Various laboratory tests are available for the assessment of liver function: 1. Tests indicative of increased membrane permeability, enzymatic induction or hepatobiliary damage: ALT (alanine aminotransferase), AST (aspartate aminotransferase), ALP (alkaline phosphatase) and GGT (gamma-glutamyl transferase). These tests are not a direct expression of liver function. 2. Tests indicative of hepatic synthesis: albumin, glucose, urea, P-ChE (pseudocholinesterase) and coagulation factors. 3. Tests indicative of uptake, conjugation and secretion: bilirubin, serum and urinary bile acids.
1)
ALT is a liver-specific enzyme located within the cytoplasm. Rare muscular dystrophies may also be the cause of increased ALT activity. Plasma half-life of 2-3 days (in the dog).
AST is an enzyme found in various tissues, particularly in the liver and muscular tissue; half-life of 12 hours (in the dog). It is located within the cytoplasm and mitochondria of hepatocytes. In order to understand the cause of increased AST activity, CPK levels may be considered: if elevated, a raised AST is likely to reflect the presence of muscular damage.
AST and ALT elevation is proportional to the severity of hepatobiliary damage: more specifically - in the absence of muscular damage - a much greater elevation of AST compared to ALT is the expression of deep liver necrosis. In the cat, being the effect of enzymatic induction on hepatic enzymes negligible, moderate increases in liver enzymes are already indicative of severe liver damage.
ALP is located on the hepatocyte membrane, towards the biliary canaliculus, and is considered the “biliary tract” enzyme. Half-life of 3 days in the dog and <8 hours in the cat. Various types of isoenzymes are present: hepatobiliary, steroid, bone, intestinal and placentar isoenzymes. Hepatobiliary isoenzymes increase following cholestasis, the second type by enzymatic induction, the third in growing subjects or in the course of bone diseases followed by remodelling, the intestinal isoenzymes - in view of their very short half-life - are negligible, and placentar isoenzymes increase during pregnancy. In the cat, an increase equal to 6-8 times the reference range is indicative of hepatic lipidosis.
GGT is located in all the excretory, principally hepatic (biliary canaliculi), ducts. Elevation is indicative of biliary stasis or enzymatic induction. In the dog, GGT is highly specific.
2)
Albumin, the most conspicuous seroprotein fraction, is mainly involved in the maintenance of colloid-osmotic pressure and in nutritional functions. As a liver function test, hypoalbuminemia may be indicative of a defect in synthesis and consequently of liver disease. Following is a list of clinical conditions associated with hypoalbuminemia, all to be necessarily included in the differential diagnosis whenever low serum albumin levels are found.
The pathogenetic mechanisms for hypoalbuminemia are:
- Lack of production: liver failure, malabsorption, negative feedback in the course of hypergammaglobulinemia;
- Loss: proteinuria, protein-losing enteropathy, cutaneous exudation;
- Consumption: fever, cachexia;
- Sequestration: body cavity haemorrhages, body cavity effusions;
- Dilution: iatrogenic following exaggerated fluid therapy, pregnancy, inappropriate ADH secretion.
Hypoglycemia may be caused by liver failure when 80% of the hepatic parenchyma is compromised; it will clearly not be the only altered value. If an adequate separation of the serum has been made, sepsis should be included in the differential diagnosis.
Decreased urea, as an expression of lack of synthesis, may again be suggestive of liver failure. High urinary flows (PU/PD) may be the cause of urea reduction, as well as prolonged fasting and low protein diets.
Cholinesterase is an enzyme with two basic types present:
- Acetylcholinesterase (true cholinesterase, AChE), located in the myoneural junction where it hydrolizes acetylcholine. The serum activity of this enzyme is extremely scarce;
- Butyrylcholinesterase (pseudocholinesterase, PChE), produced mainly in the liver but also in the pancreas and in the gastric mucosa.
PChE is usually present in serum and can easily be assayed with automated methods, which have recently been validated in the canine species. In humans, PChE is a true liver function test, since the production of the enzyme by the liver is associated with albumin production. In dogs the assay is used in the diagnosis of organophosphorus and carbamate poisoning, in which a clear reduction of its concentration is present. Decreased values may be present with liver failure, as well as with prolonged fasting and malabsorption. Cirrhosis of the liver is the exception, with marked increases usually present.
Coagulation factors (with the exception of factors V and VIII), which are exclusively synthesized in the liver, may – although nonspecifically – be suggestive of liver failure, with prolongation of the intrinsic pathway (aPTT). Fibrinogen, being a moderate, positive acute phase protein, may mask a synthesis defect in concomitant inflammatory processes.
3)
Total bilirubin deriving from the degradation of haemoglobin is the most conspicuous biliary pigment. Three fractions of bilirubin are found in blood: indirect bilirubin (bound with albumin), direct bilirubin (bound with glicuronic acid) and delta bilirubin (direct bilirubin bound with albumin) (Fig. 1).
Reduced serum bilirubin levels are of no clinical significance; the opposite is true in the presence of elevated levels, which may be found in the course of: haemolysis, liver failure, hepatic cholestasis (lipidosis, cirrhosis, cholangiohepatitis, lymphoma…) and post hepatic cholestasis (cholangitis, pancreatitis, bile duct or pancreatic neoplasms…).
Bile acids may be assayed in both serum (SBA) and urine (UBA) samples. Serum bile acids are assayed with methods specifically validated for the canine and feline species. The reference range varies depending on when the sample is taken (fasting or 2 hours after eating), with typically higher postprandial levels in view of the greater load of bile acids to be cleared by the liver. The test requires fasting of the patient for at least 12 hours, after which a serum sample is taken while the animal is fed a standard protein and fat meal. A second sample is then taken two hours after the meal, and the postprandial SBA levels are examined. These latter values are more diagnostic because with feeding there is a cholecystokinin-mediated contraction of the gallbladder, which triggers the secretion of large amounts of bile into the intestine in order to aid lipid emulsification and hence fat absorption. Most of the SBA released into the intestine are reabsorbed and undergo a highly efficient enterohepatic recirculation for hepatic extraction (portal clearance). In the presence of sub-clinical liver failure, the postprandial increase of the bile acid pool, which reaches the liver through the hepatic portal system, undergoes liver uptake but is only partially extracted (hepatic clearance), thus allowing the detection of also mild forms of liver failure.
Recently, there has been considerable interest in urine bile acids (UBA), which may represent a true time-averaged sample of the SBA serum fluctuations caused by gallbladder contractions taking place with food intake or independently from it. In addition, the test reduces costs, eliminates complications caused by feeding and by the need for a double test and is of easy interpretation. Urine may be collected by cystocentesis or by spontaneous urination at any time of day. To increase test sensitivity urine can be collected 4-8 hours after a normal meal. As the UBA assay is highly influenced by the urine sample water concentration, the urine concentration of these analytes must be normalized to urinary creatinine.
Suggested readings
- Stockham S.L., Scott M.A. Fundamentals of Veterinary Clinical Pathology, 1stedition. Blackwell, 2002.
- Willard M.D., Tveden H. Small Animal Clinical Diagnosis by laboratory Methods, 4thedition. Saunders, 2004.