Update. Diagnostic DIAGNOSTIC TECHNIQUES FOR LIVER DISEASE IN DOGS, CATS (PART 2/2) AND HORSES

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1 Diagnostic Update november 08 DIAGNOSTIC TECHNIQUES FOR LIVER DISEASE IN DOGS, CATS (PART 2/2) AND HORSES The liver is strategically positioned between the digestive tract and the systemic circulation. Arterial blood flows directly from the aorta to the liver, ensuring a good oxygen supply. The liver receives venous blood from the abdominal organs (stomach, pancreas, spleen and intestines) via the portal vein. Amongst other compounds, metabolites and foreign substances are delivered to the liver in this venous blood for detoxification or biotransformation. The liver and portal circulation also play a central role in the metabolism of bile acids, ammonia and bilirubin. This section of the diagnostic update will discuss pathophysiology and the meaning of variations in values for bilirubin, bile acid and ammonia concentrations. Following this, some principles for the interpretation of cytological and histological findings in liver samples will be discussed, as the cyto- and histopathological investigations of liver samples are key to the early diagnosis of liver disease. 1 Physiology and pathology of bilirubin, bile acid and ammonia metabolism 1.1 Bilirubin Bilirubin is a breakdown product of haemoproteins. Its main origin is the haemoglobin from aging erythrocytes. Unconjugated bilirubin is first produced by the reticuloendothelial system, which is then bound to albumin and transported in the plasma. Bilirubin is then conjugated with glucuronic acid in the liver, transported through the biliary system with other components in the bile, and excreted into the digestive system. The bilirubin is then deconjugated to urobilinogen by bacteria in the ileum and colon % of the urobilinogen is eliminated in the faeces as stercobilin, while the remaining 10 20% is reabsorbed (enterohepatic circulation). Icterus is a syndrome that is characterised by hyperbilirubinaemia and a yellow colouration of the skin, mucosa and sclera due to the deposition of bile pigments. Serum bilirubin concentration in dogs and cats should not normally exceed 7 micromol/l. The separated plasma becomes icteric once serum bilirubin concentrations exceed approximately 17 micromol/l. Obvious icterus is present when serum bilirubin concentrations exceeds approximately 30 micromol/l. The yellow discolouration is most clearly recognised under natural light and in the following organs: sclera, oral mucosa, the pinnae of the ears, the skin around the navel and the penile and vaginal mucosae. The categorisation of icterus into a pre-hepatic/haemolytic, a hepatic or a post-hepatic/obstructive icterus is of key importance in considering the possible differential diagnoses Pre-hepatic hyperbilirubinaemia Destruction of the erythrocytes (intravascular or extravascular haemolysis) is central to pre-hepatic icterus. The liver cannot cope with the uptake and processing of the additional bilirubin. Erythrocyte breakdown that is sufficiently acute to cause hyperbilirubinaemia is usually associated with an obvious anaemia (haematocrit < 25%). The anaemia is regenerative with haemolysis. Reticulocytosis may be mild at first (the first 2 4 days). Perhaps unexpectedly, dogs suffering from haemolysis often also exhibit bilirubinuria. Urine chemistry reagent strips ( DipStix ) detect only conjugated bilirubin and not the unconjugated form increased by the breakdown of the haemoproteins. Furthermore, unconjugated bilirubin cannot pass through the glomerular membrane. However, small amounts of free bilirubin may be conjugated by the renal tubules and then excreted in the urine. Causes of pre-hepatic icterus may be, for example, an immunemediated haemolytic anaemia, (either primary, or secondary to drugs, infection or neoplasia); infections (FeLV, Mycoplasma

2 False positive results can occur. Postprandial values should still be determined if preprandial values have been determined and these are normal. Higher fasting values than postprandial BA values are occasionally found. The causes of this are interdigestive gall bladder contractions during fasting prior to the conduchaemofelis, others); oxidative damage to the erythrocytes (onions, zinc, hypophosphataemia); or the resorption of blood (large haematoma). In the case of hyperbilirubinaemia, a pre-hepatic cause should always be looked for first in patients showing both hyperbilirubinaemia and anaemia. Clinical distinction between hepatic and post-hepatic icterus can be very difficult. However this is important, as the therapeutic strategies are different for the two forms of the disease. Ultrasound investigation can be a useful aid in the distinction between these forms of icterus Hepatic hyperbilirubinaemia In cases of icterus of hepatic origin, one of the stages of hepatocellular bilirubin transport is impaired. For example, inflammatory processes may result in swelling of the liver cells and impaired biliary flow, which leads to obstruction of the intra-hepatic biliary system (cholestasis). However, serum bilirubin is not a good indicator of hepatocellular function. The liver can continue to metabolise bilirubin long after other liver functions are already impaired. Thus hyperbilirubinaemia of hepatic origin indicates liver pathology is already moderate to severe. Frequent causes of hepatic icterus in cats are cholangitis/cholangiohepatitis, lymphosarcoma, hepatic lipidosis and FIP. In dogs, triggers for hepatic icterus include chronic hepatitis (idiopathic, hereditary), lymphosarcoma, acute hepatic necrosis and/ or cirrhosis. Drugs (anti-epileptic drugs, trimethoprim-sulphonamide, others) and systemic diseases with a hepatic component may also cause icterus. Bacterial toxins and antibodies against components in the bacterial cell walls can also cause cholestasis (cholestasis due to sepsis). In contrast, hepatic atrophy, as is often seen in animals with a congenital portosystemic vascular anomaly, is not normally associated with a hyperbilirubinaemia, even though other indications for impaired liver function (hypoalbuminaemia, low urea concentrations) are present Post-hepatic hyperbilirubinemia In post-hepatic icterus, intraluminal or extraluminal problems result in a mechanical obstruction to the excretion of bilirubin. Examples of this are pancreatitis, neoplasia (bile ducts, pancreas, duodenum), cholelithiasis, rupture of the gall bladder or a bile duct with biliary peritonitis. 1.2 Bile acids Bile acids (BA) are synthesised from cholesterol solely in the liver. After conjugation with taurine or glycine, the BAs are secreted into the bile and stored in a concentrated form in the gall bladder. After food intake, cholecystokinin triggers the contraction of the gall bladder and the release of BAs into the intestines. BAs play an important role in the digestion and absorption of fat in the intestines. The BAs are transported into the portal circulation in the ileum and thus return to the liver. The BAs are once more extracted from the portal circulation in the liver. Although BA synthesis can be severely compromised in patients with severe liver disease, different factors can cause increased BA concentrations in the blood. These include changes to the enterohepatic circulation, a portosystemic shunt (PSS), reduced extraction of BAs from the blood by the liver, or regurgitation of BAs into the systemic circulation due to cholestasis. This is why many animals suffering from chronic hepatitis, extensive hepatic necrosis, or liver neoplasia often exhibit raised BA values. BAs, together with ammonia, are among the most sensitive biochemical indicators of a congenital PSS. Most patients suffering from a congenital portal vascular anomaly have increased values for postprandial bile acid concentrations. In contrast, BA values are often not greatly raised in cases of a secondary involvement of the liver due to a non-hepatic primary disease, administration of glucocorticoids or anti-epileptic drugs. There are a number of factors that can result in lowered BA serum concentrations. Some examples are anorexia lasting more than 1-2 days (fasting BA value possibly lowered); delayed emptying of the stomach; changes in intestinal transit time; malabsorption, severe disease; and resection of the ileum. Determination of BA concentrations is routinely conducted as a screening test for liver function in small animal veterinary practices, as the collection of samples from the patients and laboratory determination are both straightforward. Maximum information is obtained through determination of a 12-hour fasting and a 2-hour postprandial value. Suitable food for the determination of the postprandial value in dogs and cats is canned food with a moderate fat content: two teaspoons for animals of below 5 kg or two dessertspoons of food for heavier animals.

3 tion of the test, as well as individual variation in emptying of the stomach, response to cholecystokinin secretion, or intestinal transit times. Determination of BA concentrations is not indicated in icteric patients with hepatobiliary disease as it does not provide any additional information. Severe haemolysis or severe lipaemia may result in false lowered results. Please note that Maltese dogs are distinctive in that they can exhibit raised postprandial values without suffering from hepatobiliary disease. Two qualifying points must be considered in the interpretation of BA values. Firstly, different hepatobiliary diseases cannot be distinguished from one another based on BA determinations. Secondly, there is a very poor correlation between the severity of histological lesions or the degree of a PSS, and the magnitude of increase in BA values. The only reliable indicator for clinical remission is a return to normal values, following several BA determinations conducted in a patient in order to assess disease progression or therapeutic response. 1.3 Ammonia For the purposes of detoxification, the ammonia (NH3) produced in protein metabolism is transformed into urea (urea cycle) in the mitochondria of the liver cells. Raised ammonia concentrations can occur in cases of hepatic insufficiency, when the liver can no longer detoxify the ammonia, or when the portal blood is not flowing through the liver (PSS). The significance of ammonia determination is similar to that of bile acid determination. It has recently been demonstrated that fasting ammonia values are slightly more sensitive and, above all, more specific than bile acid values for the diagnosis of a congenital or acquired PSS. There are two disadvantages of ammonia analysis compared with the bile acid analysis. Firstly, there are the more complicated handling requirements for the sample analysis is usually only practicable in the practice environment. The reader is advised to take particular care to follow the relevant instructions for the analyser. Secondly, the conduct of an ammonium chloride tolerance test in animals suffering from encephalopathy can result in a deterioration in the neurological symptoms. 2 Cytology and histology of liver samples Differentiation between different liver diseases is usually not possible based on the clinical symptoms and changes in laboratory values alone. Further information should be gained by taking liver samples for cytological and histological examinations. Such sampling is not only indicated to reach an accurate diagnosis, but also to obtain clues on the response to treatment and disease progression. The investigation of material that has been obtained using fine needle aspiration (cytology) is certainly less informative than material obtained through a biopsy (histology). However, fine needle aspiration also has advantages such as less danger of haemorrhage, and it is easier to perform. In addition, especially in cases of diffuse diseases, fine needle aspiration can result in diagnosis, e.g. for lymphosarcomas, mast cell tumours, hepatic lipidosis, corticosteroid- induced changes and amyloidosis. It also enables diagnosis of a hepatocellular carcinoma. A liver biopsy is taken using ultrasound monitoring or within the framework of a laparotomy to collect samples. Animals with hepatopathies exhibit a tendency towards coagulopathies and coagulation testing is therefore recommended prior to the procedure. Parenteral administration of vitamin K1 can be used prophylactically 24 hours before the biopsy is taken. A plasma transfusion may help if a biopsy must be taken even though a coagulopathy is present. The interpretation of the histological findings can become complicated if the sample is too small or not representative of the lesion. Even if the histological investigation of a liver sample does not always guarantee an aetiological diagnosis, as a rule, the following statements are possible: 1. Disease category: inflammatory/necrotic, neoplastic, vacuolated and vascular 2. Extent of the disease: mild/moderate/severe 3. Chronicity of the lesion: acute versus chronic Chronic inflammatory lesions pose a particular challenge to pathologists, as the liver reacts in a similar histological fashion to different types of chronic insult (whether due to toxins, infection or immune stimulation). Degenerative changes to the liver cells, inflammatory infiltrates, fibrosis and necrosis are possible manifestations of an inflammatory event. The inflammatory cell type should be noted when assessing inflammatory changes: Neutrophilic granulocytes ( acute ) are often present at the start of an inflammation, with the addition later on of lymphocytes and macrophages. The presence of fibrosis is an indicator for a chronic event. The degree of fibrosis is correlated with survival time.

4 For example, bridging fibrosis (connective tissue connections between portal triads or portal triads and the central vein) indicate a poor prognosis. Eosinophilic granulocytes often indicate an allergic or parasitic event. In summary, it should be noted that in spite of the limitations of cytological and histological investigations of liver samples, these can serve a very important role in the treatment of patients with a hepatopathy. Although histological analysis does not always permit a definitive aetiological diagnosis, it generally does provide indicators for a possible aetiology. For example, centri-lobular lesions occur in passive congestion of the liver, while infections like salmonellosis or toxoplasmosis result in (multi-) focal lesions. Vacuolated lesions indicate the presence of lipid or glycogen accumulation and PSS also exhibits specific histological characteristics. Author: Further investigations can be instigated if required. For example, specific staining can be an aid in the identification of certain infectious pathogens or permit estimates of copper content in a liver sample. In addition, samples can be obtained for aerobic and anaerobic bacterial cultures. Dr. med. vet. Cécile Rohrer Kaiser Dipl. ACVIM (Internal Medicine) and ECVIM-CA (Internal Medicine) LIVER DIAGNOSIS IN HORSES The liver is one of the main metabolic organs and, as such, is positioned between the digestive tract and the systemic circulation. The majority of compounds absorbed from the gastrointestinal tract reach the liver directly through the portal circulation. The liver is the regulatory site of carbohydrate, protein and lipid metabolism. In addition, the liver is an organ responsible for excretion (bile for the digestion of fats), a storage organ (glycogen, vitamins, trace elements), an organ where synthesis occurs (albumin, fibrinogen, prothrombin), and it participates in immune regulation (Kupffer s cells). Liver function is only impaired once more than 80% of the liver has been damaged. However, the liver does possess a unique capacity for maintaining its specific functions and simultaneously repairing and regenerating its own tissue. Aetiology Liver disease is relatively common in horses, but usually progresses without any clear clinical symptoms. Liver disease often occurs secondarily, i.e. due to other diseases (viral, bacterial, parasitic infectious disease, internal disease, fatty liver). Such diseases can, however, also be caused directly by contaminated feed (mycotoxins) or toxic plants. A full case history is therefore of great importance with regard to treatment. Clinical symptoms The clinical symptoms of liver disease are often unspecific or may be completely absent. Signs include lethargy, appetite disorders, weight loss, lowered performance, dull coat, dermatoses, neurological symptoms, icterus, photosensitisation, abdominal pain and coagulation disorders. Behavioural disorders are to be regarded as typical symptoms for advanced liver disease, caused by the malfunction of ammonia detoxification by the damaged liver (hepatoencephalopathy). The severity of the clinical symptoms and the course of a liver disease can vary substantially depending on the distribution pattern, location and extent of damage to the liver. Basically, a distinction must be made between reversible diseases (e.g., swelling, fatty degeneration) and irreversible damage (necrosis), which may both be focal (abscess, neoplasia) and/or zonal (centrilobular) in their extent. An acute generalized hepatitis causes a loss in function that is usually associated with enlargement of the liver. Clinical symptoms only appear when more than 80% of the liver has been damaged in chronic generalised fibrosis (in the final stages of cirrhosis). In this case, the liver is reduced in size.

5 Diagnosis of liver disease The permeability of the cell membrane is impaired in many pathological processes. Enzymes that are mainly intracellular can therefore be released into the blood plasma, where they can be measured. Enzymes specific to the liver in horses GGT is located in membrane structures, mainly of the biliary system. Its half-life is approximately 3 days. It is released at an early stage in liver disease and is often the only parameter that is raised in chronic metabolic disorders affecting the liver. GGT can also continue to rise for 1-2 weeks after the cause of the increase in levels has been treated. During convalescence, it may also increase if the horse is under pressure, so can be used to monitor the workload. GLDH (glutamate dehydrogenase) is an enzyme that is bound to the mitochondria of the liver cells. Most of its activity is centrilobular, i.e. it reacts highly sensitively to secondary hepatopathies (cholestasis, hypoxaemia). An increase to more than 3 times the normal value indicates the presence of an acute hepatopathy with liver cell necrosis. Milder increases are seen during infections, fever or the administration of drugs. Its half-life is approximately 3 days. Other enzymes ALKP (alkaline phosphatase) is an enzyme bound to the mitochondrial membrane and occurs in many organs (bile duct epithelia, osteoblasts). Increases in enzyme levels are observed in cholestasis, but also after the administration of drugs (corticosteroids). ALKP is higher in young animals due to their physiology, specifically the more active bone metabolism in animals during growth. AST (aspartate aminotransferase) / GOT (glutamate-oxalacetate transaminase) occurs in the mitochondria and in the cytoplasm of liver cells, but also in muscle cells and is therefore not liverspecific. Particularly high levels are observed during the course of advanced myopathies (sporadic exertional rhabdomyolysis). Assessment of liver function Bile acids are synthesised from cholesterol in the hepatocytes. These are continually released into the duodenum (approx. 3l/100kg BW) in horses, where they enable the digestion of fats. They are then partially reabsorbed in the enterohepatic cycle. In liver disease, the bile acids can no longer be adequately eliminated and therefore they accumulate. The finding of raised levels of bile acids is therefore useful to assess liver function, but does not indicate the type of liver disease. Ammonia is neurotoxic and is produced in the intestines during protein digestion. It enters the liver through the portal vein, where it is transformed into urea and is then excreted through the kidneys. This capacity for conversion to urea is restricted in functional disorders of the liver and blood ammonia concentrations rise as a result. CNS function can be impaired as ammonia is neurotoxic (hepatoencephalopathy). Determinations should only be made on blood that has been centrifuged immediately after collection and on frozen EDTA plasma, as ammonia is highly unstable in blood samples. Bilirubin is a breakdown product of haemoglobin that is insoluble in water and is transported to the liver bound to albumin. Once in the liver, it is conjugated with glucuronic acid in the hepatocytes and secreted into the intestines in a water-soluble form via the bile. From the intestines, it is either excreted in the faeces or reabsorbed (enterohepatic circulation). Bilirubin is not a sensitive indicator for liver disease, as it is not only raised in cases of hepatopathies, but also due to haemolysis (babesiosis, infectious anaemia, neonatal icterus), anorexia (inanition icterus) and colic. Persistent hyperbilirubinaemia has been described in otherwise healthy horses (Gilbert s syndrome, Crigler-Najjar syndrome). Plasma proteins are mainly synthesised in the liver (albumin is synthesised in the liver only), with the exception of the immunoglobulins. Synthesis of these proteins is limited in most severe and/or chronic liver diseases. Measuring the concentrations of these proteins can assess the liver s capacity for synthesis. Albumin concentrations are of little significance for diagnosis as synthesis is reduced only in late on in the course of liver disease.furthermore, albumin concentrations may also be lowered

6 Diagnostic Update in nephropathies, enteropathies, body cavity effusions and malnutrition. Authors: There may be reduced synthesis of coagulation factors in severe liver disorders. Further investigations Following on from the assessment of clinical symptoms and laboratory tests, ultrasound examinations and a liver biopsy can provide further information on the severity of the disease and its prognosis. The aetiology often remains unclear even after using these techniques. It is always recommended to check the coagulation factors prior to conducting a liver biopsy. Andrea Hille Expert Advisor on Horses Dr. Susi Zintner Horse Veterinary Specialist, Key Account Manager, Medical Advisor Diagnostic options for liver diseases IDEXX Reference Laboratory Liver Profile VAT Total protein, Albumin, Globulin, Albumin:Globulin Ratio, Urea, ALT, ALP, GGT, Total Bilirubin, Bile Acid, Cholesterol Liver Profile plus Bile Acid Stimulation VAT as for Liver Profile + Bile Acid Stimulation Test Liver & Kidney Profile VAT Total Protein, Albumin, Globulin, Albumin:Globulin Ratio, Urea, Creatinine, ALT, ALP, GGT, AST, Total Bilirubin, Bile Acid, Sodium, Potassium, Sodium:Potassium ratio, Cholesterol, Calcium & Inorganic Phosphate Coagulation Profile VAT Platelet Count, Morphological Assessment, PT, APTT, Fibrinogen IDEXX VetLab Suite IDEXX Catalyst Dx Chemistry Analyser IDEXX SNAPshot Dx Analyser Coag Dx Analyser VetTest Chemistry Analyser SNAP Reader Bile acid and hormone analyser Please contact our clinical pathology team for further information: ( IDEXX Reference Laboratory Southwater 4 Oakhurst, Business Park, Southwater, Horsham, West Sussex RH13 9RT Tel: , Fax: labhelp@idexx.com IDEXX Reference Laboratory Wetherby Grange House, Sandbeck Way Wetherby, West Yorkshire LS22 7DN Tel: , Fax: UK