Liver diseases often represent a real diagnostic challenge. The clinical signs present may be nonspecific and no single test is available for the identification of the underlying problem. The liver has an enormous reserve capacity and clinical signs may not appear until significant liver abnormalities are present. To further complicate things, some systemic diseases may also affect the liver and alter liver test results.
The first step to allow a correct diagnosis is the collection of a very accurate clinical history. The gathering of information on the eventual prior use of drugs (and which ones), exposure to toxic substances, presence of infectious diseases, recent surgical procedures requiring anaesthesia and information on the animal’s life style (indoor/outdoor, etc.) is extremely important.
The clinical signs that are most commonly reported in animals with liver disease include: depression, reduced appetite and lethargy, weight loss and failure to thrive, vomiting, diarrhoea, acholic stools, ascites, PU/PD, jaundice, clotting disorders, abdominal pain (rarely) and encephalopathy.
The physical examination may identify: jaundice, hepatomegaly, poor physical condition, presence of abdominal effusion and signs of systemic disease (e.g., fever, eye problems, neurological disorders, etc.).
In most cases the diagnosis may be suspected based on liver test results, however a liver biopsy (especially in the case of primary liver disease) is the only way to get a definitive answer. Before performing a liver biopsy it is first however necessary to entirely exclude all possible causes of secondary liver disease. The age, breed and sex may be suggestive of a possible cause; an example is chronic hepatitis, which is frequently present in middle-aged Dobermann females.
The diagnostic approach to liver disease must therefore include: collection of the clinical history, physical examination, laboratory tests, diagnostic imaging studies and liver biopsy. In patients with liver disease, prior to liver biopsy a minimum database is required, consisiting of a biochemistry profile, a complete blood count and urinalysis.
BLOOD CHEMISTRY PANEL
Liver enzimes
Increased liver enzymes are often considered to be indicative of liver disease. It is instead very important to remember that increased liver enyzmes are not necessarily indicative of a significant primary liver disease. Several drugs and diseases may cause the elevation of certain liver enzymes, alterations which are entirely reversible without any treatment. On the other hand, it is also absolutely necessary to understand that in the case of cirrhosis or of congenital portosystemic shunt (PSS), liver enzyme abnormalities may in fact be minimal in view of an insufficient liver mass available for the production of enzymes. It has also been hypothesized that a persistent elevation of liver enzymes in the absence of liver function abnormalities or histopathological alterations may be indicative of the presence of enzyme-antibody complexes that are very-slowly eliminated from the circulation but which are of no clinical significance.
Two types of liver enzymes are present:
• hepatocellular leakage enzymes
• cholestatic enzymes
Hepatocellular leakage enzymes
ALT
In the dog and cat, ALT is a cytosolic enzyme produced by the liver. Other isoforms are in fact present, produced by organs such as the heart and muscle, however the concentration of these other isoenzymes is low and their half-life is short. The measured ALT is therefore almost exclusively derived from the liver and for this reason ALT is considered a liver-specific enzyme. In the dog, the half-life of ALT is of 2.5 days; in the cat, no published data are available but it is considered to be much shorter (about 6 hours), hence in the cat minimal alterations are more significant. The highest ALT levels are found in acute hepatocellular necrosis or inflammation. ALT elevation is much greater in acute disorders than in chronic conditions. A common mistake is to attribute too much importance to the degree of ALT elevation. An increase of at least 2 times the upper limit considered normal by the laboratory is necessary for a liver disease to be considered, however increases beyond this value are not necessarily indicative of a more severe disease, or provide information on the prognosis and reversibility of the underlying disease process. The prognosis for acute liver disease is better than for chronic conditions. Enzymes are expected to decrease by around 50% every 3-4 days and should return to completely normal values after about 3 weeks. ALT increases rapidly in the case of trauma, but much more slowly in the case of cholestasis caused by the accumulation of bile salts and liver damage. In dogs, following trauma, ALT increases by about 1-10 times the upper limit while in the presence of cholestatic disease it increases by approximately 10-100 times. In the cat, cholangitis causes only moderate ALT elevation (about 5-10 times the upper limit). Other causes of increased ALT values include liver cancer and enzyme induction following the use of certain drugs, such as anticonvulsants, glucocorticoids, acetaminophen, antifungals, anaesthetics, sulfonamides and others. In the case of glucocorticoids, the effect of a single dose may persist for several weeks because of enzyme induction and steroid-related liver disease.
It is also important to consider that elevated ALT values may be secondary to various diseases, such as: diabetes mellitus, lipid infiltration, gastroenteropathies, dental disease, intoxication as well as hypoxia. In all these cases a biopsy is not necessary and liver function tests are within the normal range.
AST
AST is another enzyme that is released following cellular damage that increases the permeability of the hepatocellular membrane. AST is produced by the liver, but also in large quantities by the heart and muscle. AST elevation, with normal ALT values, may be indicative of muscle damage. In such cases, a creatine kinase (CK) assay is suggested; if elevated, this confirms the presence of muscle damage. AST also increases in the case of haemolysis. The specificity of AST is not high, but some specialists argue that its release occurs in cases of very severe trauma, as this enzyme is normally bound within the mitochondria and is not free in the cytoplasm. Compared to ALT, the half-life of AST is shorter (5-22 hours in the dog, 77 minutes in the cat), therefore its elevation is indicative of a more severe disease. In the presence of secondary liver disorders, AST rarely increases, and this may be helpful in discriminating between the presence of a primary or secondary liver disease. AST normalizes before ALT, and if AST levels remain elevated this is a poor prognostic sign. Mild AST elevations are characteristic of chronic hepatitis and cholestasis. In cats, being the half-life very short, even slight increases are very significant and in this species AST is considered to be a more sensitive marker of liver disease.
Cholestatic enzymes
ALP
ALP consists of a heterogeneous group of enzymes that is normally bound to cell membranes. ALP has been isolated from the: kidney, liver, placenta, bone and intestine. Two genes are necessary for its production, one specific for tissues and one for the intestine. ALP isozymes are found in the bone, liver, placenta and intestine; in addition, there is the corticosteroid-induced isoenzyme, which is found only in the dog. Only three of the various isoenzymes can be measured in the serum, thanks to their longer half-life (approximately 70 hours): the isoenzyme produced by the liver (L-ALP), by the bone (B-ALP) and the corticosteroid-induced isoenzyme (C-ALP). The half-life of the other isoenzymes is of only a few minutes and cannot be measured with currently available assays. The ALP measured is therefore the total of L-ALP, B-ALP and C-ALP. The proportion of each enzyme changes with age. In young animals (<1 year) B-ALP is prevalent, while in adult healthy animals L-ALP is the prevaling isoenzyme. C-ALP seems to prevail in elderly animals.
ALP values may be elevated in the case of: intrahepatic cholestasis caused by primary liver diseases such as nodular hyperplasia, cancer, chronic hepatitis and cirrhosis, vacuolar hepatopathy caused by toxicity and infectious diseases; extrahepatic cholestasis caused by pancreatitis, biliary tract diseases (mucocele, cholangitis/cholangiohepatitis), cancer, cholelithiasis; induction by various drugs (phenobarbital, steroids, other); and liver diseases secondary to endocrine disorders (hyperadrenocorticism, hypothyroidism, diabetes mellitus, hyperthyroidism, Addison's disease, etc.). In addition, apparently benign ALP elevations have been reported in some breeds such as the Scottish Terrier and the Siberian Husky.
The highest ALP values are usally found in the course of: focal or diffuse cholestasis, use of glucocorticoids (systemically but also locally in the form of eye drops and ear drops), chronic hepatitis and liver cancer. The concomitant increase of other liver enzymes such as ALT, AST, GGT and Tbil may help to differentiate hepatocellular damage from cholestasis. In the case of cholestasis, ALP, GGT and Tbil elevations are expected, while if ALT and AST elevations prevail, a hepatocelluar damage is more likely.
GGT
GGT is a biliary tract enzyme that increases in plasma in response to cholestasis; its rise is usually parallel to that of ALP. Most of the GGT circulating in the serum is considered of liver origin, but it can also be produced by the: kidney, pancreas, heart, intestine, muscle and red blood cells. GGT can also be found in colostrum and in milk; GGT as well can also be induced by glucocorticoids while apparently its production does not seem to be stimulated by anticonvulsants.
GGT is found in the biliary tree but it is not considered a highly specific enzyme as it cannot differentiate cholestatic diseases from hepatocellular injury. Some authors suggest that its diagnostic validity increases when assayed together with ALP. In dogs, it is more specific and less sensitive, while in cats it is more sensitive that specific; in the cat, in fact, most cholestatic diseases cause a greater increase of GGT than of ALP, with only one exception: hepatic lipidosis, in which ALP is increased while GGT is normal; it has been hypothesized that this may be due to an excessive production of ALP or because of a delay in its elimination.
Cholesterol
Cholesterol comes from the diet; it is synthesized by the liver and then enters the enterohepatic circulation. Its value as a marker of liver disease is limited; in fact, it may be normal, elevated or decreased, according to the underlying disease and the type of diet. Hypercholesterolaemia may be present in cases of bile duct occlusion but also in diseases that can affect the liver as a secondary event, such as hyperadrenocorticism, hypothyroidism, diabetes mellitus, pancreatitis, hyperlipidaemia and nephrotic syndrome. Hypocholesterolemia may instead occur in cases of PSS, cirrhosis, liver failure and malabsorption.
Triglycerides
Abnormal triglyceride levels inthe course of liver disease are not well characterized and their value can be considered comparable to that of cholesterol.
Glucose
The liver hasan enormous glucose homeostasis maintenance capacity; in fact, in order to devolop hypoglycaemia a liver function loss of at least 70% must be present. This happens more easily in cases of fulminant hepatic failure or in the presence of major vascular alterations (PSS, etc.). Hypoglycaemia can be present in the case of cancer as part of a paraneoplastic syndrome characterized by excessive glucose consumption, by sepsis or by the release of insulin-like factors. Rare diseases, such as glycogen storage disease, can be the cause of hypoglycaemia and liver glycogen accumulation.
Proteins
Albumins are made exclusivelyby the liver. Hypoalbuminaemia is present in the course of chronic diseases, when more than 70% of the liver function is lost. Hypoalbuminemia is not a specific sign of liver disease and may also be present in the course of other diseases, such as enteropathy and protein-losing nephropathy.
Globulins may be elevated in liver disease due to the inflammatory stimulus, as an acute phase reaction but also because of a reduced elimination of antigens from Kupffer cells, thus giving rise to a systemic immune response. An elevation of gamma globulins is observed especially in cats with FIP and with lymphocytic cholangiohepatitis.
Urea and creatinine
Low concentrations ofurea can be found in animals with PSS or with severe hepatic impairment due to the non-conversion of ammonia into urea and the presence of PU/PD. An increased urea and creatinine accompanied by an increased urine specific gravity (SG) are indicative of prerenal hyperazotaemia and could be the consequence of liver disease. An increase in urea alone could instead be indicative of the presence of gastrointestinal ulcers. BUN alterations are not specific for liver disease as BUN levels can be affected by the state of hydration, the diet, the presence of gastrointestinal bleeding and the use of drugs.
Bilirubin
Increased bilirubinmay result in yellowish/orange coloured body tissues (jaundice). Jaundice can be caused by various medical conditions, including liver disease, but not exclusively. Furthermore, jaundiceis not always present during liver disease; as an example, in the case of corticosteroid-induced liver disease and PSS, no increase in bilirubin is present.
Jaundice can be:
•pre-hepatic: mainly due to haemolytic anaemia;
• hepatic: due to acute and chronic hepatitis, intoxication, drugs, cholangiohepatitis, cancer, sepsis, endotoxaemia, infectious diseases (FIP, toxoplasmosis, etc.).
• post-hepatic: caused by pancreatitis, cancer, cholelitiasis, rupture of the common bile duct, etc.
Increased bilirubinis therefore not synonymous with liver disease and if present, other tests should be performed in order to rule out other diseases.
Dynamic liver function tests
Ammoniaemia
The liver is responsible for the detoxification of the ammonia produced in the gastrointestinal tract. Once taken up by the hepatocytes, it is transformed into urea or it is used for the production of glutamine.
In cases of severe hepatic impairment (loss > 60-70% of liver function) ammonia can not be eliminated and tends to accumulate in the parenchyma, leading to hyperammoniaemia. Elevated ammonia levels have a sensitivity of approximately 100% and a specificity of around 85% for congenital PSS. Hyperammoniaemia is the main cause (but not the only one) of hepatic encephalopathy; in patients with the characteristic clinical signs, high ammonia levels can thus confirm the diagnosis of encephalopathy. The test is extremely sensitive and the sample should be prepared as soon as possible and treated with care, otherwise the results cannot be considered reliable.
Hyperammoniaemia may also occur in animals with urea-cycle impairment, a phenomenon which has been described in the Border Collie, Wolfhound and in other breeds.
Bile acids
Bile acids are synthesized from cholesterol exclusively in the liver and are concentrated in the gallbladder. Following ingestion of a meal, cholecystokinin stimulates the gallbladder and bile acids are released into the intestine, where they help in the absorption of lipids. Once reached the ileum, they are transported into the portal circulation. The efficiency of this enterohepatic circulation is of about 98% and eventual alterations may cause an accumulation of bile acids in the serum.
The bile acid stimulation test is used in the diagnosis of PSS and of other liver diseases; the test gives an indication of liver function. It has a sensitivity of 50-70% and a specificity of 90-100%. The sensitivity of the test increases when two samples (pre- and postprandial) are taken, one before and one 2-hours after a meal. The high specificity makes the test ideal for liver function assessment but the sensitivity is low, thus precluding its use as a screening test.
Bile acids can also be measured in the urine, providing information on their production and excretion in the 24 hrs. Test results are expressed in relation to creatinine, in order to exclude variations influenced by the urine specific gravity and the dilution of bile acids in the urine. The test has a good specificity and sensitivity; in the cat, it is almost comparable to the blood bile acid stimulation test, while in the dog the sensitivity seems to be a bit lower. Given the ease of execution, this test can be used as a screening test for hepatobiliary disease in both the dog and the cat.
Following a recent publication, an alternative approach to the classical bile acid stimulation test has been proposed, namely the administration of ceruletide (a cholecystokinin anologue), which can stimulate gallbladder contraction about 30 minutes after its injection; this approach eliminates all the difficulties connected with the administration of a meal, especially in animals that are stressed or anorexic.
It is important to note that in some breeds, such as in the Maltese Terrier, postprandial bile acids may be elevated in the absence of any other signs of disease (the cause remains, to date, unknown).
COMPLETE BLOOD COUNT AND COAGULATION PROFILE
CBC alterations are a common finding, with the presence of non-regenerative, microcytic and hypochromic anaemia, which may be indicative of gastrointestinal bleeding. Erythrocyte microcytosis may be found in dogs with congenital vascular liver disorders or cirrhosis, as well as in cats with lipidosis. Poikilocytosis and target cells can be found in the blood smear of patients with liver problems and are probably the result of altered lipid metabolism. Schistocytes may be present in cases of microangiopathy with fibrin deposition, which may be the cause of altered RBC morphology.
The liver synthesizes fibrinogen, ATIII and all coagulation factors, apart from factor VIII, and is the site of activation of vitamin K-dependent factors: II, V , VII, IX , X and protein C. In approximately 50-70% of dogs with liver disease, PT and APTT abnormalities are present; PT is often altered as it is dependent on factor VII, which has a half-life of 48 hours. In cases of chronic biliary obstruction the circulation of bile acids is interrupted, causing the malabsorption of soluble vitamins, especially of vitamin K, which in turn leads to a factor VII deficiency.
Patients with hepatobiliary disease are at greater risk of bleeding during or shortly after invasive procedures; for this reason a coagulation profile should always be performed. Increased FDP levels have been reported in patients with liver disease; this is probably caused by a decreased elimination of degradation products by the liver. Protein C is a coagulation inhibitor, which is synthesized by the liver and is dependent on vitamin K. In patients with liver disease and especially with parenchymal vascular alterations, protein C is also reduced, but it can return to normal after surgical correction of the vascular anomaly. In these patients both qualitative and quantitative platelet alterations may be present.
URINALYSIS
Bilirubin is usually present, together with a decreased urinespecific gravity, especially in patients with PU/PD. In the dog, the presence of small amounts of bilirubin may be normal; the finding is instead not normal in the cat. Uric acid and ammonia may also be present and may favour the formation of ammonium biurate crystals and stones.
The correct diagnostic approach to liver disease therefore requires the collecction of an accurate clinical history and the performance, prior to biopsy, of a series of diagnostic tests, such as liver function tests and diagnostic imaging studies.
Suggested reading
- Balkman CE, Center SA, Randolph JF, et al. Evaluation of urine sulfated and nonsulfated bile acids as a diagnostic test for liver disease in dogs. JAVMA, 2003;222:1368.
- Battersby IA, et al. Hyperammonaemic encephalopathy secondary to selective cobalamin deficiency in a juvenile Border Collie. JSAP, 2005;46:339.
- Bigge LA, et al. Correlation between coagulation profile findings and bleeding complications after ultrasound-guided biopsies: 434 cases (1993-1996). JAAHA, 2001;37:228.
- Brady CA, et al. Severe sepsis in cats: 29 cases (1986-1998). JAVMA, 2000;217:531.
- Bridger N, et al. Comparison of postprandial and ceruletide serum bile acid stimulation in dogs. JVIM, 2008;22:873.
- Center SA, et al. Proteins invoked by vitamin K absence and clotting times in clinically ill cats. JVIM, 2000;14:292.
- Center SA. Interpretation of liver enzymes. Vet Clin North Am Small Anim Pract. 2007;37:297.
- Comazzi SC, et al. Hematological and biochemical abnormalities in canine blood: frequency and associations in 1022 samples. JSAP, 2004;45:343.
- Dail SM. Clinicopathologic evaluation of the liver. Vet Clin North Am Small Anim Pract. 1995;25:257.
- Gerritzen-Brunning MJ, et al. Diagnostic value of fasting plasma ammonia and bile acid concentration in the diagnosis of PSS in dogs. JVIM, 2006;20:13.
- Lisciandro SC, et al. Coagulation abnormalities in 22 cats with naturally occurring liver disease. JVIM, 1998;12:71.
- Nestor DD, et al. Serum alcaline phosphatase activity in Scottish Terriers versus dogs of other breeds. JAVMA, 2006;228:222.
- Taboada J, et al. Cholestasis associated with extrahepatic cholestasis in 5 dogs. JVIM, 1989;3:216.
- Tisdall P, et al. Postprandial serum bile acid concentrations and ammonia tolerance in Maltese dogs with and without hepatic vascular disease. Aust Vet J, 1995;72:121.
- Trainor D, et al. Urine sulfated and nonsulfated bile acids as a diagnostic test for liver disease in cats JVIM, 2003;17:145.
- Zandvliet MM, et al. Transient hyperammonemia due to urea-cycle enzyme deficiency in Irish Wolfhounds. JVIM, 2007;21:215.
- Zini E, et al. Paraneoplastic hypoglycemia due to an insulin-like growth factor type-II–secreting hepatocellular carcinoma in a dog. JVIM, 2007;21:193.