1. CLINICAL CHEMISTRY-2 (MLT 302) LIVER FUNCTION AND THE BILIARY TRACT LECTURE SIX
Dr. Essam H. Aljiffri

2. ASSESSMENT OF LIVER FUNCTION
Analysis of Bilirubin
Brief Review of Classical Methodology
Because its yellow color is detectable by the human eye, concentrations of serum bilirubin have been estimated for centuries.
- In 1883, Ehrlich first described a reaction in urine samples of the formation of a red or blue pigment when bilirubin was coupled with a diazotized sulfanilic acid solution.

3. Brief Review of Classical Methodology
- In 1938, Jendrassik and Grof used a procedure containing caffeine-benzoate-acetate as an accelerator for the azo-coupling reaction.

4. Brief Review of Classical Methodology
- For many years, results of bilirubin determinations were reported as direct or
indirect, it has been known that the direct reaction is given by the diglucuronide bilirubin or conjugated bilirubin, which is water-soluble.
- The indirect reaction on the other hand, is given by unconjugated bilirubin which is water-insoluble but dissolves in alcohol to couple with the diazo reagent.

5. Brief Review of Classical Methodology
- Direct and indirect bilirubin should be reported as conjugated and uncanjugated respectively.

6. JENDRASSIK METHOD FOR TOTAL AND CONJUGATED BILIRUBIN DETERMINATION
Principle
Direct (conjugated) bilirubin reacts with diazotised sulphanilic acid in alkaline medium to form a blue coloured complex.
Total bilirubin is determined in the presence of caffeine, which releases albumin bound bilirubin, by the reaction with diazotised sulphanilic acid.

7. JENDRASSIK METHOD FOR TOTAL AND CONJUGATED BILIRUBIN DETERMINATION
Principle
Serum or plasma is added to a solution of sodium acetate or and caffeine benzoate, which is then added to diazotized sulfanilic acid to form a purple azobilirubin.
- The sodium acetate buffers the pH of the diazotization reaction while the caffeine sodium benzoate accelerates the coupling of bilirubin with diazotized sulfanilic acid.

8. JENDRASSIK METHOD
- This reaction is terminated by the addition of ascorbic acid, which destroys the excess diazo reagent.
- A strongly alkaline tartrate solution is then added to convert the purple azobilirubin to blue azobilirubin and the intensity of the color is read at 600 nm.

9. JENDRASSIK METHOD Specimen Collection and Storage
A fasting serum specimen that is neither hemolyzed nor lipemic in nature is preferred.
- Prior to testing, serum should be stored in the dark and measured as soon as possible (within 2-3 h) after collection.
- Serum may be stored in the dark in a refrigerator for up to 1 week and freezer for 3 months without any change in the bilirubin concentration.

10. Calculation JENDRASSIK METHOD
- Values for total and conjugated bilirubin are obtained by comparing the absorbance read to that of a calibration curve prepared with acceptable bilirubin standards.
- Values for unconjugated (indirect) bilirubin are obtained by subtracting the conjugated (direct) bilirubin from the total bilirubin.

11. JENDRASSIK METHOD Reference Range Conjugated 0-0.2 mg/dl (0-3 µmol/L)
Unconjugated mg/dl (3-14 µmol/L)
Total mg/dl (3-17 µmol/L)

12. CASE HISTORIES
Case 1
A 24-year-old-man who was otherwise well, was thought to be mildly jaundiced and liver function tests were carried out:
Bilirubin µmol/L ( µmol/L)
ALT U/L ( U/L)
Alkaline phosphatase 85 U/L (30-95 U/L)
- Comments on the results?
- What pattern of liver function abnormality is present?
- What further analysis must be considered for diagnoses?

13. CASE HISTORIES
Case 2
The following liver function tests were obtained from a 72-year-old woman who was found to be jaundiced at a routine outpatient appointment 2 years after undergoing colectomy for a carcinoma of the colon.
Bilirubin µmol/L ( µmol/L)
ALT U/L ( U/L)
Alkaline phosphatase U/L (30-95 U/L)
- Comments on the results?
- What pattern of liver function abnormality is present?
- what is the most likely cause?