lactadherin,⁷⁶ gas-6,⁷⁷ Del-1,⁷⁸ and several complement components.⁷⁹ These proteins act as opsonins in promoting the clearance of phosphatidylserine-expressing cells by macrophages. Angiogenic endothelial cells also express several integrin associated with phagocytosis in macrophages and can engulf phosphatidylserine expressing “aged” erythrocytes and may play a role in clearance of senescent cells.⁸⁰

Eryptosis is defined as cell shrinkage and exposure of phosphatidylserine caused by entry of calcium ions followed by activation of a scramblase, an enzyme capable of randomizing the distribution of phospholipid in both membrane bilayers.⁷⁰ It may contribute to red cell clearance in diseased states,⁸¹ but its role in senescence associated clearance is not clear.⁸²

Another model that has been proposed is based upon a slight increase in green autofluorescence, believed to represent the result of oxidative damage, which has been observed in aging murine erythrocytes.⁸³ Interaction of erythrocyte cell surface antigen CD44 with hyaluronic acid may play a role in the clearance of aged erythrocytes from the circulation, but such clearance seems limited to primates, and a patient with CD44 deficiency manifested congenital dyserythropoietic anemia.⁸⁴

Hypoxia increases RBCs by enhancing hypoxia-inducible factors (HIFs). Upon return to normoxia, the secondary polycythemia is overcorrected, as the accumulated, newly formed RBCs undergo preferential destruction, a process termed neocytolysis; however, its mechanism is unclear.⁷⁴ Neocytolysis was originally observed during space travel at zero gravity wherein the mechanism is even less clear. It has been suggested that on return to normoxia, there is excessive generation of reactive oxygen species from increased mitochondrial mass correlating with decreased hypoxia controlled gene BNIP3L transcripts,^85,86 as BNIP3L mediates removal of reticulocyte mitochondria accompanied by reduced catalase activity.⁸⁷ Rapid changes in hematocrit in human newborns also suggest that neocytolysis also occurs after birth when a hypoxic fetus is polycythemic at birth, but the neonate rapidly overcorrects its increased red cell mass and becomes anemic in first 2 weeks of life.^88,89

“Senescence of Normal Erythrocytes,” above, enumerated some mechanisms that may be involved in normally terminating the life of the effete erythrocyte. It has sometimes been assumed that the mechanisms by which red cells are destroyed prematurely in disease states reflect these normal mechanisms. Although there may well be some overlap, the mechanisms of red cell destruction in disease states are likely different. The assumption that the mechanisms that bring around hemolytic anemia represent premature aging of the erythrocyte is no more logical than to suggest that an animal’s death through pneumonia, renal failure, or cancer represents premature aging.

INTRAVASCULAR DESTRUCTION

If the red cell membrane is breached in the circulation the red cell is destroyed. This mode of erythrocyte demise occurs at a low frequency, but may be the predominant mode of destruction in some hemolytic disorders, for example, ABO-incompatible transfusions (Chap. 138) and paroxysmal nocturnal hemoglobinuria (Chap. 40), where the surface complement complex creates pores in the red cell membrane, and in cardiac valve hemolysis (Chap. 51) and microangiopathic hemolytic anemia (Chaps. 51 and 132), where the shear stress may be so strong as to break open the membrane.

EXTRAVASCULAR DESTRUCTION

Most commonly, the life of the red cell comes to an end when it is ingested by a macrophage. Clearly, signals that allow the macrophage to distinguish the younger normal red cell from a damaged or senescent cell must exist. Such signals may consist of decreased deformability and/or altered surface properties.

Decreased Deformability

The red cell does not circulate as the biconcave disc customarily observed under the microscope. Instead, it is normally greatly distorted by the shear stresses in the circulation and such distortion is an absolute requirement for the red cell to be able to negotiate the narrow slits that separate the splenic pulp from the sinuses (Chaps. 6 and 56). The deformability of the erythrocyte can be measured clinically using the ektacytometer, an instrument that displays the diffraction pattern of a red cell suspension under shear stress.^90,91 The red cell membrane, a lipid bilayer, bends readily but has very little capacity to stretch. Thus, deformability is largely a function of the excess red cell membrane intrinsic to the biconcave disc shape of the cell, membrane composition, and to some extent, of the viscosity of the hemoglobin solution within the cell. As the red cell loses membrane it assumes a spherical shape and loses its ability to deform. Hereditary spherocytosis and hereditary elliptocytosis are prototypic of hemolytic anemias in which decreased deformability as a result of a decreased surface-to-volume ratio plays a key role in red cell destruction (Chap. 46). However, loss of membrane plays a role in many types of pathologic hemolysis, including autoimmune hemolytic anemia (Chap. 54). In sickle cell disease and hemoglobin C disease (Chap. 49), the internal viscosity of the cell is increased. Loss of water from the red cell, as may occur when the membrane is damaged and leaks potassium as in hereditary xerocytosis (Chap. 46), also markedly impairs the deformability of the cell.

Altered Surface Properties

The surface of the red cell membrane can be altered by binding of antibodies to surface antigens, by binding of complement components, and by chemical alterations, particularly oxidation of membrane components. Immunoglobulin (Ig) G–coated red cells⁹² and red cells coated by the third component of complement (C₃)^93,94 are bound by Fc receptors on macrophages and undergo partial phagocytosis. This results in the formation of a spherocyte.

In vitro oxidation of red cells with phenylhydrazine or adenosine diphosphate (ADP) plus iron causes clustering of band 3 protein in the membrane. Although the physiologic significance of this is far from clear, it has been suggested that the clustered protein serves as a recognition site for the binding of IgG.^9,95 Oxidative damage to the membrane may play a role in the removal of sickle cells (Chap. 49) and thalassemic cells from the circulation (Chap. 48).

INTRAVASCULAR DESTRUCTION

Hemoglobin

When red cells are destroyed in the vascular compartment the hemoglobin escaping into the plasma is bound to haptoglobin. A dimeric glycoprotein, each molecule of haptoglobin can bind two hemoglobin dimers. The binding of hemoglobin not only protects against its potential toxicity, it also triggers the second step of the scavenging process, that is, recognition by macrophage receptor CD163, and subsequent clearance of the entire complex by receptor-mediated endocytosis.⁹⁶ CD163 belongs to the scavenger receptor cysteine-rich family of proteins and the haptoglobin–hemoglobin complex is cleared from the plasma with a T_1/2 of 10 to 30 minutes. The heme of the hemoglobin is converted to iron and biliverdin by heme oxygenase and the biliverdin is further catabolized to bilirubin. CO is released (see “Indirect Methods,” above, on measuring red cell life span) in the course of cleavage of heme by heme oxygenase.⁹⁷

Haptoglobin

Free haptoglobin, in contrast to the hemoglobin–haptoglobin complex, has a T_1/2 of 5 days, and when large amounts of the rapidly turned over haptoglobin–hemoglobin complex are formed, the haptoglobin content of the plasma is depleted. The haptoglobin content of the plasma is diminished not only in the plasma of patients undergoing frank intravascular hemolysis, but also from the plasma of patients who, like those with sickle cell disease, have accelerated red cell destruction occurring primarily within macrophages. Presumably there is either enough intravascular hemolysis in such hemolytic disorders to lower the plasma haptoglobin level or sufficient leakage from the phagocytic cells into the plasma to bind to haptoglobin. Thus the measurement of plasma haptoglobin levels has usefulness in diagnosing the presence of hemolysis, although it cannot, as previously suggested, serve to clearly distinguish extravascular from intravascular hemolysis.

Heme

Free heme that is released into the circulation is bound in a 1:1 ratio to the plasma glycoprotein hemopexin,⁹⁸ which is cleared from the plasma with a T_1/2 of 7 to 8 hours.^99,100 The heme–hemopexin complex is taken up by a low-density lipoprotein-related receptor, CD91.¹⁰¹ Figure 33–3 illustrates the parallel functions of hemopexin and haptoglobin. When the capacity of hemopexin to bind heme is saturated, excess heme may bind to albumin to form methemalbumin.¹⁰² Excess heme is toxic to cells because of the ability of heme to catalyze the so-called Fenton reaction, generating hydroxyl radicals, a highly reactive oxygen species. To avoid the phenomenon and complement the negative feedback regulation of heme synthesis, the expression of heme oxygenase (HO)-1 is induced in response to an increased level of heme, which subsequently results in the degradation of excess heme not bound to proteins. In contrast to HO-1, HO-2 is constitutively expressed and participates in the regulation of a basal heme level.

EXTRAVASCULAR DESTRUCTION

Red cells that are engulfed by phagocytic cells are degraded within lysosomes into lipids, protein, and heme. The proteins and lipids are reprocessed in their respective catabolic pathways and the heme is cleaved by a microsomal HO.¹⁰³ HO catalyzes the oxygen-dependent degradation of heme to biliverdin with the release of CO and “free” iron. Biliverdin is converted into bilirubin by biliverdin reductase α (BVRα), which is expressed ubiquitously in all tissues under basal conditions with high levels in macrophages in the spleen and liver.¹⁰⁴ The overall reduction of biliverdin to bilirubin is very efficient, and under physiologic circumstances, the concentration of serum biliverdin is low.

Bilirubin Excretion

Regardless of the site of destruction of hemoglobin, one of the final products is bilirubin. Bilirubin is very insoluble and transported in plasma bound to albumin. Unconjugated bilirubin is taken up into hepatocytes by transporters of the organic anion-transporting polypeptide (OATP) family, followed by conjugation with glucuronic acid in the microsomes, and ATP-dependent transport into bile. This efflux across the canalicular membrane is mediated by multidrug resistance protein 2, which has high affinity for monoglucuronosyl bilirubin and bisglucuronosyl bilirubin.¹⁰⁵ In the gastrointestinal tract the bilirubin is converted to urobilinogens by bacterial reduction.¹⁰⁶ A small fraction of urobilinogen is reabsorbed and excreted into the urine. Thus, the fecal and urinary urobilinogen excretion have been used as an indicator of the rate of hemolysis, but are only uncommonly used for this purpose in modern practice because the collections are cumbersome and because alternative degradative pathways detract severely from the accuracy of the estimates of the rate of heme catabolism.