The document discusses the assessment of liver function through bilirubin analysis. It provides a brief history of bilirubin testing methodology and describes the Jendrassik method for determining total and conjugated bilirubin levels. This method involves a reaction between bilirubin in a serum sample and diazotized sulfanilic acid to form a colored complex that can be measured spectrophotometrically. The document also provides reference ranges for bilirubin levels and discusses two case studies involving abnormal liver function test results.

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

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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.

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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.

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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.

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

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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.

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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.

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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.

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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.

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JENDRASSIK METHOD
Calculation
- 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.

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JENDRASSIK METHOD
Reference Range
Conjugated 0-0.2 mg/dl (0-3 µmol/L)
Unconjugated 0.2-0.8 mg/dl (3-14 µmol/L)
Total 0.2-1.0 mg/dl (3-17 µmol/L)

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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 39 µmol/L (3-17 µmol/L)
ALT 35 U/L (6-37 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?

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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 75 µmol/L (3-17 µmol/L)
ALT 40 U/L (6-37 U/L)
Alkaline phosphatase 950 U/L (30-95 U/L)
- Comments on the results?
- What pattern of liver function abnormality is present?
- what is the most likely cause?