Symposium on Clinical Laboratory Medicine
Laboratory Diagnosis of Hepatic Disease William D. Schall, D.V.M.*
With respect to liver disease, the laboratory serves three basic functions for the clinician. First, the laboratory may detect hepatic disease when there is no reason to suspect that hepatic disease exists. Second, the laboratory provides the means of evaluating the patient with clinical signs or abnormal physical findings that may be referable to liver disease. Finally, the most accurate means of assessing the progress of liver disease is often by utilization of the laboratory. Widespread use of the laboratory, particularly for the detection of liver disease, is probably a result of decreased costs of biochemical profiles. Decreased cost has resulted from automation and technological advances. Serum Enzyme Activity as an Index of Hepatocellular Damage
Simplistically, increased serum activity of the commonly determined hepatic enzymes can be equated with increased cellular release due to altered membrane permeability and necrosis. Alterations in permeability may result from hypoxia or toxins, may be reversible, or may progress to necrosis. The magnitude of the increase in serum activity parallels the number of hepatocytes injured but provides no information regarding reversibility of the injury on the cellular level, regenerative capabilities on the tissue level, or the status of function at the organ level. Whether increased serum enzyme activity is sustained is a function of continued cellular injury and leakage and is influenced by the rate of plasma clearance. When enzyme leakage has begun, three factors affect release and, hence, serum enzyme activity. 6 The first of these is the concentration gradient across the cell membrane. The magnitude of the gradient varies for different enzymes and different cells. The intracellular hepatocyte concentrations of alanine transaminase (formerly serum glutamic pyruvic transaminase-SGPT) and aspartate transaminase (formerly *Associate Professor, Department of Small Animal Medicine and Surgery, College of Veterinary Medicine, University of Georgia, Athens, Georgia Veterinary Clinics of North America-Val. 6, No.4, November 1976
679
680
WILLIAM
D.
SCHALL
serum glutamic oxaloacetic transaminase-SCOT) normally exceed extracellular concentration by a factor of 10,000. The higher the concentration gradient, the more quickly enzyme leakage occurs after cell membrane injury. The second factor affecting enzyme leakage is the size of the enzyme molecules. Those with lower molecular weights generally leak more quickly because of greater diffusion rates and passage through small plasma membrane pores. The third factor affecting ease of leakage is the intracellular location of the enzymes. Cytoplasmic enzymes are released much more readily than are mitochondrial enzymes. In some instances, isoenzymes may be located in cytoplasm and mitochondria. Because of differences in the location, rate of release and the extent of injury necessary for release vary between isoenzymes. For instance, both cytoplasmic and mitochondrial isoenzymes of aspartate transaminase (formerly SGOT) exist in liver cells. Following initial cell membrane injury, the cytoplasmic isoenzyme rapidly leaks out; the mitochondrial isoenzyme, however, leaks out only after the cell membrane has deteriorated to the extent that cell death has occurred and intracellular organelles have broken down. Although individual isoenzyme activity can be determined, 5 these determinations are currently too costly to have applicability in veterinary medicine. Commonly used laboratory procedures determine the collective serum activity of isoenzymes. The duration of increased serum enzyme activity is dependent not only on leakage from dal_Ilaged cells but the rate of plasma disappearance as well. Although the plasma half-life of most proteins varies between 8 and 30 days, the plasma half-life of enzymes ranges from 2 to 4 days. The mechanism of enzyme inactivation involves steriochemical denaturation and consequential loss of catalytic capability. Renal excretion is not a significant factor because the enzymes commonly used to detect hepatic injury have a molecular weight that precludes passage into the glomerular filtrate. Following an isolated hepatic injury in the dog or cat, serum activity of the transaminases usually remains increased for about 2 weeks. 7 The magnitude of enzyme activity increase cannot be equated with reversibility because no distinction is made between degenerative permeability change and necrosis. For example, greatly increased serum enzyme activity may result from diffuse hepatocellular hypoxia due to shock. All hepatocytes would be expected to recover, however. It should be emphasized that organ function may remain near normal in spite of significant, sustained, increased serum enzyme activity. Conversely, hepatic organ failure can be present unaccompanied by increased serum enzyme activity. The number of enzymes of which serum activities are determined by medical laboratories is a reflection ofthe search for hepatic specificity. Alanine transaminase (formerly SGPT) is essentially liver specific in the
LABORATORY DIAGNOSIS OF HEPATIC DISEASE
681
dog and cat. Sorbital dehydrogenase (SDH) is liver specific in large animals as well as in dogs and cats. Aspartate transaminase (formerly SGOT), glutamic dehydrogenase, alphahydroxybutyric dehydrogenase, isocitric dehydrogenase, ornithine carbamyl transferase, and lactic dehydrogenase are present in significant quantities in other tissues, notably cardiac and skeletal musclem As a consequence, increased serum activity of these enzymes can occur with striated muscle injury. The lack of specificity does not totally negate their clinical usefulness as long as they are interpreted in the context of their nonspecificity. Although different enzymes may have different cellular concentrations depending on the location of the cells within the liver, the differences in serum concentration are not generally considered significant enough to allow localization of the lesion within the liver when enzyme leakage occurs. Following parenchymal hepatic injury, all of the previously mentioned enzymes usually increase in serum activity in a parallel fashion. Serum Enzyme Activity Due to Increased Production
Increased serum activity of the previously considered enzymes occurs primarily due to cellular damage or death of cells producing the enzyme and subsequent leakage of the enzyme into extracellular fluid. 6 Certain enzymes, however, can gain access to extracellular fluid and thence blood without apparent structural damage to the cells that produce the enzymes. In this instance, increased serum activity is an index of increased enzyme production. Alkaline phosphatase is such an enzyme.4,6,s Total serum alkaline phosphatase (SAP) activity reflects increased production of any or all of the isoenzymes of alkaline phosphatase. Tissues and cells known to produce alkaline phosphatase include osteoblasts, hepatocytes, biliary epithelial cells, intestine, placenta, and certain neoplasms. As a consequence, increased cellular production at any of these locations is associated with increased SAP activity. Increased osteoblastic activity and associated increase in SAP activity characterizes normal bone growth of the young as well as pathological causes of increased osteoblastic activity such as canine panosteitis and secondary hyperparathyroidism. The magnitude of the increase in SAP activity associated with either physiological or pathological increase in osteoblastic activity is usually moderate or on the magnitude of l Yz to 3Yz times normal SAP activity. Bile stasis, whether due to intrahepatic or extrahepatic disease, is consistently associated with increased alkaline phosphatase production by biliary epithelial cells as well as hepatocytes. Although neither the mechanism nor the reason for enzyme induction is known, the increased production causes increased SAP activity. This increased activity may be detected prior to the onset and in the absence of hyperbilirubinemia. The magnitude of the increased SAP activity varies between 2% to 30
682
WILLIAM
D.
SCHALL
times normal. The association of increased SAP activity with biliary stasis has long been recognized in the dog and recently has been established in the cat. 3 Less commonly determined enzyme activities associated with overproduction and cholestasis are 5 '-nucleotidase and leucine aminopeptidase. The extremely high increases in SAP activity commonly associated with cholestasis are occasionally present due to isoenzyme production by neoplastic tissue. Canine tumors that have been associated with increased SAP activity include adrenal cortical adenocarcinoma, mixed mammary tumor, hemangiosarcoma, lymphoma, oral carcinoma, and others. Modest increases in SAP activity may be associated with intestinal obstruction and late pregnancy. Summary: Serum Enzyme Activity and Liver Disease Increased serum activity of most enzymes found in liver cells is a reflection of leakage from damaged liver cells. The leakage of enzymes may be due to increased cell membrane permeability during reversible hepatocellular changes or due to cell necrosis. Virtually no information regarding hepatic organ function is obtained by interpretation of increased serum enzyme activity. SAP activity is consistently increased in association with both intrahepatic and extrahepatic cholestasis with and without concomitant hyperbilirubinemia. This increase is due to increased production rather than cell injury. Increases in SAP activity also occurs because of increased production of isoenzymes by other tissues. The increased production may be either physiological or pathological but seldom is of the magnitude associated with cholestasis. Drug-induced increases must always be considered in the interpretation of increased serum enzyme activity. 9 Tests Based on Bilirubin Metabolism Bilirubin is a pigment derived principally from the catabolism of senescent erythrocytes. Bilirubin thus released into plasma is attached primarily to albumin. This bonding makes plasma transport possible because bilirubin as released from erythrocytes is virtually water insoluble. Serum bilirubin determinations measure this moiety as unconjugated or indirect reading bilirubin. Hepatic parenchymal cells take up unconjugated bilirubin, cleave off albumin, and render it water soluble and lipid insoluble by conjugation to a di- or mono-glucuronide compound prior to excretion in bile. Serum bilirubin determinations measure this moiety as conjugated or direct reading bilirubin. Biliary stasis, whether due to intrahepatic or extrahepatic disease, results in the escape of conjugated bilirubin into plasma. The hyperbilirubinemia caused by cholestasis is usually characterized by co~u gated (direct reading) bilirubin increases that account for nearly 70 per
LABORATORY DIAGNOSIS OF HEPATIC DISEASE
683
cent of the total serum bilirubin concentration. In some cases of cholestasis, however, a lesser per cent of the total serum bilirubin is conjugated bilirubin. As a general rule, if 50 per cent or more of serum bilirubin is conjugated, either intrahepatic or extrahepatic cholestasis is the probable cause of hyperbilirubinemia. Cholestasis is accompanied by increased SAP activity. Disease characterized by primary hepatocellular injury of sufficient magnitude to cause icterus is typified by increased serum concentrations of both conjugated and unconjugated bilirubin. Since the fractional hyperbilirubinemia of hepatocellular injury overlaps that of primary cholestasis, increased serum transaminase activity without significantly increased SAP activity is helpful in making the distinction. Icterus due to hemolysis can also occur, but the hyperbilirubinemia is initially characterized by a preponderance of unconjugated bilirubin. Three to four days after hemolytic crisis, however, conjugated bilirubin concentration may exceed the unconjugated bilirubin concentration. Patients with hemolytic disease are usually anemic in addition and usually do not have significantly increased serum activity of leaked enzymes (transaminases) or overproduced enzymes (SAP). Conjugated bilirubin may be detected in the urine of normal cats and dogs, particularly when the urine is highly concentrated. If urine bilirubin is detected in dilute urine or if it is detected in moderate or greater amounts in concentrated urine, conjugated hyperbilirubinemia may be present. Increased urine bilirubin is usually present prior to overt icterus or hyperbilirubinemia and, for this reason, provides a sensitive means of detecting cholestasis or hepatocellular disease early. Dogs to which an intravenous arsenical has been administered for heartworm disease, for instance, usually have significant bilirubinuria prior to detectable icterus. Although the renal clearance of conjugated bilirubin in the cat and dog is not entirely understood, most data suggest that a small dialyzable fraction of conjugated bilirubin passes into the glomerular filtrate. In the dog, renal tubular cells may conjugate a minor amount of bilirubin and result in renal excretion. 2 Regardless, urine bilirubin, although often present in normal dogs, is a sensitive indicator of conjugated hyperbilirubinemia. Within the intestinal lumen, conjugated bilirubin present from bile is transformed by bacterial action into colorless urobilinogens and colored urobilins. A portion of the urobilinogens is absorbed into portal circulation. Although most of the absorbed urobilinogen is taken up by the liver, a small portion is normally excreted in urine. Urobilinogen is thought to be excreted into urine by a combination of glomerular filtration, proximal tubular secretion, and pH-dependent back diffusion in the distal tubules. Back diffusion is maximized if the urine pH is acid. Another extrahepatic variable is that the Ehrlich aldehyde reaction, which is used most commonly for urobilinogen estimation, will react with other compounds such as sulfonamides and procaine.
684
WILLIAM
D.
ScHALL
In spite of these variables, estimation of urine urobilinogen concentration is useful because of the ease with which it can be done. Complete biliary obstruction is accompanied by a lack of urine urobilinogen due to failure of bile flow. Most cholestatic diseases causing icterus in the dog and cat, however, are characterized by partial blockage, and some urine urobilinogen is present although it may be in decreased amounts. Hemolysis leading to icterus and increased bile pigment flow is often accompanied by increased quantities of urine urobilinogen. Fecal urobilinogen determinations have been utilized in human medicine but have not been extensively used in veterinary medicine. Serum Protein
Since all serum proteins other than the immunoglobulins are of hepatic origin, decreased serum concentration of protein, especially albumin, is characteristic of reduced functional hepatic mass. Liver disease must be advanced to a stage approaching failure before hypoalbuminemia is present. Inasmuch as the normal half-life of canine serum albumin is about 7 to 10 days, acute liver failure is not usually associated with hypoalbuminemia. If decreased serum albumin concentration is demonstrated, causes other than hepatic insufficiency must be ruled out. Conditions that must be ruled out are renal loss due to glomerular disease, intestinal loss due to protein-losing enteropathy, and decreased synthesis due to starvation. In contrast to other causes of hypoproteinemia, protein-losing enteropathy is usually characterized by hypoglobulinemia as well as hypoalbuminemia. Several methods are ·used to determine serum albumin concentration. Many human medical laboratories with automated equipment use a dye binding method for albumin determination. Hydroxyazobenzoic acid (HABA) dye is often used and yields erroneously low results because of incomplete binding of canine albumin. Other dyes, such as bromcresol green, may give more reliable results. Serum globulin concentration is often increased in canine patients with liver disease. The hyperglobulinemia is characteristically polyclonal but the mechanism of the increase is not precisely known. Several flocculation tests have been employed in the past to detect hepatic disease in human patients. These tests depend on alterations in serum protein fractions and have not been used to any extent in veterinary medicine. Dye Excretion Tests
The excretion of organic anion dyes, such as sulfobromophthalein (BSP) and indocyanine green, is used extensively for the detection of hepatic dysfunction. BSP is more widely used in veterinary medicine and will be discussed here. After intravenous administration, BSP is rapidly removed from blood by the liver and subsequently is excreted more
LABORATORY DIAGNOSIS OF HEPATIC DISEASE
685
slowly into bile. The most commonly used test is to determine plasma concentration 30 minutes after the single intravenous injection of 5 mg BSP/kg of body weight. It is assumed that after instantaneous mixing, a plasma concentration of 10 mg/dl is achieved. This value is considered to represent 100 per cent retention. In the dog, 5 per cent retention (or less) 30 minutes after injection is considered normal. A similar value is probably normal for the cat. After injection, BSP is rapidly bound to serum proteins and carried to the liver. Inadequate hepatic perfusion can influence hepatic uptake and must be considered in the interpretation of the results. BSP is rapidly taken up by the liver in an unknown state by a mechanism incompletely understood. The uptake process is apparently saturable and competition for uptake between BSP and bilirubin has been demonstrated.1 For this reason, BSP excretion tests are seldom done if the total serum bilirubin concentration is increased. Most of the BSP is conjugated in the hepatic cell primarily with glutathione. Conjugated and, to a lesser extent, free BSP are then excreted into the biliary tract. Biliary excretion is obviously impaired in diseases characterized by cholestasis. The primary usefulness of the BSP excretion test is the detection and assessment of reduced functional hepatic mass. Retention of BSP greater than 20 per cent is indicative of severely impaired liver function. Blood Ammonia Concentration
The deamination of amino acids by the liver and bacterial deamination of ingested amino acids within the intestine followed by absorption in portal circulation result in an ammonia load on the liver. Normally, the ammonia thus produced and absorbed enters the urea cycle in the liver, the only site of urea formation. If the functional hepatic mass is severely reduced, ammonia may not be converted to urea. As a consequence, the blood urea nitrogen (BUN) concentration may be low and blood ammonia concentration may rise. Impending hepatic encephalopathy in liver failure may be anticipated by documentation of elevated blood ammonia concentration in spite of the fact that ammonia concentration does not correlate precisely with hepatic encephalopathy. Prothrombin Time
The one stage prothrombin time is often greater than normal when the functional hepatic mass is severely reduced. Because the clotting factors assessed by prothrombin time determinations have a much shorter half-life than does serum albumin, increased prothrombin time is often present in acute hepatic failure where serum albumin concentration is normal. Although prothrombin can be used to assess hepatic function, its chief usefulness is in prebiopsy evaluation of the patient with documented liver disease. Determination of the clotting time, however, is a more practical method of prebiopsy coagulation evaluation.
686
WILLIAM
D.
SCHALL
Analysis of Ascitic Fluid The analysis of ascitic fluid is discussed in detail elsewhere in this symposium. Ascitic fluid associated with chronic reduced functional hepatic mass and associated decreased colloidal osmotic pressure is characteristically a pure transudate.
Summary of Liver Tests A summar)C- of liver tests is outlined below. It should be noted that tests which assess liver cell injury do not yield information regarding the functional state of the liver and may be normal when liver failure ts present. Cell Injury Enzyme Leakage
}
Increased Transaminase (SGPT and SGOT) and SDH
Cholestasis
}
SAP Bilirubin ESP
}
Albumin Ammonium ESP
Reduced Functional Mass
REFERENCES l. Burton, C., and Schenker, S.: Laboratory tests. In Schiff, L. (ed.): Diseases of the
Liver. Philadelphia,]. B. Lippincott Co., 1969. 2. Cornelius, C. E., and Himes, ]. A.: New concepts in canine hepatic function. J.A.A.H.A., 9:147-150, 1973. 3. Everett, R. M.: Personal communication, 1976. 4. Kaplan, M. M.: Current concepts. Alkaline phosphatase. New Eng.]. Med., 286:200202, 1972. . 5. Moore, W. E., and Feldman, B. F.: The use of isoenzymes in small animal medicine. J.A.A.H.A., 10:420-429, 1974. 6. Moss, D. W., and Butterworth, P. ].: Enzymology and Medicine. London, Pitman Medical, 1974. 7. Van Vleet,]. F., and Alberts,]. 0.: Evaluation ofliver function tests and liver biopsy in experimental carbon tetrachloride intoxication and extrahepatic bile duct obstruction in the dog. Am.]. Vet. Res., 29:2119-2131, 1968. 8. Young,]. T.: Source, fate, and possible significance of elevated serum alkaline phosphatase in nonicteric animals with partial biliary obstruction. J.A.A.H.A., 10:415419, 1974. 9. Zimmerman, H.].: Liver disease caused by medicinal agents. Med. Clin. North. Am., 59:897-907, 1975. Department of Small Animal Medicine and Surgery College of Veterinary Medicine University of Georgia Athens, Georgia 30602
Laboratory Diagnosis of Hepatic Disease William D. Schall, D.V.M.*
With respect to liver disease, the laboratory serves three basic functions for the clinician. First, the laboratory may detect hepatic disease when there is no reason to suspect that hepatic disease exists. Second, the laboratory provides the means of evaluating the patient with clinical signs or abnormal physical findings that may be referable to liver disease. Finally, the most accurate means of assessing the progress of liver disease is often by utilization of the laboratory. Widespread use of the laboratory, particularly for the detection of liver disease, is probably a result of decreased costs of biochemical profiles. Decreased cost has resulted from automation and technological advances. Serum Enzyme Activity as an Index of Hepatocellular Damage
Simplistically, increased serum activity of the commonly determined hepatic enzymes can be equated with increased cellular release due to altered membrane permeability and necrosis. Alterations in permeability may result from hypoxia or toxins, may be reversible, or may progress to necrosis. The magnitude of the increase in serum activity parallels the number of hepatocytes injured but provides no information regarding reversibility of the injury on the cellular level, regenerative capabilities on the tissue level, or the status of function at the organ level. Whether increased serum enzyme activity is sustained is a function of continued cellular injury and leakage and is influenced by the rate of plasma clearance. When enzyme leakage has begun, three factors affect release and, hence, serum enzyme activity. 6 The first of these is the concentration gradient across the cell membrane. The magnitude of the gradient varies for different enzymes and different cells. The intracellular hepatocyte concentrations of alanine transaminase (formerly serum glutamic pyruvic transaminase-SGPT) and aspartate transaminase (formerly *Associate Professor, Department of Small Animal Medicine and Surgery, College of Veterinary Medicine, University of Georgia, Athens, Georgia Veterinary Clinics of North America-Val. 6, No.4, November 1976
679
680
WILLIAM
D.
SCHALL
serum glutamic oxaloacetic transaminase-SCOT) normally exceed extracellular concentration by a factor of 10,000. The higher the concentration gradient, the more quickly enzyme leakage occurs after cell membrane injury. The second factor affecting enzyme leakage is the size of the enzyme molecules. Those with lower molecular weights generally leak more quickly because of greater diffusion rates and passage through small plasma membrane pores. The third factor affecting ease of leakage is the intracellular location of the enzymes. Cytoplasmic enzymes are released much more readily than are mitochondrial enzymes. In some instances, isoenzymes may be located in cytoplasm and mitochondria. Because of differences in the location, rate of release and the extent of injury necessary for release vary between isoenzymes. For instance, both cytoplasmic and mitochondrial isoenzymes of aspartate transaminase (formerly SGOT) exist in liver cells. Following initial cell membrane injury, the cytoplasmic isoenzyme rapidly leaks out; the mitochondrial isoenzyme, however, leaks out only after the cell membrane has deteriorated to the extent that cell death has occurred and intracellular organelles have broken down. Although individual isoenzyme activity can be determined, 5 these determinations are currently too costly to have applicability in veterinary medicine. Commonly used laboratory procedures determine the collective serum activity of isoenzymes. The duration of increased serum enzyme activity is dependent not only on leakage from dal_Ilaged cells but the rate of plasma disappearance as well. Although the plasma half-life of most proteins varies between 8 and 30 days, the plasma half-life of enzymes ranges from 2 to 4 days. The mechanism of enzyme inactivation involves steriochemical denaturation and consequential loss of catalytic capability. Renal excretion is not a significant factor because the enzymes commonly used to detect hepatic injury have a molecular weight that precludes passage into the glomerular filtrate. Following an isolated hepatic injury in the dog or cat, serum activity of the transaminases usually remains increased for about 2 weeks. 7 The magnitude of enzyme activity increase cannot be equated with reversibility because no distinction is made between degenerative permeability change and necrosis. For example, greatly increased serum enzyme activity may result from diffuse hepatocellular hypoxia due to shock. All hepatocytes would be expected to recover, however. It should be emphasized that organ function may remain near normal in spite of significant, sustained, increased serum enzyme activity. Conversely, hepatic organ failure can be present unaccompanied by increased serum enzyme activity. The number of enzymes of which serum activities are determined by medical laboratories is a reflection ofthe search for hepatic specificity. Alanine transaminase (formerly SGPT) is essentially liver specific in the
LABORATORY DIAGNOSIS OF HEPATIC DISEASE
681
dog and cat. Sorbital dehydrogenase (SDH) is liver specific in large animals as well as in dogs and cats. Aspartate transaminase (formerly SGOT), glutamic dehydrogenase, alphahydroxybutyric dehydrogenase, isocitric dehydrogenase, ornithine carbamyl transferase, and lactic dehydrogenase are present in significant quantities in other tissues, notably cardiac and skeletal musclem As a consequence, increased serum activity of these enzymes can occur with striated muscle injury. The lack of specificity does not totally negate their clinical usefulness as long as they are interpreted in the context of their nonspecificity. Although different enzymes may have different cellular concentrations depending on the location of the cells within the liver, the differences in serum concentration are not generally considered significant enough to allow localization of the lesion within the liver when enzyme leakage occurs. Following parenchymal hepatic injury, all of the previously mentioned enzymes usually increase in serum activity in a parallel fashion. Serum Enzyme Activity Due to Increased Production
Increased serum activity of the previously considered enzymes occurs primarily due to cellular damage or death of cells producing the enzyme and subsequent leakage of the enzyme into extracellular fluid. 6 Certain enzymes, however, can gain access to extracellular fluid and thence blood without apparent structural damage to the cells that produce the enzymes. In this instance, increased serum activity is an index of increased enzyme production. Alkaline phosphatase is such an enzyme.4,6,s Total serum alkaline phosphatase (SAP) activity reflects increased production of any or all of the isoenzymes of alkaline phosphatase. Tissues and cells known to produce alkaline phosphatase include osteoblasts, hepatocytes, biliary epithelial cells, intestine, placenta, and certain neoplasms. As a consequence, increased cellular production at any of these locations is associated with increased SAP activity. Increased osteoblastic activity and associated increase in SAP activity characterizes normal bone growth of the young as well as pathological causes of increased osteoblastic activity such as canine panosteitis and secondary hyperparathyroidism. The magnitude of the increase in SAP activity associated with either physiological or pathological increase in osteoblastic activity is usually moderate or on the magnitude of l Yz to 3Yz times normal SAP activity. Bile stasis, whether due to intrahepatic or extrahepatic disease, is consistently associated with increased alkaline phosphatase production by biliary epithelial cells as well as hepatocytes. Although neither the mechanism nor the reason for enzyme induction is known, the increased production causes increased SAP activity. This increased activity may be detected prior to the onset and in the absence of hyperbilirubinemia. The magnitude of the increased SAP activity varies between 2% to 30
682
WILLIAM
D.
SCHALL
times normal. The association of increased SAP activity with biliary stasis has long been recognized in the dog and recently has been established in the cat. 3 Less commonly determined enzyme activities associated with overproduction and cholestasis are 5 '-nucleotidase and leucine aminopeptidase. The extremely high increases in SAP activity commonly associated with cholestasis are occasionally present due to isoenzyme production by neoplastic tissue. Canine tumors that have been associated with increased SAP activity include adrenal cortical adenocarcinoma, mixed mammary tumor, hemangiosarcoma, lymphoma, oral carcinoma, and others. Modest increases in SAP activity may be associated with intestinal obstruction and late pregnancy. Summary: Serum Enzyme Activity and Liver Disease Increased serum activity of most enzymes found in liver cells is a reflection of leakage from damaged liver cells. The leakage of enzymes may be due to increased cell membrane permeability during reversible hepatocellular changes or due to cell necrosis. Virtually no information regarding hepatic organ function is obtained by interpretation of increased serum enzyme activity. SAP activity is consistently increased in association with both intrahepatic and extrahepatic cholestasis with and without concomitant hyperbilirubinemia. This increase is due to increased production rather than cell injury. Increases in SAP activity also occurs because of increased production of isoenzymes by other tissues. The increased production may be either physiological or pathological but seldom is of the magnitude associated with cholestasis. Drug-induced increases must always be considered in the interpretation of increased serum enzyme activity. 9 Tests Based on Bilirubin Metabolism Bilirubin is a pigment derived principally from the catabolism of senescent erythrocytes. Bilirubin thus released into plasma is attached primarily to albumin. This bonding makes plasma transport possible because bilirubin as released from erythrocytes is virtually water insoluble. Serum bilirubin determinations measure this moiety as unconjugated or indirect reading bilirubin. Hepatic parenchymal cells take up unconjugated bilirubin, cleave off albumin, and render it water soluble and lipid insoluble by conjugation to a di- or mono-glucuronide compound prior to excretion in bile. Serum bilirubin determinations measure this moiety as conjugated or direct reading bilirubin. Biliary stasis, whether due to intrahepatic or extrahepatic disease, results in the escape of conjugated bilirubin into plasma. The hyperbilirubinemia caused by cholestasis is usually characterized by co~u gated (direct reading) bilirubin increases that account for nearly 70 per
LABORATORY DIAGNOSIS OF HEPATIC DISEASE
683
cent of the total serum bilirubin concentration. In some cases of cholestasis, however, a lesser per cent of the total serum bilirubin is conjugated bilirubin. As a general rule, if 50 per cent or more of serum bilirubin is conjugated, either intrahepatic or extrahepatic cholestasis is the probable cause of hyperbilirubinemia. Cholestasis is accompanied by increased SAP activity. Disease characterized by primary hepatocellular injury of sufficient magnitude to cause icterus is typified by increased serum concentrations of both conjugated and unconjugated bilirubin. Since the fractional hyperbilirubinemia of hepatocellular injury overlaps that of primary cholestasis, increased serum transaminase activity without significantly increased SAP activity is helpful in making the distinction. Icterus due to hemolysis can also occur, but the hyperbilirubinemia is initially characterized by a preponderance of unconjugated bilirubin. Three to four days after hemolytic crisis, however, conjugated bilirubin concentration may exceed the unconjugated bilirubin concentration. Patients with hemolytic disease are usually anemic in addition and usually do not have significantly increased serum activity of leaked enzymes (transaminases) or overproduced enzymes (SAP). Conjugated bilirubin may be detected in the urine of normal cats and dogs, particularly when the urine is highly concentrated. If urine bilirubin is detected in dilute urine or if it is detected in moderate or greater amounts in concentrated urine, conjugated hyperbilirubinemia may be present. Increased urine bilirubin is usually present prior to overt icterus or hyperbilirubinemia and, for this reason, provides a sensitive means of detecting cholestasis or hepatocellular disease early. Dogs to which an intravenous arsenical has been administered for heartworm disease, for instance, usually have significant bilirubinuria prior to detectable icterus. Although the renal clearance of conjugated bilirubin in the cat and dog is not entirely understood, most data suggest that a small dialyzable fraction of conjugated bilirubin passes into the glomerular filtrate. In the dog, renal tubular cells may conjugate a minor amount of bilirubin and result in renal excretion. 2 Regardless, urine bilirubin, although often present in normal dogs, is a sensitive indicator of conjugated hyperbilirubinemia. Within the intestinal lumen, conjugated bilirubin present from bile is transformed by bacterial action into colorless urobilinogens and colored urobilins. A portion of the urobilinogens is absorbed into portal circulation. Although most of the absorbed urobilinogen is taken up by the liver, a small portion is normally excreted in urine. Urobilinogen is thought to be excreted into urine by a combination of glomerular filtration, proximal tubular secretion, and pH-dependent back diffusion in the distal tubules. Back diffusion is maximized if the urine pH is acid. Another extrahepatic variable is that the Ehrlich aldehyde reaction, which is used most commonly for urobilinogen estimation, will react with other compounds such as sulfonamides and procaine.
684
WILLIAM
D.
ScHALL
In spite of these variables, estimation of urine urobilinogen concentration is useful because of the ease with which it can be done. Complete biliary obstruction is accompanied by a lack of urine urobilinogen due to failure of bile flow. Most cholestatic diseases causing icterus in the dog and cat, however, are characterized by partial blockage, and some urine urobilinogen is present although it may be in decreased amounts. Hemolysis leading to icterus and increased bile pigment flow is often accompanied by increased quantities of urine urobilinogen. Fecal urobilinogen determinations have been utilized in human medicine but have not been extensively used in veterinary medicine. Serum Protein
Since all serum proteins other than the immunoglobulins are of hepatic origin, decreased serum concentration of protein, especially albumin, is characteristic of reduced functional hepatic mass. Liver disease must be advanced to a stage approaching failure before hypoalbuminemia is present. Inasmuch as the normal half-life of canine serum albumin is about 7 to 10 days, acute liver failure is not usually associated with hypoalbuminemia. If decreased serum albumin concentration is demonstrated, causes other than hepatic insufficiency must be ruled out. Conditions that must be ruled out are renal loss due to glomerular disease, intestinal loss due to protein-losing enteropathy, and decreased synthesis due to starvation. In contrast to other causes of hypoproteinemia, protein-losing enteropathy is usually characterized by hypoglobulinemia as well as hypoalbuminemia. Several methods are ·used to determine serum albumin concentration. Many human medical laboratories with automated equipment use a dye binding method for albumin determination. Hydroxyazobenzoic acid (HABA) dye is often used and yields erroneously low results because of incomplete binding of canine albumin. Other dyes, such as bromcresol green, may give more reliable results. Serum globulin concentration is often increased in canine patients with liver disease. The hyperglobulinemia is characteristically polyclonal but the mechanism of the increase is not precisely known. Several flocculation tests have been employed in the past to detect hepatic disease in human patients. These tests depend on alterations in serum protein fractions and have not been used to any extent in veterinary medicine. Dye Excretion Tests
The excretion of organic anion dyes, such as sulfobromophthalein (BSP) and indocyanine green, is used extensively for the detection of hepatic dysfunction. BSP is more widely used in veterinary medicine and will be discussed here. After intravenous administration, BSP is rapidly removed from blood by the liver and subsequently is excreted more
LABORATORY DIAGNOSIS OF HEPATIC DISEASE
685
slowly into bile. The most commonly used test is to determine plasma concentration 30 minutes after the single intravenous injection of 5 mg BSP/kg of body weight. It is assumed that after instantaneous mixing, a plasma concentration of 10 mg/dl is achieved. This value is considered to represent 100 per cent retention. In the dog, 5 per cent retention (or less) 30 minutes after injection is considered normal. A similar value is probably normal for the cat. After injection, BSP is rapidly bound to serum proteins and carried to the liver. Inadequate hepatic perfusion can influence hepatic uptake and must be considered in the interpretation of the results. BSP is rapidly taken up by the liver in an unknown state by a mechanism incompletely understood. The uptake process is apparently saturable and competition for uptake between BSP and bilirubin has been demonstrated.1 For this reason, BSP excretion tests are seldom done if the total serum bilirubin concentration is increased. Most of the BSP is conjugated in the hepatic cell primarily with glutathione. Conjugated and, to a lesser extent, free BSP are then excreted into the biliary tract. Biliary excretion is obviously impaired in diseases characterized by cholestasis. The primary usefulness of the BSP excretion test is the detection and assessment of reduced functional hepatic mass. Retention of BSP greater than 20 per cent is indicative of severely impaired liver function. Blood Ammonia Concentration
The deamination of amino acids by the liver and bacterial deamination of ingested amino acids within the intestine followed by absorption in portal circulation result in an ammonia load on the liver. Normally, the ammonia thus produced and absorbed enters the urea cycle in the liver, the only site of urea formation. If the functional hepatic mass is severely reduced, ammonia may not be converted to urea. As a consequence, the blood urea nitrogen (BUN) concentration may be low and blood ammonia concentration may rise. Impending hepatic encephalopathy in liver failure may be anticipated by documentation of elevated blood ammonia concentration in spite of the fact that ammonia concentration does not correlate precisely with hepatic encephalopathy. Prothrombin Time
The one stage prothrombin time is often greater than normal when the functional hepatic mass is severely reduced. Because the clotting factors assessed by prothrombin time determinations have a much shorter half-life than does serum albumin, increased prothrombin time is often present in acute hepatic failure where serum albumin concentration is normal. Although prothrombin can be used to assess hepatic function, its chief usefulness is in prebiopsy evaluation of the patient with documented liver disease. Determination of the clotting time, however, is a more practical method of prebiopsy coagulation evaluation.
686
WILLIAM
D.
SCHALL
Analysis of Ascitic Fluid The analysis of ascitic fluid is discussed in detail elsewhere in this symposium. Ascitic fluid associated with chronic reduced functional hepatic mass and associated decreased colloidal osmotic pressure is characteristically a pure transudate.
Summary of Liver Tests A summar)C- of liver tests is outlined below. It should be noted that tests which assess liver cell injury do not yield information regarding the functional state of the liver and may be normal when liver failure ts present. Cell Injury Enzyme Leakage
}
Increased Transaminase (SGPT and SGOT) and SDH
Cholestasis
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SAP Bilirubin ESP
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Albumin Ammonium ESP
Reduced Functional Mass
REFERENCES l. Burton, C., and Schenker, S.: Laboratory tests. In Schiff, L. (ed.): Diseases of the
Liver. Philadelphia,]. B. Lippincott Co., 1969. 2. Cornelius, C. E., and Himes, ]. A.: New concepts in canine hepatic function. J.A.A.H.A., 9:147-150, 1973. 3. Everett, R. M.: Personal communication, 1976. 4. Kaplan, M. M.: Current concepts. Alkaline phosphatase. New Eng.]. Med., 286:200202, 1972. . 5. Moore, W. E., and Feldman, B. F.: The use of isoenzymes in small animal medicine. J.A.A.H.A., 10:420-429, 1974. 6. Moss, D. W., and Butterworth, P. ].: Enzymology and Medicine. London, Pitman Medical, 1974. 7. Van Vleet,]. F., and Alberts,]. 0.: Evaluation ofliver function tests and liver biopsy in experimental carbon tetrachloride intoxication and extrahepatic bile duct obstruction in the dog. Am.]. Vet. Res., 29:2119-2131, 1968. 8. Young,]. T.: Source, fate, and possible significance of elevated serum alkaline phosphatase in nonicteric animals with partial biliary obstruction. J.A.A.H.A., 10:415419, 1974. 9. Zimmerman, H.].: Liver disease caused by medicinal agents. Med. Clin. North. Am., 59:897-907, 1975. Department of Small Animal Medicine and Surgery College of Veterinary Medicine University of Georgia Athens, Georgia 30602
