color indicates bilirubinuria.
p-nitrobenzene diazonium p-toluene sulfonate is the
active reagent. Place 5 drops of the urine on the mat
provided in the kit. Bilirubin, if present shall be absorbed
onto the mat surface. Place a reagent tablet on it. Let 2 drops
of water flow over the tablet. A positive test is indicated by
the appearance of a blue to purple color within 30 seconds.
Pink/red color is negative. Sensitivity > 0.1 to 0.05 mg% of
After dipping the strip in urine, match with the color chart
provided by the manufacturers.
1. Moderate to severe hepatocellular damage.
2. Obstruction of bile ducts, extrahepatic or intrahepatic.
In early hepatocellular damage and in hemolysis, urine
Urobilinogen is colorless, and on standing, it gets oxidized
to urobilin which has a brown color. It is best to perform tests
for urobilinogen on fresh specimens. If delay is inevitable,
collect the sample in a dark bottle, provide a surface layer
of petroleum ether and add sodium carbonate (5 g for
24 hours volume) and refrigerate the sample.
Ehrlich’s test: Nitrites and bilirubin interfere with this test.
Sulfonamide and procaine cause yellowish color reactions.
Pyridium, indole, porphobilinogen and PAS yield pink-red
color not different from that produced by urobilinogen.
To 10 mL of fresh sample at room temperature add 1 mL
of Ehrlich’s reagent, invert several times and let stand for
5 minutes. A pink color is normal, cherry or darker red color
indicate abnormal amounts of urobilinogen. Dilutions
may be used. Color reactions are normal in dilutions up to
Convert urobilinogen to urobilin by adding a few drops of
Lugol’s solution. Mix 10 mL of urine with an equal quantity
of saturated alcoholic solution of zinc acetate filter into a
Abnormal amounts of urobilin give the filtrate a green
fluorescence, which is best seen against a dark background
with a light source from the side, or in sunlight against a
The filtrate obtained from Harrison’s test for bilirubin
can be used for urobilinogen or urobilin.
Paper strip method (Dipstick tests are available).
Normal values: 0.1–1 Ehrlich unit/dL
Urinary urobilinogen is an important tool in routine
urinalysis since it serves as a guide in detecting and
differentiating liver disease, hemolytic disease, and biliary
obstruction. Sequential determination assists in evaluating
progress of disease and response to treatment. Both
urobilinogen and bilirubin in urine may be regarded as
bile pigments, but the tests provide different information.
Increased values of urobilinogen occur in:
¾ Cirrhosis: Bilirubin in urine may or may not be present
¾ Hemolytic jaundice: Bilirubin does not appear in urine.
A number of drugs produce false positives or negatives.
2-hour specimen: 0.1–1.0 Ehrlich units/2 hours
24-hour specimen: 1–4 mg/24 hours.
This is one of the most sensitive tests employed to
determine impaired liver function. Bilirubin, formed from
the metabolism of hemoglobin entering the intestine in
the bile, is transformed through the action of bacteria into
urobilinogen. Part of the urobilinogen formed in intestine
is excreted with the feces; another portion is absorbed into
the portal bloodstream and carried to the liver where it is
metabolized and excreted in bile. Traces of urobilinogen
that escape removal from the blood by the liver are carried
to the kidneys and excreted in the urine.
Increase in Urinary Urobilinogen
This occurs in any condition that causes an increase
in the production of bilirubin and by any disease that
prevents the liver from normally removing the reabsorbed
urobilinogen from the portal circulation.
a. Increased urobilinogen is found whenever there is
excessive destruction of RBCs as in:
b. Values above normal also occur in:
• Infectious and toxic hepatitis
• Hemolytic jaundice and anemia
• Chemical injury to liver due to chloroform and
carbon tetrachloride poisoning
c. An increased urobilinogen level is one of the earliest
signs of acute liver cell damage.
Decrease in Urinary Urobilinogen
This occurs when normal amounts of bilirubin are not
excreted into the intestinal tract. It usually indicates partial
or complete obstruction of the bile ducts. As occurs in:
¾ During antibiotic therapy. Suppression of normal
gut flora may prevent breakdown of bilirubin to
urobilinogen, leading to its absence in urine
¾ Decreased values are also associated with:
a. Drugs and foods that may cause urobilinogen to be
• Para-aminosalicylic acid (PAS)
• Drugs causing hemolysis of RBCs
b. Drugs that may cause decreased urobilinogen include
those that cause cholestasis and those that reduce
bacterial flora in the GI tract (e.g. antibiotics).
c. Peak excretion is said to occur from noon to 16:00
hours. The urinary urobilinogen is subject to diurnal
d. Strongly alkaline urine will show higher value and
strongly acid urine will show a lower level.
Perform Ehrlich’s test for urobilinogen by mixing equal
parts of urine and Ehrlich’s reagent. Add 2 parts of saturated
sodium acetate solution and mix. If turbid, filter. Shake with
a small quantity of chloroform. Urobilinogen is soluble in
chloroform, while porphobilinogen is not. If after several
extractions with chloroform the aqueous phase is still
pink, the test is positive for porphobilinogen.
Conditions producing increased levels of any of the heme
precursors are called porphyrias. The two rare major
categories of genetically determined porphyria and
erythropoietic porphyrias, in which the major diagnostic
abnormalities occur in red cell chemistry, and hepatic
porphyrias, in which heme precursors are found in urine
or feces. In acquired disorders, precursors accumulate
more in urine and feces than in red cells.
Porphobilinogens : 2 mg/24 h or negative
the formation of hemoglobin and other hemoproteins that
function as carriers of oxygen in the blood and tissues. In
health, insignificant amounts of porphyrin are excreted
in the urine. However, in conditions like porphyria
(disturbance in metabolism of porphyrin), liver disease,
lead poisoning, and pellagra, there is an increased level of
porphyrins as well as DAL and ALA in the urine. Disorders
of porphyrin metabolism also result in porphobilinogen.
In acute attacks of porphyria, the patient may suffer
skin lesions, abdominal pain, neuropathy, and mental
disturbances. The urine of patients with this disease
usually has a pinkish to reddish-black tinge and will
become darker upon standing. In the laboratory, the urine
is tested for the presence of porphyrins, porphobilinogen,
and DAL or ALA. It is also given the black light screening
test (porphyrins fluorescence when exposed to black or
to the laboratory. Porphobilinogens are always done with
porphyrin test. Should a single, fresh-voided specimen
be ordered, only a porphobilinogen will be done. Protect
specimen from light. The test must be performed within
60 minutes. Random sample should be obtained between
10:00 and 14:00 hours. Observe and record the color of
urine. If porphyrins are present, the urine may have a
grossly recognisable amber red or burgundy color. It may
vary from pale pink to almost black. Some patients will
excrete urine of normal color that turns dark after standing
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