31.2.2 Hunter’s Test
31.2.2.1 Principle
Bilirubin present in the urine sample gets adsorbed on barium sulfate precipitates.
This bilirubin reacts with diazotized sulfanilic acid to form pink-red color
azobilirubin.
31.2.2.2 Reagents
1.Diazo reagent:
Sol. A: Dissolve 0.1 g of sulfanilic acid in 1.5 ml conc. HCl, and make thefinal
volume 100 ml with distilled water.
Sol. B: 0.5% aqueous solution of sodium nitrite.
Mix 10 ml solution A with 0.3 ml solution B just before use; this is diazo reagent.
2.10% aqueous solution of BaCl 2
3.Absolute alcohol
4.6% Na 2 HPO 4
31.2.2.3 Procedure
To 5 ml of urine, add 2 ml of 10% BaCl 2 solution. Mix well and centrifuge at
3000 rpm for 5 min. Discard the supernatant and add 2 ml of distilled water to the
pellet, mix, and centrifuge at 3000 rpm for 5 min for washing the precipitates.
Discard the supernatant, and add 0.5 ml of diazo reagent. Mix and add 2 ml of
ethanol and 0.5 ml of 6% Na 2 HPO 4 , and observe the color. Purple-red color
indicates the presence of bile pigments in urine.
31.2.3 Gmelin’s Test
Add 3 ml of urine carefully over 5 ml conc. HNO 3 in a test tube. Wait for few min.
The presence of bile pigments shows a play of colors from yellow, red, violet, blue to
green due to formation of derivatives of bile pigments by action of HNO3.
31.2.3.1 Clinical Significance
Normally bile salts are absent in urine. The color of urine may be dark yellow to
greenish brown depending upon the concentration of bile pigments, and when
shaken urine becomes foamy. Bilirubin is found in urine in posthepatic or extrahe-
patic obstructive jaundice and in those types of hepatic jaundice in which there is
cholestasis. It is thus an indicator of regurgitation of conjugated bilirubin which
being water soluble and loosely attached to plasma albumin passes into the urine. It
is not present in urine in prehepatic (hemolytic) jaundice. The unconjugated biliru-
bin, circulating bound to albumin, is poorly soluble in water. The color of urine may
be dark yellow to greenish brown depending upon the amount of bile pigments. It
also forms foams readily when shaken.
31.2 Tests for Bile Pigments in Urine 125