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november 08 Diagnostic Update 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 pathophysiol- The categorisation of icterus into a pre-hepatic/haemolytic, a ogy and the meaning of variations in values for bilirubin, bile acid hepatic or a post-hepatic/obstructive icterus is of key importance and ammonia concentrations. in considering the possible differential diagnoses Following this, some principles for the interpretation of cytological and histological findings in liver samples will be discussed, as the 1.1.1 Pre-hepatic hyperbilirubinaemia cyto- and histopathological investigations of liver samples are key Destruction of the erythrocytes (intravascular or extravascular to the early diagnosis of liver disease. haemolysis) is central to pre-hepatic icterus. The liver cannot cope with the uptake and processing of the additional bilirubin. 1 Physiology and pathology of bilirubin, bile acid and ammonia metabolism Erythrocyte breakdown that is sufficiently acute to cause hyperbilirubinaemia is usually associated with an obvious anaemia (haematocrit < 25%). The anaemia is regenerative with haemolysis. 1.1 Bilirubin Reticulocytosis may be mild at first (the first 2 – 4 days). Bilirubin is a breakdown product of haemoproteins. Its main origin is the haemoglobin from aging erythrocytes. Unconjugated Perhaps unexpectedly, dogs suffering from haemolysis often also bilirubin is first produced by the reticuloendothelial system, which exhibit bilirubinuria. Urine chemistry reagent strips (“DipStix”) is then bound to albumin and transported in the plasma. Bilirubin detect only conjugated bilirubin and not the unconjugated form is then conjugated with glucuronic acid in the liver, transported increased by the breakdown of the haemoproteins. Furthermore, through the biliary system with other components in the bile, and unconjugated bilirubin cannot pass through the glomerular mem- excreted into the digestive system. The bilirubin is then deconju- brane. However, small amounts of free bilirubin may be conju- gated to urobilinogen by bacteria in the ileum and colon. gated by the renal tubules and then excreted in the urine. 80 – 90% of the urobilinogen is eliminated in the faeces as stercobilin, while the remaining 10 – 20% is reabsorbed (enterohepatic Causes of pre-hepatic icterus may be, for example, an immune- circulation). mediated haemolytic anaemia, (either primary, or secondary to drugs, infection or neoplasia); infections (FeLV, Mycoplasma 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. haemofelis, others); oxidative damage to the erythrocytes (on- Clinical distinction between hepatic and post-hepatic icterus can ions, zinc, hypophosphataemia); or the resorption of blood (large be very difficult. However this is important, as the therapeutic haematoma). In the case of hyperbilirubinaemia, a pre-hepatic strategies are different for the two forms of the disease. Ultra- cause should always be looked for first in patients showing both sound investigation can be a useful aid in the distinction between hyperbilirubinaemia and anaemia. these forms of icterus. 1.1.2 Hepatic hyperbilirubinaemia 1.2 Bile acids In cases of icterus of hepatic origin, one of the stages of hepato- Bile acids (BA) are synthesised from cholesterol solely in the liver. cellular bilirubin transport is impaired. For example, inflammatory After conjugation with taurine or glycine, the BAs are secreted processes may result in swelling of the liver cells and impaired into the bile and stored in a concentrated form in the gall blad- biliary flow, which leads to obstruction of the intra-hepatic biliary der. After food intake, cholecystokinin triggers the contraction of system (cholestasis). the gall bladder and the release of BAs into the intestines. BAs play an important role in the digestion and absorption of fat in However, serum bilirubin is not a good indicator of hepatocellular the intestines. The BAs are transported into the portal circulation function. The liver can continue to metabolise bilirubin long after in the ileum and thus return to the liver. The BAs are once more other liver functions are already impaired. Thus hyperbilirubinae- extracted from the portal circulation in the liver. mia of hepatic origin indicates liver pathology is already moderate to severe. Although BA synthesis can be severely compromised in patients with severe liver disease, different factors can cause increased Frequent causes of hepatic icterus in cats are cholangitis/cholan- BA concentrations in the blood. These include changes to the giohepatitis, lymphosarcoma, hepatic lipidosis and FIP. enterohepatic circulation, a portosystemic shunt (PSS), reduced In dogs, triggers for hepatic icterus include chronic hepatitis (idi- extraction of BAs from the blood by the liver, or regurgitation of opathic, hereditary), lymphosarcoma, acute hepatic necrosis and/ BAs into the systemic circulation due to cholestasis. This is why or cirrhosis. Drugs (anti-epileptic drugs, trimethoprim-sulphona- many animals suffering from chronic hepatitis, extensive hepatic mide, others) and systemic diseases with a hepatic component necrosis, or liver neoplasia often exhibit raised BA values. BAs, may also cause icterus. Bacterial toxins and antibodies against together with ammonia, are among the most sensitive biochemi- components in the bacterial cell walls can also cause cholestasis cal indicators of a congenital PSS. Most patients suffering from (cholestasis due to sepsis). In contrast, hepatic atrophy, as is often a congenital portal vascular anomaly have increased values for seen in animals with a congenital portosystemic vascular anomaly, postprandial bile acid concentrations. is not normally associated with a hyperbilirubinaemia, even though In contrast, BA values are often not greatly raised in cases of a other indications for impaired liver function (hypoalbuminaemia, secondary involvement of the liver due to a non-hepatic primary low urea concentrations) are present. disease, administration of glucocorticoids or anti-epileptic drugs. There are a number of factors that can result in lowered BA serum 1.1.3 Post-hepatic hyperbilirubinemia concentrations. Some examples are anorexia lasting more than In post-hepatic icterus, intraluminal or extraluminal problems 1-2 days (fasting BA value possibly lowered); delayed emptying result in a mechanical obstruction to the excretion of bilirubin. of the stomach; changes in intestinal transit time; malabsorption, Examples of this are pancreatitis, neoplasia (bile ducts, pancreas, severe disease; and resection of the ileum. duodenum), cholelithiasis, rupture of the gall bladder or a bile duct with biliary peritonitis. 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. 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 conduc- tion of the test, as well as individual variation in emptying of the 2 Cytology and histology of liver samples stomach, response to cholecystokinin secretion, or intestinal transit times. Differentiation between different liver diseases is usually not possible based on the clinical symptoms and changes in laboratory Determination of BA concentrations is not indicated in icteric values alone. Further information should be gained by taking patients with hepatobiliary disease as it does not provide any liver samples for cytological and histological examinations. Such additional information. Severe haemolysis or severe lipaemia may sampling is not only indicated to reach an accurate diagnosis, result in false lowered results. but also to obtain clues on the response to treatment and disease Please note that Maltese dogs are distinctive in that they can progression. exhibit raised postprandial values without suffering from hepatobiliary disease. The investigation of material that has been obtained using fine needle aspiration (cytology) is certainly less informative than ma- Two qualifying points must be considered in the interpretation terial obtained through a biopsy (histology). However, fine needle of BA values. Firstly, different hepatobiliary diseases cannot be aspiration also has advantages such as less danger of haemor- distinguished from one another based on BA determinations. rhage, and it is easier to perform. In addition, especially in cases Secondly, there is a very poor correlation between the severity of of diffuse diseases, fine needle aspiration can result in diagnosis, histological lesions or the degree of a PSS, and the magnitude of e.g. for lymphosarcomas, mast cell tumours, hepatic lipidosis, increase in BA values. The only reliable indicator for clinical remis- corticosteroid- induced changes and amyloidosis. It also enables sion is a return to normal values, following several BA determina- diagnosis of a hepatocellular carcinoma. tions conducted in a patient in order to assess disease progression or therapeutic response. A liver biopsy is taken using ultrasound monitoring or within the framework of a laparotomy to collect samples. Animals with 1.3 Ammonia hepatopathies exhibit a tendency towards coagulopathies and For the purposes of detoxification, the ammonia (NH3) produced coagulation testing is therefore recommended prior to the proce- in protein metabolism is transformed into urea (urea cycle) in the dure. mitochondria of the liver cells. Raised ammonia concentrations Parenteral administration of vitamin K1 can be used prophylacti- can occur in cases of hepatic insufficiency, when the liver can no cally 24 hours before the biopsy is taken. A plasma transfusion longer detoxify the ammonia, or when the portal blood is not flow- may help if a biopsy must be taken even though a coagulopathy ing through the liver (PSS). The significance of ammonia determi- is present. The interpretation of the histological findings can be- nation is similar to that of bile acid determination. It has recently come complicated if the sample is too small or not representative been demonstrated that fasting ammonia values are slightly more of the lesion. sensitive and, above all, more specific than bile acid values for the diagnosis of a congenital or acquired PSS. Even if the histological investigation of a liver sample does not always guarantee an aetiological diagnosis, as a rule, the following There are two disadvantages of ammonia analysis compared statements are possible: with the bile acid analysis. Firstly, there are the more complicated handling requirements for the sample – analysis is usually only 1. Disease category: inflammatory/necrotic, neoplastic, practicable in the practice environment. The reader is advised vacuolated and vascular to take particular care to follow the relevant instructions for the 2. Extent of the disease: mild/moderate/severe analyser. Secondly, the conduct of an ammonium chloride toler- 3. Chronicity of the lesion: acute versus chronic ance test in animals suffering from encephalopathy can result in a deterioration in the neurological symptoms. 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. For example, bridging fibrosis (connective tissue connections In summary, it should be noted that in spite of the limitations of between portal triads or portal triads and the central vein) indicate cytological and histological investigations of liver samples, these a poor prognosis. Eosinophilic granulocytes often indicate an can serve a very important role in the treatment of patients with a allergic or parasitic event. 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 le- Author: sions indicate the presence of lipid or glycogen accumulation and PSS also exhibits specific histological characteristics. 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 Dr. med. vet. Cécile Rohrer Kaiser sample. In addition, samples can be obtained for aerobic and Dipl. ACVIM (Internal Medicine) and ECVIM-CA (Internal Medicine) anaerobic bacterial cultures. 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 Clinical symptoms the digestion of fats), a storage organ (glycogen, vitamins, trace The clinical symptoms of liver disease are often unspecific or may elements), an organ where synthesis occurs (albumin, fibrinogen, be completely absent. Signs include lethargy, appetite disor- prothrombin), and it participates in immune regulation (Kupffer’s ders, weight loss, lowered performance, dull coat, dermatoses, cells). neurological symptoms, icterus, photosensitisation, abdominal pain and coagulation disorders. Behavioural disorders are to be Liver function is only impaired once more than 80% of the liver regarded as typical symptoms for advanced liver disease, caused has been damaged. However, the liver does possess a unique by the malfunction of ammonia detoxification by the damaged capacity for maintaining its specific functions and simultaneously liver (hepatoencephalopathy). repairing and regenerating its own tissue. The severity of the clinical symptoms and the course of a liver disAetiology ease can vary substantially depending on the distribution pattern, Liver disease is relatively common in horses, but usually location and extent of damage to the liver. Basically, a distinction progresses without any clear clinical symptoms. Liver disease must be made between reversible diseases (e.g., swelling, fatty often occurs secondarily, i.e. due to other diseases (viral, bacte- degeneration) and irreversible damage (necrosis), which may rial, parasitic infectious disease, internal disease, fatty liver). Such both be focal (abscess, neoplasia) and/or zonal (centrilobular) in diseases can, however, also be caused directly by contaminated their extent. An acute generalized hepatitis causes a loss in func- feed (mycotoxins) or toxic plants. A full case history is therefore of tion that is usually associated with enlargement of the liver. great importance with regard to treatment. 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. Diagnosis of liver disease in young animals due to their physiology, specifically the more The permeability of the cell membrane is impaired in many active bone metabolism in animals during growth. pathological processes. Enzymes that are mainly intracellular can therefore be released into the blood plasma, where they can be AST (aspartate aminotransferase) / GOT (glutamate-oxalacetate measured. transaminase) occurs in the mitochondria and in the cytoplasm of liver cells, but also in muscle cells and is therefore not liver- Enzymes specific to the liver in horses specific. Particularly high levels are observed during the course of GGT is located in membrane structures, mainly of the biliary advanced myopathies (sporadic exertional rhabdomyolysis). system. Its half-life is approximately 3 days. It is released at an early stage Assessment of liver function in liver disease and is often the only parameter that is raised in Bile acids are synthesised from cholesterol in the hepatocytes. chronic metabolic disorders affecting the liver. GGT can also These are continually released into the duodenum (approx. continue to rise for 1-2 weeks after the cause of the increase 3l/100kg BW) in horses, where they enable the digestion of fats. in levels has been treated. During convalescence, it may also They are then partially reabsorbed in the enterohepatic cycle. In increase if the horse is under pressure, so can be used to monitor liver disease, the bile acids can no longer be adequately elimi- the workload. nated and therefore they accumulate. The finding of raised levels of bile acids is therefore useful to assess liver function, but does GLDH (glutamate dehydrogenase) is an enzyme that is bound to not indicate the type of liver disease. the mitochondria of the liver cells. Most of its activity is centrilobular, i.e. it reacts highly sensitively to secondary hepatopathies Ammonia is neurotoxic and is produced in the intestines dur- (cholestasis, hypoxaemia). An increase to more than 3 times the ing protein digestion. It enters the liver through the portal vein, normal value indicates the presence of an acute hepatopathy with where it is transformed into urea and is then excreted through liver cell necrosis. Milder increases are seen during infections, the kidneys. This capacity for conversion to urea is restricted in fever or the administration of drugs. Its half-life is approximately functional disorders of the liver and blood ammonia concentra- 3 days. tions rise as a result. CNS function can be impaired as ammonia is neurotoxic (hepa- Other enzymes toencephalopathy). Determinations should only be made on ALKP (alkaline phosphatase) is an enzyme bound to the mi- blood that has been centrifuged immediately after collection and tochondrial membrane and occurs in many organs (bile duct on frozen EDTA plasma, as ammonia is highly unstable in blood epithelia, osteoblasts). samples. Increases in enzyme levels are observed in cholestasis, but also after the administration of drugs (corticosteroids). ALKP is higher 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 Diagnostic Update Authors: in nephropathies, enteropathies, body cavity effusions and malnutrition. There may be reduced synthesis of coagulation factors in severe liver disorders. Further investigations Andrea Hille Following on from the assessment of clinical symptoms and laboratory tests, ultrasound examinations and a liver Expert Advisor on Horses biopsy can Dr. Susi Zintner Horse Veterinary Specialist, Key Account Manager, Medical Advisor 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. Diagnostic options for liver diseases IDEXX Reference Laboratory Liver Profile IDEXX VetLab® Suite IDEXX Catalyst Dx™ Chemistry Analyser £ 20.00 + VAT Total protein, Albumin, Globulin, Albumin:Globulin Ratio, Urea, ALT, ALP, GGT, Total Bilirubin, Bile Acid, Cholesterol Liver Profile plus Bile Acid Stimulation IDEXX SNAPshot Dx™ Analyser Coag Dx™ Analyser £ 23.00 + VAT VetTest® Chemistry Analyser as for Liver Profile + Bile Acid Stimulation Test Liver & Kidney Profile 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 SNAP® Reader Bile acid and hormone analyser £ 32.50 + VAT £ 28.50 + VAT Platelet Count, Morphological Assessment, PT, APTT, Fibrinogen ( Please contact our clinical pathology team for further information: 01937 544 000 IDEXX Reference Laboratory Southwater IDEXX Reference Laboratory Wetherby 4 Oakhurst, Business Park, Southwater, Horsham, West Sussex RH13 9RT Tel: 01403 730176, Fax: 01403 732784 Grange House, Sandbeck Way Wetherby, West Yorkshire LS22 7DN Tel: 01937 544000, Fax: 01937 544001 [email protected] www.idexx.eu/united-kingdom/ UK033-1008