Role of biomarkers in diagnosis of liver diseases

Dr.Rohit Parmar
Major Guide; Dr.D.V.Joshi M.V.Sc.(Pathology)

Department of Pathology College of Veterinary Science and A.H. S. D. Agricultural University Sardarkrushinagar, Gujarat
Liver is the largest gland of the body which performs many useful functions like filtration, detoxification, storage and formation of bile. There are various types of hepatic injury which includes hepatic lipidosis, hepatic necrosis, cholestasis, glycogen accumulation, bile duct hyperplasia, hepatic fibrosis and cirrhosis. Clinical manifestation of hepatic insufficiency mostly includes jaundice which usually occurs in acute hepatitis, biliary obstruction, or advance chronic liver diseases. The liver is an important site in drug toxicity and oxidative stress because of its proximity and relationship to the gastrointestinal tract. Toxic responses occur frequently in the liver as compared to other organs because of its high metabolic capability and the portal blood supply. Liver can be damaged by overdose or toxic effect of certain drugs. The example of hepatotoxic drugs includes acetaminophen, amoxiciline, methotrexate, isoniazide, amidorane, anabolic steroids, chlorpromazine, CCl4 and various heavy metals. Although the exact mechanism of DILI remains largely unknown, it appears to involve 2 pathways direct hepatotoxicity and adverse immune reactions. In most instances, DILI is initiated by the bioactivation of drugs to chemically reactive metabolites, which have the ability to interact with cellular macromolecules such as proteins, lipids, and nucleic acids, leading to protein dysfunction, lipid peroxidation, DNA damage, and oxidative stress. According to US acute liver failure study group DILI is account for >50% of acute liver failure including hepatotoxicity caused by overdose of APAP (Acetaminophen). Various inflammatory products like cytokines and interferons produced during DILI are involved in tissue damage. (Blazka et al., 1995). The pathogenesis of the hepatic diseases of domestic animal species is remarkably complex, involving acute and chronic forms of hepatitis, cirrhosis, bile duct obstruction, intrahepatic forms of cholestasis, neoplasia, and disorders of hepatic vasculature. The frequency of these diseases varies with species, breed, age, and, in some cases, by environment (diet, geographical location).Diagnosis of hepatic disease involves the evaluation of clinical history, physical examination, biochemical tests, hepatic imaging and histopathological examination of hepatic biopsies. The laboratory testing of liver function usually involves some aspect of the livers role in intermediary metabolism. A good understanding of the livers basic functions will assist interpretation of results. In animals with suspected liver disease, the minimum database should include complete blood count, serum chemistry, urinalysis, and fecal flotation. In clinical patients with a history and signs suggestive of hepatic disease, laboratory tests are used for confirmation. Laboratory tests are used to assess the severity of liver injury, to establish prognosis, to define treatable complications of hepatic insufficiency and to monitor clinical progress. Finally, biochemical tests of hepatic function may be performed on clinically healthy patients that are known to be at high risk of developing liver disease. Consensus conference statements recommended liver biopsy in the management of almost all patients with hepatitis C and B. Liver biopsy is
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considered as gold standard for diagnosis of liver injury but it has certain disadvantages which include percuteneous insertion of needle, chance of damage to organs other than liver, significant bleeding and the size of tissue sample collected (Guido and Rugge, 2004). Hence, laboratory tests especially clinical biochemistry tests are most commonly employed on the patient with hepatic disease. Biomarker, in general, is a substance used as an indicator of a biological state. It is a characteristic that is objectively measured and evaluated as an indicator of normal biological process, pathogenic process or pharmacological responses to a therapeutic intervention. Those substances used as liver biomarker should be liver specific, easy to perform, measurable, have capacity to reflect stage of liver disease and most importantly it should be inexpensive. Certain criteria are used to classify biomarker such as their characteristics, application, or their use in fibrosis i.e. direct and indirect biomarker. The presence of liver disease often is recognized on the basis of elevated serum activities of enzymes of hepatic origin. serum enzymes do not measure hepatic function directly but indicate alteration in the integrity of the cell membrane of the hepatocyte, necrosis of hepatocytes or biliary epithelium, impeded bile formation or bile flow (cholestasis), or the induction of enzyme synthesis ( Center, 2007 ). Among the different biomarkers of hepatocellular injury, the most commonly employed are serum Alanine and Aspartate Aminotransferases. Measurement of serum ALT is used routinely for the assessment of hepatocellular injury in dog, cat, human and rodents (Kaneko et al., 2008). Hepatic ALT activity is lower in horses, cattle, sheep, and swine, and in these species, serum ALT is not measured routinely. The Level of ALT guides the urgency and extent of further investigation. ALT also has significant value in hepatitis, auto immune and cholestatic liver diseases. ALT rises dramatically in acute liver damage, such as all types of hepatitis (viral, drug-induced etc) or paracetamol (acetaminophen) overdose. Acute Auto immune hepatitis, hepatic lymphoma, or acute billiary occlusion than highly elevated ALT activity found. Serum Alanine Aminotransferase (ALT) as a general rule ALT level greater than 3 times the upper limit of normal has been considered as a marker of liver diseases. In all domestic animals, AST activity is higher in liver and serum activity characteristically increases in acute and chronic liver diseases. AST level is dramatically affected by shock, low blood pressure or any other condition that may deprive the liver of blood or oxygen. AST is also increase in centrilobular and periportal necrosis. Because of the relatively low ALT activity in the livers of large domestic species (Keller et al., 1985), other liver-specific enzymes have been validated for clinical use. One of these, SDH, is a zinc metalloenzyme. The serum activity of SDH has been shown to be useful in assessment of hepatocellular injury in most domestic species including the dog (Valentine et al., 1990), horses (Asquith et al., 1980), and ruminants (Kalaitzakis et al., 2007). Following acute liver injury, serum arginase increases and then decreases rapidly (Aminlari et al., 1994). Elevations in serum arginase have been demonstrated in naturally occurring liver disease of horses (Wolf et al., 1967), cattle, sheep (Ross, 1966), goats, and dogs. Elevated GD (Glutamate Dehydrogenase) activity has been reported in ruminants with hepatic necrosis (Fowler, 1971), associated with parturition (Treacher and Collis, 1977) and with obstruction of the bile duct (Ford and Gopinath, 1976). Several investigators have used AST/ALT ratio of >1 with specificity of 60.0% and positive predictive value of 87.3 % and negative predictive value of 48%. to predict fibrosis and cirrhosis in chronic hepatitis C patients. Combined assessment of AST/ALT ratio and platelet count has high diagnostic value in cirrhosis. Acute and chronic liver diseases may be accompanied by (i) prolongations in prothrombin and partial thromboplastin times and (ii) associated coagulopathies. Prothrombin index reflects the synthetic capacity of liver and is the earliest indicator of liver fibrosis, (Croquet et al., 2002). Increased serum ALP
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activity also occurs in canine glucocorticoid hepatopathy, however serum ALP activity increases without concurrent increases in hepatic bile acids concentrations or other evidence of cholestasis (Solter et al., 1994).Biliary tract diseases, especially obstruction cause massive increase in plasma ALP activity-5000oiu/l may be reached.GGT is an enzyme produced in bile duct and may be elevated in bile duct illness. GGT activity is relatively high in the livers of cows, horses, sheep, and goats, but GGT activity is considerably lower in the livers of dogs and cats. In experimental bile duct obstruction, serum GGT activity is increased significantly in the dog (Noonan and Meyer, 1979), sheep (Ford, 1974), and cattle. The sensitivity of GGT has been reported to be similar to that of ALP as an indicator of cholestasis in the cat (Spano et al., 1983). The liver is the exclusive site of synthesis of albumin, the most abundant of the plasma proteins. Hypoalbuminemia may be caused by defective albumin synthesis associated with severe hepatocellular disease. In severe, chronic hepatopathy, there is a tendency for elevations in IgM, IgG, and IgA. In cholestasis, bile acids may accumulate in hepatocytes and their toxicity may cause cell death (Webster et al., 2002). Removal of bile acids from the hepatic portal vein may be diminished by impaired hepatocellular function. The predictive value of the serum bile acid test is remarkably high in the dog (Center, 1993). The rate of removal of organic anions can be measured and used to assess the functional capacity of the liver and hepatic blood flow. Sulfobromophthalein (BSP) and indocyanine green have been used most frequently to assess hepatic function. In the dog and cat, a standard dose of 5mg/kg of BSP is administered as an intravenous bolus. A sample of blood is removed at 30 min, and the BSP concentration is determined spectrophotometrically. The BSP test is safe although the dye is irritating if infiltrated perivascularly. The test should be used only when cardiovascular function is normal. If hypovolemia or hypotension is present, hepatic perfusion will be reduced and erroneously prolong the rate of BSP clearance. Other biomarkers used are serum bilirubin, PIIINP and Laminin P1. Acute and chronic liver diseases may be accompanied by (i) porphyrin accumulation and the syndrome of porphyria, but more often (ii) bilirubin accumulation and icterus. PIIINP and Laminin P1 markers have good correlation with histological stages of hepatic fibrosis in viral hepatitis and primary billiary cirrhosis. Serum level of hyluronate and YKL-40 also has diagnostic value for liver injury. MMPs and TIMPs markers are associated with matrix degradation and are elevated in cirrhosis. Excess production of TIMPs relative to MMPs may be an important factor for progression of liver fibrosis (Iredale et al., 1992).Various cytokines like TGF-1 and PDGF are also indicators of liver injury. TGF- (Transforming Growth Factor Beta) in hepatic pathology is the most important stimulus for production of extra cellular matrix. Platelet Derived Growth Factor (PDGF) has been linked to several diseases such as fibrosis and malignant diseases. Serum level of PDGF-BB was found to have highest value for assessment of hepatic fibrosis. The combination of serum PDG-BB, TIMP-1 mRNA and MMP-1 mRNA in PBMCs was the best test in screening of liver fibrosis (Zhang BB et al., 2003)

CONCLUSIONS:Conventional tests for hepatic disease provide information about the integrity of the hepatocytes (ALT, AST, and SDH) and the status of the billiary system (ALP, GGT). Hepatic function can be assessed by estimating the excretory capacity (bilirubin, bile acids) and

synthetic functions (prothrombin) of the liver. Tests for hepatic disease are performed for a variety of purposes including to confirm the existence of liver disease, to assess the nature (e.g., hepatocellular injury, cholestasis) and severity of the disease to determine prognosis, to monitor the clinical course and response to therapy, and to screen individuals at risk for the existence of occult liver disease. Many of the standard tests for liver disease are based on rather simple biochemical procedures that have been automated for use in multichannel autoanalyzers. One seldom obtains the result of a single test for liver disease but rather a panel of results or a liver profile. The combined results of a panel of tests often provide increased sensitivity and specificity and improved predictive value in assessing severity or in differentiating between acute and chronic forms of liver disease. Hepatic tests, however, continue to have an important place in evaluating and monitoring clinical patients with liver disease and in understanding the underlying pathophysiological mechanisms essential for successful treatment.

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