- Email: [email protected]
The diagnostic utility of haptoglobin
Vol. 11, No. 9, 1991
identified by immunofixation electrophoresis with the use of antiserum specific for CRP to rule out the possibility of an otherwise occult monoclonal gammopathy. In our laboratories, we have used SPE routinely as part of our biochemistry profile over the past 10 years. By far, the most common dysproteinemias we have observed are those of the acute and chronic inflammatory types. As mentioned previously, others have found this to be the case as well. 2'5'6 Occult acute and chronic inflammatory conditions have been detected in otherwise healthy-
CLINICAL IMMUNOLOGY Newsletter 135
appearing patients in our experience. In our hands, we feel the SPE procedures to be simple and cost effective not only for the detection of inflammatory conditions but for the many other pathophysiological states mirrored by dysproteinemia. The use of specific protein procedures acts to complement and verify these studies. 1 CrN
References 1. Johnson AM: Plasma protein assays in clinical diagnosis and management. Bulletin 6215. Brea, Beckman Instruments, 1981.
2. Jolliff CR: Classification and interpretation of paragon serum protein electrophoresis patterns. EP-1. Brea, Beckman Instruments, 1983. 3. Kawai T: Clinical aspects of the plasma proteins. Philadelphia, Lippincott, 1973. 4. Keren DF: High-resolution electrophoresis and immunofixation. Techniques and interpretation. Stoneham, Butterworth, 1987. 5. Ritzman SE, Daniels JC: Serum protein abnormalities. Diagnostic and clinical aspects. Boston, Little Brown, 1975. 6. Sun T: Interpretation of protein and isoenzyme patterns in body fluids. New York, Igaku-Shoin, 1991.
The Diagnostic Utility of Haptoglobin Frederick Van Lente Department of Biochemistry, Cleveland Clinic Foundation, Cleveland, Ohio
'aptoglobin, or more correctly haptoglobins, are a family of .alpha-2 globulins of dramatically variable size that are inherent constituents of the classic acute phase response. The description of haptoglobin types refers back to the original description of their banding patterns on starch gels. They are designated as haptoglobins 1-1, 1-2, and 2-2 and are phenotypes of the expression of two genetic alleles, Hp 1 and Hp 2. Serum from individual homozygotes for Hp 1 demonstrates a single band on starch gel (haptoglobin 1-1). Serum from individuals with homozygous Hp 2, however, exhibits a series of bands on starch gel of decreasing mobility which has been interpreted as indicating a series of haptoglobin polymers rather than a collection of distinct polypeptide chains. The phenotype expressed by Hp 1/Hp 2 heterozygotes is a series of polymers of intermediate size relative to either homozygous phenotype. This is consistent with a combination of equal amounts of Hp 1 and Hp 2 haptoglobin products and can be duplicated in vitro. This somewhat peculiar behavior is better understood after an analysis of certain aspects of the structure of the haptoglobin molecule which is quite similar to that of hemoglobin. The fundamental
H
haptoglobin molecule is a monomer of about 85,000 kDa molecular weight consisting of two alpha chains and two beta chains connected by disulfide linkages as does hemoglobin. These chains have different compositions and are dissociable with use of mercaptoethanol and urea. Interestingly, the beta chains are identical in the three major haptoglobin types but the alpha chains are different. It is clear that the ability to aggregate the basic monomer to higher molecular weight polymers must be a characteristic of the alpha chain expressed by the Hp 2 genotype alone. In the haptoglobin 1-2 phenotype, equal amounts of monomeric and polymerizing alpha chains are present. The variation in haptoglobin types, therefore, resides in the alpha polypeptide chain. There is additional variation of the gene expression of the alpha chain but these are applicable to genetic studies only and have little relevance to the discussion here. One haptoglobin molecule (monomertwo alpha and two beta chains) binds two alpha/beta dimers of hemoglobin. This binding is essentially irreversible, although there is no evidence that covalent bonding is involved. In fact, this binding is one of the strongest noncovalent bonds known. Neither isolated heme or myo© 1991 ElsevierSciencePublishingCo., Inc.
globin can be bound by haptoglobin, and several abnormal hemoglobins such as hemoglobin H and hemoglobin Barts are also not bound. The bound complex does retain pseudoperoxidase activity. This specific and avid binding of hemoglobin is apparently the sole physiological role for this interesting protein. Serum haptoglobin is synthesized and secreted as a glycoprotein by the liver. In the circulation it serves as a reservoir of hemoglobin binding capacity. Although free hemoglobin dimers can pass through the renal glomeruli and be lost (along with the associated iron) in the urine, the hemoglobin-haptoglobin complex (recall that the size of this complex will depend on the haptoglobin phenotype) is too large to be filtered by the kidney. The renal threshold for hemoglobin is almost entirely determined by the plasma hemoglobin binding capacity or haptoglobin concentration. Free hemoglobin that finds its way into the intravascular space is bound by haptoglobin and the resultant complex is removed primarily by the liver. Hepatic parenchymal cells contain a specific receptor for this complex in the plasma membrane. The complexes initially internalized by the receptor-mediated process are first concentrated in the Golgi or0197-1859/91/$0.00 + 2.20
136 C L I N I C A L I M M U N O L O G Y
Newsletter
ganelles and, subsequently, heme is detached for further processing intracellularly where it is converted to bilirubin. 5 The globulin or protein portion of the complex is degraded separately. It is of some interest why a hemoglobin-scavenging protein should contribute to the biology of the acute phase response in humans. It is obvious that the retention of iron is important to an organism so dependent upon iron as a constitutent of the oxygen-carrying hemoglobin molecule. But what is the relationship of iron to the changes that occur during acute inflammation? The standard explanation for this phenomenon is that the fundamental attribute of this response is the defense against bacterial infection. The deprivation of circulating " f r e e " hemoglobin-iron, known to be a potent bacterial growth factor--inhibits the spread of a bacterial infection, l However, the recent focus on the role of free radicals in the pathophysiology of diseases as well as inflammation itself may provide an additional explanation for haptoglobin's role in the acute phase response. Free hemoglobin can stimulate the process of lipid peroxidation that leads to free radical chain reactions that are damaging to tissues. Also, free iron released from hemoglobin can actively promote hydroxyl radical formation via the infamous Fenton reaction (Figure 1). Hydroxyl radical is the most potent reactive species in biological systems. When viewed in context, haptoglobin shares much in common with several other well-known plasma proteins, all of which may provide an antioxidant defense system in the peripheral circulation. Proteins, such as transferrin, metallothionein, and ceruloplasmin, are all active in preventing the generation of active, transitional, metal irons in an unbound state by a combination of binding and enzymatic activity. This, in turn, minimizes the risk of uncontrolled free radical generation. It has been demonFe z+ + O z ~ F e 3 + + O z ' 2Oz'- + 2H +---~ H2Oz + Oz Fe z+ +
HzOz ~
O H - + Fe 3+ + "OH
Figure 1. The Fenton reactions. 0197-1859/91/$0.00 + 2.20
Vol. 1 1, No. 9, 1991
strated that hemoglobin-haptoglobin binding effectively prevents hemoglobinstimulated peroxidaton of fatty acid. 3 Therefore, haptoglobin may prevent damage to tissue via three mechanisms: preventing release of free hemoglobin--iron that would be available for bacterial growth, preventing free, iron-induced radical reactions, and preventing free hemoglobin-induced radical reactions. .
.
.
.
.
.
.
.
.
.
.
.
.
.
With the advent of specific polyclonal antibodies, most laboratories began determining serum haptoglobin immunochemically, initially with radial immunodiffusion (RID).
Several techniques have been employed to measure haptoglobin in serum. Early methods were based on properties of the hemoglobin-haptoglobin binding itself, in particular, the effect on the peroxidase activity of hemoglobin. The binding enhances this activity and several methods have been described including those that allow the use of standard automatic analyzers. Other methods involve the detection of altered spectrophotometric characteristics or separation of the complex by physical means or by electrophoresis. The quantitation of haptoglobin using most of these methods was in terms of grams per deciliter of hemoglobin binding capacity (HBC). There was no direct assessment of, or relationship determined to, the actual mass concentration of haptoglobin in the sample; that is, these measurements were assessing an overall characteristic of the serum in question and not the direct mass measurement of haptoglobin itself. With the advent of specific polyclonal antibodies, most laboratories began determining serum haptoglobin immunochemically, initially with radical immunodiffusion (RID). This technique proved to be somewhat slow and may have contributed to the lack of enthusiasm many clinicians displayed toward this © 1991 ElsevierScience Publishing Co., Inc.
laboratory determination. Nonetheless, the application of immunochemical techniques raised some significant issues regarding what was actually quantified. Recall that haptoglobin is not a homogenous protein among all individuals. Rather it varies normally as a function of phenotype, and that variation is in the sequence of the alpha chain and, therefore, in the molecular weight of the haptoglobins present in serum. Immunochemical methods are not easy to standardize as a rule, and a heterogenous protein, such as haptoglobin, makes that task more difficult. It has been shown that haptoglobin phenotypes are expressed by different immunologic determinants but most commercial antisera are raised against pooled sera, and antisera can be raised against isolated haptoglobin beta chains that are identical in all phenotypes. All these considerations can make the immunochemical determination of haptoglobin phenotype dependent. This variation is most obvious in radial immunodiffusion where the larger polymers of haptoglobins 1-2 and 2-2 form immuno complexes of very high molecular weight and their diffusion is retarded significantly when compared with the smaller monomers of haptoglobin 1-1. Correction factors have been described that adjust for these differences. Nephelometric and turbidimetric methods are presently the techniques most commonly employed to measure serum haptoglobin concentrations. Under optimal circumstances, these methods demonstrate minimal phenotypic variation, although some variation is to be expected due to the difference in size of the immuno complexes and, therefore, the resultant light scatter. Because the results of most commercially available methods for haptoglobin correlate with haptoglobin concentrations expressed as hemoglobin binding capacity, these assays demonstrate adequate clinical applicability. 6 Arguments advocating the correct mass measurement of haptoglobin protein are valid but not germane to the potential usefulness of these methods. These methods are precise and rapid, which is important considering their clinical usage. There have been several reports of the reference range for serum haptoglobin
C L I N I C A L I M M U N O L O G Y Newsletter 137
Vol. 11, No. 9, 1991
TABLE 1. HAPTOGLOBIN REFERENCE INTERVALS Haptogiobin All Types All Types All Types 1-1 1-2 2-2
Range (g/L)
Method
Reference
0.40-2.40 0.412-2.00 0.56-3.76 0.77-2.49 0.86--4.76 0.48-3.15
Nephelometry Enzymatic Turbidometric
6 9 7
and it is clear that the range of values found in apparently healthy individuals depends on the phenotype as shown in Table 1. However, most agree that the determination of phenotype is quite impractical and useful only in exceptional circumstances. It is the opinion of this author that haptoglobin determinations must be interpreted using a total population reference interval as is done with other analyses with genetic variability and the usefulness of the test evaluated in that context. As will be discussed below, the major clinical application of this determination has little relevance to reference intervals per se. During the acute phase response to inflammation, haptoglobin concentrations in serum will increase from two- to fourfold. Concentrations are correlated with other acute phase proteins including alpha-1 antitrypsin and alpha-1 acid glycoprotein. Because it migrates in the alpha-2 region on serum protein electrophoresis, haptoglobin contributes to the increased alpha-2 band associated with the traditional acute phase pattern. In addition, haptoglobin can contribute to the markedly abnormal protein electrophoresis pattern associated with the Nephrotic Syndrome. The degree of relative retention of haptoglobin in this condition is a function of phenotype and, therefore, molecular weight. Haptoglobins 1-2 and 2-2 are retained to a greater extent than the smaller haptoglobin 1-1. The haptoglobin-hemoglobin complex migrates more cathodic than haptoglobin itself, and this complex may be present on electrophoresis as a result of hemolysis induced by specimen collection. To the accomplished eye, these variations may be detected, but qualitative evaluations should not be substituted for quantification when clinically indicated.
The main diagnostic application of serum haptoglobin measurement is the diagnosis of hemolysis. As shown in Table 2, an active hemolytic process is present in a variety of conditions. Because the hemoglobin-haptoglobin complex is cleared rapidly by the hepatocytes (half life = 10 min) when compared to its rate of synthesis by the liver, the initial serum haptoglobin concentration should fall rapidly with the onset of hemoglobin release from any source and especially when release occurs directly in the circulation. It has been shown that the upper limit of the reference interval for haptoglobin is about 2.5 g/L. Assuming equivalency with hemoglobin binding capacity, the complete lysis of only 1.7% of the erythrocytes in an equal volume of blood would completely exhaust the available haptoglobin. Theoretically, only at this
point could free hemoglobin appear in the urine. A reduction of erythrocyte survival time to one-half of normal can result in a severe reduction of haptoglobin concentrations. It is difficult to assess the diagnostic performance of serum haptoglobin in the diagnosis of hemolytic disorders as there is no convenient gold standard, and the administration of red cell survival studies in large groups of patients is problematical. Nonetheless, using total clinical information, we have shown that decreased serum haptoglobin determinations demonstrate an 87% predictive value (83% sensitivity) for the hemolytic conditions listed in Table 2 at a cutoff of 0.25 g/L. 6 It should be emphasized that this concentration is clearly below the reference interval obtained in normal individuals. We observed a variation in the degree of abnormality among the various disease states with the most frequent "normal" values being observed in megaloblastic anemias where the degree of intravascular release of free hemoglobin is not clear. The overall specificity of serum haptoglobin in conditions not considered hemolytic, such as iron deficiency and anemia of chronic disease, was 96%. In our study all cases of autoimmune and microangiopathic anemias had haptoglobin values
TABLE 2. CONDITIONS ASSOCIATED WITH CHANGES IN SERUM HAPTOGLOBIN CONCENTRATION A. IncreasedHaptoglobinValues Inflammation Malignancy Infection Post trauma B. DecreasedHaptoglobinValues Acute hemolyticdisease Autoimmune hemolyticanemias Microangiopathichemolyticanemias Hemolytictransfusionreaction Severeexercise Acute toxic hemolyticanemia Hypersplenism Megaloblasticanemia Hemoglobinopathyhemolysis Other nonimmunehemolyticanemias Chronic hemolyticdisease Prosthetic valvereplacement Hematoma Severe liver disease Genetic deficiency © 1991 Elsevier Science Publishing Co., Inc.
0197-1859/91/$0.00 + 2.20
138 C L I N I C A L I M M U N O L O G Y Newsletter
below 0.25 g/L. 6 Serum haptoglobin concentrations are also depressed as a result of acute hemolysis due to toxic exposure (e.g., arsine gas) as well as severe exercise. 2 Serum haptoglobin may also be depressed in patients with chronic, compensated hemolysis, or with clinically significant hematomas. The former is often present in patients with prosthetic heart valve replacements. As is usually the case, there are confounding situations that must be kept in mind when interpreting decreased haptoglobin concentrations and many of these occur when multiple conditions exist or when tests are requested when they are not indicated. A total, genetically determined haptoglobin deficiency has been described in a small percentage of the black population, and a combined alpha-1 antitrypsin and haptoglobin deficiency has also been reported in a European family. 4 The author is unaware of any pathophysiology that has been definitely linked to these deficiencies. The liver is the synthetic site of haptoglobin and other acute phase proteins, and failure of protein synthesis in end-stage liver disease is undoubtedly the most common cause of a decreased serum haptoglobin concentration, and not a direct result of hemolysis. Haptoglobin demonstrates a fairly close correlation with other acute phase proteins, such as alpha-1 acid glycoprotein, in these conditions, although the latter has been shown to be a more sensitive indicator of liver failure. None of these proteins appears to have displaced the more widely used protein markers of liver synthetic capacity such as transferrin, prealbumin, or retinal binding protein. Haptoglobin is similar to ferritin in that decreases in both are used in the differential diagnosis of anemia but both are acute phase reactants and their synthesis and serum concentrations rise in response to acute inflammation. This combination of changes has led to skepticism regarding the usefulness of a parameter that may be doing two things at once. Reasonably designed studies of the diagnostic performance of a test such as haptoglobin, however, must include the confounding variables that are present in the very patients studied. We extended this approach in order to investigate the worst I 0197-1859/91/$0.00 + 2.20
case scenario and studied the decline in serum haptoglobin secondary to mechanical hemolysis caused by the blood oxygenator used in cardiopulmonary bypass surgery. 8 These patients also undergo a marked acute phase response secondary to surgery that is easily documented by increases in C-reactive protein concentrations. It was found that all patients exhibited a fall in serum haptoglobin after surgery and 71% exhibited concentrations <0.25 g/L, even though C-reactive protein demonstrated a concurrent sixfold increase in the same time period. Therefore, although a concurrent acute phase response can affect the decline in haptoglobin during inflammation, the effect is minimal. In summary, hypohaptoglobinemia, except when inherited, is usually always clinically significant after infancy. In some cases, the cause may be decreased synthesis due to severe hepatic failure, but these cases are evident, and haptoglobin should only be used as a prognostic indicator in these situations when evidence for active hemolysis is lacking. Haptoglobin measurements are best utilized when there is an indication of active hemolysis or unexplained anemia. These indications include a decreasing hemoglobin concentration with evidence of reticulocytosis and/or red cell destruction as well as an unexplained increase in serum LDH-I activity or unconjugated bilirubin concentration. The finding of a significantly depressed serum haptoglobin (<0.25 g/L) with a concurrent increased LDH-1 isoenzyme activity or reticulocytosis in these settings is extremely predictive of hemolytic disease. In these
Vol. 11, No. 9, 1991
situations serum haptoglobin is a useful adjunct to the differential diagnosis of hemolytic disorders, c~
References 1. Eaton J W , Brandt P, Mahoney JR, Lee JT: Haptoglobin: A natural bacteriostat. Science 215:691-3, 1982. 2. Egan LM, Watts PB, Silta BC: Changes in serum haptoglobin as an acute response to a marathon road race. J Sports Med 5:55--60, 1987. 3. Gutteridge JMC: The antioxidant activity of haptoglobin towards haemoglobin-stimulated lipid peroxidation. Biochim Biophys Acta 917:219-23, 1987. 4. Kalsheker NA, Kingston JE, Moore CJ, Sarjant JA: A family with deficiencies of both alpha-l-antitrypsin and haptoglobin. Clin Chem 28:381-2 (letter), 1982. 5. Nakajima H, Satom S: Intracellular site of the catabolism of heme and globin moiety of hemoglobin-haptoglobin after intravenous administration to rats. J Biol Chem 263:16032-6, 1988. 6. Van Lente F, Marchand A, Galen RS: Evaluation of a nephelometric assay for haptoglobin and its clinical usefulness. Clin Chem 25:2007-10, 1979. 7. Van Rijn HJM, Van Der Witt W, Stroes TW, Schrijver T: Is the turbidimetric immunoassay of haptoglobin phenotypedependent. Clin Biochem 20: 245-8, 1987. 8. Warkentin DL, Van Lente F: Serum haptoglobin concentrations in concurrent hemolysis and acute-phase reaction. Clin Chem 33:1265-6, 1987. 9. Weissman N, Weinstein D: Haptoglobin reference intervals and clinical significance of serum levels. Clin Chem 28:1556 (abstract), 1982.
C.Reactive P r o t e i n : C l i n i c a l Monitoring Disease Activity Sharad D. Deodhar
ications i n
Department of lmmunopathology, Cleveland Clinic Foundation, Cleveland, Ohio
-reactive protein (CRP) is probably the best known of the acute phase proteins produced by liver parenchymal cells in response to any tissue injury or inflammation. CRP was
C
© 1991 Elsevier Science Publishing Co., Inc.
first described in 1930 by Tillet and Francis44 as a protein present in the sera of patients with penumococcal pneumonia, that formed a complex in a flocculation reaction with the C-polysaccharide iso-
identified by immunofixation electrophoresis with the use of antiserum specific for CRP to rule out the possibility of an otherwise occult monoclonal gammopathy. In our laboratories, we have used SPE routinely as part of our biochemistry profile over the past 10 years. By far, the most common dysproteinemias we have observed are those of the acute and chronic inflammatory types. As mentioned previously, others have found this to be the case as well. 2'5'6 Occult acute and chronic inflammatory conditions have been detected in otherwise healthy-
CLINICAL IMMUNOLOGY Newsletter 135
appearing patients in our experience. In our hands, we feel the SPE procedures to be simple and cost effective not only for the detection of inflammatory conditions but for the many other pathophysiological states mirrored by dysproteinemia. The use of specific protein procedures acts to complement and verify these studies. 1 CrN
References 1. Johnson AM: Plasma protein assays in clinical diagnosis and management. Bulletin 6215. Brea, Beckman Instruments, 1981.
2. Jolliff CR: Classification and interpretation of paragon serum protein electrophoresis patterns. EP-1. Brea, Beckman Instruments, 1983. 3. Kawai T: Clinical aspects of the plasma proteins. Philadelphia, Lippincott, 1973. 4. Keren DF: High-resolution electrophoresis and immunofixation. Techniques and interpretation. Stoneham, Butterworth, 1987. 5. Ritzman SE, Daniels JC: Serum protein abnormalities. Diagnostic and clinical aspects. Boston, Little Brown, 1975. 6. Sun T: Interpretation of protein and isoenzyme patterns in body fluids. New York, Igaku-Shoin, 1991.
The Diagnostic Utility of Haptoglobin Frederick Van Lente Department of Biochemistry, Cleveland Clinic Foundation, Cleveland, Ohio
'aptoglobin, or more correctly haptoglobins, are a family of .alpha-2 globulins of dramatically variable size that are inherent constituents of the classic acute phase response. The description of haptoglobin types refers back to the original description of their banding patterns on starch gels. They are designated as haptoglobins 1-1, 1-2, and 2-2 and are phenotypes of the expression of two genetic alleles, Hp 1 and Hp 2. Serum from individual homozygotes for Hp 1 demonstrates a single band on starch gel (haptoglobin 1-1). Serum from individuals with homozygous Hp 2, however, exhibits a series of bands on starch gel of decreasing mobility which has been interpreted as indicating a series of haptoglobin polymers rather than a collection of distinct polypeptide chains. The phenotype expressed by Hp 1/Hp 2 heterozygotes is a series of polymers of intermediate size relative to either homozygous phenotype. This is consistent with a combination of equal amounts of Hp 1 and Hp 2 haptoglobin products and can be duplicated in vitro. This somewhat peculiar behavior is better understood after an analysis of certain aspects of the structure of the haptoglobin molecule which is quite similar to that of hemoglobin. The fundamental
H
haptoglobin molecule is a monomer of about 85,000 kDa molecular weight consisting of two alpha chains and two beta chains connected by disulfide linkages as does hemoglobin. These chains have different compositions and are dissociable with use of mercaptoethanol and urea. Interestingly, the beta chains are identical in the three major haptoglobin types but the alpha chains are different. It is clear that the ability to aggregate the basic monomer to higher molecular weight polymers must be a characteristic of the alpha chain expressed by the Hp 2 genotype alone. In the haptoglobin 1-2 phenotype, equal amounts of monomeric and polymerizing alpha chains are present. The variation in haptoglobin types, therefore, resides in the alpha polypeptide chain. There is additional variation of the gene expression of the alpha chain but these are applicable to genetic studies only and have little relevance to the discussion here. One haptoglobin molecule (monomertwo alpha and two beta chains) binds two alpha/beta dimers of hemoglobin. This binding is essentially irreversible, although there is no evidence that covalent bonding is involved. In fact, this binding is one of the strongest noncovalent bonds known. Neither isolated heme or myo© 1991 ElsevierSciencePublishingCo., Inc.
globin can be bound by haptoglobin, and several abnormal hemoglobins such as hemoglobin H and hemoglobin Barts are also not bound. The bound complex does retain pseudoperoxidase activity. This specific and avid binding of hemoglobin is apparently the sole physiological role for this interesting protein. Serum haptoglobin is synthesized and secreted as a glycoprotein by the liver. In the circulation it serves as a reservoir of hemoglobin binding capacity. Although free hemoglobin dimers can pass through the renal glomeruli and be lost (along with the associated iron) in the urine, the hemoglobin-haptoglobin complex (recall that the size of this complex will depend on the haptoglobin phenotype) is too large to be filtered by the kidney. The renal threshold for hemoglobin is almost entirely determined by the plasma hemoglobin binding capacity or haptoglobin concentration. Free hemoglobin that finds its way into the intravascular space is bound by haptoglobin and the resultant complex is removed primarily by the liver. Hepatic parenchymal cells contain a specific receptor for this complex in the plasma membrane. The complexes initially internalized by the receptor-mediated process are first concentrated in the Golgi or0197-1859/91/$0.00 + 2.20
136 C L I N I C A L I M M U N O L O G Y
Newsletter
ganelles and, subsequently, heme is detached for further processing intracellularly where it is converted to bilirubin. 5 The globulin or protein portion of the complex is degraded separately. It is of some interest why a hemoglobin-scavenging protein should contribute to the biology of the acute phase response in humans. It is obvious that the retention of iron is important to an organism so dependent upon iron as a constitutent of the oxygen-carrying hemoglobin molecule. But what is the relationship of iron to the changes that occur during acute inflammation? The standard explanation for this phenomenon is that the fundamental attribute of this response is the defense against bacterial infection. The deprivation of circulating " f r e e " hemoglobin-iron, known to be a potent bacterial growth factor--inhibits the spread of a bacterial infection, l However, the recent focus on the role of free radicals in the pathophysiology of diseases as well as inflammation itself may provide an additional explanation for haptoglobin's role in the acute phase response. Free hemoglobin can stimulate the process of lipid peroxidation that leads to free radical chain reactions that are damaging to tissues. Also, free iron released from hemoglobin can actively promote hydroxyl radical formation via the infamous Fenton reaction (Figure 1). Hydroxyl radical is the most potent reactive species in biological systems. When viewed in context, haptoglobin shares much in common with several other well-known plasma proteins, all of which may provide an antioxidant defense system in the peripheral circulation. Proteins, such as transferrin, metallothionein, and ceruloplasmin, are all active in preventing the generation of active, transitional, metal irons in an unbound state by a combination of binding and enzymatic activity. This, in turn, minimizes the risk of uncontrolled free radical generation. It has been demonFe z+ + O z ~ F e 3 + + O z ' 2Oz'- + 2H +---~ H2Oz + Oz Fe z+ +
HzOz ~
O H - + Fe 3+ + "OH
Figure 1. The Fenton reactions. 0197-1859/91/$0.00 + 2.20
Vol. 1 1, No. 9, 1991
strated that hemoglobin-haptoglobin binding effectively prevents hemoglobinstimulated peroxidaton of fatty acid. 3 Therefore, haptoglobin may prevent damage to tissue via three mechanisms: preventing release of free hemoglobin--iron that would be available for bacterial growth, preventing free, iron-induced radical reactions, and preventing free hemoglobin-induced radical reactions. .
.
.
.
.
.
.
.
.
.
.
.
.
.
With the advent of specific polyclonal antibodies, most laboratories began determining serum haptoglobin immunochemically, initially with radial immunodiffusion (RID).
Several techniques have been employed to measure haptoglobin in serum. Early methods were based on properties of the hemoglobin-haptoglobin binding itself, in particular, the effect on the peroxidase activity of hemoglobin. The binding enhances this activity and several methods have been described including those that allow the use of standard automatic analyzers. Other methods involve the detection of altered spectrophotometric characteristics or separation of the complex by physical means or by electrophoresis. The quantitation of haptoglobin using most of these methods was in terms of grams per deciliter of hemoglobin binding capacity (HBC). There was no direct assessment of, or relationship determined to, the actual mass concentration of haptoglobin in the sample; that is, these measurements were assessing an overall characteristic of the serum in question and not the direct mass measurement of haptoglobin itself. With the advent of specific polyclonal antibodies, most laboratories began determining serum haptoglobin immunochemically, initially with radical immunodiffusion (RID). This technique proved to be somewhat slow and may have contributed to the lack of enthusiasm many clinicians displayed toward this © 1991 ElsevierScience Publishing Co., Inc.
laboratory determination. Nonetheless, the application of immunochemical techniques raised some significant issues regarding what was actually quantified. Recall that haptoglobin is not a homogenous protein among all individuals. Rather it varies normally as a function of phenotype, and that variation is in the sequence of the alpha chain and, therefore, in the molecular weight of the haptoglobins present in serum. Immunochemical methods are not easy to standardize as a rule, and a heterogenous protein, such as haptoglobin, makes that task more difficult. It has been shown that haptoglobin phenotypes are expressed by different immunologic determinants but most commercial antisera are raised against pooled sera, and antisera can be raised against isolated haptoglobin beta chains that are identical in all phenotypes. All these considerations can make the immunochemical determination of haptoglobin phenotype dependent. This variation is most obvious in radial immunodiffusion where the larger polymers of haptoglobins 1-2 and 2-2 form immuno complexes of very high molecular weight and their diffusion is retarded significantly when compared with the smaller monomers of haptoglobin 1-1. Correction factors have been described that adjust for these differences. Nephelometric and turbidimetric methods are presently the techniques most commonly employed to measure serum haptoglobin concentrations. Under optimal circumstances, these methods demonstrate minimal phenotypic variation, although some variation is to be expected due to the difference in size of the immuno complexes and, therefore, the resultant light scatter. Because the results of most commercially available methods for haptoglobin correlate with haptoglobin concentrations expressed as hemoglobin binding capacity, these assays demonstrate adequate clinical applicability. 6 Arguments advocating the correct mass measurement of haptoglobin protein are valid but not germane to the potential usefulness of these methods. These methods are precise and rapid, which is important considering their clinical usage. There have been several reports of the reference range for serum haptoglobin
C L I N I C A L I M M U N O L O G Y Newsletter 137
Vol. 11, No. 9, 1991
TABLE 1. HAPTOGLOBIN REFERENCE INTERVALS Haptogiobin All Types All Types All Types 1-1 1-2 2-2
Range (g/L)
Method
Reference
0.40-2.40 0.412-2.00 0.56-3.76 0.77-2.49 0.86--4.76 0.48-3.15
Nephelometry Enzymatic Turbidometric
6 9 7
and it is clear that the range of values found in apparently healthy individuals depends on the phenotype as shown in Table 1. However, most agree that the determination of phenotype is quite impractical and useful only in exceptional circumstances. It is the opinion of this author that haptoglobin determinations must be interpreted using a total population reference interval as is done with other analyses with genetic variability and the usefulness of the test evaluated in that context. As will be discussed below, the major clinical application of this determination has little relevance to reference intervals per se. During the acute phase response to inflammation, haptoglobin concentrations in serum will increase from two- to fourfold. Concentrations are correlated with other acute phase proteins including alpha-1 antitrypsin and alpha-1 acid glycoprotein. Because it migrates in the alpha-2 region on serum protein electrophoresis, haptoglobin contributes to the increased alpha-2 band associated with the traditional acute phase pattern. In addition, haptoglobin can contribute to the markedly abnormal protein electrophoresis pattern associated with the Nephrotic Syndrome. The degree of relative retention of haptoglobin in this condition is a function of phenotype and, therefore, molecular weight. Haptoglobins 1-2 and 2-2 are retained to a greater extent than the smaller haptoglobin 1-1. The haptoglobin-hemoglobin complex migrates more cathodic than haptoglobin itself, and this complex may be present on electrophoresis as a result of hemolysis induced by specimen collection. To the accomplished eye, these variations may be detected, but qualitative evaluations should not be substituted for quantification when clinically indicated.
The main diagnostic application of serum haptoglobin measurement is the diagnosis of hemolysis. As shown in Table 2, an active hemolytic process is present in a variety of conditions. Because the hemoglobin-haptoglobin complex is cleared rapidly by the hepatocytes (half life = 10 min) when compared to its rate of synthesis by the liver, the initial serum haptoglobin concentration should fall rapidly with the onset of hemoglobin release from any source and especially when release occurs directly in the circulation. It has been shown that the upper limit of the reference interval for haptoglobin is about 2.5 g/L. Assuming equivalency with hemoglobin binding capacity, the complete lysis of only 1.7% of the erythrocytes in an equal volume of blood would completely exhaust the available haptoglobin. Theoretically, only at this
point could free hemoglobin appear in the urine. A reduction of erythrocyte survival time to one-half of normal can result in a severe reduction of haptoglobin concentrations. It is difficult to assess the diagnostic performance of serum haptoglobin in the diagnosis of hemolytic disorders as there is no convenient gold standard, and the administration of red cell survival studies in large groups of patients is problematical. Nonetheless, using total clinical information, we have shown that decreased serum haptoglobin determinations demonstrate an 87% predictive value (83% sensitivity) for the hemolytic conditions listed in Table 2 at a cutoff of 0.25 g/L. 6 It should be emphasized that this concentration is clearly below the reference interval obtained in normal individuals. We observed a variation in the degree of abnormality among the various disease states with the most frequent "normal" values being observed in megaloblastic anemias where the degree of intravascular release of free hemoglobin is not clear. The overall specificity of serum haptoglobin in conditions not considered hemolytic, such as iron deficiency and anemia of chronic disease, was 96%. In our study all cases of autoimmune and microangiopathic anemias had haptoglobin values
TABLE 2. CONDITIONS ASSOCIATED WITH CHANGES IN SERUM HAPTOGLOBIN CONCENTRATION A. IncreasedHaptoglobinValues Inflammation Malignancy Infection Post trauma B. DecreasedHaptoglobinValues Acute hemolyticdisease Autoimmune hemolyticanemias Microangiopathichemolyticanemias Hemolytictransfusionreaction Severeexercise Acute toxic hemolyticanemia Hypersplenism Megaloblasticanemia Hemoglobinopathyhemolysis Other nonimmunehemolyticanemias Chronic hemolyticdisease Prosthetic valvereplacement Hematoma Severe liver disease Genetic deficiency © 1991 Elsevier Science Publishing Co., Inc.
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below 0.25 g/L. 6 Serum haptoglobin concentrations are also depressed as a result of acute hemolysis due to toxic exposure (e.g., arsine gas) as well as severe exercise. 2 Serum haptoglobin may also be depressed in patients with chronic, compensated hemolysis, or with clinically significant hematomas. The former is often present in patients with prosthetic heart valve replacements. As is usually the case, there are confounding situations that must be kept in mind when interpreting decreased haptoglobin concentrations and many of these occur when multiple conditions exist or when tests are requested when they are not indicated. A total, genetically determined haptoglobin deficiency has been described in a small percentage of the black population, and a combined alpha-1 antitrypsin and haptoglobin deficiency has also been reported in a European family. 4 The author is unaware of any pathophysiology that has been definitely linked to these deficiencies. The liver is the synthetic site of haptoglobin and other acute phase proteins, and failure of protein synthesis in end-stage liver disease is undoubtedly the most common cause of a decreased serum haptoglobin concentration, and not a direct result of hemolysis. Haptoglobin demonstrates a fairly close correlation with other acute phase proteins, such as alpha-1 acid glycoprotein, in these conditions, although the latter has been shown to be a more sensitive indicator of liver failure. None of these proteins appears to have displaced the more widely used protein markers of liver synthetic capacity such as transferrin, prealbumin, or retinal binding protein. Haptoglobin is similar to ferritin in that decreases in both are used in the differential diagnosis of anemia but both are acute phase reactants and their synthesis and serum concentrations rise in response to acute inflammation. This combination of changes has led to skepticism regarding the usefulness of a parameter that may be doing two things at once. Reasonably designed studies of the diagnostic performance of a test such as haptoglobin, however, must include the confounding variables that are present in the very patients studied. We extended this approach in order to investigate the worst I 0197-1859/91/$0.00 + 2.20
case scenario and studied the decline in serum haptoglobin secondary to mechanical hemolysis caused by the blood oxygenator used in cardiopulmonary bypass surgery. 8 These patients also undergo a marked acute phase response secondary to surgery that is easily documented by increases in C-reactive protein concentrations. It was found that all patients exhibited a fall in serum haptoglobin after surgery and 71% exhibited concentrations <0.25 g/L, even though C-reactive protein demonstrated a concurrent sixfold increase in the same time period. Therefore, although a concurrent acute phase response can affect the decline in haptoglobin during inflammation, the effect is minimal. In summary, hypohaptoglobinemia, except when inherited, is usually always clinically significant after infancy. In some cases, the cause may be decreased synthesis due to severe hepatic failure, but these cases are evident, and haptoglobin should only be used as a prognostic indicator in these situations when evidence for active hemolysis is lacking. Haptoglobin measurements are best utilized when there is an indication of active hemolysis or unexplained anemia. These indications include a decreasing hemoglobin concentration with evidence of reticulocytosis and/or red cell destruction as well as an unexplained increase in serum LDH-I activity or unconjugated bilirubin concentration. The finding of a significantly depressed serum haptoglobin (<0.25 g/L) with a concurrent increased LDH-1 isoenzyme activity or reticulocytosis in these settings is extremely predictive of hemolytic disease. In these
Vol. 11, No. 9, 1991
situations serum haptoglobin is a useful adjunct to the differential diagnosis of hemolytic disorders, c~
References 1. Eaton J W , Brandt P, Mahoney JR, Lee JT: Haptoglobin: A natural bacteriostat. Science 215:691-3, 1982. 2. Egan LM, Watts PB, Silta BC: Changes in serum haptoglobin as an acute response to a marathon road race. J Sports Med 5:55--60, 1987. 3. Gutteridge JMC: The antioxidant activity of haptoglobin towards haemoglobin-stimulated lipid peroxidation. Biochim Biophys Acta 917:219-23, 1987. 4. Kalsheker NA, Kingston JE, Moore CJ, Sarjant JA: A family with deficiencies of both alpha-l-antitrypsin and haptoglobin. Clin Chem 28:381-2 (letter), 1982. 5. Nakajima H, Satom S: Intracellular site of the catabolism of heme and globin moiety of hemoglobin-haptoglobin after intravenous administration to rats. J Biol Chem 263:16032-6, 1988. 6. Van Lente F, Marchand A, Galen RS: Evaluation of a nephelometric assay for haptoglobin and its clinical usefulness. Clin Chem 25:2007-10, 1979. 7. Van Rijn HJM, Van Der Witt W, Stroes TW, Schrijver T: Is the turbidimetric immunoassay of haptoglobin phenotypedependent. Clin Biochem 20: 245-8, 1987. 8. Warkentin DL, Van Lente F: Serum haptoglobin concentrations in concurrent hemolysis and acute-phase reaction. Clin Chem 33:1265-6, 1987. 9. Weissman N, Weinstein D: Haptoglobin reference intervals and clinical significance of serum levels. Clin Chem 28:1556 (abstract), 1982.
C.Reactive P r o t e i n : C l i n i c a l Monitoring Disease Activity Sharad D. Deodhar
ications i n
Department of lmmunopathology, Cleveland Clinic Foundation, Cleveland, Ohio
-reactive protein (CRP) is probably the best known of the acute phase proteins produced by liver parenchymal cells in response to any tissue injury or inflammation. CRP was
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© 1991 Elsevier Science Publishing Co., Inc.
first described in 1930 by Tillet and Francis44 as a protein present in the sera of patients with penumococcal pneumonia, that formed a complex in a flocculation reaction with the C-polysaccharide iso-