CLINICAL CHEMISTRY-2 (MLT 302) LIVER FUNCTION AND THE BILIARY TRACT LECTURE SIX

1
CLINICAL CHEMISTRY-2 (MLT 302)LIVER FUNCTION AND
THE BILIARY TRACTLECTURE 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
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.

11
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)

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 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?

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 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?