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Liver Function Test Prepared by: Siti Norhaiza Binti Hadzir Liver Function Test Prepared by: Siti Norhaiza Binti Hadzir

Objectives By the end of this lecture, students should know how to § § Objectives By the end of this lecture, students should know how to § § Describe the anatomy of liver, blood supply and the function of liver. Outline the component of liver function test (LFT) and its clinical use.

Anatomy of liver Anatomy of liver

Normal histology of the liver Normal histology of the liver

The Function of Liver • • • 1. 2. Liver is largest and most The Function of Liver • • • 1. 2. Liver is largest and most complex internal organ All blood flow fm intestine and pancreas reaches liver via portal venous system Liver is a multifunctional organ that is involved in diverse body functions. Metabolic Functions Liver actively participates in carbohydrate metabolism, lipid, protein, mineral and vitamin metabolisms. Excretory Functions Bile pigments, bile salts and cholesterol are excreted in bile into intestine.

3. Protective functions & detoxification Kupffer cells of liver perform phagocytosis to eliminate foreign 3. Protective functions & detoxification Kupffer cells of liver perform phagocytosis to eliminate foreign compounds. For example ammonia is detoxified to urea and metabolism of xenobiotics (detoxification). Clearance of hormones such as insulin, parathyroid hormone, oestrogen, cortisol 4. Hematological and synthetic functions Liver participates in formation of blood (particularly in embryo) Synthesis of plasma proteins (albumin and prothrombin), hormones e. g angiotensinogen, insulin-like growth factor • and triiodothyronine. • Destruction of erythrocytes (Bilirubin)

5. Storage functions Glycogen, vitamins A, D and B 12 6. Serum enzymes Acting 5. Storage functions Glycogen, vitamins A, D and B 12 6. Serum enzymes Acting as markers of liver damage

Test to assess liver function • Liver function tests(LFT) are helpful to detect the Test to assess liver function • Liver function tests(LFT) are helpful to detect the • • abnormalities and extent of liver damage. LFT assays are frequently more sensitive than clinical signs and symptoms. Typically the LFT comprises of: - Total protein • • • Albumin and globulin (Prothrombin Time) Transaminases – AST & ALT Alkaline PO 4 ase Bilirubin, usually fractionated Gamma Glutamyl Transpeptidase (GGT)

Total protein • Not a very useful measure, non-specific; only provides information on: • Total protein • Not a very useful measure, non-specific; only provides information on: • General nutritional status • Severe organ disease (esp protein losing d/s) • Fractionated values of greater use • Methods of measurement: • • Electrophoresis followed by, Precipitation, followed by, Column separation. Nitrogen content is usual method in automation

Total protein • Note! Measures of protein are in serum – avoid • dilution Total protein • Note! Measures of protein are in serum – avoid • dilution of proteins fm anticoagulant Precipitation is used to fractionate proteins into albumin and globulin • Addn of Na. SO 4, Na sulfite, Ammonium SO 4, methanol will ppt globulins • A/G ratio is a frequently used value in determining serum protein abnormalities • Albumin changes [see later] • Globulin changes: in disease fm increased synthesis

Total protein • Nitrogen content measurement is reference • method (Kjeldahl technique) This technique Total protein • Nitrogen content measurement is reference • method (Kjeldahl technique) This technique uses acid digestion of proteins to release ammonium ions, which are quantified by nesslerisation to form a coloured complex in an alkali environment

Total protein • Refractive index (useful if level > 2. 5 g/d. L) • Total protein • Refractive index (useful if level > 2. 5 g/d. L) • SG – pipetting serum into graded Cu. SO 4 soln • UV absorption (280 nm) • Tubidimetric methods • Colourimetric – biuret method, most common method in automated instruments. Can be made more sensitive using Folin-Ciocalteu reagent

 • Albumin and globulin • Usu most abundant protein in serum [120 mg/kg/day] • Albumin and globulin • Usu most abundant protein in serum [120 mg/kg/day] • ↓albumin • Impaired synthesis (malnutrition, malabsorption, hepatic dysfunction, cirrhosis) • Loss (ascites, protein losing-nephropathy, enteropathy) • May result in peripheral oedema • Up to 25% of albumin in hyperglycaemia becomes glycosylated with Hb. A 1 c – aka fructosamine useful in monitoring DM

 • Albumin and globulin • albumin • Unusual – can occur in dehydration • Albumin and globulin • albumin • Unusual – can occur in dehydration or as artifact fm tourniquet use • Types of globulin of clinical significance: • • • 1 -antitrypsin (AAT) 2 -macroglobulin Haptoglobin Transferrin Ceruloplasmin

 1 -antitrypsin (AAT) • • Most abundant 1 -globulin Inhibits trypsin Several genetic 1 -antitrypsin (AAT) • • Most abundant 1 -globulin Inhibits trypsin Several genetic variations May be associated with incidence emphysema and neonatal jaundice

 2 -macroglobulin • • Largest non-immunoglobulin protein in plasma in nephrotic syndrome 2 -macroglobulin • • Largest non-immunoglobulin protein in plasma in nephrotic syndrome

Haptoglobin • • • Another major 2 protein Function – to combine with Hb Haptoglobin • • • Another major 2 protein Function – to combine with Hb released by RBC lysis to preserve Fe and protein stores Circulating half-life approx 4 days in stress, infection, acute inflamm, tissue necrosis ↓ post haemolytic episode Useful to monitor slow rate of haemolysis i. e. fm mechanical valves, exercise associated trauma, haemoglobinopathies

Ceruloplasmin • Cu containing enzyme (ferroxidase) in serum • ↓ in Wilson’s d/s • Ceruloplasmin • Cu containing enzyme (ferroxidase) in serum • ↓ in Wilson’s d/s • Associated with chronic hepatitis (occ acute) and may have neurologic/ psychiatric sequelae

 -Fetoprotein • • One of the major plasma proteins in foetal life Function -Fetoprotein • • One of the major plasma proteins in foetal life Function not known, similar structure to albumin Falls thru-out gestation (~10, 000 ng/m. L at birth) and by age one yr (<10 ng/m. L – adult levels) In acute hepatic injury AFP 10 – 20 X upper ref limits Abt 10% pt with viral hepatitis have AFP Fibrosis post chronic liver d/s, AFP Used to screen and diagnose HCC & hepatoblastoma

Prothrombin Time • Most coag factors made in liver (particularly those assoc with vitamin Prothrombin Time • Most coag factors made in liver (particularly those assoc with vitamin K) • Hence in liver d/s, coagulopathies are common • Commonly PT is used for detecting liver assoc coagulopathies • Best PT method not clear • PT INR is useful for monitoring oral anti-coag therapy, not very useful for liver disease • An indirect test of hepatic synthetic function includes administration of vitamin K (10 mg) subcutaneously over three days. Several days later, the prothrombin time may be measured. If the prothrombin time becomes normal, then hepatic synthetic function is intact. This test does not indicate that there is no liver disease, but is suggestive that malnutrition may coexist with (or without) liver disease.

Transaminases • • • Tests of liver injury Hepatocytes contain high levels of enzymes Transaminases • • • Tests of liver injury Hepatocytes contain high levels of enzymes that can leak into the plasma when there is liver injury Enzymes found in hepatocytes are: • Cytoplasmic = LDH, AST, ALT • Mitochondrial = ASTm • Canalicular = ALP, GGT

Alanine Aminotransferase (ALT) • • • The test is primarily used to diagnose liver Alanine Aminotransferase (ALT) • • • The test is primarily used to diagnose liver disease, to monitor the course of treatment for hepatitis, active postnecrotic cirrhosis, and the effect of drug therapy. The level of ALT abnormality is increased in conditions where cells of the liver have been inflamed or undergone cell death As the cells are damaged, the ALT leaks into the bloodstream leading to a rise in the serum levels Any form of hepatic cell damage can result in an elevation in the ALT level may or may not correlate with the degree of cell death or inflammation ALT is the most sensitive marker for liver cell damage. ALT differentiates between hemolytic jaundice and jaundice due to liver disease.

Alanine Aminotransferase (ALT) • Increased ALT levels are found in the following conditions: - Alanine Aminotransferase (ALT) • Increased ALT levels are found in the following conditions: - Hepatocellular disease - Active cirrhosis (mild increase) - Metastatic liver tumor - Obstructive jaundice or billiary obstruction (mild to moderate increase) - viral, infectious or toxic hepatitis (30 -50 x normal) - infectious mononucleosis)

Aspartate Aminotransferase (AST) • • • Also reflects damage to the hepatic cell It Aspartate Aminotransferase (AST) • • • Also reflects damage to the hepatic cell It is less specific for liver disease It may be elevated and other conditions such as a myocardial infarct and muscle disease Although AST is not a specific for liver as the ALT, ratios between ALT and AST are useful to physicians in assessing the aetiology of liver enzyme abnormalities Viral heptitis, mononucleosis, and acute hepatotoxicity typically show elevations in ALT that are equal to or greater than AST elevations (AST/ALT less than or equal to 1. 0) ALT is elevated to a lesser degree than AST in alcoholic liver disease and cirrhosis, passive congestion, bile duct obstruction, or metastatic tumor to the liver (AST/ALT greater than 1. 0)

Measurement • • • Uses coupled enzymatic reactions with NADH as final reaction product Measurement • • • Uses coupled enzymatic reactions with NADH as final reaction product measured Reagents with NH 4+ should be avoided as it may artificially the AST/ALT values Values also affected by buffers

Specimens • • Stable in whole blood for 24 hrs (then gradually from release Specimens • • Stable in whole blood for 24 hrs (then gradually from release fm RBC) AST/ALT stable at 4 o. C for up to 3 weeks AST stable indefinitely with freezing ALT may show ↓ with freezing depending on buffer used

Alkaline Phosphatase • Source: liver, bone, placenta and intestine. • ↑ ALP activity in Alkaline Phosphatase • Source: liver, bone, placenta and intestine. • ↑ ALP activity in liver disease are the result of increased synthesis of the enzymes by cells lining the bile canaliculli, usually in response to cholestasis (intra or extra-hepatic). • ALP ↑ 2 x the reference interval in cholestasis. • Also ↑ in infiltrative diseases of liver, when space occupying lesions (e. g tumours) are present. • Growing bones need ALP. • ↑ serum ALP by osteoblast-rapid growth of bone (growth, healing of fracture, bone cancer, Paget’s disease, rickets). • For pregnant women, ALP is produce by the placenta. • ALP from the intestine is increased in a person with inflammatory bowel disease such as ulcerative colitis.

Gamma Glutamyl Transferase (GGT) Enzyme • • • GGT is used by the body Gamma Glutamyl Transferase (GGT) Enzyme • • • GGT is used by the body to synthesize glutathione tri peptide GGT is present in liver, kidney, pancreas, intestinal cells and prostrate glands Elevated levels (> 10 - 30 IU/l) are observed in : chronic alcoholism, pancreatic disease, myocardial infarction, renal failure, chronic obstructive pulmonary disease and in diabetes mellitus In liver diseases, GGT elevation parallels that of ALP In alcoholic liver disease GGT levels may be parallel to alcohol intake

Bilirubin • • Normal serum bilirubin levels: Total bilirubin: 4 to 19 mol/L Conjugated Bilirubin • • Normal serum bilirubin levels: Total bilirubin: 4 to 19 mol/L Conjugated bilirubin (Direct ; glucuronide): 0 to 4 mol/L Unconjugated bilirubin ( Indirect; bilirubin albumin complex): up to 12 mol/L

BILIRUBIN is estimated by van den Bergh reaction • • Principle of this test BILIRUBIN is estimated by van den Bergh reaction • • Principle of this test is the reaction between sulfanilic acid (sulfanilic acid in HCl and sodium nitrate) with bilirubin Aforementioned reaction forms a purple coloured complex, azobilirubin

BILIRUBIN is estimated by van den Bergh reaction • • • Conjugated bilirubin produces BILIRUBIN is estimated by van den Bergh reaction • • • Conjugated bilirubin produces purple color immediately on mixing with reagent This response is known van den Bergh as direct positive Unconjugated bilirubin gives purple color only on addition of alcohol This response is called as indirect positive If both, conjugated and unconjugated bilirubin, are present in increased amounts, a purple color is produced immediately The purple color, so obtained, is intensified on adding alcohol. The the reaction is called biphasic

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