1. Liver Function Tests (LFTs) Measurement of Serum Bilirubin (Total, direct &indirect) T.A. Bahiya Osrah

2. Bilirubin Bilirubin is the product of heme degradation (80% hemoglobin, 20% other hemo-protein as cytochrome, myoglobin). Elevated levels of bilirubin in blood and urine indicate certain diseases.

3. Bilirubin Bilirubin Structure: Bilirubin consists of four open chain pyrrols, unlike heme which consists of four rings pyrrols called (porphyrin). Bilirubin Heme

4. Types of bilirubin in serum Indirect bilirubin: is unconjugated or water insoluble, it is called indirect because it reacts slowly, so it indicates indirect reaching to reagent. Direct bilirubin: is conjugated or water soluble it is called direct because it reacts faster, so it indicates direct reaching to reagent. Note: Total bilirubin = D+ ID Knowing the level of each type of bilirubin has diagnostic important

5. Bilirubin Production After approximately 120 days in the circulation, red blood cells are taken up and degraded by the reticuloendothelial (RE) system, particularly in the liver, spleen and in the bone marrow. hemoglobin destroyed to the heme + globin amino acid Iron is removed from the heme molecule, porphyrin ring is opened to form bilirubin

6. Bilirubin Transportation Bilirubin is insoluble in water and is carried in plasma bound to albumin On reaching the liver, the bilirubin is taken into the hepatocyte by specific carrier mechanism Specific carrier mechanism

7. Conjugation of bilirubin and secretion into bile In the liver: Glucouronic acid + un-conjugated bilirubin (water insoluble) UDP-glucuronyltransferase Bilirubin diglucuronides (water soluble) Bilirubin diglucuronides are water soluble and readily transported into bile.

8. Further metabolism of bilirubin in the gut In the intestine: Bilirubin diglucuronides Bacteria Glucouronic acid + un-conjugated bilirubin

9. Further metabolism of bilirubin in the gut Reabsorption Into the blood

10. SUMMARY

11. Measurements of plasma bilirubin: Serum bilirubin concentration depends on the rate of removal of bilirubin from destruction of hemoglobin. A bilirubin test measures the amount of bilirubin in a blood sample. Types of Bilirubin: Bilirubin is present in plasma as: Indirect Bilirubin (unconjugated bilirubin) Direct Bilirubin (conjugated bilirubin) Total and direct bilirubin levels can be measured from the blood, but indirect bilirubin is calculated from the total and direct bilirubin.

12. Jaundice Jaundice: Is a term used in clinical medicine to describe a condition in which the skin and sclera appear yellow caused by increased amounts of bilirubin in the blood Classification of the causes of Jaundice: 1. Prehepatic jaundice 2. Hepatic jaundice 3. Posthepatic jaundice

13. Pre-hepatic (before bile is made in the liver) Jaundice in these cases is caused by rapid increase in the breakdown and destruction of the red blood cells (hemolysis), overwhelming the liver's ability to adequately remove the increased levels of bilirubin from the blood. Examples of conditions with increased breakdown of red blood cells include: Malaria, sickle cell crisis, thalassemia, glucose-6-phosphate dehydrogenase deficiency (G6PD), drugs or other toxins, and autoimmune disorders

14. Hepatic (the problem arises within the liver) Jaundice in these cases is caused by the liver's inability to properly metabolize and excrete bilirubin. Results from: Impaired cellular uptake. Defective conjugation. Abnormal secretion of bilirubin by the liver cell.

15. Post-hepatic (after bile has been made in the liver) Jaundice in these cases, also termed obstructive jaundice, is caused by conditions which interrupt the normal drainage of conjugated bilirubin in the form of bile from the liver into the intestines. This may due to: gallstones in the bile ducts, tumor Rise in the serum conjugated bilirubin level and stool becomes clay-colored. Why? Because of the normal drainage interruption of conjugated bilirubin in the form of bile from the liver into the intestines urine urobilinogen levels got decreased therefore the secretion of sterocobilin resulted to a clay-colored stool

16. Physiologic jaundice of the newborn High bilirubin levels are common in newborns age (1-3 days old). It is happened because after birth the newborns breaking down the excess RBCs they are born with and, because the newborn’s liver is not fully mature, it is unable to process the extra bilirubin, leads to elevate its level in blood and other body tissues. This situation usually resolves itself within a few days. Usually newborn is treated by phototherapy which breakdown bilirubin (ID<<<<D) and convert it to the photo isomer form which is more soluble. Very high bilirubin is danger and toxic. It may cause brain damage and affect on muscles, eyes and even death.

17. SUMMARY

18. The Lab practice Calculations The absorbance of bilirubin equivalent standard represents: 1.Direct bilirubin=2.5 mg/dl 2.Total bilirubin= 5 mg/dl 3.Direct bilirubin after 1min= (abs test- abs test blank/abs std )* 2.5 4.Total bilirubin after 5 min= (abs test- abs test blank/abs std )* 5 5.To convert mg/dl into µmol/l, multiply the final results by 17.1