Liver disease is often first suspected on the basis of increased liver enzyme activity on health screening profiles. However, abnormally increased liver enzyme activity exceeds the prevalence of liver disease because a wide spectrum of nonhepatic disorders may influence liver enzyme activity.
Liver enzyme measurements are not liver function tests but rather reflect hepatocyte membrane integrity, hepatocyte or biliary epithelial necrosis, cholestasis, or induction phenomenon. Liver enzyme activity also may be attenuated secondary to critical loss of functional hepatic parenchyma. Up to 2.5% of clinically normal animals have borderline abnormal liver enzyme activities.
Recognizing whether enzyme abnormalities are persistent or cyclic helps categorize likely causes. The pattern of liver enzyme abnormalities must be considered in relation to patient signalment, history, total bilirubin concentration, serum bile acid concentrations, comorbid conditions, and medications.
Assessment of liver enzyme activity should consider the following:
predominant pattern of enzyme change (hepatocellular leakage enzymes vs cholestatic enzymes)
magnitude of increase in enzyme activity relative to the reference range (mild, < 3 times; moderate, 3–9 times; marked, > 10 times)
rate of change (increase or resolution) with sequential sample assessments
nature of the course of change (fluctuation vs progressive increase or decrement)
Serum liver enzyme activity usually reflects hepatocyte death corresponding to features evident on histologic evaluation. However, disease processes in which hepatocyte death is orchestrated by apoptotic pathways may not show corresponding enzyme activity.
Toxic phenomena impairing hepatocyte synthetic capabilities blunt enzyme activity. This can occur with specific toxins such as microcystin (blue-green algae hepatotoxicosis) and aflatoxicosis as well as in chronic late-stage liver injury when there is noteworthy decrease in viable hepatic mass. Some toxicoses causing fulminant hepatic failure lead to death before escalating liver enzymes can be documented.
Investigating liver function by measuring paired pre- and postprandial total serum bile acid (TSBA) concentrations may expedite the decision to pursue liver biopsy in patients with vague clinical signs and vacillating enzyme activity. The shortcoming of this approach is that numerous small-breed dogs with microvascular dysplasia have increased TSBA concentrations that do not warrant liver biopsy. Imaging studies help detect primary underlying disorders secondarily affecting the liver and causing increased enzyme activity. Ultrasonographic imaging may help determine utility of liver biopsy methods, needle biopsies being ill advised in animals with microhepatia, ascites, or difficult-to-sample focal liver lesions.
Age-appropriate reference ranges for serum liver enzyme activity are essential for interpretation of laboratory values in puppies and kittens. Plasma enzyme activities of alkaline phosphatase (ALP) and gamma-glutamyl transferase (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 hours after birth. In kittens, serum activities of ALP, CK, and LDH exceed adult values through 8 weeks. Serum ALP activity increases remarkably in day-old puppies and kittens after colostrum ingestion, as also observed in neonatal calves, lambs, pigs, and foals.
Aminotransferases in Enzyme Activity in Hepatic Disease
AST and ALT are aminotransferases commonly used to infer liver injury in questions of hepatic disease. 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 injury or lesions.
The cytosolic location of transaminases allows their immediate release with even minor alteration in hepatocyte cell membrane integrity. Unfortunately, indiscriminate leakage limits the specificity of their diagnostic utility. Nonetheless, duration and magnitude of serum transaminase activity measured sequentially can predict disease activity and severity and roughly estimate the magnitude of hepatocyte involvement.
Hepatic transaminase activity also increases 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 circulating enzyme activity in patients with hepatic insufficiency (ie, those with substantial loss of functional hepatic parenchyma, regenerative nodules, dissecting fibrosis, and acquired portosystemic shunts).
The greatest increase in circulating ALT activity develops with hepatocellular necrosis and inflammation. After acute severe hepatocyte necrosis, serum ALT activity increases sharply within 24–48 hours to values often > 100-fold above normal, peaking during the first 5 days of injury. If the injurious event resolves, ALT activity gradually declines to normal over 2–3 weeks.
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). However, with many acute severe injuries, a declining ALT also may reflect an acquired paucity of viable hepatocytes at end-stage status.
Examples of classic necrotizing hepatotoxins are carbon tetrachloride, nitrosamine, and repeated acetaminophen exposure. Single exposure to carbon tetrachloride causes an acute sharp increase in ALT that resolves over the ensuing week. Hepatocellular necrosis induced by nitrosamines increases plasma ALT activity, but not notably until 1 week of intermittent chronic exposure. Increased ALT activity thereafter persists for weeks in the absence of recurrent exposure until necrosis resolves.
Hepatotoxicity induced by acetaminophen causes a marked increase in ALT and AST within 24 hours that may decline within 72 hours to near-normal values. This toxin is highly dose dependent in dogs and cats, with cats exceedingly susceptible to hematologic toxicity (methemoglobinemia, severe hemolysis) after ingesting as little as 125 mg. However, in dogs, ingesting 200 mg/kg may provoke lethal liver failure, with susceptibility heightened by antecedent exposure to P450 enzyme-inducing drugs (eg, phenobarbital) and repeated dose administration.
Low-grade hepatocellular degeneration, observed in some dogs with congenital portosystemic shunts, reflects delayed enzyme clearance and slow hepatocyte dropout. Many of these dogs develop small lipogranulomas, presumably reflecting hepatocyte injury associated with altered sinusoidal perfusion and inflammatory foci that may reflect impaired Kupffer cell scavenging of enteric-derived toxins and debris.
Acute hepatic necrosis due to 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 above normal.
Enzyme fluctuations contrast with sequential enzyme profiles associated with single injurious events. In dogs with chronic hepatitis, serum ALT activity declines as injury resolves; however, serum ALP activity may increase as a result of regenerative responses associated with progenitor cell proliferation (ductural reaction). Dogs treated with glucocorticoids may develop mildly increased ALT activity that resolves within several weeks of glucocorticoid withdrawal.
Despite the high sensitivity of ALT for identification of a liver disorder, its lack of specificity does not allow it to differentiate clinically significant liver disease or specific histologic abnormalities or to detect hepatic dysfunction. Its best utility is 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 hepatocyte cell membrane permeability, hepatocyte mitochondrial membrane damage, 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 weeks. 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 extrahepatic bile duct obstruction [EHBDO]).
A similar trend is evident in some dogs. Because AST is located within the mitochondria and free within the cytosol of hepatocytes, AST fold increases greater than or equal to ALT fold increases may reflect mitochondrial injury. Dogs treated with glucocorticoids may develop mildly increased AST activity; however, this resolves within several weeks of glucocorticoid withdrawal.
Alkaline Phosphatase in Hepatic Disease in Small Animals
Increased alkaline phosphatase (ALP) activity in dogs is the most common abnormality on routine biochemical testing for hepatic disease; its high sensitivity and low specificity can defy diagnostic interpretation without liver biopsy. ALP activity in dogs has the lowest specificity of routinely used liver enzymes because of the 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 chronicity, with the 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 the 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 considered.
Utility of serum ALP activity as a diagnostic indicator in dogs is complicated by the common accumulation of L-ALP and G-ALP isozymes. While each of these can be induced by steroidogenic hormones, G-ALP predominates. Unfortunately, the clinical utility of determining which isozyme contributes to ALP activity is low, because even in dogs with liver disease driving ALP release, the G-ALP eventually predominates.
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 may reflect 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, including EHBDO, pancreatitis-associated liver disease (mostly EHBDO), massive hepatocellular carcinoma (induction phenomenon), and bile duct carcinoma, and in dogs exposed to steroidogenic hormones (endogenous or exogenous).
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 ~50% of dogs with malignant or benign mammary tumors, with highest ALP activity reported 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 weeks. Sustained ALP activity usually correlates with a reparative ductal response (progenitor cell hyperplasia, otherwise referred to as a ductular reaction). In cats, EHBDO Extrahepatic Bile Duct Obstruction in Small Animals Extrahepatic bile duct obstruction is the blockage of the normal flow of bile from the liver to the intestinal tract. Obstruction of the common bile duct is associated with a number of diverse... read more results in a 2-fold increase in ALP within 2 days, as much as a 4-fold increase within 1 week, and up to a 9-fold increase within 2–3 weeks. Thereafter, activity stabilizes and gradually declines but usually not into the normal range; the declining enzyme activity coordinates with developing biliary cirrhosis.
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 that facilitates ALP release from membranes. 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, feline hepatic lipidosis Feline Hepatic Lipidosis Feline hepatic lipidosis (FHL), the most common acquired and potentially lethal feline liver disease, is a multifactorial syndrome. A primary disease process causing anorexia or food deprivation... read more is associated with marked increases in ALP activity and jaundice. The increased ALP seemingly reflects canalicular dysfunction or compression based on ultrastructural study of hepatocytes in affected cats. 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 diffuse, severe, and degenerative, VH impairs transhepatic perfusion and provokes a cholestatic effect that may cause 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. These dogs often demonstrate normal dexamethasone suppression and adrenocorticotropic hormone (ACTH) response tests. However, in some dogs, high ALP associated with glycogen-associated VH signals the presence of atypical adrenal hyperplasia, a disorder associated with abnormal sex hormone production.
Unfortunately, there is no consistent relationship between the magnitude of serum ALP activity, predominance of G-ALP activity, or hepatic histologic lesions. Determining G-ALP isozyme is not useful for syndrome characterization as this isozyme is also prominent in dogs treated with glucocorticoids and in dogs with spontaneous or iatrogenic hyperadrenocorticism, hepatic or nonhepatic neoplasia, hepatic inflammation, and 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. Typically prednisone or prednisolone increases ALP activity within 5–7 days with gradual sequential escalation with dosing of 2–4 mg/kg, PO, every 24 hours. 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 occasional cats developing a glycogen-associated VH or accelerated hepatocyte lipid vacuolation.
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, 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, every 12 hours) for 30 days.
Gamma-Glutamyl Transferase in Hepatic Disease
Gamma-glutamyl transferase (GGT) is a membrane-bound glycoprotein that plays a critical role in cellular detoxification because it is involved with glutathione availability. 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 increase (1- to 3-fold normal) in GGT activity that resolves in ~10 days after a single event. 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 weeks. 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 week, and 4- to 16-fold within 2 weeks.
Glucocorticoids and certain other microsomal enzyme inducers may stimulate GGT production in dogs; however, response is variable. Administration of many drugs (eg., glucocorticoids, carbamazepine, cimetidine, furosemide, heparin, isotretinoin, methotrexate, estrogens, phenobarbital, phenytoin, and valproic acid) may increase GGT activity significantly above baseline. Dogs treated with phenytoin or primidone developed a modest increase in serum GGT activity (up to 2- to 3-fold), unless they were manifesting anticonvulsant hepatotoxicosis that often associated with marked enzyme activity. Currently, these drugs are seldom used as anticonvulsants in dogs.
Cats with advanced necroinflammatory liver disease, EHBDO, or inflammatory intrahepatic cholestasis can develop a larger increase in GGT activity relative to ALP activity. Glucocorticoids and other enzyme inducers in dogs do not clinically influence serum GGT activity 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, and canine reference intervals should not be applied to feline measurements.
GGT activity can be markedly increased in dogs and cats with primary hepatic or pancreatic neoplasia. However, GGT activity does not appear to be suitable for surveillance of hepatic metastasis in either species.
Like ALP activity, GGT activity lacks specificity in differentiating between parenchymal hepatic disease and obstructive biliary disease. It is not as sensitive in dogs as ALP activity but does have higher specificity. In cats with inflammatory liver disease, it is more sensitive but less specific than ALP activity.
These two enzyme activities should be interpreted simultaneously. The likelihood that hepatic lipidosis (HL) has developed secondary to necroinflammatory liver disease, EHBDO, or pancreatic disease can be predicted by examining the relative increases in GGT and ALP activity.
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.