Dennis J. Meyer1 and Raquel M. Walton2 1 Charles River Laboratories, Reno, NV, USA 2 IDEXX Laboratories, Inc., Langhorne, PA, USA While the diagnostic and prognostic gold standard for hepatic disease is histological evaluation, biopsy is an invasive procedure. Diagnosis of hepatic disease can be achieved with noninvasive procedures such as evaluation of serum biochemical data in conjunction with clinical findings, history, and ultrasound examination. Clinical signs of hepatic disease in horses can include dullness, anorexia, abdominal pain, encephalopathy, weight loss, jaundice, diarrhea, photosensitization, and coagulopathy [1]. In one study, the diagnostic specificity of clinical signs for the presence of hepatic disease was good (81%) but sensitivity was poor (28%), while the most useful noninvasive prognostic test in mature horses with suspected liver disease was the severity of clinical signs [2, 3]. Certain biochemical tests have better sensitivity than clinical signs in predicting the presence of liver disease when they are abnormal, but normal test results do not preclude the presence of liver disease. Liver enzymes are generally categorized as hepatocellular and hepatobiliary for diagnostic purposes. The hepatocellular enzymes are aspartate aminotransferase (AST), sorbitol dehydrogenase (SDH), and glutamate dehydrogenase (GLDH). Alanine aminotransferase (ALT), a hepatocellular enzyme that is identified as “liver specific” in humans, dogs, and cats, has limited activity in the horse liver and therefore no correlative diagnostic utility. Alkaline phosphatase (ALP) and gamma‐glutamyltransferase (GGT) constitute the hepatobiliary enzymes. In contrast to the use of ALP in humans, dogs, and cats, GGT is considered the preferable hepatobiliary enzyme in the horse. The location of these enzymes is illustrated in Figure 5.1. Patterns of enzyme changes are associated with different types of liver pathology. An acute, severe, predominantly hepatocellular injury is initially associated with high activity of hepatocellular enzymes with increased values for GGT gradually developing over days and even persisting after the hepatocellular enzymes have returned within the reference interval after complete recovery. Persistent injury is associated with minimal to mild changes in both hepatocellular and hepatobiliary enzymes and requires other diagnostics to define the causation of their persistent increase. A disorder that causes predominantly cholestasis is associated with increases in GGT with low elevations for the hepatocellular enzymes developing over days to weeks. Sufficiently severe liver pathology, acute or chronic and persistent, is likely to alter one or more tests of liver function. The hepatocellular enzymes are preformed and located in high activity within the hepatocyte. Altered membrane permeability, generally some type of injury, permits increased leakage into the circulation; the magnitude of increase generally correlates with the number of affected hepatocytes when the pathology is acute and severe. The time to return to reference values following complete recovery is dependent on their respective plasma half‐life; the half‐life of SDH is shorter than that of AST and GLDH (Figure 5.2). Incomplete return to reference values after several weeks of repeated testing suggests persistent pathology, referred to as chronic persistent (active) hepatitis. Figure 5.1 The cells and the structures they form along with their constituent enzymes are illustrated. The hepatocellular enzymes, AST, SDH, and GLD, are preformed and located within the hepatocyte. Altered integrity of the hepatocyte membrane, generally due to some type of injury, allows increased “leakage” of these enzymes into the circulation as a reflection of the underlying pathology. The hepatobiliary enzymes, ALP and GGT, are associated with the bile canaliculi and biliary epithelium. Minimal activity is preformed and a stimulus is required to increase their production. An increase in their synthesis and release into the circulation is generally due to impairment of bile flow (cholestasis). Bile acids and bilirubin are eliminated via bile. An increase in their concentrations in the circulation occurs if the cause of the cholestasis is acute and severe (attendant high activities of SDH, GLD, and AST) or chronic and diffuse (high activities of ALP and/or GGT). An increase in bile acids generally precedes an increase in bilirubin. AST, aspartate aminotransferase; GLD, glutamate dehydrogenase; SDH, sorbitol dehydrogenase; ALP, alkaline phosphatase; GGT, gamma‐glutamyltransferase; BA, bile acids; BR, total bilirubin. Source: Modified from Meyer and Harvey [4]. Figure 5.2 The approximate magnitude and duration of SDH, AST, and GLD increases in the circulation following severe hepatic injury with recovery are illustrated. Persistent increases for several weeks in one or more of these enzymes with repeat testing, especially if accompanied by an increase in hepatobiliary enzymes, are suggestive of persistent, progressive (chronic) liver pathology. AST, aspartate aminotransferase; SDH, sorbitol dehydrogenase; GLD, glutamate dehydrogenase. Source: Modified from Meyer and Harvey [4]. Sorbitol dehydrogenase is considered a liver‐specific enzyme, present in high concentrations in the equine liver but in low concentrations in other tissues. The enzyme is free within hepatocyte cytoplasm and is more specific than AST or GLDH in detecting hepatocellular damage. The half‐life is less than 12 hours, so serum concentrations can return to reference values within days after a single insult. The in vitro stability is much less than other diagnostic enzymes, which can limit the utility of the test; it should be performed on serum within five hours if stored at room temperature, and within 48 hours if frozen [5]. While SDH is very specific for liver disease, its sensitivity is lower than either GLDH or AST for detection of hepatic lipidosis, cirrhosis, and necrosis in horses [6]. Similar to SDH, GLDH is a liver‐specific enzyme present in high concentrations in hepatocytes but in low concentrations in other tissues. It is located primarily in the central lobular region. In contrast to SDH, it is mitochondrial rather than cytosolic, and is therefore typically released only with irreversible cell injury. The half‐life of GLDH in the horse is about 14 hours, slightly longer than for SDH but shorter than AST. The in vitro stability of enzyme activity is considered greater than SDH. The activity of bovine GLDH is reported to be stable for a month at −20 °C [7]. The sensitivity of GLDH for diagnosis of hepatic disease in mature horses is reported to be 63%, which exceeds the reported sensitivity of SDH [2]. As a liver‐specific enzyme with greater stability than SDH, GLDH is a valuable tool for the diagnosis of acute liver injury in horses but the limited availability of the assay in the US restricts its use in practice. Aspartate aminotransferase is present in high concentrations in hepatocytes and myocytes (see Chapter 10). Creatine kinase (CK) has high activity in skeletal muscle but is not present in the liver. An enzyme profile of marked increases in AST and CK (without increases in SDH and GLDH) is indicative of primarily skeletal muscle injury. Erythrocytes also contain AST so the presence of hemolysis in samples will increase AST activity. The enzyme is present in both the cytosol and mitochondria of hepatocytes, but in higher concentrations in the mitochondria. In theory, higher magnitude increases in AST would be expected with irreversible than reversible hepatocyte (or myocyte) injury, but this has not been proven. In vitro stability at room temperature or refrigerated is days rather than hours, in contrast to SDH or GLDH. Marked increases in serum AST and SDH or GLDH suggest acute or active hepatocellular injury, whereas marked increases in serum AST with mild to moderate increases in SDH or GLDH suggest chronic hepatic injury or recovery from acute liver injury (Figure 5.2). In one study, the diagnostic sensitivity of AST was determined to be 100% for hepatic lipidosis and 72% for hepatic necrosis in horses [6]. The hepatobiliary enzymes require a stimulus for increased production with a resultant increase in the circulation. Retained bile is generally considered the primary stimulant for increased production by the liver, with GGT having relatively greater responsiveness and magnitude of production compared to ALP in the horse. Both GGT and ALP, when increased, have high diagnostic value for the presence of cholestatic liver disease. Alkaline phosphatase is present in multiple tissues, but serum ALP activity does not necessarily correlate with tissue concentrations. ALP is present in relatively low concentrations in liver and yet liver ALP is found in serum, whereas intestinal ALP, present in high tissue concentrations in horses, is not. In hepatocytes, the majority of ALP is bound to bile canalicular surface membranes. In health, serum ALP originates primarily from liver and bone and is therefore higher in growing horses. In foals, serum ALP may be l00‐fold greater than in adults due to bone ALP. In mature horses, roughly 80% of serum ALP is liver ALP and 20% is bone ALP. The ALP isoforms can be distinguished on the basis of differences in heat stability, electrophoretic migration, and wheat germ lectin precipitation. The bone isoform is more susceptible to wheat germ lectin precipitation than the liver, has slower anodal electrophoretic migration, and is more sensitive to heat inhibition. Bone ALP can be directly measured using commercially available antibodies for human bone ALP that have been validated for use in the horse [8]. Often, evaluation of ALP in conjunction with another biliary‐specific enzyme such as GGT is sufficient in determining the origin and significance of increased serum ALP concentrations. The most common cause of increased serum ALP concentrations is cholestasis. Cholestasis induces the synthesis of ALP, which concentrates on the basolateral hepatocyte membrane where it is released into blood or lymphatics [9]. The highest concentrations of serum ALP are associated with cholangitis, biliary cirrhosis, or extrahepatic bile duct obstruction. Gamma‐glutamyltransferase (also known as gamma‐glutamyltranspeptidase) is a membrane‐bound enzyme that is primarily associated with biliary epithelial cells in horses, like other domestic animals. Most serum GGT activity originates from the liver, although it is present in high concentrations in the kidney, intestine, and pancreas. This is attributable to its presence on the luminal surfaces in these organs. Serum half‐life of GGT in horses is thought to be around three days, similar to ALP [9]. Increases in GGT are associated with cholestasis and biliary hyperplasia. Serum GGT concentrations are more sensitive than ALP in the diagnosis of cholestatic diseases; in horses with cholestasis, GGT increased ninefold whereas ALP activity only increased twofold [9]. GGT activity is high in colostrum and increased serum GGT concentrations are present in calves and goats during suckling, but not in foals. There is no significant difference in GGT concentrations before and after suckling in foals, but GGT concentrations are higher in foals relative to adults [10]. Total bilirubin has two forms: unconjugated and conjugated. The primary source of bilirubin is from the degradation of hemoglobin from aged erythrocytes, termed unconjugated (indirect‐reacting) bilirubin. Unconjugated bilirubin is transported to the liver via albumin for receptor‐mediated uptake by hepatocytes, internalized, and altered to form conjugated (direct‐reacting) bilirubin, and actively secreted by the specialized portion of the hepatocyte membrane (canalicular membrane) into bile for delivery to the small intestine (Figure 5.3). A relatively unique feature of bilirubin removal by the horse occurs at the site of uptake by the hepatocyte. Lack of food intake is commonly associated with moderate to marked increases in total bilirubin due to increases in the unconjugated form. It is likely that free fatty acids and other metabolic constituents that are increased in the fasted state compete for uptake of unconjugated bilirubin; it decreases once appetite returns. Figure 5.3 Bilirubin metabolism. Unconjugated bilirubin is released into the plasma from macrophages after the breakdown of hemoglobin to heme, which is further degraded into iron and bilirubin. Unconjugated bilirubin is bound by albumin and transported to the liver where it is conjugated to glucuronic acid. Conjugated bilirubin is excreted into bile via biliary canaliculi and then into the intestinal tract, where it is reduced to urobilinogen or stercobilinogen by the action of bacteria. About 10% of urobilinogen returns to the liver via the portal system and the majority is excreted into bile; a small proportion (1–5%) enters the circulation to be excreted via the kidneys. In cases of biliary obstruction or impaired bilirubin excretion due to hepatocellular dysfunction, conjugated bilirubin will efflux into the plasma (dotted line). Another cause of increased unconjugated bilirubin is acute, severe hemolysis. Concomitant moderate to marked decreases in circulating red cell mass as measured by the hematocrit are supportive of a hemolytic process. Intrahepatic diseases and extrahepatic obstruction can both cause increases in total bilirubin, with conjugated bilirubin comprising less than half of the total bilirubin. The circulating concentrations of bile acids are dependent on an intact enterohepatic circulation. Bile acids are efficiently removed from the intestines into the portal circulation, transported to the liver, efficiently extracted from the blood by receptor‐mediated uptake by the hepatocyte, processed by the hepatocyte, and actively excreted by the canalicular membrane into bile for delivery to the intestine where the process is repeated (Figure 5.1). Serum bile acid concentrations in foals are significantly higher than in adult horses in the first six weeks of life and age‐specific reference values should be used in interpreting results [11]. Bile acids increase in the circulation before an increase in total bilirubin due to liver disease. In contrast to total bilirubin, serum bile acid concentrations in the horse are not affected by short‐term fasting (<3 days) [6, 9]. Postprandial increases are also not observed given the lack of gallbladder in equids. The measurement of bile acids is a diagnostic adjunct when abnormal liver enzyme tests are detected; a concomitant increase in bile acids suggests substantial liver pathology precluding their removal from the sinusoidal blood and supports further diagnostics such as liver biopsy. Congenital abnormalities of the portal circulation, often manifested as encephalopathy, cause bile acids to by‐pass the liver, resulting in increases in the circulation [12]. Albumin is produced exclusively by hepatocytes. The formation of albumin is relatively well preserved and decreases are not significant until there is substantial loss of hepatocellular mass due to disease (>60–80% of functional mass). Moreover, the half‐life of albumin in horses is longer than in other species (19.4 days). Hypoalbuminemia is an infrequent finding with severe acute or chronic liver disease, occurring in only 13–16% of cases in two studies [13, 14]. Hypoalbuminemia occurred more frequently in chronic than acute liver disease.
5
The Liver
5.1 Liver Enzymes
5.1.1 Hepatocellular Enzymes
5.1.1.1 Sorbitol Dehydrogenase
5.1.1.2 Glutamate Dehydrogenase
5.1.1.3 Aspartate Aminotransferase
5.1.2 Hepatobiliary Enzymes
5.1.2.1 Alkaline Phosphatase
5.1.2.2 Gamma‐Glutamyltransferase
5.2 Liver Function Tests
5.2.1 Excretory Function Tests
5.2.1.1 Total Bilirubin
5.2.1.2 Bile Acids
5.2.2 Tests Dependent on Synthetic/Metabolic Functions
5.2.2.1 Albumin
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