مرحلة ثالثة الدراسة الصباحية Clinical Biochemistry Liver function tests The liver is the largest organ in the body It is located below the diaphragm in the right upper quadrant of the abdominal cavity and extended approximately from the right 5th rib to the lower border of the rib cage. The liver is separated into a right and left lobe, separated by the falciform ligament. The right is much larger than the left. Functions of liver ① Excretory function: bile pigments, bile salts and cholesterol are excreted in bile into intestine. ② Metabolic function: liver actively participates in carbohydrate, lipid, protein, mineral and vitamin metabolisms. ③ Hematological function: liver is also produces clotting factors like factor V, VII. Fibrinogen involved in blood coagulation is also synthesized in liver. It synthesizes plasma proteins and destruction of erythrocytes. ④ Storage functions: glycogen, vitamins A, D and B12, and trace element iron are stored in liver. ⑤ Protective functions and detoxification: Ammonia is detoxified to urea. kupffer cells of liver perform phagocytosis to eliminate foreign compounds. Liver is responsible for the metabolism of xenobiotic. 1 م.م. وفاء حفظي عجام Classification of liver functions test Classified based on the major functions of liver: ① Excretion: Measurement of bile pigments, bile salts. ② Serum enzymes: Transaminase (ALT, AST), alkaline phosphate (ALP), 5’-nucleotidase, LDH isoenzyme. ③ Synthetic function: Prothrombin time, serum albumin. ④ Detoxification : Excretion: Bilirubin Bilirubin is the main bile pigment that is formed from the breakdown of heme in red blood cells. The broken down heme travels to the liver, where it is secreted into the bile by the liver. Serum bilirubin Normally, a small amount of bilirubin circulates in the blood. 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. A. indirect bilirubin (Normal value = 0.3 - 1.2 mg/dl) B. direct bilirubin (Normal value ≤ 0.4 mg/dl) C. total bilirubin Normal value for = 0.3- 1.2 mg/dl. A. urobilinogen : Conjugated bilirubin is excreted via bile salts to intestine. Bacteria in the intestine break down bilirubin to urobilinogen for excretion in the feces (normal value for fecal urobilinogen = 40 - 280 mg/day) Normally there are mere traces of urobilinogen in the urine. Average is 0.64 mg , maximum normal 4mg/24hours. 2 م.م. وفاء حفظي عجام B. Urobilin Urobilin is the final product of oxidation of urobilinogen by oxygen in air. The amount change with the amount of urobilinogen excretion. B. bilirubinurine: Bilirubin is not normally present in urine and feces since bacteria in intestine reduce it to urobilinogen. The kidneys do not filter unconjugated bilirubin because of its avid binding to albumin. Conjugated bilirubin can pass through glomerular filter. Bilirubin is found in the urine in obstructive jaundice due to various causes and in cholestasis. Note: Bilirubin in the urine may be detected even before clinical jaundice is noted. Who is a candidate for the test? Bilirubin is used to diagnosis of jaundice. Abnormal bilirubin levels can be found in many disorders, including: blocked bile ducts, cirrhosis, hepatitis and other liver diseases or immature liver development in newborns. Hemolytic Jaundice Hepatic Jaundice Obstructive jaundice ( Cholestasis) Congenital Jaundice 3 م.م. وفاء حفظي عجام Serum enzymes A large number of enzyme estimations are available which are used to ascertain liver function. They are being divided into two groups: I: most commonly and routinely done in the laboratory. Serum transaminase(ALT/AST) Serum alkaline phosphate (ALP) II: not routinely done in the laboratory. Alanine transaminase (ALT) ALT or sGPT (serum glutamate pyruvate transaminase Aspartate aminotransferase (AST) AST or sGOT (serum glutamate oxaloacetate transaminase) Therefore, when the liver is injured, GPT is released into the bloodstream. 4 م.م. وفاء حفظي عجام Elevated levels of GPT may indicate: alcoholic liver disease cancer of the liver cholestasis or congestion of the bile ducts cirrhosis or scarring of the liver with loss of function death of liver tissue Hepatitis or inflammation of the liver noncancerous tumor of the liver Use of medicines or drugs toxic to the liver GOT also reflects damage to the hepatic cells and is less specific for liver disease. It can also be released with heart, muscle and brain disorders. Therefore, this test may be ordered to help diagnose various heart, muscle or brain disorders, such as a myocardial infarct (heart attack). Elevated levels of GOT may indicate : acute hemolytic anemia, Acute pancreatitis or inflammation of the pancreas. Acute renal failure or loss of kidney function. Cirrhosis of the liver. Hepatitis heart attack primary muscle disease recent surgery severe burns Muscle injury Although GOT is not a specific for liver as the GPT, ratios between GPT and GOT are useful to physicians in assessing the etiology of liver enzyme abnormalities. ◆ Normally : GPT is normal, GOT is normal, GPT/GOT is about 1.15. ◆ Virus hepatitis: GPT↑, GOT is normal, GPT/GOT＞1, even more than 2.5； ◆ Chronic hepatitis: GPT↑, GOT ↑GPT/GOT is about 1. 5 م.م. وفاء حفظي عجام ◆ Liver cancer, cirrhosis, Alcohol-induced hepatitis: GPT↑, GOT ↑ < 1, about 0.6~0.7. ◆ Accute myocardial infarct :< 1 GPT and GOT is in the different distribution of the hepatocytes. GPT exists primarily in the cytoplasm of liver cell. If there is a slight liver cell damage, GPT firstly leak into the bloodstream, so that the serum GPT increased. The GOT mainly in the "mitochondria“of liver cells, the mitochondria are "bubble" in the liver cell cytoplasm. If there is a slight liver cell damage, GOT don`t leak into the bloodstream. Alkaline phosphatase (ALP) ALP occurs in all tissues, especially liver and bone. The alkaline phosphatase test is often used to help diagnose certain liver diseases and bone disorders. Nor mal range: 30 - 95 IU/L (3-13 kings unit) ALP is a hydrolase enzyme responsible for removing phosphate groups from many types of molecules, including nucleotides and proteins. most effective in an alkaline environment In humans, alkaline phosphatase is present in all tissues throughout the entire body, but is particularly concentrated in liver, bile duct, kidney, bone, and the placenta. Levels are significantly higher in children and pregnant women. Higher levels of ALP than normal may indicate: liver disease bone disease leukemia, a cancer of the blood and bone marrow various hormone problems Pregnancy 6 م.م. وفاء حفظي عجام Lower levels of ALP than normal may indicate: anemia, or a low red blood cell count malnutrition Various hormone problems Determination of total plasma proteins/ albumin/ globulin/ A : G ratio This yields most useful information in chronic liver disease. Liver is the site of albumin synthesis and also possibly of some of α and β globulins. Normal value: Total plasma proteins: 80~110mg/dl Albumin: 40-50mg/dl Globulin: 25~35mg/dl A: G ratio: 1.5~2.5 Serum Albumin Albumin is an important blood 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. ! Low albumin level produce ascites. ★ Albumin is also very important in the transportation of many substances such ! as drugs, lipids, hormones and toxins that are bound to albumin in the bloodstream. This test is normally performed to assist in diagnosing diseases that affect proteins ! in the body, such as cancer, liver disease, renal or intestinal problems, and immune disorders. Normal range: 34 - 54 g/L ! Abnormally low contents of albumin may indicate: ascites extensive burns kidney disease liver disease malabsorption syndromes 7 م.م. وفاء حفظي عجام Formation of prothrombin by liver At least 12 different proteins are involved in clotting. Blood clotting factors are proteins made by the liver and are associated with the incorporation of vitamin K metabolites into a protein. When the liver is significantly injured, these proteins are not normally produced. PT (Prothrombin time) Estimation of plasma fibrinogen APTT(activated partial thromboplastin time) Prothrombin time (protime or PT) Prothrombin is a plasma protein that is converted into thrombin during blood clotting. ★ Prothrombin is formed in the liver from inactive “preprothrombin” in presence of vitamin K. What is prothrombin time? prothrombin time is measured as prothrombin activity. The term prothrombin time was given to time required for clotting to take place in plasma to III factor and Ca+ have been added. PT is used to assess the activity of extrinsic blood clotting pathway. ★ PT is also a useful test of liver function, since there is a good correlation between abnormalities in coagulation measured by PT and the degree of liver dysfunction. PT is usually expressed in seconds and compared to a normal control patient’s blood. 8 م.م. وفاء حفظي عجام Formation of Bilirubin from Heme• Heme is degraded in RE system (esp. liver & spleen) 85% from RBCs 15% from turnover of immature RBCs & cytochromes Hemoglobin (Hb) consists of four (tetramer) sub-units held together by multiple non- covalent interactions. Each subunit consists of heme (Protoporphyrin IX and ferrous ion (Fe+2) and globin protein. Globin protein folds around heme group forming hydrophobic pocket that protects the heme , which is the site of oxygen binding. RBC is largest repository of heme in humans which it is life span about 120 days. Heme ↓ hemeoxygenase biliverdin(green) ↓ bilirubin (red-orange) bile pigments ↓ In Blood with albumin UNCONJUGATED BILIRUBIN (Or INDIRECT BILITUBIN) 9 م.م. وفاء حفظي عجام Bilirubin Metabolism in the Liver• Uptake of Bilirubin by hepatocytes:• Bilirubin dissociates from its carrier albumin & enters hepatocytes Conjugation of Bilirubin:• In hepatocytes, bilirubin is conjugated with two molecules of glucuronic acid by the enzyme glucuronyltransferase Excretion of bilirubin into bile:• Conjugated bilirubin (bilirubin diglucuronide) is transported into bile canalculi& then into bile.