REACTIONS OF NORMAL URINE

BI 11.4 Perform urine analysis to estimate and determine normal

constituents.

At the end of the session the phase I MBBS student must be

able to correctly:
➢ List the chemical components in the normal urine.
➢ Write the normal concentration of the chemical
constituents in normal urine.
➢ Perform the tests to detect the normal constituents in the
given urine sample.
➢ List the pathological conditions associated with increased
or decreased concentrations of these chemical
constituents of urine.

1. SPECIFIC GRAVITY

Fill ¾ of a measuring jar with urine and gently float the urinometer in
it without touching the sides of the container. Note the reading on the
scale on the urinometer, which corresponds to the upper meniscus of the
level of urine. The numbers found on the scale represent the second and
third decimal places of the specific gravity value. Thus, if the upper
meniscus of the urine level is at the number 20 on the scale, this reading
of 20 is recorded as a specific gravity 1.020. Note the temperature of the
specimen, using a thermometer. The urinometer is calibrated at 15°C.
Find out the room temperature. For every 3° C rise, add 0.001 to the
observed reading to get a real specific gravity of the urine. Measure the
specific gravity of the given sample of urine.

Long’s coefficient : The total solids normally excreted in the urine

may be calculated using Long’s Coefficient that is 2.6. The solid content
of 1000ml of urine is calculated by multiplying last two digits of specific
gravity at 25˚C by 2.6 and is expressed in g/l.
2. pH REACTION
Test the reaction of urine with litmus paper. Note the color change.

3. INORGANIC CONSTITUENTS
a. Chlorides
Take 3 ml of urine in test tube and add 1 ml of conc. Nitric acid and 2
ml of 3 % silver nitrate solution.
Principle: A white precipitate of AgCl 2 is formed when acidified urine
reacts with AgNO3 solution. Nitric acid prevents the precipitation of
urates and phosphates as silver salts.
b. Earthly Calcium & Phosphates
To 8 ml of urine, add 0.5 ml of strong ammonia and boil, cool, filter and
discard filtrate. Then add 2 ml of dilute acetic acid on the filter paper and
collect the solution in a test tube. Divide this into two parts A and B.
To A, add 2 ml of 5 % ammonium oxalate solution.
To B, add 6 drops of nitric acid and 2 ml of ammonium molybdate and
boil.
Principle:
A. Calcium present in the urine is precipitated as calcium oxalate.
B. Phosphates of calcium and magnesium are precipitated by ammonium
hydroxide on boiling and the phosphates are dissolved in hot dil. acetic acid.
With ammonium molybdate, phosphates form a yellow precipitate of
ammonium phosphomolybdate.
c. Ammonia
To 10 ml of urine, add 2 drops of phenolphthalein and N/10 NaOH drop wise
till the color becomes faintly pink. Boil and hold a glass rod dipped in
phenolphthalein to the evolved steam.
Principle: Ammonium salts on decomposition evolve ammonia, which
changes the color of the rod to pink.
d. Inorganic Sulphate
To 5 ml urine in the test tube, add 1 ml conc. HCl and 5 ml of 10 % barium
chloride solution. Mix well.
Principle: Acidified urine reacts with BaCl 2 to form BaSO4. HCl prevents
precipitation of phosphates.

4. ORGANIC CONSTITUENTS
a. Ethereal Sulphates
The filtrate from the above experiments already contains HCl and excess
barium chloride. Boil the solution.
Principle: The inorganic sulphate is precipitated as barium sulphate and the
organic sulphates present in the filtrate are liberated as free sulphates on
boiling with HCl. The free sulphate reacts with barium chloride to form a
white precipitate of barium sulphate.

b. UREA
i. Hypobromite test:
To 5 ml of urine, add 1 ml of alkaline sodium hypobromite solution.
Principle: Alkaline hypobromite decomposes urea to nitrogen, carbon
dioxide and hydrogen. The brisk effervescence is due to the liberation of
nitrogen gas.
ii. Specific Urease Test:
To 3 ml of urine in a test tube; add one spatula of horse-gram powder and 5
drops of phenol red. Mix well. In another test tube, take 3 ml of water; add
one spatula of horse-gram powder and 5 drops of phenol red. Mix well.
Formation of pink color in the urine sample shows the presence of urea.
Principle: Horse gram powder is a source of enzyme urease which acts on urea
to form ammonium carbonate which makes the solution alkaline. Phenol red
is an indicator which works between pH 6.6 and 8.2 and ammonium
carbonate increases the pH and yellow color changes to red.

c. URIC ACID
i. Schiff’s Test
Moisten a filter paper with silver nitrate solution and add a few drops of urine
on it.
Principle: Uric acid reduces salts of silver nitrate to metallic silver.
ii. Benedict’s Uric Acid Test
To 3 ml of urine, add 1 ml of Benedict’s uric acid reagent and 1ml of 20 %
sodium carbonate.
Principle: Uric acid in alkaline conditions reduces phosphotungstic acid to
tungsten blue.

d. CREATININE

Jaffe’s Test
Take 5 ml of water in a test tube and 5 ml urine in another tube. Add to both
tubes, 1 ml of saturated picric acid and 10 drops of 10 % NaOH.
Principle: Creatinine reacts with picric acid in the presence of alkali to form
creatinine-picrate complex which is reddish orange in color.
 Normal Constituents Urine

1. What is the normal urine pH?

Normal pH of urine : 4.8 – 7.5

2. What is normal specific gravity of urine?

Normal specific gravity is between 1.010-1.025

3. Define specific gravity of urine.

Urinary specific gravity (SG) is a measure of the concentration of solutes in the
urine. It measures the ratio of urine density compared with water density and
provides information on the kidney’s ability to concentrate urine.
4. Name the Non-protein nitrogenous substances.
Urea, uric acid, creatinine.
5. Why is the normal urine amber yellow in color?
Fresh normal urine is amber yellow in color. The coloring pigment is
urochrome, a compound of urobilin or urobilinogen.
6. What is Urea?
Urea is the end product of protein metabolism.
7. What is creatinine?
Anhydrous form of creatine is creatinine.
8. Name the aminoacids which form creatine.
Glycine, arginine, methionine
9. How is creatinine formed in the body?
Glycine + arginine + methionine (Liver & kidney)→ creatine + ATP →
Creatine PO4 (present in muscles).
10. How much of creatinine is excreted in urine per day?
1 – 2 g/ day.
11. What is uric acid?
Uric acid is end product of purine metabolism.
12. What are the common causes of turbidity seen in a fresh urine sample? Why
sometimes a normal appearing urine sample becomes turbid on standing?
Turbidity could be due to the presence of

➢ Pus cells links in infections

➢ Fat globules link in chyluria or nephrotic syndrome
➢ Phosphates precipitation. Urine become turbid on standing due
to phosphate precipitation which could be seen during
prolonged convalescence.