PHTH1011 011 Chemical Pathology of the Liver (Liver Function Test I) - McGrowder.pdf

Chemical Pathology of the Liver - Liver Function Tests Dr Donovan McGrowder Department of Pathology University of the West Indies Liver Function Tests Overview q Functions of the Liver. q Bilirubin production and metabolism. q Routine liver function test (enzymes – ALT, AST, ALP, GGT, albumin along...

Chemical Pathology of the Liver - Liver Function Tests Dr Donovan McGrowder Department of Pathology University of the West Indies Liver Function Tests Overview q Functions of the Liver. q Bilirubin production and metabolism. q Routine liver function test (enzymes – ALT, AST, ALP, GGT, albumin along with direct and total bilirubin). Liver Function Tests q Liver function tests (LFTs), are groups of clinical biochemistry laboratory blood assays designed to give information about the state of a patient's liver. Liver Function Tests q q q q q Liver has considerable functional reserve. Most liver diseases cause only mild symptoms initially. True tests provide assessment of functional hepatic cell activity – non routine. Routine LFTs indicate nature of disease but less often a specific diagnosis. LFTs are cheap, non-invasive and widely available. Liver Function Tests q (i) (ii) (iii) (iv) These tests can be used to: Detect the presence of liver disease, Distinguish among different types of liver disorders, eg - differentiating between acute viral hepatitis, chronic liver disease and various cholestatic disorders, Assess the severity and predict the outcome, and Follow up/monitoring – evaluate response to therapy. Routine LFTs Serum enzymes (i) Biomarkers of liver injury – Transaminases: Alanine aminotransferase (ALT) and aspartate aminotransferase (AST), q (ii) Disease linked to biliary tract Alkaline phosphatase (ALP), gamma glutamyl transferase (GGT), 5’nucleotidase. q q Excretion: Direct (conjugated) and total bilirubin. Synthetic function: Albumin. Bilirubin q q q Serum bilirubin is considered a true test of liver function, as it reflects the liver's ability to take up, process, and secrete bilirubin into the bile. Normal serum bilirubin levels: Total bilirubin: 4 - 18 µmol/L; conjugated bilirubin (direct): 2 - 6 µmol/L; unconjugated bilirubin (indirect; bilirubin - albumin complex). Indirect bilirubin = Total bilirubin – direct bilirubin. Bilirubin n n n Bilirubin is the yellow breakdown product of normal haem catabolism. About 300 mg of bilirubin is formed per day. Bilirubin is excreted in bile and urine, and elevated levels may indicate certain diseases. Conjugated (direct) - water soluble, so it is excreted by kidneys; unconjugated (indirect) insoluble in water, bound to albumin in blood. Bilirubin Production and Metabolism Bilirubin Production q q Bilirubin is the yellow breakdown product of normal haem catabolism. Bilirubin is the breakdown product of the haem moiety of haemoglobin, other haemoproteins, such as cytochromes, catalase, peroxidase and tryptophan pyrrolase, and a small pool of free haem. Bilirubin Production q q q Haem oxygenase converts haem to biliverdin, which is reduced to unconjugated bilirubin by biliverdin reductase. The α-methene bridge carbon is eliminated as CO and the iron molecule is released. Three molecules of oxygen are consumed in this reaction and NADPH is required. Transport to the liver q q q There is the formation of unconjugated bilirubinalbumin complex. Unconjugated bilirubin circulates in bound tightly but reversibly to albumin. plasma Unconjugated bilirubin is transported to the liver via the plasma bound to albumin (unconjugatedalbumin complex). Hepatic uptake q q At the sinusoidal surface of the hepatocyte, unconjugated bilirubin dissociates from albumin and is taken up by the hepatocyte by facilitated diffusion that requires inorganic anions, such as Cl-. Ligandin carries bilirubin from the cytosol into the smoooth endoplasmic reticulum of the hepatocytes. Conjugation of Bilirubin (unconjugated) q In the smooth endoplasmic reticulum, bilirubin is conjugated with glucoronic acid, a reaction catalyzed by uridine diphosphate (UDP)glucuronosyltransferase to form mono- and diglucuronides of bilirubin (conjugated bilirubin). Bilirubin monoglucoronide acid Glucoronic Secretion of Conjugated Bilirubin q q q Mono- and di- glucuronides of bilirubin (conjugated bilirubin) are water soluble & secreted (along with bile salts) into the bile canaliculi, eventually reaching the small intestine via the ducts of the biliary system. Conjugated bilirubin undergoes unidirectional transport into the bile against a concentration gradient. Secretion into the bile canaliculi is the rate-limiting step in bilirubin metabolism. Fate of Bilirubin in the GI Tract n n Bilirubin mono and di-glucuronides (conjugated bilirubin) are degraded by bacterial action (bacterial proteases; beta-glucoronidase) mainly in the colon, to a mixture of colourless water-soluble compounds collectively called urobilinogen. Urobilinogen is further metabolized to stercobilinogen, and further oxidized to stercobilin in the large intestine. This gives faeces its brown colour. Fate of Bilirubin in the GI tract q A small amount of urobilinogen (10%) is reabsorbed by the intestines (terminal ileum) and reaches the liver by portal blood supply and is then resecreted by the liver into the small intestine. The process is known as enterohepatic circulation). q A small amount of reabsorbed urobilinogen (about 5%) is transported by the blood to the kidneys where it is oxidized to urobilin and excreted. Urobilin gives the urine its yellow/straw colour. Bilirubin Metabolism Review Question q Briefly describe metabolism. bilirubin production and Review Question q Where do excretion and metabolism of bilirubin occurs? A. Liver B. Spleen C. Intestine D Both A and C Review Question n Where do conjugation of bilirubin in liver takes place? A. Nucleus B Endoplasmic reticulum C. Cytoplasm D. Mitochondria Review Question q Bilirubin is bound to which plasma protein for its transport to peripheral tissues? A. Globulin B Albumin C. Fibrinogen D. All of the above Review Question n In the liver, bilirubin binds to which intracellular protein? A Ligandin B. Apolipoprotein C. Both D. None Review Question q Which of the following is the intermediate of bilirubin metabolism and excretion? A. Bilirubin-diglucuronide B. Urobilinogen C. Stercobilinogen D All of the above Fate of Bilirubin in the GI Tract q q During severe intrahepatic cholestasis or complete obstruction of the bile duct, urobilinogen/urobilin and stercobilin are absent in urine and stool respectively. The stool will have a pale colour (so-called clay-coloured). In liver disease and states of increased bilirubin production, urinary urobilinogen/urobilin and stercobilin excretion is increased. EVALUATION OF LIVER FUNCTION TESTS Hyperbilirubinaemia q q Jaundice (also known as icterus) is a yellowish pigmentation of the skin, the conjunctival membranes over the sclerae (whites of the eyes), and other mucous membranes caused by hyperbilirubinaemia. Jaundice becomes clinically apparent when the total serum [bilirubin] exceeds about 50 µmol/L (Normal , 4 - 18 µmol/L). Biochemical tests for liver function Routine liver function tests, Transaminase activities in human tissues, relative to serum as unity Heart Liver Skeletal Muscle Kidney Pancreas Spleen Lung Erythrocytes Serum AST ALT 7800 450 7100 2850 5000 300 4500 1200 1400 130 700 80 500 45 15 7 1 1 Alanine aminotransferase (ALT) q q q q Widely distributed, although the largest amounts found in the liver. Also found in kidney and heart. Smaller amounts occur in the heart but usually remains normal after myocardial infarction. Excellent marker of hepatacellular injury. Hepatocellular injury trigger the release of ALT into the circulation. More specific for liver disease than AST. Alanine Aminotransferase n n n n Normal reference interval in serum (4-17 IU/mL for females and 6-21 IU/mL for males). Up to 300 IU/ml: non-specific, any type of liver disorder (chronic liver disease, cirrhosis, malignancy). >1,000 IU/mL: extensive hepatocellular damage (acute viral hepatitis, ischemic liver injury , toxin /drug induced liver injury). Acute hepatitis : ALT is more increased than AST (20 -100 x the upper limit of normal). Aspartate aminotransferase q This enzyme is widely distributed in the body. q Normal reference interval in serum (7 - 32 U/L). q q Main sources: Heart, liver, skeletal muscle, and kidney. Useful in the diagnosis of MI, liver disorders and muscle damage. Aspartate aminotransferase q Causes of ­serum AST levels: 1. Liver diseases: Hepatitis, hepatic necrosis, cholestasis. 2. Cardiac disease: Myocardial Infarction. 3. Diseases of skeletal muscle: Crush injury, trauma, myopathy. 4. From Erythrocytes: Haemolysis. AST and ALT n n n ALT and AST are useful indicator of hepatocellular damage. AST is present in both the mitochondria and cytosol of liver cells, whilst ALT is found in the cytosol only. The relative activities of the enzymes give some index of the underlying pathology and severity of the disease process. Transaminases In Hepatobiliary Diseases Hepatocellular injury AST ALT Cytoplasmic AST & ALT released into serum. Mild: Plasma membrane damaged More severe: Mitochondrial membrane damaged AST Mitochondrial AST released into serum: AST 80% Disproportionate elevation of AST. AST and ALT n n In inflammatory or infective conditions, the cytoplasmic membrane sustains the main damage. Leakage of cytoplasmic contents causes a relatively greater increase in plasma ALT than AST activities. In infiltrative disorders in which there is damage to both mitochondrial and cytoplasmic membrane, there is a proportionately greater increase in plasma AST activity than ALT. Review Questions q q What are constituents of the routine liver function test profile? What is the clinical implication of the abnormalities? How useful are ALT and AST in evaluating hepatocellular damage? Review Question n Which of the following samples is usually taken for the liver function test? a) Blood sample b) Urine sample c) Intestine Biopsy sample d) Sputum Review Question q a. b. c. d. In infiltrative diseases such as colon cancer that spread to the liver. what is the relationship between ALT and AST? AST > ALT ALT = AST ALT > AST None of the above Review Question q a. b. c. d. Wilson disease is a genetic disorder that prevents the body from removing extra copper, causing copper to build up in the liver, brain, eyes, and other organs. The AST/ALT ratio in patients with Wilson’s disease is: Between one and two Equal to one Greater than two Less than one Review Question q a. b. c. d. Acute viral hepatitis is diffuse liver inflammation caused by specific hepatotropic viruses. The AST/ALT ratio in patients with acute viral hepatitis is: Between one and two Equal to one Greater than two Less than one Serum Enzymes – that reflect cholestasis n Two enzymes: (i) (ii) Alkaline Phosphatase (ALP) Gamma glutamyl tranferase (GGT) Alkaline phosphatase q In the liver, alkaline phosphatase (ALP) is found at two distinct sites – on the sinusoidal surface of hepatocytes, and in the microvilli of the bile canaliculi. Alkaline phosphatase (ALP) Alkaline phosphatase Causes of increased serum alkaline phosphatase(ALP) enzyme activity: - Infancy - Puberty Physiological : - Pregnancy - Intestinal isoenzymes Bone disease: - Hepatobiliary disease: Others: Hyperparathyroidism Osteomalacia, rickets Paget’s disease of bone Osteomyelitis - Hepatitis - Cholestasis - Cirrhosis Carcinoma of the bronchus Alkaline phosphatase n q n Produced by biliary epithelial cells. n Non-specific to liver: bone, intestine, placenta, kidney. Liver: exists predominantly in biliary tract & is a marker for biliary dysfunction. Elevations: Biliary duct obstruction, Primary biliary cirrhosis, infiltrative liver disease, hepatitis/cirrhosis and medications. Alkaline phosphatase In obstructive cholestasis disease the plasma ALP is usually increased to levels greater than three times ULN (Normal range, 15-105 U/L). q Non-pathological ­ Age >60 yrs Bld group – O & B Growing children & adolescents Late in normal pregnancy Pathological ­ 1° biliary cirrhosis Choledocholithiasis Hepatic malignancy 1° & 2° Paget’s disease Alkaline phosphatase In hepatocellular diseases the level may be normal or slightly increased, but any increase is usually less than three times ULN. q This reflects mild cholestasis due to obstruction of bile canaliculi by swollen hepatocytes & inflammation/necrosis of the ductular lining cells. q Clinical application There is increased synthesis of ALP (enzyme induction). q g GLUTAMYL TRANSFERASE (GGT) q q A microsomal enzyme its synthesis induced by ethanol and anticonvulsant drugs. Found mainly in the kidney and significant amounts in liver, brain, prostate, and pancreas. However, elevated plasma levels usually only indicate a hepatic origin – sensitive indicator of liver disease. GGT GGT q This enzyme is present in the bile canaliculi, the epithelial cells lining the bile ducts and, to a certain extent, in the periportal hepatocytes. GGT Gamma glutamyl transferase q Normal reference interval in serum (10 – 70 U/L). q Used primarily for diagnosis of hepatobiliary diseases. q q q q Although reasonably specific to the liver and a more sensitive marker for cholestatic damage than ALP. In biliary obstruction, plasma GGT activity may increase before that of alkaline phosphatase. Marked elevation of serum GGT level is seen in alcoholic liver disease. Elevated serum GGT activity sometimes following myocardial infarction or congestive cardiac failure. Causes of raised serum gammaglutamyl transferase (SGGT) 5’-Nucleotidase q q An intrinsic membrane glycoprotein that is present as an enzyme in a wide variety of mammalian cells. Catalyze the reaction: 5'-ribonucleotide + H2O → ribonucleoside + phosphate q q Normal reference interval (2 – 15 U/L). This enzyme is released by the liver when the liver is injured due to bile duct obstruction or impaired bile flow. 5’-Nucleotidase n n n n Specific and sensitive for hepatobiliary disease; highly elevated in biliary obstruction. May be used to confirm hepatic origin of elevated serum ALP. Moderate elevated – hepatitis. Unlike ALP , the level is unrelated with osteoblastic activity (i.e. unaffected by bone diseases). Review Questions q What tests in the liver function test profile are used to assess cholestasis/biliary obstruction? How useful are they? Review Questions q What tests in the liver function test profile are used to assess cholestasis/biliary obstruction? How useful are they? Review Question q Which of the following are the nonfunctional plasma enzymes increased in alcoholic subjects? q a) Alkaline phosphatase b) Acid Phosphatase c) Lactate dehydrogenase d) Gamma-glutamyltransferase Review Question q Inflammatory hepatobiliary diseases refers to chronic autoimmune disorders with predominant hepatic or biliary manifestations. Which of the following pairs of plasma enzymes are the most sensitive in diagnosing hepatobiliary diseases? a) Alkaline phosphatase and 5’-nucleotidase b) Acid Phosphatase and 5’-nucleotidase c) Alkaline phosphatase and Gamma-glutamyltransferase d) Gamma-glutamyltransferase and and 5’-nucleotidase Review Question q Gamma glutamyl transferase is an enzyme found in cell membranes of many tissues mainly in the liver, kidney, and pancreas. Gamma glutamyl transferase is elevated in which of the following group of disorders? a) Hypertension, diabetes mellitus and end stage renal disease b) Diabetes mellitus, myocardial infarction and hypertension c) Myocardial infarction, diabetes mellitus and pancreatic disease d) Myocardial infarction, stomach cancer and diabetes mellitus Albumin q q q q Albumin is a protein that is made only by the liver and excreted by the kidneys. Albumin is essential for maintaining the osmotic pressure in the vascular system. Albumin is also very important in the transportation of many substances such as drugs, lipids, hormones. Albumin has a half-life of approx. 20 days. Serum Albumin q q q Normal in early stages of acute hepatitis (poor indicator of acute hepatitis disease). Significantly decreases in chronic liver disease (e.g. cirrhosis). Abnormally low contents of albumin may indicate: kidney disease, malnutrition, extensive burns, kidney disease and malabsorption syndromes etc. Review Question q How useful is albumin as part of the repertoire of liver function tests? Review Question q a) b) c) d) Albumin is most likely decreased in which of the following disorders? Myocardial infarction Diabetes mellitus Acute viral hepatitis Chronic active hepatitis Liver Function Tests Summary q q Bilirubin production and metabolism involves the reticuloendothelial system, liver, small and large intestine, kidney etc. Routine liver function test (enzymes – ALT, AST (hepatocellular damage), ALP, GGT (hepatobiliary obstruction), albumin (decreased synthesis along with direct and total bilirubin (cholestasis). Thank you