Liver Function Tests Lecture (BC10A) 2018-04-10 PDF

Liver Function Tests Dr Donovan McGrowder Department of Pathology University of the West Indies Liver Function tests I Overview ❑ Functions of the Liver. ❑ Bilirubin production and metabolism. ❑ ❑ ❑ Hyperbilirubinaemia – Prehepatic, hepatic and posthepatic. Routine liver function test (enzymes – AST...

Liver Function Tests Dr Donovan McGrowder Department of Pathology University of the West Indies Liver Function tests I Overview ❑ Functions of the Liver. ❑ Bilirubin production and metabolism. ❑ ❑ ❑ Hyperbilirubinaemia – Prehepatic, hepatic and posthepatic. Routine liver function test (enzymes – AST, ALT, ALP, GGT & albumin). True and special tests of liver function (Alphafetoprotein & immunoglobulin quantification). Liver Function Tests ❑ 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 ❑ ❑ ❑ ❑ ❑ 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 ❑ These tests can be used to: (i) (ii) (iii) (iv) detect the presence of liver disease, distinguish among different types of liver disorders, eg - differentiating between acute viral hepatitis, various cholestatic disorders and chronic liver disease, Assess the severity and predict the outcome, and follow up – evaluate response to therapy. Routine LFTs ❑ Serum enzymes (i) Biomarkers of liver injury – Transaminases: Alanine and aspartate aminotransferase (ALT & AST respectively), (ii) Disease linked to biliary tract Alkaline phosphatase (ALP), gamma glutamyl transferase (GGT), 5’nucleotidase. ❑ Excretion: Direct and total bilirubin. ❑ Synthetic function: Albumin. Bilirubin ◼ ◼ ◼ Bilirubin is the yellow breakdown product of normal heme 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 - water soluble, so it is excreted by kidneys; unconjugated - insoluble in water, bound to albumin in blood. Bilirubin ❑ 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: < 18 mol/L; conjugated bilirubin (direct): < 6 mol/L; unconjugated bilirubin (indirect; bilirubin - albumin complex). ❑ ❑ Indirect bilirubin = Total bilirubin – direct bilirubin. Bilirubin Production and Metabolism Bilirubin ❑ ❑ 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 ❑ ❑ ❑ Haem oxygenase converts Haem to biliverdin, which is reduced to bilirubin by biliverdin reductase. Three molecules of oxygen are consumed in this reaction and NADPH is needed. The α-methene bridge carbon is eliminated as CO and the iron molecule is released. Transport to the liver ❑ ❑ Formation of unconjugated bilirubinalbumin complex. Unconjugated bilirubin circulates in plasma bound tightly but reversibly to albumin. Transport of unconjugated bilirubin bound to albumin to the liver. Hepatic uptake ❑ ❑ At the sinusoidal surface of the hepatocyte, bilirubin dissociates from albumin and is taken up by the hepatocyte by facilitated diffusion that requires inorganic anions, such as Cl-. Ligandin carries bilirubin into the smoooth endoplasmic reticulum of the hepatocytes. Conjugation of Bilirubin ❑ 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 Glucoronic acid Secretion of Conjugated Bilirubin ❑ ❑ ❑ Mono- and di- glucuronides of 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 ◼ ◼ Bilirubin mono and di-glucuronides are degraded by bacterial action (bacterial proteases) mainly in the colon, to a mixture of colourless watersoluble 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 ❑ ❑ Urobilinogen 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). 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 Plasma Fragile RBCs BILIRUBIN METABOLISM RE System unconjugated bilirubin (protein – bound) Liver Urobilinogen Kidneys Liver Conjugated bilirubin Absorbed Bacterial action Urobilinogen Oxidation Urobilin Urobilinogen Stercobilinogen Oxidation Stercobilin Intestinal Contents Urine Fate of Bilirubin in the GI tract ❑ ❑ During severe intrahepatic cholestasis or complete obstruction of the bile duct, urobilin/urobilinogen and stercobilin are absent in urine and stool respectively. The stool will have a pale colour (so-called claycoloured). In liver disease and states of increased bilirubin production, urinary urobilinogen excretion is increased. EVALUATION OF LIVER FUNCTION TESTS Hyperbilirubinaemia ❑ ❑ 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. Hyperbilirubinaemia ❑ Three types of hyperbilirubinaemia: prehepatic, hepatic and posthepatic. Category Definition Pre-hepatic/ hemolytic The pathology is occurring prior to the liver. Hepatic/ hepatocellular The pathology is located within the liver. Post-Hepatic/ cholestatic The pathology is located after the conjugation of bilirubin in the liver. Prehepatic Hyperbilirubinaemia  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. ❑Prehepatic hyperbilinaemia also occurs in: ineffective erythropoiesis, and bleeding into tissues (e.g. sports injuries). Prehepatic Hyperbilirubinaemia ❑ (i) (ii) ❑ Laboratory findings include: Urine: Urobilinogen may be increased. Serum: increased unconjugated (indirect) bilirubin in the blood. The combination of increased urine-urobilinogen with no bilirubin (conjugated) in urine is suggestive of haemolytic jaundice. Hepatic Hyperbilirubinaemia 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. Hepatic Hyperbilirubinaemia ❑ ❑ ❑ Cell necrosis which reduces the liver's ability to metabolize and excrete bilirubin leading to a build up of unconjugated bilirubin in the blood. Hepatocellular (hepatic) hyperbilirubinaemia can be caused by acute hepatitis, hepatotoxicity, and alcoholic liver disease. Other causes: primary biliary cirrhosis, Gilberts’s syndrome (Low UDP-glucuronosyltransferase activity). Hepatic Hyperbilirubinaemia ❑ (i) (ii) Laboratory findings include: Urine: Conjugated bilirubin; urobilinogen (x 2 ULN) variable. Serum: increased conjugated (direct) bilirubin in the blood. Posthepatic Hyperbilirubinaemia Caused by an obstruction of the biliary tree. Plasma conjugated and other biliary metabolites, such as bile acids accumulate in the plasma. Characterized by pale coloured stools (absence of fecal bilirubin or urobilin), and dark urine (increased conjugated bilirubin). In a complete obstruction, urobilin is absent from the urine. Posthepatic Hyperbilirubinaemia ❑ ❑ Caused by an interruption to the drainage of bile in the biliary system. The most common causes are gallstones in the common bile duct, and pancreatic cancer in the head of the pancreas. Cholestatic hyperbilirubinaemia intrahepatic or extrahepatic. can be Posthepatic Hyperbilirubinaemia ❑ ❑ Intrahepatic hyperbilirubinaemia occurs in acute hepatocellular cholestasis, cirrhosis, intrahepatic carcinoma, primary biliary cirrhosis etc. Extrahepatic hyperbilirubinaemia occurs in gallstones, carcinoma of the head of the pancreas, carcinoma of the biliary tree, bile duct compression etc. Posthepatic Hyperbilirubinaemia ❑ Laboratory findings include: (i) Urine: Dark (increased conjugated bilirubin), (ii) Serum: increased conjugated bilirubin, (iii) Decreased urobilinogen, in urine. (iv) Stool colour: pale. ❑ The presence of pale stools and dark urine suggests an obstructive or post-hepatic cause as normal feces get their color from bile pigments. Biochemical tests for liver function ❑ Three main groups: (i) Routine liver function tests, (ii) True liver function tests, (iii) Special laboratory tests. Alanine aminotransferase (ALT) ❑ ❑ ❑ ❑ 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. 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 20 16 Alanine Aminotransferase ◼ ◼ ◼ ◼ Normal reference interval in serum (4-17 IU/ml for females and 6-21 IU/ml for males). Up to 300 U/L: non-specific , any type of liver disorder (chronic liver disease, cirrhosis, malignancy). >1,000 U/L: extensive hepatocellular damage (viral acute 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 ❑ This enzyme is widely distributed in the body. ❑ Normal reference interval in serum (7 - 32 U/L). ❑ ❑ Main sources: Heart, liver, skeletal muscle, and kidney. Useful in the diagnosis of MI, liver disorders and muscle damage. 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 Aspartate aminotransferase ❑ 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 ◼ ◼ ◼ 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 ◼ ◼ 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 membranes, there is a proportionately greater increase in plasma AST activity than ALT. AST/ALT ratio ◼ ◼ ◼ ◼ AST/ALT (DeRitis) ratio: to discriminate alcoholic hepatitis vs other liver diseases. AST/ALT > 2: likely to be associated with alcoholic hepatitis or hepatocellular carcinoma. AST/ALT between 1 and 2: likely to be associated with cirrhosis. AST/ALT 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 disease the level may be normal or slightly increased, but any increase is usually less than three times ULN. ❑ This reflects mild cholestasis due to obstruction of bile canaliculi by swollen hepatocytes & inflammation/necrosis of the ductular lining cells. ❑ There is increased synthesis of ALP (enzyme induction). ❑ Clinical application  GLUTAMYL TRANSFERASE (GGT) ❑ ❑ 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 ❑ 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 ❑ Normal reference interval in serum (10 – 70 U/L). ❑ Used primarily for diagnosis of hepatobiliary diseases. ❑ ❑ ❑ ❑ 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) Synthetic Function Albumin ❑ 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 ❑ ❑ ❑ 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. Disorders of the Liver Acute Hepatitis ❑ ❑ ❑ ❑ Hepatitis is defined by the inflammation of the liver and characterized by the presence of inflammatory cells. Hepatitis is acute when it last for less than six months. Acute hepatitis is usually caused by viral infection (particularly viruses A, B, C, D) or toxins (alcohol, paracetamol and various fungal toxins). There is cell membrane damage (cell necrosis & cell swelling) with an increase in plasma ALT activity greater than those of AST. Acute Hepatitis ◼ ◼ ◼ In acute hepatitis the areas of abnormal tissue predominantly contain diffuse sinusoidal and portal mononuclear infiltrates (lymphocytes, plasma cells) and swollen hepatocytes. Necrosis and inflammation of the biliary tree occurs. There is no evidence of fibrosis or cirrhosis (fibrosis plus regenerative nodules) and normal architecture is preserved but there may be some lobular disarray. Acute Hepatitis ❑ ❑ Cell necrosis causes the release of intracellular enzymes (ALT & AST), which are then absorbed by the blood vessels, leading to increased plasma enzyme activity. The plasma level of ALT rises early in the disease, prior to the onset of jaundice and reaches a peak [10 – 100 times upper limit of normal (ULN)] when jaundice appears. Acute Hepatitis ❑ ❑ The swollen hepatocytes distort the small intrahepatic bile passages and produce cholestasis, resulting in retention of bile pigments and increased plasma ALP levels (enzyme induction). The elevated serum bilirubin is mainly conjugated because of the obstruction and the liver being unable to excrete all the pigments. Acute Hepatitis ❑ ❑ Unless cholestasis is severe the serum bilirubin concentrations rarely exceed 350 µmol/L. Depending on the severity of the cholestasis the plasma ALP rarely rises beyond three times the ULN. Acute Hepatitis ❑ ❑ AST/ALT ratio < 1.0 (albeit the high activities of ALT and AST). Most cases of viral hepatitis resolve completely, and the chemical indices of abnormality revert to normal within a few weeks. Chronic Hepatitis ❑ ❑ Hepatic inflammation persisting for more than six months. Long lasting inflammation of the liver due to viruses or other causes. ❑ Two types: (i) Chronic active hepatitis (CAH) (ii) Chronic persistent hepatitis (CPH). Causes of chronic hepatitis ◼ Viral hepatitis: Hepatitis B with or without hepatitis D, hepatitis C (neither hepatitis A nor hepatitis E causes chronic hepatitis) ◼ Autoimmune: Autoimmune hepatitis ◼ Drugs: methyldopa, nitrofurantoin, isoniazid, ketoconazole. ◼ Heredity: Wilson's disease, alpha 1-antitrypsin deficiency. ◼ Others: Inflammatory bowel disease, ulcerative colitis and alcohol. Chronic Active Hepatitis ❑ ❑ ❑ It is caused by active hepatocellular destruction with episodes of relapses and remissions. It may follow acute hepatitis, or it may develop insidiously. There is an active, continuing inflammation of the liver associated with liver necrosis and fibrosis. Chronic Active Hepatitis ❑ ❑ ❑ A potentially fatal form of hepatitis complicated by portal inflammation and extending into the parenchyma. There may be progressive destruction of the liver lobule with necrosis and fibrosis leading to scarring, cirrhosis and liver decompensation. The possible causes include viral infections, drugs, and autoimmune reactions. Chronic Active Hepatitis Biochemistry (i) (ii) (iii) (iv) (v) As the disease progresses more cells are destroyed and the plasma AST activity may rise to or exceed that of ALT. Elevated ALP – cholestasis due to fibrosis. Elevated Bilirubin (mild to moderate in 70%) – cholestasis. Elevated plasma globulins – chronic inflammatory response. Decreased albumin – severe liver dysfunction. Primary Biliary Cirrhosis ❑ ❑ ◼ Primary biliary cirrhosis is an autoimmune disease of the liver. It results from a slow, progressive destruction of the small bile ducts of the liver, causing bile and other toxins to build up in the liver, a condition called cholestasis. Further slow damage to the liver tissue can lead to scarring and fibrosis. Primary Biliary Cirrhosis ❑ ◼ ◼ Abnormalities in liver enzyme tests are usually present and elevated gamma-glutamyl transferase and alkaline phosphatase (ALP) are found in early disease. Elevations in bilirubin occur in advanced disease. There is also elevation of ALT and AST due to cell necrosis. Thank you