Liver disease is often first suspected based on increased liver enzyme activity. However, abnormally increased liver enzyme activity is considerably more common than the prevalence of liver disease. A wide spectrum of nonhepatic disorders may influence liver enzyme activity. It is important to recognize that liver enzyme measurements are not liver function tests but rather reflect hepatocyte membrane integrity, hepatocyte or biliary epithelial necrosis, cholestasis, or induction phenomenon.
The pattern of liver enzyme abnormalities in relation to the signalment, history, total bilirubin concentration, serum bile acid values, and comorbid conditions/medications provides the first indication of a liver-specific disorder. A full assessment of liver enzyme aberration considers: 1) the predominant pattern of enzyme change (hepatocellular leakage enzymes vs cholestatic enzymes), 2) the magnitude of increase of enzyme activity above the normal reference range (mild is <3 times the upper reference range, moderate is 3–9 times, marked is >10 times), 3) the rate of change (increase or resolution) with sequential sampling, and 4) the nature of the course of change (fluctuation vs progressive increase or decrement). Up to 2.5% of clinically “normal” animals can have borderline abnormal enzyme values.
Recognizing whether enzyme abnormalities are persistent or cyclic helps categorize likely causes. Investigating liver function with paired fasting and postprandial total serum bile acids (TSBAs) or urine bile acid/creatinine measurements (urine collected 4–8 hr after meal ingestion) may expedite a decision to pursue liver biopsy when clinical signs remain vague and enzymes are only mildly increased. Imaging studies help detect primary underlying disorders that have secondarily influenced the liver, causing increased enzyme activity. Ultrasonographic assessment may help determine the method of liver biopsy; needle biopsies are ill advised in animals with microhepatica, ascites, or difficult-to-sample focal liver lesions.
Age-appropriate reference ranges for serum liver enzyme activity are essential to interpret laboratory values in puppies and kittens. Plasma enzyme activities of ALP and GGT in neonatal dogs and cats are remarkably higher than those of adults. Differences reflect physiologic adaptations during the transition from fetal and neonatal life stages, colostrum ingestion, maturation of metabolic pathways, growth effects, differences in volume of distribution and body composition, and nutritional intake. Serum activities of ALP, AST, CK, and LDH in neonates usually increase greatly during the first 24 hr of life. In kittens, serum activities of ALP, CK, and LDH exceed adult values through 8 wk of age. Serum ALP increases remarkably in day-old puppies and kittens after colostrum ingestion, as also observed in neonatal calves, lambs, pigs, and foals.
AST and ALT are commonly measured to detect liver injury; however, both enzymes are present in high concentrations in liver and several other tissues. AST activity is higher in kidney, heart, and skeletal muscle than liver, whereas ALT activity is highest in liver. Because hepatic ALT activity is 10,000-fold greater than plasma enzyme activity in healthy animals, it has high diagnostic utility to detect “liver lesions.” The cytosolic location of transaminases allows their immediate release with even minor change in hepatocellular membrane integrity. Unfortunately, indiscriminate leakage limits their diagnostic utility. Nonetheless, duration and magnitude of transaminase activities measured sequentially can predict disease activity and severity and roughly estimate the number of involved cells.
Hepatic transaminases increase with muscle injury as well as vigorous physical activity in dogs. Persistence of transaminases in plasma contributes to their sustained high activities in certain disorders. Because transaminase catabolism occurs by absorptive endocytosis at the hepatocyte sinusoidal border, slow enzyme clearance may sustain plasma enzyme activity in hepatic insufficiency associated with liver fibrosis, nodular regeneration, and development of APSSs.
The largest increases in ALT develop with hepatocellular necrosis and inflammation. After acute severe hepatocyte necrosis, serum ALT activity increases sharply within 24–48 hr to values often >100-fold normal, peaking during the first 5 days of injury. If the injurious event resolves, ALT activity gradually declines to normal over 2–3 wk. Although this pattern is considered classic, some severe hepatotoxins are not associated with increased ALT activity, because they inhibit gene transcription or interfere with ALT biosynthesis (eg, aflatoxin B1 hepatotoxicity, microcystin hepatotoxicity). A declining ALT also may represent a paucity of viable hepatocytes in end-stage chronic hepatitis or severe acute liver disease.
Examples of classic necrotizing hepatotoxins are carbon tetrachloride, acetaminophen, and nitrosamine. A single exposure to carbon tetrachloride causes an acute sharp increase in ALT that resolves over the ensuing week. Hepatotoxicity induced by acetaminophen causes a marked increase in ALT and AST within 24 hr that may decline within 72 hr to near normal values. This toxin is highly dose dependent in dogs and cats. Cats are exceedingly susceptible, with hematologic signs dominating after ingestion of as little as 125 mg. However, in dogs, a dosage of 200 mg/kg may be life-threatening, with susceptibility heightened by antecedent exposure to phenobarbital. Hepatocellular necrosis induced by nitrosamines increases plasma ALT activity, but not significantly, until after 1 wk of intermittent chronic exposure. The ALT activity persists for weeks until necrosis resolves. Low-grade hepatocellular degeneration, observed in some dogs with congenital portosystemic shunts, reflects delayed enzyme clearance and low-grade hepatocyte dropout; most of these dogs have small lipogranulomas reflecting single hepatocyte dropout/necrosis in the absence of an inflammatory response.
Acute hepatic necrosis caused by infectious canine hepatitis increases plasma ALT activity by 30-fold, peaking within 4 days. Thereafter, chronic sustained ALT activity persists as chronic hepatitis develops in dogs unable to clear the virus. Hepatic injury induced by toxins usually causes plasma ALT activity to increase, peak, and return to normal sooner than it does in infectious viral hepatitis. Chronic hepatitis, an idiopathic or copper-associated persistent or cyclic necroinflammatory liver injury in dogs is associated with varying severities of necrosis and fibrosis. Cyclic disease activity is reflected by plasma enzyme “flares.” At times, plasma ALT activity is >10-fold normal. Enzyme fluctuations contrast with profiles associated with single injurious events. In dogs with hepatitis, serum ALT activity declines as injury resolves, but serum ALP activity may increase as a result of regenerative responses (progenitor cell proliferation, ductal or oval cell response). Dogs treated with glucocorticoids may develop mildly increased ALT activity that resolves within several weeks of glucocorticoid withdrawal.
Despite high sensitivity of ALT to identify liver disorders, its lack of specificity to differentiate clinically significant liver disease, specific histologic abnormalities, or hepatic dysfunction requires that it be interpreted in conjunction with other diagnostic tests.
AST is present in substantial concentrations in a wide variety of tissues, especially muscle. Increased AST activity can reflect reversible or irreversible changes in hepatocellular membrane permeability, cell necrosis, hepatic inflammation, and in dogs, microsomal enzyme induction. After acute diffuse severe hepatic necrosis, serum AST sharply increases during the first 3 days to values 10- to 30-fold above normal in dogs and up to 50-fold above normal in cats. If necrosis resolves, AST activity gradually declines over 2–3 wk. In most cases, AST parallels changes in ALT activity.
Although increased AST activity in the absence of abnormal ALT activity implicates an extrahepatic enzyme source (notably in muscle injury), there are clinical exceptions that may relate to severity and zonal location of hepatic damage. In some cats with liver disease, AST is a more sensitive marker of liver injury than ALT (eg, hepatic necrosis, cholangiohepatitis, myeloproliferative disease, hepatic infiltrative lymphoma, and EHBDO). A similar trend is evident in some dogs. Because AST is located within the mitochondria and free within the cytosol of hepatocytes, AST in fold increases greater than those of ALT may reflect mitochrondrial injury. Dogs treated with glucocorticoids may develop mildly increased AST activity that resolves within several weeks of glucocorticoid withdrawal.
Increased ALP activity in dogs is the most common abnormality on routine biochemical testing; its high sensitivity and low specificity can defy diagnostic interpretation without a liver biopsy. ALP activity in dogs has the lowest specificity of routinely used liver enzymes as a result of its complexity associated with induction of different isozymes.
In dogs and cats, tissues containing highest ALP activity (in descending order) are intestine, kidney (cortex), placenta (dogs only), liver, and bone. Distinct serum ALP isozymes can be extracted from some of these tissues in each species; eg, bone (B-ALP), liver (L-ALP), and glucocorticoid-induced (G-ALP) isoenzymes in canine serum. In dogs, L-ALP and G-ALP are primarily responsible for high serum ALP activity, whereas L-ALP is primarily responsible in cats. Increased ALP activity develops in up to 75% of hyperthyroid cats, depending on the chronicity of the condition, with B-ALP substantially contributing.
The comparably small magnitudes of ALP activity in cats with liver disease (2- to 3-fold normal) relative to dogs (usually >4- to 5-fold) reflect the lower specific activity of ALP in feline liver and its shorter half-life. Nevertheless, ALP activity remains clinically useful in the diagnosis of feline liver disease when the species-appropriate perspective is maintained.
The utility of serum ALP activity as a diagnostic indicator in dogs is complicated by the common accumulation of L-ALP and G-ALP isozymes, which can both be induced by steroidogenic hormones.
Because the B-ALP isozyme increases secondary to osteoblast activity, it is detected in young growing animals and in animals with bone tumors, secondary renal hyperparathyroidism, and osteomyelitis. However, the minor contribution of B-ALP to total serum ALP activity usually does not lead to an erroneous diagnosis of cholestatic liver disease. Bone remodeling secondary to neoplasia may not substantially affect serum ALP activity or may cause only a trivial increase (2- to 3-fold) in dogs. In young growing cats, increased B-ALP activity may simulate enzyme activity seen in hepatobiliary disease.
Although ALT is immediately released from the hepatocellular cytosol in acute hepatic necrosis, the small quantities of membrane-bound ALP are not. It takes several days for induction of membrane-associated enzyme to “gear up” and spill into the systemic circulation. Increased serum ALP reflects enhanced de novo hepatic synthesis, canalicular injury, cholestasis, and solubilization of its membrane anchor (by bile salts). The largest increases in serum ALP activity (L-ALP and/or G-ALP ≥100-fold normal) develop in dogs with diffuse or focal cholestatic disorders, massive hepatocellular carcinoma, bile duct carcinoma, and those exposed to steroidogenic hormones.
Although serum activity of ALP may be normal or only modestly increased in dogs with metastatic neoplasia involving the liver, it may also increase dramatically in dogs with mammary neoplasia. High serum ALP activity develops in ~55% of dogs with malignant and 47% with benign mammary tumors, with highest ALP activity seen in dogs with malignant mixed tumors. Nevertheless, serum ALP has no value as a diagnostic or prognostic marker in mammary cancer; it remains unclear whether disease remission (surgical, chemotherapy) is followed by a regression in serum ALP activity or whether serum ALP activity functions as a paraneoplastic marker.
After acute severe hepatic necrosis, ALP activity increases 2- to 5-fold in dogs and cats, stabilizes, and then gradually declines over 2–3 wk. Sustained ALP activity usually correlates with a reparative ductal response (progenitor or oval cell hyperplasia). In cats, EHBDO results in a 2-fold increase in ALP within 2 days, as much as a 4-fold increase within 1 wk, and up to a 9-fold increase within 2–3 wk. Thereafter, activity stabilizes and gradually declines but usually not into the normal range; the declining enzyme activity coordinates with developing biliary cirrhosis (see Extrahepatic Bile Duct Obstruction in Small Animals). Inflammatory disorders involving biliary or canalicular structures or disorders compromising bile flow increase serum ALP activity secondary to membrane inflammation/disruption and local bile acid accumulation. In both dogs and cats, similar increases in serum ALP activity develop in intrahepatic (metabolic, biochemical, sepsis) associated cholestasis or obstruction involving the extrahepatic biliary structures. Consequently, ALP activity cannot differentiate between intra- and extrahepatic cholestatic disorders.
Many extrahepatic and primary hepatic conditions are associated with increased L-ALP. In cats, HL (see Feline Hepatic Lipidosis) is associated with marked increase in ALP activity and jaundice. The increased ALP seemingly reflects canalicular dysfunction or compression. Although ALP in cats is rarely affected by anticonvulsants or glucocorticoids, it can increase with diabetes mellitus, hyperthyroidism, and pancreatitis.
In dogs, primary hepatic inflammation as well as systemic infection or inflammation and exposure to steroidogenic hormones may induce a glycogen-associated vacuolar hepatopathy (VH). When severe, VH has a cholestatic effect that seemingly causes canalicular compression. Although glycogen-associated VH was initially characterized as a glucocorticoid-initiated lesion, it is now established that nearly 50% of dogs with glycogen-associated VH lack overt exposure to steroidogenic substances. Chronically ill dogs may produce the G-ALP isozymes secondary to stress-induced glucocorticoid release. Such dogs with glycogen-associated VH (lacking exogenous glucocorticoid exposure) may demonstrate normal dexamethasone suppression and adrenocorticotropic hormone (ACTH) response tests. However, in some dogs, high ALP with a glycogen-associated VH signals the presence of atypical adrenal hyperplasia associated with abnormal sex hormone production. There is no consistent relationship between the magnitude of serum ALP activity, the presence of high G-ALP activity, or histologic lesions. Unfortunately, G-ALP is not useful for syndrome characterization because it can become the predominant ALP isoenzyme in dogs treated with glucocorticoids and in dogs with spontaneous or iatrogenic hyperadrenocorticism, hepatic or nonhepatic neoplasia, hepatic inflammation, or numerous diverse chronic illnesses, including primary liver disease.
The magnitude of ALP activity induced by glucocorticoid administration depends on the type of drug and dose given, as well as the individual’s response. The production of G-ALP does not imply that a dog treated with cortisone has iatrogenic hyperadrenocorticism, a suppressed pituitary-adrenal axis, or a clinically important glycogen-associated VH. By comparison, the feline liver is relatively insensitive to glucocorticoids, with rare development of a glycogen-associated VH or acceleration of hepatocyte lipid vacuole accumulation.
In dogs, serum total ALP activity and L-ALP isozyme also may be induced by administration of certain anticonvulsants (phenobarbital, primidone, and phenytoin) and other drugs; in this circumstance, the ALP activity usually increases 2- to 6-fold normal. In contrast, serum ALP and L-ALP did not increase in cats after administration of phenobarbital (0.25 grain, bid) for 30 days.
Gamma-glutamyl transferase (GGT) is a membrane-bound glycoprotein that plays a critical role in cellular detoxification (involved with glutathione availability), conferring resistance against a number of toxins and drugs. Tissue concentrations of GGT in dogs and cats are highest in the kidney and pancreas, with lesser amounts in the liver, gallbladder, intestines, spleen, heart, lungs, skeletal muscle, and erythrocytes. However, serum GGT activity is largely derived from the liver, although there is considerable species variation in its localization within this organ.
Acute, severe, diffuse necrosis is associated with either no change or only mild increases (1- to 3-fold normal) in GGT activity that resolve in ~10 days. In dogs with EHBDO, serum GGT activity increases 1- to 4-fold above normal within 4 days, and 10- to 50-fold within 1–2 wk. Thereafter, values may plateau or continue to increase as high as 100-fold. In cats with EHBDO, serum GGT activity may increase up to 2-fold within 3 days, 2- to 6-fold within 5 days, 3- to 12-fold within 1 wk, and 4- to 16-fold within 2 wk. Glucocorticoids and certain other microsomal enzyme inducers may stimulate GGT production in dogs, similar to their influence on ALP. Administration of dexamethasone (3 mg/kg/day) or prednisone (4.4 mg/kg/day, IM) may increase GGT activity within 1 wk to 4- to 7-fold above normal and up to 10-fold within 2 wk. Dogs treated with phenytoin or primidone develop only a modest increase in serum GGT activity (up to 2- to 3-fold), unless they develop anticonvulsant hepatotoxicosis that is often associated with marked enzyme activity.
Cats with advanced necroinflammatory liver disease, EHBDO, or inflammatory intrahepatic cholestasis can develop a larger increase in GGT activity relative to ALP. Glucocorticoids and other enzyme inducers in dogs do not clinically influence serum GGT in cats. The normal range for serum GGT activity in cats is much narrower and lower than that in dogs; therefore, assays must be sensitive enough to detect low GGT activity.
GGT values can be markedly increased in dogs and cats with primary hepatic or pancreatic neoplasia. However, GGT does not appear to be suitable for surveillance of hepatic metastasis in either species.
Like ALP, GGT lacks specificity in differentiating between parenchymal hepatic disease and obstructive biliary disease. It is not as sensitive in dogs as ALP but does have higher specificity. In cats with inflammatory liver disease, it is more sensitive but less specific than ALP; these two enzymes should be interpreted simultaneously. The likelihood that HL has developed secondary to necroinflammatory liver disease, EHBDO, or pancreatic disease can be predicted by examining the relative increases in GGT and ALP. Necroinflammatory disorders involving biliary structures, the portal triad, or pancreas are often associated with a greater fold increase in GGT than in ALP. With the exclusion of these underlying disorders, cats with HL usually have a higher fold increase in ALP relative to GGT; this has important diagnostic utility in discerning the underlying cause of HL.
Neonatal animals of several species, including dogs but not cats, develop high serum GGT activity secondary to colostrum ingestion.