Wintrobe’s Clinical Hematology, 12th Edition

Chapter 8

Destruction of Erythrocytes

Bertil Glader

The mature erythrocyte lacks the organelles that make protein synthesis possible, and thus it is incapable of self-repair. Its life-span is finite and is shortened further when the cell’s environment becomes hostile or when the cell’s ability to cope with damaging extracellular influences becomes impaired. Excessive destruction of red cells plays a major role in hemolytic diseases. This chapter focuses on the following issues: How long do normal red cells live? Why do red blood cells (RBCs) die? Where do red blood cells die? What are the biochemical changes associated with red cell death?

Methods for Estimating Erythrocyte Lifespan

Valid measurements of erythrocyte survival in the intact animal became possible in 1919 when Ashby devised the differential agglutination technique (1). Since then, other less cumbersome methods have been introduced. Studies with these techniques have demonstrated that after a finite lifespan, the erythrocyte disappears from the circulation. In humans, this lifespan averages approximately 120 days. In other mammals, values range from 40 days in mice to 225 days in the llama (2). Still longer survivals, 600 to 1,400 days, are observed in poikilotherms with low metabolic rates, such as toads and turtles (3).

There are a number of direct methods for measuring erythrocyte lifespan (2,4). Cohort methods depend on the incorporation of an isotopically labeled substance into a group (“cohort”) of newly formed cells. If exposure to the label is brief and there is no reuse of label, the tagged cells will be of very nearly the same age. In contrast, random-labeling methods use tracers that bind with all cells in the circulation regardless of age. The patterns of time-dependent change in circulating red cell label produced by these two procedures are quite different (Fig. 8.1). Cohort labels result in a pattern characterized by a plateau, the length of which is a measure of erythrocyte lifespan. In contrast, random labels begin to disappear from the circulation immediately, and erythrocyte lifespan is related to the time when all label has vanished, the so-called extinction time. In the past these procedures helped define normal and abnormal RBC survival in various anemias; however, from a clinical perspective, red cell survival studies seldom are done nowadays and largely are of historical interest.

Cohort Methods

The ideal cohort label would produce labeling in the shortest possible time, would label the erythrocyte and no other cells or proteins, would not injure the labeled cell or alter its survival, would remain with the cell throughout its life, would not be reused, would present no risks to the recipient, would be easily assayed, and would not be excessively expensive. Some of the isotopes used throughout the years include 59Fe (5), and glycine tagged with 15N, 14C, or 3H (2). These are incorporated into hemoglobin when it is synthesized by the erythrocyte precursors and remain with the cell throughout its lifespan. To determine the erythrocyte lifespan with cohort labels, assays must be performed for over 120 days if the red cell lifespan is normal. Of the various cohort labels, 59Fe is reasonably safe and relatively easy to assay.

Random-labeling Methods

Random-labeling methods have proved to be much more useful than cohort methods for both clinical and research applications because accurate information is made available in a relatively short time. The time at which half of the label has disappeared from the circulation (t1/2) is recorded. When there is neither a significant degree of random destruction nor elution of label, the shape of the disappearance curve is linear, and the mean erythrocyte lifespan is equal to twice the value for t1/2. Random methods include those based on the Ashby differential agglutination technique as well as those in which cells are labeled with isotopes, the most common of which is 51Cr.

The Ashby method is of historical interest. This procedure utilizes the transfusion of compatible but immunologically identifiable blood, for example, group O cells into a group A, B, or AB recipient (1,6). At appropriate intervals, the donor red cells are enumerated following agglutination or hemolysis (7) of the recipient’s cells by appropriate antisera. When properly performed, the differential agglutination technique yields accurate results. The observed disappearance curves are linear in normal human subjects, and deviation from linearity indicates random destruction. Inability to measure the survival of a subject’s cells in his or her own circulation, as well as the hazards of transfusion and the exacting requirements of technique, obviously limited the usefulness of this method.

Radioactive chromium was introduced as a red cell label in 1950 and was utilized in survival studies in vivo shortly thereafter (8,9). Anionic (hexavalent) chromium in the form of the chromate ion (51CrO4-2) can penetrate the red cell membrane. Once inside the cell it is converted to the trivalent cation (Cr+3) and in this form becomes firmly, but not irreversibly, bound to hemoglobin. The chromium attaches to the β-chains of hemoglobin A and, to a lesser extent, to the γ-chains of hemoglobin F (10). The principal disadvantage of the chromium label is that it is slowly eluted from the cell. The rate of elution in normal subjects was found to be 0.57 to 1.28% per day and averaged 0.93% per day (8). An assumed rate of 1% per day is often used for analysis of chromium survival curves. The disappearance of 51Cr from the circulation thus reflects not only the red cell lifespan, but also the elution rate; as a consequence the chromium disappearance curve is nonlinear. However, if the data are plotted on a semilogarithmic scale, or if appropriate correction factors (11) are applied, the pattern approximates a straight line (Fig. 8.2). From such a plot, the disappearance half-time (t1/2 Cr) may be derived. The chromium elution rate depends in part on the techniques used in the labeling procedure (12). Consequently, reported average normal values for t1/2 Cr have varied widely from 25 to 33 days.

As mentioned above, red cell survival studies now are infrequently done, but if performed, the chromium method is used more than any of the other available methods. Because it emits a high-energy γ ray, radioassay is comparatively easy and requires little sample preparation. Also, radioactivity monitored over the body surface can be used to determine the principal sites of destruction (13). Because labeling usually is performed ex vivo, cross-transfusion studies also are possible; thus, survival of a patient’s cells in a normal recipient or of normal cells in a patient can be evaluated by this method. Previously these procedures yielded much information regarding cell survival in various hemolytic anemias, helping to define intrinsic versus extrinsic hemolytic disorders. Safety considerations nowadays do not allow for similar studies.

Figure 8.1. Comparison of idealized patterns produced by cohort and random methods of labeling circulating erythrocytes. These curves are constructed on the assumption that no elution of label occurs and that all erythrocyte destruction is senescent. Bars below the main graphs, distribution of labeled cells (shaded areas) by cell age. These labeled cells can be thought of as being forced to the right by a piston made of newly produced, unlabeled cells. (Modified from Berlin NI, et al. Life span of the red blood cell. Physiol Rev 1959;39:577.)

Diisopropylfluorophosphate labeled with 32P (DF32P) reacts with esterases, especially cholinesterase, and becomes firmly bound to the cell (14). DF32P labels leukocytes and platelets as well as erythrocytes. In contrast to chromium, little DF32P is eluted after the first 24 to 48 hours and none after 10 days. As a result, the normal disappearance curve is nearly linear and many complexities of interpretation are avoided (15,16). For these reasons, DF32P is a highly satisfactory label for determining lifespan. However, sample preparation is somewhat complicated, surface counting is not possible, and it is no longer commercially available (17).

Figure 8.2. 51Cr erythrocyte survival curve in a normal subject before (A) and after (B) correction for elution (see text). Half of the radioactivity is gone at 30 days (T1/2Cr), but the radioactivity does not disappear completely until 115 days, the so-called extinction time.

The lifespan of the normal human erythrocyte as measured by the various procedures described above is essentially the same, namely, 117 (110 to 135) days by the Ashby method, 113 (108 to 120) days when the 51Cr label is used, 118 (109 to 127) days with 15N-glycine, and 124 days with DF32P. Thus, it appears that approximately 0.83% of the circulating red cells are replaced each day.

Erythrocyte Aging

Once the erythrocyte has lost its nucleus and ribosomes, it can no longer synthesize protein. With the loss of its mitochondria, it is dependent upon anaerobic glycolysis to provide energy. In humans, it must then survive 4 months and hundreds of miles of hazardous travel, subjected to oxidant and osmotic stresses, with very limited equipment with which to maintain itself. Nevertheless, it does retain significant repair capacity. In addition to its ability to sustain effective levels of adenosine triphosphate (ATP), nicotinamide adenine dinucleotide (NADH), and nicotinamide adenine dinucleotide phosphate (NADPH), and to maintain its ionic composition, it can also reduce methemoglobin and oxidized glutathione, synthesize new glutathione, and reseal its membrane if a portion of it is lost. Despite these and other capabilities, the cell ultimately is unable to sustain its existence, and after 120 days is removed from the circulation. However, the mechanisms that determine red blood cell senescence and cell death remain unclear despite many years of study.

In attempting to identify the changes that occur in the erythrocyte as it ages, the typical approach has been to separate young and old red cells, to measure a chemical or physical feature of the old cells, and to ascribe the observed difference to the effect of cell age. One of the problems with this approach is that it has been common to assume that any observed change thought to be due to age will continue to worsen and will ultimately be the cause of death of the erythrocyte; however, as discussed below (Erythrocyte Enzymes and Energy Depletion), this assumption may not be correct.

Another major issue has been the difficulty in isolating pure populations of old erythrocytes. The most commonly used method for separating young and old cells makes use of the observation that erythrocyte density increases as a function of age (18,19,20,21,22,23). Although this change occurs with considerable consistency, some mixture of young and old cells almost always has been demonstrated in each density fraction (24,25,26,27), and thus the different compartments actually represent mostly “young” or mostly “old” cells. Other approaches to study red cell aging have been based on examining surviving RBCs from patients with suppressed erythropoiesis, such as in transient erythroblastopenia of childhood (TEC) (28,29). Also, in animal studies it has been possible to label RBCs with N-hydroxysuccinimide biotin, and then, at a later date, separate the aged biotinylated red cells on an avidin matrix (30,31,32). This technology makes it possible to reliably isolate aged red cells for biochemical analysis, and the biotin-labeled cells have been shown to have a normal survival in vivo. Both of the approaches discussed here avoid the problems inherent in the methods based on density separation.

Through the years various hypotheses have been proposed to explain normal red cell death, and these have included changes in red cell enzymes and energy depletion with age; alterations in calcium balance; changes in membrane surface charge; oxidative injury; development of autologous antibodies to membrane antigens; and changes in membrane phospholipid asymmetry. Most of these changes probably do not explain RBC senescence today; they are briefly discussed here for historical reasons.

Erythrocyte Enzymes and Energy Depletion

The activities of erythrocyte enzymes have been measured in cells separated by age on the basis of cell density or osmotic lysis. The activities of a number of enzymes have been reported to decline with the age of the erythrocyte, including all glycolytic enzymes. Based on this it had been suggested by many that red cell senescence and death may be related to this lost metabolic potential (33,34,35,36). However, the assumptions upon which these studies are based have been challenged (29). It is commonly recognized that the activities of certain age-dependent enzymes, such as glucose-6-phosphate dehydrogenase (G6PD), hexokinase, and pyruvate kinase, are greatly increased in red cells of individuals with hemolytic anemia and reticulocytosis. It also is agreed that the metabolic activity of reticulocytes decreases during their short maturation. However, it is not clear that the same decrease in enzyme activity continues during the erythrocytes’ subsequent lifespan or that metabolic failure is the cause of red cell senescence and death. As Beutler has pointed out, if enzyme activity were lost rapidly during the first few days of the red cell’s life but remained essentially stable thereafter, contamination of samples of old cells with progressively smaller numbers of reticulocytes would result in gradually decreasing enzyme activity in those samples (29).

Of interest, in studies using methods in which contamination of older cells with reticulocytes is less likely to occur, the loss of enzyme activity with age has not been confirmed (30,37,38,39,40). One particularly interesting study examined red cells from patients with TEC, a disorder characterized by an older population of red cells at diagnosis (Chapter 45). In this study, no difference in enzyme activity in TEC or control red cells was noted, thus suggesting there is minimal loss of enzyme activity beyond the first days of red cell life (37).

Early studies based on density separation suggested that ATP levels decreased in older red cells (41,42). However, in studies utilizing biotinylated rabbit red cells, the level of ATP in pure old cells was not decreased, but in fact was elevated 75% over control values (32). Moreover, the ATP level in RBCs from patients with TEC also was markedly increased above normal (43). Together, these observations of increased ATP in aged erythrocytes are a surprise, and contrary to traditional concepts of cell aging. The basis of this increased ATP content of older cells is thought to be due to an age-related decrease in adenylic acid deaminase (adenosine monophosphate [AMP] deaminase), an enzyme that irreversibly degrades AMP to inosine 5-monophosphate. In contrast to AMP deaminase, other enzymes involved with maintaining the adenylic acid pool (i.e., adenosine kinase, adenylate kinase, adenosine deaminase) do not change with age (43). Thus, normal ATP synthesis with decreased irreversible breakdown of the adenine nucleotide pool could result in a net increase in ATP (43,44). This relationship of decreased AMP deaminase activity with elevated ATP levels now has been observed in three different situations: In biotinylated separated old rabbit red cells (44), in anemic children with TEC (43), and in red cells from nonanemic individuals with hereditary AMP deaminase deficiency (45).

Calcium Balance

Senescence of erythrocytes has been ascribed to a change in calcium balance resulting in an increasing concentration of calcium in older red cells, a change that might reduce erythrocyte deformability. It has been proposed that a major consequence of ATP depletion in aging cells is decreased activity of membrane-bound (Ca2+-Mg2+)-ATPase and, therefore, calcium accumulation. Increased cell calcium would, in turn, result in potassium loss, dehydration, increased cell density, increased viscosity, and diminished cell deformability (46). It also has been proposed that calcium-activated transglutaminases can cross-link membrane or skeletal protein in senescent cells (47,48). There are no data to support any of the above assumptions and, as discussed above, ATP depletion probably does not occur during erythrocyte aging.

A decrease in (Ca2+-Mg2+)-ATPase activity in denser, and thus potentially older, red cells has been demonstrated (49,50), but normal activity has also been reported (51), and once again the doubtful reliability of the density gradient separation method makes the significance of these observations uncertain. More central to the hypothesis is the concentration of cell calcium, and measurements of the concentration of erythrocyte calcium have yielded inconsistent results (52,53,54,55,56).

Membrane Carbohydrates and Surface Charge

It has been proposed that a diminished negative charge on the erythrocyte membrane would significantly reduce the repulsive interaction between red cells and tissue macrophages and thus facilitate red cell phagocytosis. Specifically, it has been shown that sialic acid, the principal source of membrane negative charge, is lost from membranes of high-density red cells and the total surface negative charge is reduced. Desialylation of red cells decreases their lifespan in vivo (57) and increases phagocytosis by autologous macrophages in vitro (58,59). It has been proposed that desialylation of membrane glycophorin occurs as the red cell ages and creates a “senescence factor glycopeptide” that is the signal for sequestration of moribund red cells (60). However, electrophoretic mobility of high- and low-density cells is not different (61,62) and the difference in charge and sialic acid content is explainable by a loss of membrane surface area in the high-density cells (41,63). In addition, erythrocytes from individuals with recessively inherited deficiencies of red cell sialoglycoproteins survive normally (64). It would seem that if destruction of aging red cells is dependent upon loss of sialic acid and of charge, the relationship is not a simple one.

Oxidative Injury

The role of the erythrocyte as an oxygen-carrying device repeatedly exposes it to the risk of oxidative injury. In addition, intermediate products of oxidative denaturation of hemoglobin, the hemichromes (65), interact with superoxide and hydrogen peroxide to generate hydroxyl radicals (66), and thus create another source of potential damage to the cell. Increased amounts of methemoglobin (67), hemichromes (68), and denatured hemoglobin (Heinz bodies) (68,69) and increased thiobarbituric acid-reactive substances (70,71), an indicator of lipid peroxidation, all have been reported as evidence of oxidation damage in high-density red cells. Further evidence is inferred from similarities between aged cells and those exposed to oxidants in vitro. In red cells incubated with H2O2 or with an end-product of fatty acid peroxidation, malonyldialdehyde, polypeptide polymers are found that may result from cross-linking of the cytoskeletal protein spectrin (72). This oxidant-induced polymerization of spectrin results in decreased cellular deformability and increased adherence to and phagocytosis by monocytes (73,74). Similar abnormalities were found in high-density red cells (73), but not all investigators have found a change in oxidative state of membrane proteins with age using the density-gradient method (75). Most importantly, however, no abnormalities of spectrin are found in aged biotinylated erythrocytes (76) or in red cells separated by methods other than the density-gradient method (25,77).

Autologous Antibodies to Membrane Antigens

Immunoglobulin G (IgG) antibodies that bind to the membranes of some erythrocytes are present in the serum of normal humans. One of these identified antibodies binds to an altered form of band 3 protein (58). It has been proposed that binding of these autologous antibodies provides a senescence signal that is responsible for directing the phagocytosis of aged cells, presumably by recognition of the Fc portion of the bound antibody by the Fc receptor of the macrophage. The apparent anti–band 3 antibody has been demonstrated in a variety of mammals (58,78,79,80), but the amount is small. It is possible that during the aging process, modification of band 3 occurs, perhaps by clustering of band 3 molecules as a consequence of oxidative injury (81). The antibody binds to high-density but not low-density red cells (58,80). However, the number of IgG molecules per biotinylated red cell does not increase with increasing red cell age (82). Despite the large literature on the characteristics of the anti–band 3 antibody, there is little evidence that it is of physiologic importance. Erythrocyte lifespan in mice with severe combined immunodeficiency disease is normal, despite the absence of anti–band 3 antibody in the animals (83).

Change in Membrane Phospholipid Asymmetry

Phospholipids are asymmetrically distributed across the membrane of all eukaryotic cells (84). The outer leaflet contains the cholinephospholipids, phosphatidylcholine and sphingomyelin, while the inner leaflet contains the aminophospholipids, phosphatidylserine (PS) and phosphatidylethanolamine (PE). This asymmetric distribution of lipids across the cell membrane is stable and is maintained by active ATP-dependent processes. Whenever the phospholipid distribution is altered, such that PS or PE are on the outer leaflet, an aminophospholipid translocase (“flipase”) transfers the aminophospholipid from the outer to the inner bilayer, thereby restoring the normal phospholipid asymmetry (85,86). This process is important for many cells because PS exposure is an important signal for macrophages to ingest and remove apoptotic nucleated cells (87,88,89). The mechanisms by which normal phospholipid asymmetry is lost are poorly understood (90).

Several years ago it was observed that phospholipid asymmetry was altered in dense old red cells (91). It was suggested that anucleate red cells with exposed PS are more likely to be ingested by macrophages (92) and that PS exposure may be the signal for macrophages to remove aged red cells from the circulation (83). In studies with biotinylated aged rabbit red cells, it now has been shown that PS exposure does increase during normal red cell aging and thus may be the long-sought signal for red cell senescence (93).

Summary

A clear explanation for the mechanism of red cell senescence remains elusive. It may be that human erythrocyte aging and destruction results from a combination of the abnormalities described in the preceding paragraphs, or from some as yet unrecognized phenomenon. Contrary to long-held concepts, it probably is not due to enzyme or energy depletion. The role of membrane protein alterations also has not been documented. The one intriguing possibility relates to changes in RBC membrane phospholipid asymmetry associated with phosphatidyl serine exposure on the external bilayer. This mechanism would fit into a general concept of how cells are removed from the circulation. However, the factors that might lead to programatic expression of PS on the external RBC membrane remain to be defined (90).

Mechanisms and Site of Red Cell Destruction

The specific determinants of red cell survival are unknown. Much of what is believed to be true is based on inference or on observations made in individuals with hemolytic anemia. Under normal conditions approximately 80 to 90% of normal erythrocyte destruction occurs without release of hemoglobin into plasma (94,95). Because of this fact, the major part of the destructive process is considered to be extravascular, within macrophages of the spleen and, to a lesser extent, the liver and bone marrow. Only 10 to 20% of normal destruction occurs intravascularly, and this mode of destruction has special characteristics.

Extravascular Hemolysis

It has long been assumed that erythrophagocytosis is the primary mode of extravascular destruction of senescent red cells in normal subjects (96). This is supported by two fundamental observations. First of all, the heme oxygenase system responsible for hemoglobin degradation is located primarily in the phagocytic cells of the spleen, liver, and bone marrow, and also in hepatocytes. Secondly, iron derived from heme degradation is largely stored within the macrophage. Thus, it seems probable that hemoglobin degradation and hence red cell destruction normally takes place within these phagocytes. This clearly is true when red cell destruction is greater than normal, as in many types of hemolytic anemia. Likewise, when red cells are damaged in vitro and then reinfused, they are mostly removed from the circulation by the fixed macrophages of the spleen and liver (97).

The relative importance of the spleen and liver in erythrocyte destruction is influenced by the degree of cell damage (97). Severe degrees of damage lead to destruction in all macrophage-containing organs, but especially in the liver because of its relatively great blood flow. The spleen, in contrast, is more sensitive to lesser degrees of cell injury; cells only very minimally damaged are preferentially removed by that organ (98). It is probable that effete red cells are destroyed primarily in the spleen; however, if this organ is removed from normal subjects, macrophages found in other organs, especially the liver, rapidly assume this function, and there is no increase in normal red cell survival following splenectomy (99).

Intravascular Hemolysis

Analysis of haptoglobin kinetics in normal human subjects demonstrates that 10 to 20% of erythrocyte destruction occurs intravascularly (94,95). The mechanisms by which intravascular red cell destruction can occur are osmotic lysis or red cell fragmentation. However, it is unlikely that osmotic lysis has any significant role in normal red cell destruction because there is no site within the vascular compartment where the plasma is sufficiently hypotonic to bring lysis about. Fragmentation is defined as the loss of a portion of the erythrocyte membrane, usually accompanied by loss of cellular contents, including hemoglobin. Fragmentation is the characteristic mode of cell destruction in the “microangiopathic” hemolytic anemias (Chapters 36 and 52). In the blood smear, there are small, misshapen, often triangular or helmet-shaped structures (schistocytes or schizocytes). Fragmentation of red cells usually is a result of erythrocyte interactions with altered endothelium, fibrin deposition, or increased shear stresses. After a portion of the cell is lost, the membrane is capable of self-repair. Fragmentation also occurs within the spleen when the reticulocyte is pitted of red cell inclusions, such as residual organelles and hemosiderin granules. To what extent this accounts for the small fraction of intravascular destruction seen in normal erythrocyte aging is not known.

Fate of Intravascular Hemoglobin

Special features characterize those situations in which red cells are destroyed within the circulation rather than within macrophages. This occurs to a small extent with normal red cell aging, but it is increased significantly in certain hemolytic anemias. When intravascular breakdown of red cells happens, hemoglobin is discharged directly into the circulation. Free hemoglobin can be harmful to the body, promoting the formation of hydroxyl radicals via the Fenton reaction (H2O2 + Fe2+ → Fe3+ + OH + OH), thereby resulting in oxidative tissue damage (100). Fortunately, there are several physiologic mechanisms to remove free hemoglobin from the circulation (Fig. 8.3).

Haptoglobin

At low rates of release of hemoglobin into plasma, all of the hemoglobin is attached to haptoglobin (Hp) (101,102,103), and, once irreversibly bound to haptoglobin, free hemoglobin loses its oxidizing ability (100,104). Haptoglobin and hemoglobin bind within the vascular compartment in an essentially irreversible, noncovalent complex. Haptoglobin binds αβ hemoglobin dimers. Thus, dissociation of tetrameric hemoglobin is the necessary rate-limiting step in the reaction (105). Haptoglobin binds oxygenated free hemoglobin with a very high affinity (106). Heme-free globin is bound but heme is not (107). The role of haptoglobin as a hemoglobin-binding protein and as the principal factor affecting the apparent “renal threshold” for hemoglobin was described by Laurell and Nyman (108).

Haptoglobins are a family of α2-glycoproteins that bind hemoglobin (109). The tetrameric molecule resembles certain immunoglobulins in that it has two light (α) chains and two heavy (β) chains linked in humans by disulfide bonds (110). Haptoglobin is synthesized as a single polypeptide chain that is cleaved posttranslationally within the endoplasmic reticulum to generate its α and β subunits (111,112). The structural gene for haptoglobin has been localized to the long arm of chromosome 16 (16q22) (113,114,115). Transcriptional activity of the haptoglobin gene is promoted by interleukin-1, interleukin-6, and glucocorticoids as a part of the acute-phase response to systemic inflammation and related physiologic disturbances (116,117,118,119,120), thereby explaining why haptoglobin levels are increased with inflammation. An increased haptoglobin level is recognized as a nonspecific sign of disease with much the same significance as an accelerated sedimentation rate.

Figure 8.3. Pathways for the disposal of hemoglobin in plasma. Hemoglobin freely dissociates into αβ dimers. These are bound by haptoglobin with subsequent removal of the hemoglobin–haptoglobin complex by hepatic parenchymal cells. Hemoglobin in excess of the haptoglobin-binding capacity circulates as the unbound (free) protein. In this form it is partially removed by hepatic cells, but it may also follow two other pathways; it may be excreted by the kidney or oxidized to methemoglobin, from which heme is easily dissociated. Heme is initially bound to hemopexin, which transports it to the hepatic parenchymal cell. Heme may also be bound nonspecifically by albumin, forming methemalbumin. This complex probably transfers its heme to hemopexin as the latter becomes available.

Haptoglobin was first described as polymorphic by Smithies et al., who used starch gel electrophoresis to separate the various types. It was proposed that there were two genetic alleles that gave rise to three major phenotypes in man referred to as Hp1-1, Hp2-1, and Hp2-2. They can be distinguished using starch gel or polyacrylamide gel electrophoresis according to their different sizes and band patterns (Hp1-1 = 100 kDa; Hp2-1 = 120 to 220 kDa; Hp2-2 = 160 to 500 kDa) (121). Differences in the haptoglobin phenotypes observed in different racial groups and in association with various diseases has been of interest (109).

Early methods for the clinical determination of haptoglobin concentration were based on the hemoglobin-binding capacity of the plasma. Measured in this way, the normal range was 0.4 to 2.0 g Hb/L (94). Nowadays, most clinical laboratories measure haptoglobin directly by radial immunodiffusion or immunonephelometric methods, and phenotyping can be performed employing monoclonal antibodies and immunoblotting (122,123). Normal concentrations differ substantially with technique; ranges such as 0.5 to 1.6 g/L (124) are representative, but each clinical laboratory should establish its own reference values. The concentration is also influenced by age. Haptoglobin levels are very low in newborns, but there are measurable levels by about 3 months of age, and these increase gradually throughout childhood (125). Decreased haptoglobin concentrations also may be observed in disorders associated with hemolytic anemia, ineffective erythropoiesis, liver disease, and pregnancy and estrogen therapy. Increased concentrations may be present in any of those diseases in which concentrations of acute-phase reactants are increased, such as infections and malignancies.

Haptoglobin is synthesized in the parenchymal cells of the liver (126,127). When not bound to hemoglobin, it leaves the plasma with a half-disappearance time of 3.5 to 5 days (95,102,128). The haptoglobin–hemoglobin (Hp–Hb) complex leaves much more rapidly, with a half-disappearance time of 9 to 30 minutes. In hemolytic anemias characterized by intravascular hemolysis, catabolism of Hp is so rapid that it essentially disappears from the plasma, a change that is not accompanied by a compensatory increase in haptoglobin synthesis. Hypohaptoglobinemia also occurs in hemolytic states associated with predominantly extravascular hemolysis (129,130). The explanation for this observation is not established, but it has been suggested that some hemoglobin may be regurgitated from macrophages when the rate of phagocytosis of erythrocytes or erythrocyte fragments reaches a maximum (94).

The Hp–Hb complex is removed from the circulation after binding to a receptor (CD163) found on the cell surface of monocytes and tissue macrophages (131,132). After binding to the receptor, the Hb–Hp complex is endocytosed into the macrophage. Subsequently, iron is released by heme oxygenase and transported back to the bone marrow via plasma transferrin, for synthesis of new hemoglobin. Haptoglobin is not returned to the plasma, and when serum haptoglobin is depleted, it can take 5 to 7 days to recover, because synthesis is not increased by low haptoglobin levels.

Hemoglobin and the Kidney

The hemoglobin–haptoglobin complex is too large (molecular weight approximately 150 kDa) to pass into the glomerular filtrate. Thus, the level of circulating haptoglobin is the most important determinant of the apparent renal threshold (108). When haptoglobin is saturated, free (unbound) hemoglobin circulates briefly in plasma. The hepatic parenchymal cell is responsible for removal of some of the free hemoglobin from plasma (128). The free hemoglobin dissociates into αβ dimers, which have a molecular weight of about 32 kDa, and readily pass through the glomerulus (133). There is a low (<0.6 g/L) renal threshold for free hemoglobin present after haptoglobin saturation that is related to renal tubular reabsorption (134). If this tubular reabsorption capacity is exceeded, hemoglobin appears in the urine.

Hemoglobinuria, when it is of considerable magnitude, can cause precipitation of heme pigment as casts in the distal tubules, proximal tubule cell necrosis, and acute renal failure. The mechanism is disputed, but several theories have been proposed: (a) that hemoglobin or hemoglobin products are directly toxic to proximal tubule cells, (b) that precipitation of hemoglobin results in tubular obstruction and renal failure, and (c) that direct renal injury by hemoglobin does not occur but instead products of intravascular hemolysis result in hypotension and other systemic and local vascular events, and these lead to renal failure (135,136,137,138).

Within the tubular epithelial cell, hemoglobin iron is rapidly extracted and stored in the cell as ferritin and hemosiderin (133). Some of the tubular epithelial iron may be reused for hemoglobin synthesis, but its mobilization for this purpose occurs only at a very slow rate. When iron-laden tubular cells are sloughed into the urine, the urine iron concentration increases and both ferritin and hemosiderin may be detected (138). Clinically, hemosiderinuria is usually detected by the Prussian blue stain of the urinary sediment (139). Detectable hemosiderin usually does not appear in the urine for 48 hours after a specific episode of hemoglobinuria (138) and may persist for more than a week (135). In chronic intravascular hemolysis, such as occurs in red cell fragmentation associated with abnormal prosthetic heart valves, hemosiderinuria is continuous (140) and can result in iron deficiency.

Plasma Heme, Hemopexin, and Methemalbumin

Heme

Free hemoglobin in plasma is readily oxidized to methemoglobin. The latter dissociates easily and nonenzymatically into heme and globin (141). Hemopexin and albumin are able to bind heme, maintain it in a soluble form, and thereby prevent the strong oxidative and proinflammatory effects of free heme. Heme is removed from these proteins by hepatocytes.

Hemopexin

Hemopexin is a heme-binding glycoprotein in plasma that, after haptoglobin, forms the second line of defense against hemoglobin-mediated oxidative damage during intravascular hemolysis or internal hemorrhage (142). Hemopexin is a β1-glycoprotein, consisting of a single polypeptide chain and containing 20% carbohydrate, with a molecular weight of about 70 kDa. It binds heme with the highest known affinity of any heme-binding protein and plays an important role in receptor-mediated hepatocyte heme uptake. The human gene encoding hemopexin has been localized to the region p15.4-p15.5 of chromosome 11 and is thus at the same site as the β-globin gene cluster, genes that also code for polypeptides that share the property of binding heme (143,144,145).

Hemopexin is synthesized in the liver (146) and is found in the plasma in a concentration of 40 to 150 mg/dl. Each hemopexin molecule avidly binds one molecule of heme (Kd ∼10-13 M). Of all the body heme proteins, hemoglobin and myoglobin are the most abundant, and thus hemopexin is most important in conditions such as intravascular hemolysis, internal hemorrhage, and rhabdomyolysis. In addition to heme, this protein also binds to other porphyrins and bilirubin, but with less avidity (147,148). Binding is through the heme iron to two histidine residues of hemopexin (149,150,151,152).

The half-life of hemopexin in normal subjects is about 7 days (153), whereas the heme–hemopexin complex is removed from the circulation with a half-disappearance time of 7 to 8 hours (140). Hepatocyte uptake of this complex is by receptor-mediated endocytosis (154,155). The receptor is a high-density lipoprotein (HDL) receptor–related protein (LRP), also known as CD91, that has been purified (156). This receptor is located on many different cells and heme–hemopexin complexes are taken up by hepatocytes, macrophages, fibrocytes, neurons, and other cells. After endocytosis, the heme–hemopexin complex dissociates and the released hemopexin is returned to the plasma as an intact protein (150,154,157,158). Transport of heme within the cytoplasm occurs by means of an intrinsic heme-binding membrane protein (153,154), and iron is rapidly released by heme oxygenase.

Plasma hemopexin values may be reduced following intravascular hemolysis because of increased catabolism (153). The depletion is less pronounced than is true of haptoglobin, and low values imply a relatively severe degree of hemolysis. Hemopexin, like haptoglobin, is an acute-phase reactant and plasma levels rise with an inflammatory stimulus (159).

Methemalbumin

Each mole of human albumin can bind several moles of heme to form methemalbumin (160). The disappearance of methemalbumin from the circulation is kinetically complex (161). Heme added to human serum is initially associated primarily with albumin, presumably because the molar concentration of albumin is considerably greater than that of hemopexin (147,162).

Extravascular Hemoglobin Degradation

Formation of Bilirubin

Within 30 minutes of injection of either hemoglobin or nonviable red cells, there is an increased concentration of bilirubin in both plasma and bile, as well as an increase in carbon monoxide (CO) production. The last component, derived from the α-methene carbon of heme (162), is exhaled from the lungs. These substances represent the products of heme degradation by a ternary enzyme complex that is composed of heme oxygenase, NADPH-cytochrome c (P450) reductase, and biliverdin reductase (163). Almost all endogenously derived CO comes from this source (162,164). In most mammals, the heme oxygenase reaction is coupled with a second step in which biliverdin IXα is reduced to bilirubin IXα (Fig. 8.4).

Figure 8.4. Formation of bilirubin IXα from heme. The initial reactions are catalyzed by microsomal heme oxygenase and require nicotin-amide adenine dinucleotide phosphate (NADPH) as a cofactor. The reaction has a high specificity for the α-methene bridge, and α-oxyheme is a probable intermediate that is oxidized by molecular oxygen to biliverdin. In mammals, biliverdin is converted to bilirubin by biliverdin reductase. At bottom, alternative methods of representing the structure of bilirubin. The intramolecular hydrogen bonding that occurs with the Z,Z configuration is less extensive in the geometric isomers designated E,Z, and E,E (not shown); hence, the latter are more soluble in water.

Heme oxygenase activity is present in many tissues (165). The enzyme is inducible by its substrates (164,166) and increased heme oxygenase activity is observed in the spleen and liver during hemolytic anemia (166,167,168), in the liver following splenectomy (167), and in renal tubule cells in association with hemoglobinuria (133,168). The enzyme also is induced by metals, hormones, various drugs, organic compounds, and fever, starvation, and stress (169,170,171,172,173,174). Moreover, there also are agents that inhibit heme oxygenase activity (175) and the most studied of these is tin (Sn)-protoporphyrin (176). This substance acts as a competitive inhibitor of heme oxygenase, and thus, its administration results in a marked decrease in the enzyme activity (177). The effect of Sn-protoporphyrin is to inhibit the degradation of heme and hence the formation of bilirubin (171,178,179,180). It has been useful in the treatment of hyperbilirubinemia of newborns (181,182,183,184,185,186).

The process of heme degradation is a series of autocatalytic oxidations, catalyzed by heme oxygenase, with the reaction intermediates serving as cofactors (187) (Fig. 8.4). Overall, the oxidation of heme utilizes oxygen and NADPH, with the equimolar production of biliverdin, iron, and carbon monoxide (188). In mammals, the heme oxygenase reaction is coupled with a second step in which biliverdin is converted to bilirubin. This reaction is brought about by an enzyme, biliverdin reductase, which catalyzes the reduction of the central methene bridge of biliverdin. In birds, amphibians, and reptiles, biliverdin represents the principal end product of hemoglobin degradation. Mammals have developed bilirubin as the end product, a change with important biologic consequences. Bilirubin is highly lipophilic and virtually insoluble in water, it is toxic to the developing brain, and it requires conjugation to make biliary excretion efficient (189). However, because of its lipophilicity, it can cross the placenta from fetus to mother with greater facility and thus the fetus can utilize the mother’s excretory mechanisms.

If isotopically labeled glycine is administered to a normal person, the label is incorporated into the porphyrin ring and ultimately makes its appearance in bilirubin (190) (Fig. 8.5). Most of the label appears about 120 days after administration of the isotope and therefore is presumed to be derived from the destruction of senescent red cells; however, a substantial fraction of the pigment is labeled within several days and hence is often referred to as the “early-labeled” bilirubin. The original studies estimated that about 10 to 20% of bilirubin was early labeled (190). This fraction is made up of at least two components (191). The first, normally the larger one, is labeled maximally at 24 hours and is of hepatic origin, chiefly from the heme of cytochrome P450, but also from other hemoproteins (192). The second is labeled maximally at about 3 to 4 days and is a by-product of erythropoiesis (191). An isotopic label from administered aminolevulinic acid, to which erythroid cells are relatively impermeable, appears primarily in the hepatic fraction, whereas that from glycine appears in both (191). The erythropoietic component is probably the product of “ineffective erythropoiesis” and results from the intramedullary destruction of defective red cells. In illnesses associated with exaggerated ineffective erythropoiesis, such as thalassemia or pernicious anemia, there is a marked increase in the early-labeled bilirubin fraction.

Figure 8.5. The incorporation of isotopically labeled glycine into heme and bile pigments. Most of the bilirubin labeling occurs at about 120 days, at the time of destruction of senescent red cells. About 25% of bilirubin, however, is labeled within the first several days and is therefore called the “early-labeled” fraction.

Bilirubin Transport

After being released from sites of heme catabolism, bilirubin appears in the plasma. The normal concentration of plasma bilirubin is <1.0 mg/dl. At equilibrium, the concentration is directly related to bilirubin production and is inversely related to hepatic clearance (193,194).

The structure of the bilirubin IXα molecule is asymmetric and several isomeric forms exist depending upon the orientation of the outer pyrrole rings (rings A and D) in relation to the inner pyrrole rings (rings B and C) (Fig. 8.4) (195). In the naturally occurring configuration, all pyrrole rings are similarly rotated, representing the Z,Z or trans configuration. If either of the outer rings is rotated, the E,Z or Z,E geometric isomers are formed. Photoisomerization of the naturally occurring Z,Z configuration of bilirubin forms the photoisomers of the E,Z and E,E configurations in which intramolecular hydrogen bonding is less extensive. Thus, the E,Z and E,E isomers are more soluble and can be excreted without conjugation (195,196,197), and this is the basis for using phototherapy to prevent neurotoxicity in newborns with hyperbilirubinemia (195,196).

Bilirubin is normally present in plasma in several forms (198,199). Although unconjugated bilirubin is essentially insoluble in water, it combines reversibly with albumin in neutral or alkaline solution. At normal plasma albumin concentrations, the theoretical bilirubin-binding capacity is of the order of 70 mg/dl, of which half is tightly bound. These values are reduced by a decrease in plasma albumin concentration or by the presence of one of many organic, anionic substances that compete for albumin-binding sites, such as heme, fatty acids, sulfonamides, and salicylates (200). When the binding capacity is exceeded, bilirubin diffuses into the tissues. The tendency of bilirubin to bind to tissues, such as brain, may be due to complex formation with cell membrane polar groups, such as phosphatidylcholine (197).

In normal adults, <5% of measurable bilirubin is of the conjugated form (201,202), but under certain pathologic circumstances, the proportion of conjugated bilirubin is greater. This relatively soluble bilirubin derivative may also be bound to albumin. Most is less tightly bound than the unconjugated form, but a portion is covalently and irreversibly bound (203). That portion of esterified bilirubin that is reversibly bound to albumin is ultrafilterable. In contrast to the other forms of bilirubin present in the plasma, this complex enters the glomerular filtrate, is not reabsorbed in the tubules, and is excreted in the urine.

Figure 8.6. Normal and abnormal pathways of bilirubin excretion by the hepatic cell. The normal pathways (solid arrows) include uptake and conjugation of bilirubin and excretion of the conjugated derivative. Abnormal pathways (dashed arrows) include regurgitation of bilirubin glucuronide into plasma and excretion of unconjugated bilirubin into bile.

Hepatic Bilirubin Metabolism

The processing of bilirubin by the liver is one aspect of a general mechanism whereby plasma protein-bound, organic anions are metabolized and excreted. The process of hepatic bilirubin metabolism may be divided into three distinct phases: Uptake, conjugation, and excretion (204) (Fig. 8.6). All three phases must be operative if bilirubin is to be excreted at a normal rate; however, in the normal person the excretion step is the slowest, and therefore the rate-limiting, step.

Within the hepatic sinusoids, the albumin–bilirubin complex dissociates, and bilirubin, but not albumin, passes into the hepatocyte. The entry of bilirubin into the hepatocyte takes place via a bidirectional energy-dependent process (205,206,207). Conjugated bilirubin uses this mechanism as well. The bidirectional movement of bilirubin is extensive, with 40% of pigment extracted by hepatocytes in a single pass refluxing back into the plasma without change (208). In addition, a part of the bilirubin formed within the liver cell from degradation of hepatic heme, such as cytochrome P450, refluxes into the plasma in the unconjugated form (209).

Within the liver cell, bilirubin is conjugated with glucuronic acid to form bilirubin diglucuronide (Fig. 8.7). In normal human bile, 80% of the bilirubin is present as the diglucuronide; the two monoglucuronide isomers comprise most of the remainder in approximately equal amounts (210). Bilirubin diglucuronide and other conjugated bilirubins are considerably more water soluble than unconjugated bilirubin, in part because the carbohydrate moieties prevent intramolecular hydrogen bonding. The increased solubility makes biliary excretion of the pigment possible. Little or no unconjugated bilirubin is found in bile, and if conjugation is seriously impaired, bile bilirubin content is low.

 

Figure 8.7. Bilirubin diglucuronide. The two glucuronic acid molecules are emphasized by bolder type.

The conjugation reaction is catalyzed by uridine diphosphate-glucuronyl transferase (UGT). Hepatocyte UGT exists in several isoforms, catalyzing the glucuronidation of a variety of substrates, such as steroid hormones, and a variety of drugs. Bilirubin serves as the substrate for two of these isoforms (B-UGT 1 and B-UGT 2) (211), but only B-UGT 1 is quantitatively significant in humans (212). B-UGT activity is also modulated by a variety of drugs, such as phenobarbital, and hormones, such as cortisol and thyroxine (213).

In hepatocellular disease or biliary obstruction, some conjugated bilirubin may be “regurgitated” into the plasma. In addition, a small portion of conjugated bilirubin within the hepatocyte is deconjugated and the unconjugated bilirubin in part refluxes into the plasma (209). These pathways in part explain the increase of both fractions of bilirubin in patients with cholestatic liver disease. In other clinical situations, hyperbilirubinemia is mainly due to an increase in unconjugated bilirubin. The most common of these is hemolytic disease, in which hemoglobin catabolism and therefore bilirubin production are increased. However, there also are several inherited disorders in which unconjugated hyperbilirubinemia occurs because of impaired capacity for bilirubin conjugation.

Decreased ability to conjugate bilirubin is the common feature of three hereditary disorders that differ from one another primarily in the severity of the conjugation impairment. In Crigler-Najjar syndrome type I, severe unconjugated hyperbilirubinemia is present from birth and kernicterus is common. In Crigler-Najjar syndrome type II, less severe jaundice occurs, and in Gilbert syndrome, the jaundice is quite mild, often not obvious clinically (214,215). These disorders are recessively transmitted, and most patients are therefore homozygous for the mutant gene, although occasionally heterozygotes are minimally jaundiced. Mutations in the gene for B-UGT 1 occur in all three diseases. In the two Crigler-Najjar syndromes, the mutation most often involves exon 1, the exon that confers substrate specificity to the enzyme, resulting in a structurally abnormal protein. In Crigler-Najjar syndrome type I, and in the Gunn rat, an animal model of this disease, the gene product is entirely nonfunctional, while in type II, activity of the product is variably reduced (212,216,217,218,219). In Gilbert syndrome, the mutation in some patients has been shown to affect the promoter sequence of exon 1 (218). Gilbert syndrome is very common, occurring in as many as 10% of the general population, but both Crigler-Najjar syndromes are quite rare. In Crigler-Najjar type I, Sn-protoporphyrin may be effective in reducing the bilirubin concentration. Liver transplantation provides a more permanent benefit (220). No treatment is generally necessary for the other two disorders. However, it is now recognized that the magnitude of hyperbilirubinemia seen in children with different chronic hemolytic states is influenced by the simultaneous inheritance of the gene for Gilbert disease (184,221). In infants with hereditary spherocytosis who also are homozygous for the mutation responsible for Gilbert syndrome, hyperbilirubinemia almost always requires phototherapy (221). It also is thought the variable degree of hyperbilirubinemia in G6PD-deficient neonates reflects the presence or absence of the variant form of uridine-diphosphoglucoronylsyl-transferase responsible for Gilbert syndrome (184). In infants known to be G6PD deficient, prevention of severe hyperbilirubinemia by administration of a single intramuscular dose of Sn-mesoporphyrin, an inhibitor of heme oxygenase, has been demonstrated to be highly effective and safe (185,222).

Excretion of conjugated bilirubin from the hepatic cell into the bile canaliculus proceeds against a 40:1 gradient, when concentration in the bile is compared with that in plasma (223). From animal studies, it has been demonstrated that excretion of bilirubin from hepatocytes is mediated by an ATP-dependent transport system, a canalicular multispecific organic anion transporter (cMOAT) in the apical or canalicular membrane of hepatocytes (224). The ATP-dependent carrier system is at least partially shared by a variety of organic anions (225). This is normally the rate-limiting step in overall hepatic bilirubin transport. The Dubin-Johnson syndrome (226,227) is a disorder transmitted by autosomal recessive inheritance and characterized by mild conjugated hyperbilirubinemia, and by impaired biliary secretion of non–bile-acid organic anions. Bilirubin uptake and conjugation are normal. This defect is similar to that of the TR-rat, in which the ATP-dependent transport system is absent (157,214). Dubin-Johnson syndrome is now thought to be due to mutations in the canalicular multispecific organic anion transporter gene (228).

Intestinal Bile Pigment Metabolism

Bilirubin diglucuronide is excreted into the duodenum with other constituents of bile (229). There is little or no intestinal absorption of the conjugated pigment, although unconjugated bilirubin is readily absorbed. Bilirubin diglucuronide probably remains in the conjugated form during its transit through the small intestine. However, during intestinal stasis, and in newborns, increased deconjugation of bilirubin occurs and intestinal absorption takes place. This enterohepatic circulation of bilirubin may contribute to the severity of jaundice associated with the physiologic hyperbilirubinemia of the newborn (230).

When bilirubin diglucuronide reaches the terminal ileum and colon, it is hydrolyzed by bacterial β-glucuronidases (Fig. 8.8). The two methene bridges and usually the two vinyl groups are then reduced by bacterial flora to form a series of colorless tetrapyrroles called urobilinogens (231). Since urobilinogen formation is accomplished by bacteria, it does not occur in newborns or in germ-free animals, and it may be markedly affected by administration of broad-spectrum antibiotics. The urobilinogens are easily dehydrogenated across the two middle rings to form the orange-yellow pigments, urobilins, that contribute to the color of feces.

About 10 to 20% of the urobilinogen formed in the gut is reabsorbed and the remainder is lost with the feces. The reabsorbed fraction is efficiently excreted by the normal liver without being conjugated (229). This sequence of events is referred to as the enterohepatic recirculation of urobilinogen. A portion of the reabsorbed pigment may also be excreted into the urine. Urobilinogen is filtered by the glomerulus, secreted by the renal tubule, and reabsorbed. If the liver’s capacity to excrete urobilinogen is impaired, a disproportionate amount appears in the urine.

Also found in the colon are a group of poorly characterized dipyrroles known as mesobilifuscins. These brown pigments are partly responsible for the color of normal feces (232). Most of these dipyrroles do not appear to be derived from the degradation of bilirubin; instead, they probably are anabolic by-products of heme synthesis (233). However, in conditions associated with Heinz bodies, excessive amounts of such dipyrroles are excreted in the urine. These are thought to be derived from the heme freed from globin when the Heinz body forms. Furthermore, a dipyrrole is formed from the photodegradation of bilirubin (234), as discussed in the following section.

Figure 8.8. Bile pigment metabolism in the gut. These reactions are carried out by bacteria. d-urobilinogen, mesobilirubinogen, and stercobilinogen are members of the urobilinogen group, which is characterized structurally by saturation of the carbon bridges connecting the pyrrol rings. The urobilins are derived from urobilinogens by oxidation and have one or more double bonds in the connecting carbon bridge. (From Lester R, Troxler RF. Recent advances in bile pigment metabolism. Gastroenterology 1969;56[1]:143–169, with permission.)

Alternate Pathways of Heme and Bilirubin Catabolism

Several lines of evidence suggest that some heme may be degraded by pathways other than those previously described. On a stoichiometric basis, it can be calculated that 35 mg of bilirubin should be produced from degradation of 1 g of hemoglobin. However, recovery of fecal urobilinogen is substantially less than expected, suggesting that as much as 20 to 40% of heme may be degraded by some other pathway (234). Only minimal amounts of unconjugated bilirubin are excreted in the urine. Excretion of conjugated bilirubin is substantial despite complete biliary obstruction. In rats with biliary fistulas, only 60 to 80% of administered radioactive hemoglobin or heme is recovered in bilirubin; a portion of the remainder is found in nonbilirubin fractions of bile (235,236). Radioactive bilirubin in patients and rats with prolonged complete biliary obstruction gradually disappears via an unidentified route (236). Furthermore, in severe, inherited defects of bilirubin conjugation, such as those found in the Gunn rat and in human infants with the Crigler-Najjar syndrome, the alternate pathways appear to be increased (237).

One explanation for these observations is that bilirubin is converted by a series of light-stimulated reactions to a variety of water-soluble derivatives, including hydroxyrubins, bilichrysins, and a dipyrrole (234). These light-induced products, which are colorless and do not react with diazo reagents, have been found in the bile of the Gunn rat. A microsomal P448-dependent monooxygenase may contribute to these alternate pathways because inducers of this enzyme increase the turnover of bilirubin in the Gunn rat and reduce the plasma bilirubin concentration. In addition, a mitochondrial bilirubin oxygenase has been identified that in vitro degrades bilirubin to a variety of products (238).

Photodegradation of bilirubin, such as described above, has been applied to the treatment of unconjugated hyperbilirubinemia in infants (Chapter 34) (195,196,234,239). Exposure to light brings about increased bilirubin excretion and a decrease in the serum bilirubin concentration to nontoxic levels. The photodegradation products are excreted promptly in the bile and may cause the stool to turn to a green or darker brown color. No toxicity of these products has been demonstrated.

Laboratory Evaluation of Hemoglobin Catabolism and Bile Pigments

The serum bilirubin concentration is an important marker of the rate of bilirubin production and of hepatobiliary function. Traditionally, it has been measured by the van den Bergh test described in 1916 (240). It is based on Ehrlich’s observation that a mixture of sulfanilic acid, hydrochloric acid, and sodium nitrite (diazo reagent) yields a reddish-violet color with a maximum absorption at a wavelength of 450 nm when added to plasma or other solutions containing bilirubin. This reaction remains the basis for most automated clinical measurements of bilirubin (241). The color may appear and reach its maximum intensity at once (direct reaction). A direct reaction is obtained with bilirubin in bile and in the plasma and urine from patients with obstructive jaundice. The direct-reacting bilirubin approximately corresponds to conjugated bilirubin. The bilirubin in plasma of patients with hemolytic disease does not react directly with the diazo reagent but requires addition of an accelerator, such as alcohol (indirect reaction). Indirect-reacting bilirubin corresponds to unconjugated bilirubin.

The measurement of fecal urobilinogen excretion over several days is a crude test previously used to quantify heme breakdown, but this rarely ever is utilized nowadays. More precise measurements of heme catabolism can be determined from measuring endogenous bilirubin production or generation of carbon monoxide. As discussed previously, the principal catabolic products of heme are iron, carbon monoxide, and bilirubin. There are no other significant endogenous sources of the last two compounds; thus, the breakdown of ne mole of heme yields precisely one mole of carbon monoxide and one mole of bilirubin.

Engstedt (242) and, later, others (243,244) called attention to the fact that blood carboxyhemoglobin (COHb) levels are increased in hemolytic disease. However, precise interpretation of such static measurements is complicated by exogenous exposure to CO and by variations in CO excretion. In contrast, however, the endogenous rate of CO production also can be measured by a rebreathing method that circumvents these problems (162). The subject breathes into a closed system from which CO2 is absorbed and to which O2 is added. CO excretion is thereby prevented and the blood level of COHb increases. Endogenous CO production is calculated from the rate of increase over a 2-hour period and from the body CO dilution. With this method, the rate of CO production has been found to be increased severalfold from normal in patients with a variety of hemolytic anemias (162) and similarly elevated in conditions associated with ineffective erythropoiesis (243). For newborn infants with increased jaundice it is important to know whether the hyperbilirubinemia is a consequence of accelerated red cell breakdown or due to some other cause. For this purpose a noninvasive instrument to measure exhaled carbon monoxide now is commercially available, and appears to be a reliable marker of increased hemoglobin breakdown (245,246).

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