Diagnostics for hepatobiliary disease (Proceedings)

Clinical signs of gastrointestinal disease, such as vomiting and diarrhea are extremely common in small animal patients. When seeking advice from a veterinarian pet owners expect an accurate diagnosis and definitive therapy of the problem. In very few cases the diagnosis can be made by history and physical examination alone and the veterinarian has to utilize diagnostic tests to arrive at the diagnosis. The challenge for the veterinarian is to choose the most appropriate diagnostic test to arrive at the most accurate diagnosis.

In general, the clinician is guided by the golden rule to only perform a diagnostic test when the outcome of the test will have an impact on the management of the patient. This golden rule can be expressed more scientifically by stating that a diagnostic test should only be performed if the post-test probability for a disease is either significantly smaller or significantly larger than the pre-test probability. This means that the probability of a certain disease either needs to increase or decrease after the results of a diagnostic test have been received and interpreted.

While some diagnostic tests are either negative or positive, most diagnostic tests will yield a quantitative result that needs to be interpreted. This is achieved by comparison of the result found in a diseased animal to that found in a healthy animal (reference range, control range, or normal range). However, a result outside the reference range does not necessarily indicate disease and cut-off values need to be determined that afford the best test characteristics for the diagnosis of a disease. A result outside the reference range may have different implications for different tests. For example if the upper limit of the reference range for serum creatinine concentration is 133 µmol/L (1.5 mg/dL), a result of 150 µmol/L (1.7 mg/dL) may very well be significant, while a serum ALT activity of 76 U/L in a dog may be of no significance when the reference range is 0-65 U/L. Therefore, a cut-off value for each diagnostic test must be identified, that determines whether an abnormality is significant enough to warrant further clinical work-up or even a specific diagnosis of a disease.

Imaging

Abdominal radiography has a limited usefulness for the diagnosis of hepatic disease in dogs and cats. Abdominal radiographs are most useful to evaluate hepatic size. Both hepatomegaly, for example due to hyperadrenocorticism or hepatic masses, and microhepatica, for example due portosystemic vascular anomalies or cirrhosis in dogs, can be appreciated on an abdominal radiograph, hepatomegaly should also be apparent on abdominal palpation. Also, calcifications of the hepatic parenchyma can be diagnosed by abdominal radiography.

In contrast to abdominal radiographs, abdominal ultrasound is very useful to evaluate the architecture of the hepatic parenchyma and the hepatic vasculature. Ultrasound can be used to evaluate the overall echogenicity of the liver and is helpful to compare the echogenicity to other abdominal organs. Abdominal ultrasound is also useful to identify isolated masses or lesions within the parenchyma. Abdominal ultrasound is also a great diagnostic tool to identify and localize portosystemic shunts. However, the success of the evaluation is greatly dependant on the expertise of the ultrasonographer and also on the patience in trying to identify the shunt vessel.

Transcolonic and transsplenic scintigraphy are very useful to definitively diagnose a portosystemic vascular anomaly and to determine the shunt fraction. However, they provide little information about the exact location of the shunt. A transsplenic approach is superior to a transcolonic approach as a smaller amount of radioactive tracer can be used and also there are less factors that might negatively influence the outcome of the examination.

Serum chemistry profile

Serum activities of hepatic enzymes are analyzed as markers for hepatobiliary disease in both dogs and cats. Unfortunately, some of these enzymes are also synthesized by other tissues. Therefore, increases of serum activities of hepatic enzymes outside the control range can be seen with many non-hepatic conditions.

Alanine amino transferase (ALT) is a cytosolic enzyme of hepatocytes and leaks into the vascular space during hepatocellular damage. The serum half-life for ALT is rather short (1-2 days). Mild increases of serum ALT activities are considered to be non-specific for hepatocellular injury but moderate to severe increases are cause for concern and should prompt immediate further work-up of the patient for possible hepatobiliary disease.

Alkaline phosphatase shows species-specific differences. In the dog, increased serum ALP activities are most commonly due to hyperadrenocorticism, iatrogenic drug therapy (glucocorticoids, phenobarbital, or other), or biliary disease. However, primary hepatic disease can also be associated with increases of serum ALP activities. In cats, serum ALP activities are more specific for hepatobiliary disease.

Gamma glutamyl transferase is also non-specific for liver disease in dogs and cats but is more sensitive for liver disease in cats than it is in dogs.

Serum blood urea nitrogen, cholesterol, albumin, and glucose concentrations can all be decreased in dogs and cats with hepatic failure. However, these findings are rather insensitive and are also not specific for hepatic failure. Also, the liver synthesizes several coagulation factors and hepatic failure can thus lead to coagulopathies and abnormalities on a coagulation profile. It should be noted that the synthesis of these various molecules does not require the same degree of hepatic function. Serum albumin and cholesterol concentrations can be abnormal early during hepatic failure, while coagulopathies and hypoglycemia only occur in end-stage hepatic failure.

Plasma ammonia concentration

Plasma ammonia concentration can be used as a crude indicator for the presence of hepatic encephalopathy. The plasma sample must be placed on ice immediately after collection and needs to be analyzed within about 30 minutes from collection for accurate results. However, a plasma ammonia concentration within the reference range does not exclude hepatic encephalopathy as many other substances have been implicated in the pathogenesis of hepatoencephalopathy.

Some clinicians have recommended the use of an ammonia tolerance test. However, the author does not believe that an ammonia tolerance test yields any further information compared with pre- and postprandial serum bile acids concentrations.

Also, a recent study has suggested that the measurement of plasma ammonia concentration is superior to that of serum bile acids concentrations for the diagnosis of portosystemic shunts in dogs, but this needs to be confirmed by further studies.

Serum bile acids concentrations

Bile acids are metabolites of cholesterol degradation. They are formed and conjugated in the liver and secreted in the bile. After a meal cholecystokinin stimulates gall bladder contraction and release of bile into the duodenum. Conjugated bile acids play a crucial role in fat absorption as they help to emulsify fat. A small amount of bile acids are deconjugated by bacteria of the small intestinal microbiota. These unconjugated bile acids are absorbed in the small intestine and are no longer available for fat emulsification. In contrast, conjugated bile acids are absorbed in the large intestine, reach the vascular space, and are extracted from the portal blood by the liver.

Pre- and postprandial bile acids concentrations are used for the diagnosis of hepatic impairment and portosystemic shunting. Food is withheld from the patient for 12 hours and a serum sample is collected. A small amount of food, rich in fat, is fed to stimulate gall bladder contraction and another serum sample is collected 2 hours later.

When hepatic function is significantly impaired extraction of bile acids from the portal blood becomes less efficient and both pre- and postprandial serum bile acids concentrations increase. In patients with portosystemic vascular anomalies pre-prandial bile acids concentrations maybe only slightly increased, while post-prandial serum bile acids concentrations are often severely increased. However, the pattern of serum bile acids concentrations is not diagnostic of a specific hepatobiliary disorder and can only suggest the likelihood of one over another disorder. In some normal patients paradoxical results are observed in that pre-prandial bile acids concentrations are higher than post-prandial concentrations. It has been speculated that this finding is due to gall bladder contraction without food intake. However, it is important to note that this interpretation requires that both pre- and post-prandial serum bile acids concentrations are below the upper limit of the reference range for post-prandial serum bile acids concentrations. Also, increased pre-prandial bile acids concentrations have also been found in dogs with evidence of small intestinal dysbiosis.

Recently, the use of sulfated and non-sulfated urinary bile acids concentrations in dogs and cats with suspected hepatic disease has been described. However, further studies are necessary before their routine use can be suggested instead of serum bile acid concentrations.

13C-aminopyrine demethylation blood test

The aminopyrine demethylation breath test (ABT) has been shown to be useful in quantifying hepatic microsomal enzyme function in humans and laboratory animals. Aminopyrine, a compound chemically similar to the non-steroidal anti-inflammatory drugs antipyrine and phenylbutazone, is demethylated by microsomal enzymes in the liver. The liberated methyl groups are oxidized to CO2, which diffuses into the blood stream, reaches the pulmonary alveoli, and is released into the expiratory air. The administration of aminopyrine labeled with either 13C or 14C isotopes allows for the specific measurement of CO2 derived from aminopyrine, by detection of CO2 isotopes released in the expiratory air. Reproducible collection of breath samples can be difficult in veterinary species. Therefore, a 13C-aminopyrine demethylation blood test has been developed and is currently being evaluated in dogs and cats that undergo hepatic biopsy. Initial results have been promising.

Cytology

Cytology can be useful in arriving at a specific diagnosis in patients with a hepatopathy. However, cytology does not allow evaluation of hepatic architecture. Thus, the number of conditions where hepatic cytology is clinically useful is limited to hepatic neoplasia (if cells exfoliate) and hepatic lipidosis.

Liver biopsy

A liver biopsy is the best diagnostic test to arrive at a definitive diagnosis in pets with liver disease. A biopsy can be collected by use of an ultrasound-guided true-cut biopsy needle, by laparotomy, or by laparoscopy. The advantage of laparoscopy and laparotomy are that bigger pieces can be collected under direct visualization and that complications, such as bleeding, can be identified more rapidly. However, laparoscopy and laparotomy are also more invasive than a true-cut biopsy. Also, localized lesions in the center of a liver lobe may be missed by laparoscopy and laparotomy, but may easily be identified during abdominal ultrasonography and may be emendable to biopsy by use of a true-cut biopsy instrument. No matter what method is chosen, the patient must be carefully evaluated before collection of a hepatic biopsy. This evaluation should include complete blood work, but also a coagulation profile, a platelet count, and a buccal mucosal bleeding time. Multiple biopsies must be taken. One biopsy should be cultured and in dogs a second one should immediately frozen and stored for copper analysis if the histopathologist raises any questions concerning copper accumulation in the liver. After a biopsy is collected the patient should be carefully evaluated to identify any potential bleeding. It should also be noted that there can be disagreement between histopathologists as to the diagnosis. Thus, the diagnosis should be critically considered, especially if the patient does not respond to appropriate therapy for the condition diagnosed.