Canine liver enzymes – FAQs

Introduction

Most small animal clinicians will take blood samples several times a day for assessment of their patients (Figure 1). However, interpretation of the results is not always straightforward, especially when it comes to the various liver parameters, and it can be useful to know what tests are the most valuable when seeking a diagnosis or monitoring certain conditions. This paper offers a question-and-answer approach to some of the most common queries around liver enzymes. 

When is a high ALT/AST/ALP significant?

Typically, a two-fold increase in alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels is considered significant. In terms of the pathophysiology, increased hepatocellular enzyme activities are the result of enzyme leakage from cells (ALT, AST) or induction of enzymes (alkaline phosphatase (ALP)). However, the tests should always be interpreted in light of the patient’s history, clinical signs, and additional diagnostic findings; for example, the results can vary depending on whether an acute or a chronic disease is present. Chronic disease may go along with liver atrophy or fibrosis, and subsequently liver enzyme activity may be within the reference interval or show only a mild increase. As liver function will be impaired in a severe disease, normal liver enzyme levels in combination with changes in liver function parameters (i.e., hypoalbuminemia, decreased blood urea nitrogen (BUN), hypoglycemia, hyperbilirubinemia, changes in cholesterol and triglyceride concentration, prolonged coagulation times) are the classic picture of a severe disease such as a portosystemic shunt. The conclusion that normal liver enzyme activity is indicative of a healthy liver is, therefore, clearly wrong. Interpretation of liver enzyme results always warrants concurrent evaluation of liver function parameters and correlation to the history and clinical signs of the patient.

What are the best laboratory tests for a liver shunt?

Patients which have a portosystemic shunt suffer from vascular anomalies, whereby a vein from the portal system is directly linked to either the caudal vena cava or azygos vein. Because of this bypass, blood does not reach the hepatocytes in sufficient quantities, leading to a small, atrophied liver. The loss of hepatocytes may be associated with a wide variety of laboratory changes. Liver enzymes such as the cytosolic enzyme ALT, or AST, which are predominantly present in the hepatocyte mitochondria, may show normal or increased levels in affected patients. However, if the number of hepatocytes has declined significantly, the remaining cells may not release significant amounts of these enzymes, leading to low or normal serum levels. When only 20-30% of the liver mass remains, signs of liver insufficiency will become apparent. In such cases the liver can no longer maintain its physiologic functions, leading to changes in carbohydrate, lipid, vitamin and protein metabolism, as well as impaired detoxification abilities. The results of this insufficiency can include hypoglycemia, changes in cholesterol and triglyceride concentrations, hyperbilirubinemia, hypoalbuminemia, prolonged coagulation times, decreased urea concentrations and increased bile acids and/or hyperammonemia. Alongside these alterations, microcytic anemia and lower urine specific gravity can often be observed. 

So what is the best test to use if a liver shunt is suspected and the aforementioned tests do not provide a clear diagnosis? If liver insufficiency is present, evaluating fasting and postprandial/ stimulated bile acids is of high value. Note that if hyperbilirubinemia is present, increased bile acid concentrations are to be expected, and a bile acid stimulation test may not add much more information for the patient. The mechanism is that diseases which lead to impaired excretion of conjugated bilirubin from hepatocytes into the bile canaliculi also cause impaired bile acid excretion, and a subsequent increase in parameter concentrations. 

If neurologic signs indicative of hepatic encephalopathy are present (e.g., stupor or tremor) evaluation of ammonia levels is most helpful. However, this is a highly delicate parameter, and falsely high results can easily occur if samples are not handled properly. Immediate centrifugation of the sample with separation of cells from plasma, measurement within one hour post sampling, and limiting exposure to air are all very important to limit variability of results and a possible subsequent false diagnosis in a patient.

Why would a dog with a primary liver tumor have normal liver enzymes?

This can happen! To understand which mechanisms lead to increased enzyme activity, it is helpful to go back to the pathophysiology. Liver enzymes are not a homogeneous group; typically, ALT and AST are regarded as “liver enzymes”, whilst ALP and gamma glutamyltransferase (GGT), although often included in this category, also originate from the cell membrane of the biliary epithelial cells, and, therefore, are classic markers for intra- or extrahepatic cholestatic disturbances. Increased ALT and AST activity occurs due to reversible or irreversible (necrosis) hepatocellular damage. A wide variety of tumors can affect the liver; primary liver neoplasia may be a focal, nodular tumor (i.e., most hepatocellular carcinomas), or grow in a diffuse pattern, infiltrating the hepatic tissue in a more disseminated manner. Focal lesions may cause significant increases in liver enzyme activity, due to severe hepatocellular destruction and tissue necrosis. Depending on the degree of intrahepatic cholestasis, ALP levels may be normal or increased. Diffuse liver infiltrates by round cell tumors (e.g., lymphoma or mast cell tumors) may not be associated with significant hepatocellular damage, and in such cases liver enzymes may, therefore, only show mild or no increase in activity. 

In summary, any overall increase in liver enzyme activity with a hepatic neoplasm depends on the degree of hepatocellular damage and subsequent release of enzymes and/or the amount of tissue necrosis associated with the neoplastic lesion. Focal or diffuse neoplastic infiltrates may or may not lead to increased liver enzymes, so diagnostic imaging (abdominal ultrasound) and fine-needle aspirates are, therefore, important additional steps to identify hepatic disease (Figure 2). 

What non-hepatic diseases can significantly affect liver enzymes?

As the liver is the central organ in the body responsible for regulating many metabolic functions, secondary hepatopathies are a frequent finding. For example:

• Cholestasis, either intra- or post-hepatic, classically leads to increased liver enzyme activity. Due to their physiological location in the biliary epithelial cells, ALP and GGT enzymes are sensitive indicators for this condition. Intrahepatic cholestasis as a result of hepatocellular swelling and subsequent obstruction of the biliary canaliculi can be seen in hepatitis secondary to any cause. In such diseases ALT and AST enzyme levels are also frequently increased. Post-hepatic cholestasis originates from conditions where obstruction of the bile duct occurs. These can include inflammation, neoplasia or choleliths. Due to the close proximity of the two organs, pancreatitis is a frequent reason for post-hepatic cholestasis, and evaluation of DGGR lipase is helpful in such cases. 
• Systemic diseases which may have an impact on the liver are multiple, and include inflammatory conditions such as gastrointestinal diseases, pancreatitis and sepsis. Not only nutrients, minerals, trace elements and vitamins are absorbed from the intestines via the portal vein and delivered to the liver; in gastrointestinal diseases bacteria, toxins, drugs, and/or cytokines are also transported. Associated cytological changes in hepatocytes are water and/or glycogen accumulation and are usually mild in nature. Consequently, increased ALT enzyme activity is usually also only mild. Infectious conditions including bacterial (e.g., leptospirosis), viral (e.g., canine adenovirus 1 infection), or fungal (e.g., histoplasmosis) can all be associated with markedly increased liver enzyme levels. Oxygen supply is vital to the liver and hypoxia, which can be secondary to anemia, hemolysis or cardiovascular disease, can lead to hepatocellular damage and abnormal liver enzyme activities. 
• Muscle damage should also be considered; AST enzyme activity is not liver-specific, and it may also increase after muscle damage due to its location in cardiac and skeletal muscle tissue. Due to the unspecific nature of AST, it is helpful evaluating this enzyme together with other laboratory parameters. ALT may also increase in some rare muscle dystrophies; active muscle disease is mostly not associated with ALT enzyme leakage. Where there is a simultaneous increase in ALT levels, a hepatic origin is, therefore, likely. If history and anamnesis of the patient reveals possible muscle trauma or inflammation, measurement of creatine kinase (CK) enzyme activity can help identify muscle disease as the reason for the increased AST. 
• Hemolytic disruption of red blood cells (RBCs) can produce mild increases in AST activity, as the enzyme is also present in erythrocytes. This example shows that it is vital to evaluate the hematologic assessment along with biochemical results.
• Toxicities are a frequent reason for increased liver enzymes, and in particular administered medical drugs. The most important mechanism is induction of enzyme production by these substances, and is one of the main causes for increased ALP activity. Notable drugs are the anticonvulsants phenobarbital and primidone. Glucocorticoids (especially prednisolone and prednisone) also need to be mentioned, as even topical ointments can influence ALP activity (Figure 3). Meticulous survey of the patient’s history is of highest importance here, as owners may not consider the influence of such therapeutics and may only mention them after being specifically asked. Glucocorticoids stimulate production of the canine-specific corticoid-induced ALP (C-ALP) isoform. Increases in ALP enzyme activity may be observed within seven days after initiation of treatment and may only decline six weeks after therapy cessation. Interpretation of ALP activity in response to treatment is further complicated as enzyme changes will depend on the type of steroid and dosage applied, duration of treatment and route of administration (orally, intravenously or topically). To further complicate interpretation, some patients may show a highly individualized response to certain treatments.
• Bone conditions can also affect liver enzyme activity, as ALP exists in various isoforms. Bone-ALP originates from osteoblasts, and up to a 3-fold increase in this isoform can, therefore, be observed in young, growing animals due to their increased osteoblast activity. Osteosarcoma may go along with increased bone remodeling; therefore, neoplasia of the bone is one of the differentials if adult patients show increases in ALP (Figure 4). Other bone disorders (e.g., necrosis, fractures and orthopedic interventions) should also be considered.
• Endocrinopathies often also produce increased liver enzyme activity. High levels of endogenous glucocorticoids, as occurs with hyperadrenocorticism (Cushing’s disease), induce C-ALP enzyme activity, which is a characteristic finding in this disorder. In fact, if an increase in this enzyme is absent, Cushing’s disease is less likely, but diagnosis still needs to be based on matching clinical signs, laboratory findings and results of specific tests, such as the low-dose dexamethasone suppression test. Other endocrine disorders, such as hyperparathyroidism and diabetes mellitus, are also potential candidates that can increase liver enzyme levels.

How useful are liver enzyme tests when monitoring diabetes mellitus?

The cornerstone of diabetes mellitus monitoring is evaluation of glucose levels. However, diabetic patients show disturbed lipid metabolism and subsequent increased lipid metabolization in the liver, making monitoring of liver enzymes a helpful tool to assess disease status. Cytologically, hepatic steatosis may be observed, although this is more prominent in cats than in dogs. Lipid accumulation in hepatocytes leads to hepatocellular damage and increased ALT or ALP enzyme levels can be seen (the latter being a particularly sensitive marker to detect hepatic lipidosis in cats), and a blood sample may show marked lipemia (Figure 5). 

What tests should I do for a dog on long-term medication for seizures?

As described above, anticonvulsant drugs such as phenobarbital can induce ALP enzyme production. Evaluating phenobarbital levels during therapy is important, as drug levels > 35 µg/mL are hepatotoxic, and monitoring of the liver’s general health is, therefore, also recommended twice yearly for a dog on chronic phenobarbital treatment. As noted above, liver enzyme levels within the reference interval do not rule out significant liver insufficiency; therefore, a bile acid stimulation test should be considered if liver dysfunction is suspected in a dog that is on anticonvulsant therapy.

Do I need to do clotting factors if a dog has liver disease?

The liver not only produces clotting factors but also anticoagulant proteins, such as Protein S and Protein C, antithrombin and plasminogen. Alterations in liver function thereby influence pro- and anticoagulant protein production and function, and the subsequent clinical picture of coagulopathies can range from severe, such as spontaneous bleeding, to only subclinically prolonged clotting times. How an individual patient reacts is difficult to predict. Typically, a profound reduction in hepatic mass and subsequent severe liver failure needs to be present to significantly impair synthesis of the described factors and proteins. As factor VII has the shortest half-life (6 hours) of all clotting factors, prothrombin time (PT) is expected to change initially. 

In many cases of suspected hepatopathy, fine-needle aspirates or biopsy of the liver are warranted to further characterize the nature of the disease, but assessment of clotting factors such as PT and activated partial thromboplastin clotting time (aPTT) prior to biopsy sampling facilitates risk assessment in patients with hepatic failure – although correlation of results to possible clinical bleeding tendencies are not straightforward. Profound prolongation of clotting times can be associated with spontaneous or biopsy-related bleeding, but such cases have also been observed to sometimes have only mildly prolonged or even normal coagulation times.

What’s the best protocol for a bile acid stimulation test? 

The highest sensitivity when assessing bile acids for liver insufficiency is achieved by performing a dynamic bile acid stimulation test. This involves measuring fasting bile acids (typically after a 12-hour fast) and the results of this baseline value compared to a sample taken 2 hours post-ingestion of food containing a moderate fat content. Food consumption stimulates gallbladder contraction and release of bile acids into the enterohepatic circulation. While the timing of gallbladder contraction is unclear with the baseline value, it is more likely to be stimulated by food intake and thereby allows a better view of hepatic function.

Interpretation of the stimulation test results can be challenging, as firstly ingestion of food needs to be assured, and gastrointestinal diseases which can lead to delayed gastric emptying, or decreased ileal absorption of bile, must be excluded. Gallbladder contraction may occur at unpredictable time points, and not all the stored bile may be released, or the bladder may not fill completely with bile after release, which can also influence the results. Enterohepatic circulation of bile acids is further influenced by metabolism of the intestinal bacteria. A general rule of thumb in interpreting the results is evaluating the highest value gained, irrespective of whether this is the baseline or the stimulated sample. If both samples show bile acids to be > 25 µmol/L this supports a diagnosis of liver insufficiency. Fasting bile acids may also be increased in cholestasis or other secondary hepatopathies. Results > 50 µmol/L are still likely for dysfunction related to primary hepatopathies. Results of pre- and postprandial bile acids between 20-50 µmol/L are equivocal and should prompt reanalysis in 2-3 weeks, along with correlation to other laboratory and/or diagnostic imaging findings. Finally, it should be noted that abnormal bile acid results may occur due to various underlying hepatobiliary diseases and are a non-specific finding. 

Conclusion

Given that almost any biochemistry profile conducted on a dog will routinely include a variety of parameters that can be used to assess liver function, it is important to know when an abnormal result is significant, and when a result within the normal reference range is not sufficient to exclude a potential diagnosis. The clinician is advised to always interpret liver enzyme results alongside the patient’s history and clinical signs, and to repeat certain tests – and also to seek additional diagnostic options – whenever appropriate.

Further reading 

• E. Villiers, Ristić J. BSAVA Manual of Canine and Feline Clinical Pathology, 3^rd ed. Gloucester, British Small Animal Veterinary Association, 2016
• Stockham SL, Scott MA. Fundamentals of Veterinary Clinical Pathology, 2^nd edition. Ames, IA; Blackwell Publishing, 2008
• Lawrence YA, Steiner JM. Laboratory evaluation of the liver; a review. Vet. Clin. North Am. Small Anim. Pract. 2017;47(3):539-553. Doi: 10.1016/j.cvsm.2016.11.005. Epub 2017 Jan 4.
• Konstantinidis AO, Patsikas MN, Papazoglou LG, et al. Congenital portosystemic shunts in dogs and cats: classification, pathophysiology, clinical presentation and diagnosis. Vet. Sci. 2023;10(2):160. Doi: 10.3390/vetsci10020160.
• Charalambous M, Muñana K, Patterson EE, et al. ACVIM Consensus Statement on the management of status epilepticus and cluster seizures in dogs and cats. J. Vet. Intern. Med. 2024;38(1):19-40. Doi: 10.1111/jvim.16928. Epub 2023 Nov 3.