Recommended
More Related Content
What's hot
What's hot (20)
Viewers also liked
Similar to going to linked in
Similar to going to linked in (20)
going to linked in
- 1. Quantitative Determination of Total and Direct Bilirubin in Serum By Femi Emmanuel Ojo Department of Biochemistry 2016
- 2. What is BILIRUBIN ? • Bilirubin is the yellow breakdown product of normal heme catabolism. • Bilirubin determination is among the panel of tests done when a liver function test is been carried out • Bilirubin is excreted into the bile duct and urine, and elevated levels may indicate certain diseases. • Bilirubin excretion is responsible for the amber color of our urine and the brown color of faeces • High level of bilirubin in the blood hyperbilirubinaemia is responsible for the yellow discoloration in jaundice.
- 3. Principle Of Serum Bilirubin Determination • Direct bilirubin reacts with diazotised sulphanilic acid to form azobilirubin complex which gives a blue color in alkaline medium • Total bilirubin can be determine by the addition of caffeine which releases albumin bound bilirubin by the reaction with diazotised sulphanilic acid • Indirect bilirubin can be determine by subtracting direct bilirubin from total bilirubin
- 4. Materials • Apparatus ; micro-pipette, tips, spectrophotometer, test-tubes, rack, tissue paper, water bath. • Reagents ; sulphanilic acid, nitrite, caffeine, sodium hydroxide
- 6. Procedure TOTAL BILIRUBIN • Pipette 200µl of reagent 1 into two test tubes (one for blank, the other for sample). • Pipette 50µl (1 drop) of reagent 2 into the sample labeled test tube but non into the blank labeled test tube. • Pipette 1000µl of reagent 3 into both the blank and sample labeled test tube • Pipette 200µl of serum into both test tube • Incubate for 10 minutes at 20-25 degrees • Pipette 1000µl of reagent 4 into both test tubes . • Incubate for 30 minutes and read absorbance at 578nm against the blank MANUAL CALCULATION Total bilirubin (µmol/l) = 185 x ATB(578 nm) Total bilirubin (mg/dl) = 10.8 x ATB(578 nm)
- 7. Procedure contd DIRECT BILIRUBIN • Pipette 200µl of reagent 1 into two test tubes (one labeled blank, the other labeled sample) • Pipette 50µl of reagent 2 into the sample labeled test tube but none into the blank labeled test tube • Pipette 2000µl of 0.9% of NaCl into both test tubes • Pipette 200µl of serum into both test tubes • Incubate for 10 minutes at 20-25 degrees Celsius • Read the absorbance at 546nm against the blank MANUAL CALCULATION Direct bilirubin (µmol/l) = 246 x ADB (546 nm) Direct bilirubin (mg/dl) =14.4 x ADB (546 nm)
- 8. Bilirubin Metabolism Bilirubin formation Transportation of Bilirubin in the plasma Hepatic circulation of bilirubin Conjugation of bilirubin Excretion of bilirubin
- 9. BLOOD CELLS LIVER Bilirubin diglucuronide (water-soluble) 2 UDP-glucuronic acid via bile duct to intestines Stercobilin excreted in feces Urobilinogen formed by bacteria KIDNEY Urobilin excreted in urine CO Biliverdin IX Heme oxygenase O2 Bilirubin (water-insoluble) NADP+ NADPH Biliverdin reductase Heme Globin Hemoglobin reabsorbed into blood Bilirubin (water-insoluble) via blood to the liver INTESTINE Catabolism of bilirubin
- 12. Structure Of Bilirubin Diglucoronide H N H N H N H NO O HH H2C CH2 CH2 CO O CH2 CO O O H HO H H OH COOH H H OH O H OH HO H H H OH H COOH
- 14. Clinical Significance • Icterus, a condition caused by hyperbilirubinaemia can be treated by phototherapy and EBT (Exchange blood transfusion). • Kernicterus, damages the brain passing through the BBB (Blood Brain Barrier), causing mental retardation. • When bilirubin is detected in urine, it may infer a liver damage but it (bilirubin) not a solely dependent test to examine the liver function.
- 15. Conclusion The normal value for Total bilirubin is up to 1mg/dl and for direct bilirubin is 0.25mg/dl
- 16. Recommendation Base on my experience, I recommend the following; • Parents and intending parents take this test seriously when it is required of them during ANTENATAL for their neonates. • Medical firms/clinic should make it an obligatory, compulsory and important test to be done immediately after birth to save the Neonates from parental negligence and future damages such as Icterus, Kernicterus and liver damage.