ANALYSIS OF

URINE
ACTIVITY NO. 13
 OBJECTIVES

AT THE END OF THE EXPERIMENT, THE STUDENTS MUST HAVE BEEN ABLE TO:
1. IDENTIFY THE NONPATHOLOGICAL AND PATHOLOGIC CONSTITUENTS OF THE
URINE AND THEIR BASIC CHEMICAL REACTIONS USING FUNDAMENTAL
CHEMICAL PROCEDURES.
2. PERFORM THE EXPERIMENT WITH CARE AND UTMOST RESPONSIBILITY SINCE
NUMBER OF CHEMICALS ARE BEING USED IN THIS ACTIVITY, AND
3. NOTE THE POSITIVE AND NEGATIVE RESULTS OBTAINED IN ALL TESTS.
 MATERIALS/ EQUIPMENT

PIPET BEAKER 250 ML

ASPIRATOR STIRRING ROD
10 TEST TUBES LITMUS PAPER BUNSEN
TEST TUBE HOLDER BURNER

TEST TUBE RACK TRIPOD WATER BATH

GRADUATED CYLINDER
URINE
ANALYSIS
Beginning of
Laboratory
Medicine

Routine Testing

Richard Bright
 RICHARD
BRIGHT
Richard Bright introduced the
concept of urinalysis as part of a
doctor’s routine patient
examination in 1827. By the
1930s, however, the number and
complexity of the tests performed
in a urinalysis had reached a point
of impracticality, and urinalysis
began to disappear from routine
examinations
 Two unique characteristics of a urine specimen
account for this continued popularity:
1. Urine is a readily available and easily collected
specimen.
2. Urine contains information, which can be
obtained by inexpensive laboratory tests, about
many of the body’s major metabolic functions.
These characteristics fit in well with the current
trends toward preventive medicine and lower
medical costs. In fact, the Clinical and Laboratory
Standards Institute (CLSI) defines urinalysis as “the
testing of urine with procedures commonly
performed in an expeditious, reliable, accurate, safe,
and cost-effective manner.”
 URINE
• THE KIDNEYS CONTINUOUSLY FORM URINE
AS AN ULTRAFILTRATE OF PLASMA.
REABSORPTION OF WATER AND FILTERED
SUBSTANCES ESSENTIAL TO BODY
FUNCTION CONVERTS APPROXIMATELY
170,000 ML OF FILTERED PLASMA TO THE
AVERAGE DAILY URINE OUTPUT OF 1200 ML.
 TESTS FOR NORMAL
ORGANIC CONSTITUENTS
 60%–90% of nitrogenous material;
UREA derived from the metabolism
of amino acids into ammonia

Procedure
1. Place 10 drops of urine in a test
tube
2. Add 5 drops of 10% HCl, then,
3. Add a few crystals of sodium
nitrite.
4. Note the evolution of N2 gas.
 URIC ACID
COMMON COMPONENT OF KIDNEY STONES;
DERIVED FROM CATABOLISM OF NUCLEIC ACID
IN FOOD AND CELL DESTRUCTION

PROCEDURE
1. PLACE 5 ML OF THE URINE IN A BEAKER.

2. ADD 1 ML CONCENTRATED HCL

3. STIR WELL AND SET ASIDE UNTIL THE NEXT LABORATORY

PERIOD.

4. DESCRIBE THE APPEARANCE OF THE CRYSTALS THAT

DEPOSIT ON THE SIDES OF THE BEAKER.

5. DISSOLVE A FEW OF THE URIC ACID CRYSTALS IN 2 ML OF 5%

SODIUM CARBONATE SOLUTION.

6. TEST ITS REDUCING PROPERTY BY ADDING 0.05% CUSO4

(BENEDICT’S TEST).

7. BOIL IN A WATER BATH FOR 15 MINUTES.

 CREATININE
DERIVED FROM CREATINE,
NITROGENOUS SUBSTANCE
IN MUSCLE TISSUE
PROCEDURE
1. PLACE 10 DROPS OF URINE IN A
BEAKER, THEN ADD 5 DROPS
SATURATED PICRIC ACID.
ALKALINITY WITH 10% NAOH.
2. A RED COLOR IS PRODUCED WHICH
TURNS YELLOW WHEN ACIDIFIED.
 Chloride
PRINCIPAL SALT; VARIES
WITH INTAKE

PROCEDU
RE
1.TO 3 ML OF URINE, ADD
SEVERAL DROPS OF HNO3
AND 5 DROPS OF 5%
AGNO3.
 PHOSPHATES
OCCURS PRIMARILY AS SODIUM
COMPOUNDS THAT SERVE AS BUFFERS
IN
THE BLOOD

PROCEDURE
1. TO 5 ML OF URINE, ADD DILUTE
NH4OH.
2. OBSERVE THE COLOR OF THE
SOLUTION.
3. WARM THE MIXTURE GENTLY AND
FILTER.
4. TO THE FILTRATE, ADD A FEW DROPS
PATHOLOGICAL CONSTITUENTS

Simulate a pathological sample of urine by adding 5 mL each

of 5% glucose, 5% albumin, acetone, bile, and blood
solution. Test for the presence of the pathological constituents
as follows:
 ACETONE
The term ketones represents three intermediate
products of fat metabolism, namely, acetone,
acetoacetic acid, and betahydroxybutyric acid.
Normally, measurable amounts of ketones do
not appear in the urine, because all the
metabolized fat is completely broken down into
carbon dioxide and water. However, when the
use of available carbohydrate as the major
source of energy becomes compromised, body
stores of fat must be metabolized to supply
energy. Ketones are then
detected in urine.
Rothera’s Nitroprusside Test
 GLUCOSE
Under normal circumstances, almost all the glucose filtered by the glomerulus is reabsorbed in the
proximal convoluted tubule; therefore, urine contains only minute amounts of glucose.

Benedict’s Test
Procedure
1. Place 2-3 mL of Benedict’s reagent in a test
tube, then add 5 mL of urine.
2. Mix thoroughly.
3. Boil it in a water bath for 10-15 minutes
and allow to cool.
4. Take note of the color of the precipitate
formed.
 Benedict’s Test
BENEDICT’S TEST IS A VERY SENSITIVE TEST DONE
UNDER MILDLY ALKALINE CONDITIONS.
THE REAGENT CONTAINS CUSO4, NA2CO3, AND SODIUM
CITRATE.
THE FORMATION OF A BRICK RED PRECIPITATE OF CU2O
IS CONSIDERED POSITIVE.
MOST ALDEHYDES HAVE THE ABILITY TO REDUCE
BENEDICT’S REAGENT. OTHER COMPOUNDS LIKE
FORMIC ACID, HYDRAZOBENZENE, PHENOLS,
PHENYLHYDRAZINE, PYROGALLOL, AND URIC ACID WILL
ALSO GIVE A POSITIVE RESULT IN THIS TEST.
 ALBUMIN
Albumin is the major serum protein found in normal
urine. Even though it is present in high
concentrations in the plasma, the normal urinary
albumin content is low because the majority of
albumin presented to the glomerulus is not filtered,
and much of the filtered albumin is reabsorbed by
the tubules.
1. Albumin: Heller’s Ring test

Heller’s test is a biochemical test performed to detect proteins

in a sample by the denaturation of those proteins by the
addition of strong acids. Heller’s test usually uses concentrated
nitric acid for the denaturation of proteins.

Heller’s test is a type of precipitation test where the

precipitation is brought about by denaturation

POSITIVE REACTION
1.The test tube is then observed for the formation of a white
ring at the junction of the two layers.
PROCEDURE
1. To 1.5 mL concentrated HNO3 in a test tube, deliver 1
mL urine down the side of the tube such that it forms a
separate layer.
2. The presence of protein is shown by a fluffy zone
(sometimes a white precipitate) at the urine-acid
interface.
 BLOOD
Hematuria is most closely related to disorders of renal or
genitourinary origin in which bleeding is the result of trauma or
damage to the organs of these systems. Major causes of hematuria
include renal calculi, glomerular diseases, tumors, trauma,
pyelonephritis, exposure to toxic chemicals, and anticoagulant
therapy. The laboratory is frequently requested to perform a urinalysis
when patients presenting with severe back and abdominal pain are
suspected of having renal calculi. In such cases, hematuria is usually
of a small to moderate degree, but its presence can be essential to the
diagnosis. Hematuria of nonpathologic significance is observed
following strenuous exercise and during menstruation.
 Benzidine Test
a sensitive test for the presence of blood (as in urine
or feces) based on the production of a blue color upon
contact with a solution of benzidine, hydrogen
peroxide, and glacial acetic acid

PROCEDURE
a. Place about 3 mL of urine suspected of containing blood in
a porcelain evaporating dish and add 5 drops of benzidine
reagent (a saturated solution of benzidine in glacial HAc).
(Caution: Benzidine is mutagenic and carcinogenic.)
b. Mix, then add 2 drops of 3% H2O2.
c. Spread this solution over the surface of the dish and
describe the color that soon forms.
 
 BILE PIGMENTS
Bilirubin is a yellowish pigment found in bile, which is a fluid
produced by the liver and stored in the gallbladder. Bilirubin, a
highly pigmented yellow compound, is a degradation product of
hemoglobin.
 Bile Pigments: Gmelin’s Test
Gmelin's test is a chemical test used for detecting the presence of bile pigments in urine. It is named after
Leopold Gmelin, who introduced the test. Five millilitres of urine is slowly added to five millilitres of
concentrated nitric acid in a test-tube. Different coloured rings between the two layers are visible if bile
pigments are present as they are oxidised to various chemical products. Nitric acid is used as the
oxidising agent. Blue, green and violet rings are seen if bilirubin is present.

PROCEDURE
1. Place 1 mL concentrated HNO3 in a test tube.
2. By means of a pipet, deliver down the side of
the tube 5 mL of urine. Do not shake.
3. Note the color of the ring which appears at
the interface of the 2 liquids.
4. Green, blue, or violet rings appear if
bilirubin is present.
 BILE ACIDS
Bile is generated by the liver and is stored by
the gall bladder. Whenever food is ingested,
bile is released into the duodenum. Bile salts
formation is initiated with the disintegration of
red blood cells. Rupturing of the damaged and
old red blood cells is brought about as they
pass through the liver or spleen.
 Pettenkofer’s Test
Bile salt will reacts with hydroxymethylenephurphural to forms red solution.
Hydroxymethylenephurphural is formed of sugar that dehydrated by sulphuric acid.

Procedure
1. Place 20 drops of urine in a test tube, then add 5 drops of
5% sucrose.
2. Let 2 mL concentrated H2SO4 slide down the side of the test
tube.
3. Do not shake.
4. A red ring develops at the point of contact of the two
solutions.
5. Do not mistake the brown color resulting from the charring
of sucrose by H2SO4 for the red ring.
6. Stir the mixture, and note the spreading of the red color
throughout the liquid.
The End