D- LIVER FUNCTION TESTS

1. D- LIVER FUNCTION TESTS

2. Introduction • Liver has to perform different kinds of biochemical, synthetic and excretory functions • no single biochemical test can detect the global functions of liver. • All laboratories usually employ a battery of tests for initial detection and management of liver diseases • These tests are frequently termed “Liver function tests”.

3. Normal liver function • The various functions of the liver are carried out by the liver cells or hepatocytes a) Metabolic function • carbohydrate metabolism • Gluconeogenesis • Glycogenolysis • Glycogenesis • protein metabolism, synthesis as well as degradation

4. lipid metabolism: • Cholesterol synthesis • Lipogenesis, the production of triglycerides (fats) b) Storage functions • glucose (in the form of glycogen), • Vitamins A , D, E, B12 • iron, and copper. .

5. c) Synthetic function • coagulation factors I (fibrinogen) , II (prothrombin), V, VII, IX, X and XI, as well as protein C, protein S and antithrombin • produces and excretes bile (a yellowish liquid) required for emulsifying fats. • Synthesizes albumin

6. d) Detoxification : • ammonia to urea • Drugs and drug metabolites e)Immunological - the reticuloendothelial system of the liver contains many immunologically active cells, acting as a 'sieve' for antigens carried to it via the portal system.

7. Functions of liver

8. Uses of liver function tests • Screening : non-invasive yet sensitive • Pattern of disease : eg - differentiating between acute viral hepatitis and various cholestatic disorders and chronic liver disease. (CLD). • Assess severity and predict the outcome . • Follow up : and also helpful in evaluating response to therapy

9. Liver function tests in practice • Markers of liver dysfunction serum bilirubin ( excretory function) urine bilirubin,urobilinogen and bile salts • Markers of synthetic dysfunction Total protein, albumin, A/G ratio Prothrombin time & INR • Detoxification markers Blood ammonia Exogeneous dye tests – BSP test indocyanine green test

10. Markers of hepatocellular injury • Alanine aminotransferase ( ALT) • Aspartate transaminase (AST) • Alkaline phosphatase • Gamma glutamyl transferase • Leucine aminopeptidase • 5’ nucleotidase

11. Other special tests Ceruloplasmin ( Wilson’s disease) Ferritin Alpha-1-antitrypsin Alphafetoprotein ( hepatocellular carcinoma) • Immunological tests Antimitochondrial antibodies ( Pri biliary cirrhosis) ANCA ( pri sclerosing cholangitis )

12. Bilirubin • Bilirubin is a breakdown product of haem (a part of haemoglobin in red blood cells). • The liver is responsible for clearing the blood of bilirubin. • Reference range : 0.2–0.8 mg/dL • Increased total bilirubin causes jaundice ( levels greater than 2mg/dl)

14. Bilirubin ( contd) • Unconjugated (indirect)- insoluble • ↑ in hemolysis, Gilbert syndrome • Unconjugated hyperbilirubinemia when >80% of total bilirubin is unconjugated • Conjugated (direct)- soluble • ↑ in obstruction, cholestasis, cirrhosis, hepatitis, primary biliary cirrhosis, etc. • Conjug hyperbilirubinemia> 50% is conj bilirubin

15. Methods for bilirubin • Van den bergh reaction • Jendrassik- grof bilirubin method • Dry chemistry slide methods • Methods for determination of serum bilirubin are based on coupling of bilirubin pigments with diazo compounds to form azobilirubin • Conjugated bilirubin - immediate colour reaction (direct) • Unconj bili – reaction on addition of alcohol/caffeine benzoate ( indirect)

16. Urine bilirubin & urobilinogen • Urine bilirubin : only conjugated bilirubin is excreted in urine • Urobilinogen: • increased in haemolytic anaemias • Decreased in hepatic diseases • Ref range – 0 – 4 mg/day • Lab determination is based on Ehlrich reaction using para-dimethyl aminobenzaldehyde to give a red colour

17. Bile acid assays • Are the final products of metabolism of cholesterol and play a role in overall digestion of lipids • Serum concentration are microgram/ml amounts even after a meal • Increased levels seen in liver diseases • Most helpful in differentiation of hepatobiliary disease from congenital hyperbilirubinemia or hemolysis. • Lab methods: gas chromatography, Enzymatic methods or immunoassays

18. Serum albumin • Albumin is the most significant protein synthesized by the liver and is an indicator of overall function. • The half-life of albumin is approximately 20 days. • Albumin levels are decreased in chronic liver disease, such as cirrhosis • Normal albumin concentration does not rule out liver disease as an acute condition may exist • Ref range 3.5 – 5 g/dl • Poor marker of liver function- decreased by trauma, inflammatory conditions, malnutrition

19. Prothrombin time and INR • The liver is responsible for the production of coagulation factors • The prothrombin time is the time it takes plasma to clot after addition of tissue factor(obtained from animals) • This measures the quality of the extrinsic pathway(greatly affected by levels of factor VII in the body) • poor factor VII synthesis (due to liver disease) may prolong the PT. • Normal : 12 – 14 seconds • .

20. . The international normalized ratio (INR) measures the speed of a particular pathway of coagulation, comparing it to normal • Normal range for a healthy person is 0.9–1.3 • high INR level INR=5 high chance of bleeding • The INR will be increased only if the liver is so damaged that synthesis of vitamin K-dependent coagulation factors has been impaired; • it is not a sensitive measure of liver function. • It is very important to normalize the INR before operating on people with liver problems as they could bleed

21. Grading Liver Function Using the Child-Turcotte Class as Modified by Pugh • The Child-Turcotte class, as modified by Pugh, often known simply as the "Child class," is calculated by adding the points as determined by the patient's laboratory results: class A=0 to 1; class B=2 to 4; class C=5 and higher. • The classes indicate severity of liver dysfunction: class A is associated with a good prognosis, and class C is associated with limited life expectancy

22. Hepatocellular enzymes

23. Major diagnostic hepatocellular enzymes are located at various sites in the hepatocyte giving rise to different patterns of enzyme release depending on the cause. • ALT & ASTc – cytosol Released in membrane injury in viral or toxin induced hepatitis. • ASTm – mitochondria , alc hepatitis • ALP & GGT – canalicular side of hepatocyte Bile acids accumulate in cholestasis dissolve memb fragments and release enzymes into plasma

24. Transaminases • Located in hepatocytes • Released after hepatocellular injury • 2 Forms • AST ---ref range 10 – 35 IU/L • Non-specific to liver: heart, skeletal muscle, blood • ALT ----- ref range 9 – 40 IU/L • More specific

25. Very high levels ( > 1000) – acute hepatitis • Moderate elevation ( 100-300) – alc hepatitis, wilson’s disease, nonalcoholic cirrhosis • Mild elevation ( < 100) – chr viral hepatitis • AST:ALT ratio Elevated in alcoholic disease (2: 1)

26. AST & ALT assays • There are several variants of assays for transaminases • In one of them alanine for ALT or aspartate for AST is added to yield glutamate. • This is then coupled to enzyme glutamate dehydrogenase ( indicator reaction) yeilding alpha keto glutarate. • In this reaction NAD is converted to NADH which is measured as increase in absorbance at 340 nm

27. Aminotransferases • normal in patients with cirrhosis. • uncomplicated alcoholic hepatitis, the AST value is rarely greater than 500 U per L and is usually no more than 200 to 300 U per L. • The highest peak values are found in patients with acute ischemic or toxic liver injury

28. Alkaline phosphatase • Produced by biliary epithelial cells • Non-specific to liver: bone, intestine, placenta • Elevations • Biliary duct obstruction • Primary biliary cirrhosis • Primary sclerosing cholangitis • Infiltrative liver disease Highest elevationsI( more than 3 times) represent obstructive disease • Ref range –30 -120 IU/L

29. Gammaglutamyl transpeptidase • Among the most sensitive tests in detecting early hepatobiliary disease • More sensitive marker for cholestatic damage than ALP • may be elevated with even minor, sub-clinical levels of liver dysfunction. • It can also be helpful in identifying the cause of an isolated elevation in ALP • Very sensitive in detecting chronic alc liver disease.

30. Leucine aminopeptidase and 5’nucleotidase • Used to increase the specificity of ALP enzyme for liver dysfunction. • Not increased in bone diseases. • Lactate dehydrogenase: LD-5 isoenzyme is liver specific indicates hepatocellular necrosis or metastatic liver disease.

31. Detoxification and excretion markers Ammonia : plasma ammonia concentration reflects the liver’s ability to detoxify ammonia into urea and excrete urea. • Mainly elevated in advanced liver disease • Useful in evaluating patients who are comatose or with altered mental status • Most common lab determination is based on enzymatic reaction using glutamate dehydrogenase • Ref range : 15 – 45 µg/dl

32. Exogeneous dye tests : Evaluate liver’s ability to transport exogeneous substances into hepatocyte, metabolize and finally excrete into bile. Bromosulphopthalein test –a normally functioning liver should remove > 95% of dye in 45 min and retain only 5% in plasma Determined spectrophotometrically

33. Indocyanine green test : dye is not conjugated in the liver almost all of it is recovered in the bile • Disappearence is direct function of blood flow • Disadv of dye tests : problems associated with exogeneous substances so infrequently performed.

34. Metabolic function tests Lipid metabolism : hepatic disorders oten cause derangements in lipoprotein metabolism. • In liver diseases there is deficiency of LCAT and lipoprotein lipase • Resulting in reduced HDL, altered lipoprotein distribution and hypertriglyceridemia. Carbohydrate metabolism: Glucose , galactose and fructose tolerance test

35. Limitations • Lack sensitivity: The LFT may be normal in certain liver diseases like cirrhosis, non cirrhotic portal fibrosis, congenital hepatic fibrosis, etc. • Lack specificity : • not specific for any particular disease. • Serum albumin may be decreased in chronic disease and also in nephrotic syndrome. • Aminotransferases may be raised in cardiac diseases and hepatic diseases.

36. Except for serum bile acids the LFT are not specific for liver diseases and • all the parameters may be elevated for pathological processes outside the liver. • Thus, we see that LFT have certain advantages as well as limitations at the same time. • Thus, it is important to view them keeping the clinical profile of the patient .

37. Jaundice • Jaundice (also known as icterus) is a yellowish pigmentation of the skin, the conjunctival membranes over the sclerae, and other mucous membranes caused by hyperbilirubinemia (increased levels of bilirubin in the blood).

38. Types of jaundice • Increased plasma concentrations of bilirubin (> 2 mg/dL) occurs when • there is an imbalance between its production and excretion • Recognized clinically as jaundice

39. Prehepatic • Results from excess production of bilirubin (beyond the livers ability to conjugate it) following hemolysis • Excess RBC lysis the result of -autoimmune disease; hemolytic disease of the newborn • structurally abnormal RBCs (Sickle cell disease); or breakdown of extravasated blood • High plasma concentrations of unconjugated bilirubin (normal concentration ~0.5 mg/dL)

40. Hepatic • Impaired uptake, conjugation, or secretion of bilirubin • Reflects a generalized liver (hepatocyte) dysfunction • In this case, hyperbilirubinemia is usually accompanied by other abnormalities in biochemical markers of liver function

41. Post hepatic • Caused by an obstruction of the biliary tree • Plasma bilirubin is conjugated, and other biliary metabolites, such as bile acids accumulate in the plasma • Characterized by pale colored stools (absence of fecal bilirubin or urobilin), and dark urine (increased conjugated bilirubin) • In a complete obstruction, urobilin is absent from the urine

43. Neonatal jaundice • Common, particularly in premature infants • Transient (resolves in the first 10 days) • Due to immaturity of the enzymes involved in bilirubin conjugation • High levels of unconjugated bilirubin are toxic to the newborn – due to its hydrophobicity it can cross the blood-brain barrier and cause kernicterus • Serum levels > 15mg/dl are usually seen • Jaundice within the first 24 hrs of life or which takes longer then 10 days to resolve is usually pathological and needs to be further investigated

44. Summary • Individual LFT results are not usually specific to a particular disease; but the pattern of alteration can be more useful. • Abnormal liver function tests must be interpreted with regard to clinical context and results of previous tests • levels of derangements in LFTs are not always indicative of the severity of the disease and clinical context is necessary to determine the underlying aetiology

45. Take home message • Despite the development of highly sensitive laboratory tests, clinical assessment and judgement remain paramount1