Practical 3

PRACTICAL 3 DETERMINATION OF SERUM AND URINARY BILIRUBIN (JENDRASSIK METHOD) Objectives 1. Understand the principles of bilirubin measurements in serum and urine. 2. Perform the test on determination of total bilirubin, conjugated bilirubin and non- conjugated bilirubin in serum. 1. Perform the test on bilirubin determination in urine. 2. Understand the clinical conditions that arise due to impairment of bilirubin metabolism. Introduction Bilirubin is the end product of red blood cell (haemoglobin) catabolism in reticuloendothelial system (spleen, liver and bone marrow). Bilirubin is waste product that must be excreted by the liver through the conjugation process. Conjugated bilirubin is formed in the liver through a UDP glucoronyltransferase enzyme. It is transported to the intestine through the biliary system. In the intestine, conjugated bilirubin is transformed into urobilinogen by bacteria. Uribilinogen is further transformed into urobilin and stercobilin and finally excreted in bile. Urobilinogen is further filtered in the kidney and upon oxidation will be converted to urobilin, which is responsible for the yellow color of urine. In the normal individuals, concentration of bilirubin does not exceed 1 mg/dI ( (-20pmol/L) and almost 85 – 90% of this bilirubin are non-conjugated, whereas 10 – 15 % are conjugated bilirubin. Jaundice or icterus is a condition that causes yellowness of the skin and sclera of the eyes when the serum bilirubin exceeds 2 – 3mg/dI (>35 – 40 pmol/L). Hyperbilirubinaemia or increased blood bilirubin levels due to liver disease or haemolysis of red blood cells. Laboratory investigation could differentiate the source of hyperbilirubinaemia – either due to the impairment of the liver function or to excessive degradation of erythrocytes.

Chemical 1. 2. 3. 4. 5. 6.

Reagent 1 – Sulphanilic acid Reagent 2 – Sodium nitrate Reagent 3 – Caffeine Reagent 4 – Tartrate Sodium chloride (9 g/1) Dipstick

Method A. Determination of Conjugated Bilirubin in serum 1. Test tube is prepared as follows : CBA = Blank for sample A CSA = Sample A CBB = Blank for sample B CSB = Sample B

REAGENT 1 REAGENT 2 SODIUM CHLORIDE (9g/L) SAMPLE

CBA (ml) 0.20 2.00 0.20

A CSA (ml) 0.20 0.05 2.00 0.20

CBB (ml) 0.20 2.00 0.20

B CSB (ml) 0.20 0.05 2.00 0.20

The test tubes is shaked and leave for 5 minutes at room temperature. 2. The absorbance of the sample against blank at 546nm wavelength is measured. 3. the concentration of conjugated bilirubin is calculated in µmol/L unit. 1.

B. Determination of total serum bilirubin 1. Four test tubes is prepared as follows: TBA = Blank for sample A TSA = Sample A TBB = Blank for sample B TSB = Sample B

REAGENT 1 REAGENT 2 REAGENT 3 SAMPLE

TBA (ml) 0.20 1.00 0.20

A TSA (ml) 0.20 0.05 1.00 0.20

TBB (ml) 0.20 1.00 0.20

B TSB (ml) 0.20 0.05 1.00 0.20

2. the test tubes is shaked and is leaved for 10 minutes at room temperature.

1.

1.00 ml of reagent 4 is added into all test tubes. 3. The test tubes is shaked and leaved for 15 minutes at room temperature. 4. Measure the absorbance of the sample against blank at 578 nm wavelength. 5. Calculate the concentration of conjugated bilirubin in pmol/L unit. 1.

Calculation For Conjugated Bilirubin

Conjugated Bilirubin = 246 x Abs (546 nm) 2.

Calculation For Total Bilirubin

Total Bilirubin = 186 x ATB (578 nm) 3.

Calculation For Non-Conjugated Bilirubin

Non-Conjugated Bilirubin = Total Bilirubin – Conjugated Bilirubin 2. Determination of Urine Bilirubin Using Dipstick Dipstick

1. Perform the dipstick test on two urine samples given A and B. Record your results. Note : Sample A is your urine collection Sample B contains standard bilirubin

Questions Name the type of bilirubin that is present in the urine and what is the colour of urine if bilirubinis present? Why these occur? Give examples of disease for such conditions.

It is the conjugated bilirubin and it is present in urine due to high conjugated bilirubin in the blood (Hyperbilirubinemia). The colour of urine will be dark yellow. This occur because the conjugated bilirubin in the urine was oxidized by the oxygen in the air. An example of disease for such conditions is haemolytic anemia or sickle cell anemia. What is the additional non-biochemical test that could be performed to confirm the diagnosis of the disease in (1) Clinical test for jaundice. Check the skin and sclera colour. If it is yellow, then it is a bilirubin disease. Hyperbilirubinemia can occur in haemolytic anemia patient. Explain the reason for this. Bilirubin is one of the product of red blood cell (rbc) degradation. In haemolytic anemia patient, the rbc degradation rate is high. Thefore, the production rate of bilirubin is higher than the excretion rate. This will lead to the increase bilirubin in blood which is also known as Hyperbilirubinemia.