Urinalysis Part 2

1. Detection of Sugar in Urine

Objective: To detect the presence of sugar in 4 different samples of urine.

A large volume of volatile and non-volatile waste products is produced by different metabolic
processes in the body. Urine is a liquid by-product produced in the animal and human bodies. It
is produced in the kidneys through a process called urination and is excreted through the
urethra. Urination is the primary method of excreting water-soluble chemicals from the body.

Normal urine usually has a light yellow color, due to the presence of a yellow pigment called
urochrome. Average production of urine in adult humans is 2 liters per day, depending upon
various conditions. The pH of urine varies between 4.6-8 and the specific gravity of urine varies
between 1.010-1.40.

Normal Constituents of Urine

Normal urine is a highly complex aqueous solution of organic and inorganic substances. Urine
consists of about 95-96% water. The most important nitrogenous organic substances present in
urine are urea, uric acid and creatine. The other organic substances are oxalic acid and lactic
acid. The principle inorganic constituents of urine are sodium chloride, potassium chloride,
sulphates and phosphates.

Abnormal Constituents of Urine

The abnormal constituents of urine are sugar (glucose), ketone bodies, blood, protein and
bile. Ordinarily, glucose (sugar) is absent in normal urine. But when the glucose level in blood
exceeds the renal threshold of glucose (160 – 180 mg /dl), glucose starts to appear in urine. The
presence of glucose in urine is called glucosuria and is usually an indication of diabetes mellitus.

Generally the following two tests are used to test the presence of sugar in urine sample.
• Benedict’s Test
• Fehling’s Test

In Benedict’s test, Benedict’s solution is used as the reagent. Benedict’s reagent is a

combination of sodium carbonate, sodium citrate and copper (II) sulphate pentahydrate
(CuSO4.5H2O). In Fehling’s test, Fehling’s solution-A and Fehling’s solution-B are used as the
reagents. Fehling’s solution-A is an aqueous solution of copper (II) sulphate, having blue colour,
while Fehling’s solution-B is clear colourless aqueous solution of sodium potassium tartrate.
On boiling the urine sample with the reagents, the copper (II) sulphate (CuSO4) present in the
Benedict’s solution and Fehling’s solution is reduced by the reducing agent, glucose (sugar), to
form a colored precipitate of cuprous oxide.
 • Depending upon the concentration of glucose, green, yellow and brick red precipitates of
cuprous oxide are formed. Below is the table showing the color sequence depending upon
the concentration of glucose level.

Color of precipitate Percentage of sugar present

Blue sugar absent

Green 0.5 to 1%

Yellow 1 to 2 % sugar

Brick Red 2 % or more sugar

Benedict's Test

Materials required:

Test tube, test tube holder, urine sample, measuring cylinders, Benedict’s solution and burner.

Procedure:

• Take 2 ml urine sample in a measuring cylinder from the urine sample bottle.
• Take a test tube and pour the urine sample in it.
• Take 5 ml Benedict’s reagent in a measuring cylinder.
• Add Benedict’s reagent to the test tube that contains urine sample.
• Using a test tube holder, hold the test tube firmly and heat it for 2 minutes on the burner.
• Keep shaking the test tube while heating.
• Observe the color formed and record.
Fehling's test

Materials required

Test tube, test tube holder, urine sample, measuring cylinders, Fehling’s solution A, Fehling’s
solution B and burner.

Procedure

• Take 2 ml urine sample in a measuring cylinder from the urine sample bottle.
• Take a test tube and pour the urine sample in it.
• Take 2 ml Fehling’s solution A in a measuring cylinder.
• Add Fehling’s solution A to the test tube that contains urine sample.
• Take 2 ml Fehling’s solution B in a measuring cylinder.
• Add Fehling’s solution B to the test tube that contains urine sample.
• Using a test tube holder, hold the test tube firmly and heat it gently for 2 minutes on the
burner.
• Keep shaking the test tube while heating.
• Observe the color formed and record.

2. Detection of Albumin in Urine

Objective: To detect the presence of albumin in urine.

Albumin is a protein produced by the liver and its mainly found in the blood. Its main functions
are the ability to maintain intravascular oncotic pressure, meaning it keeps the fluid pressure
stable within the blood vessels. It is also a carrier protein for steroids, fatty acids, and thyroid
hormones in the blood.

What is albuminuria?

A healthy kidney does not let albumin pass into the urine. A trace of protein which is less than
250 mg is found in normal urine daily. Under pathological conditions like albuminuria, level of
albumin found in urine will be way above the normal level. Albuminuria indicates some damage
to the kidneys, as it is starting to pass albumin into the urine. In kidney disturbance and high
blood pressure, albumin level in urine is significantly high.
We can test for the presence of albumin in urine using Heller’s test and Sulphosalicylic acid test.
Heller’s test causes the precipitation of albumin and forms a white ring in the solution. When
heated or treated with sulphosalicylic acid, albumin undergoes coagulation and forms a white
turbidity (cloudiness) in the solution.

Sulphosalicylic acid test

Materials required:

Burner, test tube holder, test tube, measuring cylinder, dropper, urine sample, 30%
sulphosalicylic acid

Procedure

• Take 2 ml urine sample in a measuring cylinder from the urine sample bottle.
• Take a test tube and pour the urine in the test tube.
• Using a dropper, take some sulphosalicylic acid.
• Add few drops of sulphosalicylic acid in the test tube containing urine. A whitish color
appears in the solution.
• Using a test tube holder, hold the test tube firmly and heat it gently upon a burner.
• A whitish or cloudy turbid solution indicates the presence of albumin in the urine sample.

Heller’s Test

Materials required: Burner, test tube, test tube holder, measuring cylinder, dropper,
concentrated nitric acid (HNO3)
Procedure

• Take 5 ml concentrated nitric acid in a measuring cylinder from the reagent bottle.

• Pour the concentrated nitric acid in a test tube from the measuring cylinder.

• Using a dropper, take some urine from the urine sample bottle.

• Now incline the test tube and add sample of urine by means of a dropper along the inner
side of the test tube so that it forms a layer over the nitric acid.

• The presence of white ring at the junction of two layers indicates the presence of albumin
in the urine.

3. Detection of Urea in Urine

Objective: To detect the presence of urea in the given sample of urine.

Urine is a liquid produced by the kidneys to remove waste products from the bloodstream.
Human urine is yellow in color and contains various chemical components. It primarily consists
of water, organic substance such as urea, uric acid, trace amounts of enzymes, carbohydrates
and hormones. The handling of urea by the kidneys is an important part of mammalian
metabolism.

Urea is present mainly in the urine of all mammals, but it also occurs in blood, bile and milk.
Urea is naturally produced during the process of breakdown of proteins. Due to this process,
amino groups are removed from the amino acid present in the proteins. These amino groups
are converted to highly toxic ammonia (NH3), and the ammonia thus produced is finally
converted to urea by the liver. The urea thus formed then passes to the kidneys and is finally
excreted from the body through the urine.
A healthy adult person normally excretes about 15g of nitrogen per day. Among this, about 95%
of this nitrogen is excreted as urea through urine. If the liver is not healthy, it may not
efficiently break down proteins. Similarly, if the kidneys are not healthy, they may not properly
filter urea. Either of these problems leads to changes in the amount of urea nitrogen in our
body. If the urine is kept exposed to the atmosphere, it splits, and ammonia gets released due
to bacterial activity. Due to this process the stored urine becomes alkaline.
Generally, urease tests are used to detect the presence of urea in the urine sample. The
enzyme urease decomposes urea into ammonia and carbon dioxide. On adding an alkaline
substance, ammonium carbonate to it, changes the slightly acidic urine to an alkaline
solution. The color of phenol red indicator changes from yellow to red in this reaction mixture.

Sodium Hypobromite Test

Materials required: test tube, dropper, measuring cylinder, urine sample, sodium hypobromite
solution

Procedure:

• Take 2 ml urine sample in a measuring cylinder from the urine sample bottle.
• Take a test tube and pour the urine sample into the test tube.
 • Using a dropper, take sodium hypobromite solution.
• Add few drops of sodium hypobromide solution to the urine sample.
• Brisk effervescence of nitrogen appears in the test tube which indicates presence of urea
in the urine.
Urease Test

Materials required: dropper, spatula, measuring cylinder, urine sample, 2% Na2CO3, 1% acetic
acid, phenol red indicator, urease powder

Procedure:

• Take 5 ml urine sample in a measuring cylinder from the urine sample bottle.
• Take a test tube and pour the urine sample into the test tube.
• Using a dropper, take some phenol red indicator.
• Add 4-5 drops of phenol red indicator to the test tube.
• Use a fresh dropper to take few drops of 2% Na2CO3.
• Add Na2CO3 solution drop by drop until a pink colour develops in the test tube.
• Take few drops of 1% acetic acid using a fresh dropper.
• Add 1% acetic acid to the test tube drop by drop until the pink colour disappears.
• Using a spatula, take some urease powder from the watch glass.
• Add urease powder to the test tube and shake it well.
• The pink or red colour appearing in the solution indicates the presence of urea in urine.

4. Detection of Bile Salts in Urine

Objective: to detect the presence of bile salt in urine.

Bile is a yellow-green fluid that contains water and organic molecules such as cholesterol, bile
acids, and bilirubin. In humans, the two main function of bile are digestion and absorption of
fats and eliminating bile salts from the body by secretion into bile. Adult humans produce
around 400 to 800 ml of bile daily.

In humans and most vertebrates, bile is produced by the liver. The gall bladder holds the bile
produced in the liver and when the organism eats, bile is discharged into the duodenum. The
formation of bile salts starts with the breakdown of red blood cells. The old red blood cells
become more fragile and may be damaged while they are passing through the small blood
vessels. These old and damaged red blood cells rupture as they pass through the spleen or liver.
The macrophages break down hemoglobin in the red blood cells and remove iron from the
heme component. The iron-free portion of heme is converted to biliverdin, a green pigment,
and then into bilirubin, a yellow orange pigment. In the liver, bilirubin are excreted in the bile as
bile pigments, which passes into the small intestine and then into the large intestine. Bilirubin is
detected in urine in certain pathological conditions only. Bilirubin is not found in urine. It is
present in urine during jaundice or because of liver damage.

We can test for the presence of bile salt in urine using Smith’s reagent and Pattern Raffo’s test.
On adding urine to the Smith’s reagent, a green ring is formed in the presence of bile salt in urine.
Pattern Raffo’s test gives a red colour in the presence of bile salt.

Smith’s Test

Materials required: test tube, dropper, measuring cylinder, urine sample, Smith’s reagent

Procedure:

• Take 1 ml Smith’s reagent in a measuring cylinder from the reagent bottle.

• Pour the Smith’s reagent from the measuring cylinder into a test tube.
• Using a dropper, take some urine from the urine sample bottle.
• Tilt the test tube and pour the urine along the side of the test tube.
• A green ring is formed at the junction of two layers indicating the presence of bile salt in
urine.
Pettenkofer’s Test

Materials required: test tube, dropper, spatula, measuring cylinder, urine sample, sucrose,
concentrated H2SO4

Procedure

• Take 2 ml urine in a measuring cylinder from the urine sample bottle.

• Pour the urine from the measuring cylinder into a test tube..
• Take some sucrose using a spatula.
• Put the sucrose into the test tube containing urine.
• Take 2 ml H2SO4 in a measuring cylinder from the reagent bottle.
• Tilt the test tube and pour the H2SO4 along the side of the test tube.
• Appearance of red colour indicates the presence of bile salt in urine.