Estimation of Bilirubin by spectrophotometer, normal values and interpretation of the results. Conjugated Vs Unconjugated hyperbilirubinemia

1. 2ND YEAR MBBS
BIOCHEMISTRY PRACTICAL
NO. 4
Friday, 31st July, 2020
Dr. Sana Samad Khan
MBBS,
Demonstrator/ Facilitator
Biochemistry Department,
PMC AJK

2. ESTIMATION OF BILIRUBIN BY
SPECTROPHOTOMETER

3. OBJECTIVES
• What is bilirubin?
• Method for estimation of bilirubin levels
• Normal values
• Clinical interpretation of the results

4. BILIRUBIN
• A yellow pigment that is a breakdown product of
heme catabolism.
• It is in two forms; conjugated and unconjugated.
• It is excreted as bile in faeces and in urine.
• It imparts yellow colour to urine and bruises.
• Gives brown colour to faeces.
• Elevated levels result in condition called jaundice.
• Daily synthesis in adult: average 250 – 350mg.

5. Formation of Bilirubin
• Water insoluble bilirubin is converted to water soluble
form for excretion through following steps.
1. Formation: Heme is converted to bilirubin.
2. Transport: Bilirubin is transported from peripheral tissues
to liver bound to plasma albumin.
3. Uptake: Bilirubin is picked up by liver parenchymal cells.
4. Conjugation: It is conjugated with glucuronate in the
endoplasmic reticulum via UDP-gluocoranyl transferase.
5. Secretion: Conjugated bilirubin is secreted into the bile.
Converted to stercobilin/urobilin and excreted.
1 gm of hemoglobin yields 35 mg of bilirubin.

6. BILIRUBIN FORMATION AND EXCRETION

7. COLORIMETRIC TEST FOR DIRECT AND
TOTAL BILIRUBIN (Modified Jeandrassik/
Grof Method)
• Principle
• Bilirubin reacts with diazotized sulphanilic acid (DSA)
to form a red azo dye. Absorbance of dye at 546 nm is
directly proportional to the bilirubin concentration.
• Water-soluble bilirubin glucoronides react directly
with DSA whereas the albumin conjugated indirect
bilirubin will react with DSA only in the presence of an
accelerator (caffeine).
• Total bilirubin = Direct + Indirect bilirubin

8. • REACTIONS:
Sulphanilic acid + sodium nitrite DSA
Bilirubin + DSA Direct Azobilirubin
Bilirubin + DSA + Accelerator Total Azobilirubin

9. REQUIREMENTS
1. Total Bilirubin Reagent (TBR)
• Sulphanilic acid
• Hydrochloric acid
• Caffeine
• Sodium benzoate
2. Total Nitrite Reagent
• Sodium nitrite
3. Direct Bilirubin Reagent (DBR)
• Sulphanilic acid
• Hydrochloric acid
4. Direct Nitrite Reagent
• Sodium nitrite
5. Spectrophotometer (Wavelength = 546 nm)
6. Specimen
7. Measure against sample blank

10. PROCEDURE
• For Total Bilirubin:
1. Put 1000 μL (1ml) of TBR into both the sample and
sample blank cuvettes.
2. Now put only 1 drop (40 μL) of TNR into the sample
cuvette.
3. Mix thoroughly and incubate for 5 mins.
4. Now put 1000 μL of the sample into both the
cuvettes.
5. Mix and incubate at room temperature for 10 – 30
mins.
6. Measure the sample against sample blank at 546 nm.

11. • For Direct Bilirubin:
1. Put 1000 μL (1ml) of DBR into both the sample and
sample blank cuvettes.
2. Now put only 1 drop (40 μL) of DNR into the sample
cuvette.
3. Mix thoroughly.
4. Add sample within 2 mins.
5. Now put 1000 μL of the sample into both the cuvettes.
6. Mix and incubate at room temperature for 5 mins.
7. Measure the sample against sample blank at 546 nm.

12. • Calculations:
• Calculate both total and direct bilirubin by using
factor 13.
• Bilirubin concentration = ΔA546 × 13 = .... mg/dL
Or
• Bilirubin concentration = mg/dL × 17.1 μmol/L

13. NORMAL VALUES
Total Bilirubin mg/dL μmol/L
At birth up to 5 85.5
5 days up to 12 205
1 month up to 1.5 25.6
Adults up to 1.1 18.8
Direct Bilirubin
Adults up to 0.25 4.3

14. PRECAUTIONS
1. Avoid hemolysis by shaking.
2. Hemolysis will also lower the bilirubin due to
inhibitory effect of hemoglobin on the diazo
reaction.
3. Do not expose the contents to sunlight.
4. Obtain fresh sample for the experiment.
5. Do not try to ingest or inhale the reagent. In
case of contact, wash thoroughly.

15. CLINICAL INTERPRETATION

16. HYPERBILIRUBINEMIA
• Two types:
1. Conjugated hyperbilirubinemia
2. Unconjugated hyperbilirubinemia

17. Unconjugated Hyperbilirubinemia
1. Overproduction
• Hemolysis (Intra and extravascular)
2. Decreased hepatic uptake
3. Decreased bilirubin conjugation (transferase
defeciency)
• Gilbert syndrome
• Crigler-Najjar syndrome
• Neonatal jaundice
4. Acquired transferase defeciency
• Drug inhibition (chloramphenicol and pregnanediol)
• Breast milk jaundice
• Sepsis
• Hepatocellular Disease (hepatitis, cirhosis).

18. Conjugated Hyperbilirubinemia
1. Impaired hepatic excretion
• Dubin-Johnson syndrome
• Recurrent (benign) intrahepatic
cholestasis
• Cholestatic jaundice of
pregnancy
2. Acquired disorders
• Hepatocellular disease (e.g.
Viral or drug induced hepatitis,
cirrhosis)
• Drug induced cholestasis (e.g.
Oral contraceptives, androgens,
chlorpromazine)
• Alcoholic liver disease
• Sepsis
• Biliary cirrhosis
3. Extra-hepatic Biliary
obstruction
• Gallstones
• Biliary malformation
• Infection
• Hemophilia (trauma, tumor)
• Sclerosing cholangitis
• Malignancy
• Inflammation (pancreatitis)

19. RESOURCES
1. Harper’s Illustrated Biochemistry
2. Experimental Biochemistry manual by Prof.
Muhammad Shoaib Khan
3. Slideshare (Online)

20. Thank you for your time and patience!
Please feel free to contact me in case of
any questions.
sanasamadkhan@gmail.com

21. STAY HOME
STAY SAFE