Republic of the Philippines

CEBU TECHNOLOGICAL UNIVERSITY

BIOCHEMISTRY LABORATORY
Experiment No. 7
BIOCHEMICAL TEST OF URINE

Name: _Pila, Mary Ella Mae C. _ Course/yr/sec.: _BSN I-B_ Date: _June 05, 2021_
Group No. _3_

INTRODUCTION
Urine is a liquid byproduct of the body secreted by the kidneys through a process called urination
and excreted through the urethra. The normal chemical composition of urine is mainly water content, but
it also includes nitrogenous molecules, such as urea, as well as creatinine and other metabolic waste
components. In humans, the formation of urine begins in the nephrons of the kidneys by filtration of blood
plasma into the nephron; the fluid found within the nephron is essentially the same as blood plasma
without the macromolecules (e.g., proteins). As the fluid passes along the nephron tube, water and useful
plasma components such as amino acids, glucose, and other nutrients are reabsorbed into the
bloodstream, leaving a concentrated solution of waste material called final, or bladder, urine. It consists
of water, urea (from amino acid metabolism), inorganic salts, creatinine, ammonia, and pigmented
products of blood breakdown, one of which (urochrome) gives urine its typically yellowish color. In
addition, any unusual substances for which there is no mechanism of reabsorption into the blood remain
in the urine. 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.

PREPARATION
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1. Detection of sugar in urine

Objective:
To be able to detect the presence of sugar in the given sample of urine.

A. Benedict’s Test

I. Materials:
Test tube, test tube holder, urine sample, measuring cylinders, Benedict’s solution, and
burner.
• Benedict’s reagent is a combination of sodium carbonate, sodium citrate and copper (II)
sulphate pentahydrate (CuSO4.5H2O).

II. 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.
• A yellow precipitate appears which indicates the presence of sugar in urine.
• Depending upon the concentration of sugar in the urine, either green, yellow, or brick
red precipitates are formed.

Reaction:
The contents of the test tube become turbid due to a precipitate that ranges from green to
brick red in color, depending on the amount of sugar present in the urine. The copper sulphate in
Benedict's solution reacts with reducing sugars and the cupric ions to cuprous ions, these are
precipitated as red copper oxide, which is insoluble in water. Alkaline medium is provided to the
reaction by sodium carbonate present in the reagent. The original color of Benedict's reagent is
blue. It changes to green, yellow, orange, or red, according to the concentration of glucose
present in urine.

III. Observation and result

Upon adding the Benedict's reagent to the test tube that contains the urine sample and
heating it with Bunsen burner, a yellow precipitate appeared on the test tube that indicates the
presence of sugar in the given sample of urine. Depending upon the concentration of sugar in the
urine, either green, yellow, or brick red precipitates of cuprous oxide are formed. A green
precipitate appears when there is 0.5 to 1% of sugar is present, a yellow precipitate appears when
there is 1 to 2& of sugar is present, and a brick red precipitate appears when there is 2% or more
sugar is present. However, a blue precipitate appears or the color of the solution within the test
tube does not change if sugar is absent.

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

II. 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.
• A green precipitate appears which indicates the presence of traces of sugar in urine.
• Depending upon the concentration of sugar in the urine, either green, yellow or brick red
precipitates are formed.

Fehling’s solution-A is an aqueous solution of copper (II) sulphate, having blue color.
Fehling’s solution-B is clear colorless aqueous solution of sodium potassium tartrate.

Note: 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.

Reaction:
When the urine sample was heated with the Fehling's solution, the CuSO4 found in Fehling's
solution was reduced by the reducing agent, glucose for the formation of a colored cuprous oxide
precipitate. This is due to the reducing sugar having a free aldehyde or ketone group so when
glucose reacts with Fehling's solution, it forms red precipitate or depending upon the sugar
concentration in the urine and gluconic acid.

III. Observation and result

• What is the condition called when a person has a high level of sugar in urine?
• What does the presence of sugar in urine indicates?

Upon heating the test tube with the given sample that was added with Fehling's solution A
and Fehling's solution B, gradually, a green precipitate was formed in the test tube that indicates
the presence of sugar in the given urine sample. Different precipitates are formed depending
upon the sugar concentration in urine which can be yellow, green, brick red. The same as with the
Benedict's test, a green precipitate appears when there is 0.5 to 1% of sugar is present, a yellow
precipitate appears when there is 1 to 2& of sugar is present, and a brick red precipitate appears
when there is 2% or more sugar is present. However, a blue precipitate appears or the color of
the solution within the test tube does not change if sugar is absent.

When a person has a high level of sugar in urine, glycosuria occurs. It is where the urine
contains more blood sugar than usual. The kidneys normally absorb blood sugar back into the
blood vessels from any liquid that passes through them. However, with glycosuria, the kidneys
might not take enough blood sugar out of the urine before it passes out of the body.

Healthy individuals should not have glucose in their urine at all therefore the presence of
sugar in the urine indicates a health problem especially if there is an abnormally high levels of
glucose in the urine. The normal amount of glucose in urine is 0 to 0.8 mmol/L. Such health
problems might be diabetes, kidney problems, urinary tract infection, renal glycosuria and in
some cases, it could also be due to pregnancy.
2. Detection of Albumin in urine
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Albumin is a protein that is produced by the liver. It is a typical constituent of blood that is filtered
by the kidney. It acts as a carrier protein for fatty acids, thyroid hormones, steroids in the blood. A
normally functioning kidney has very less to no traces of albumin in the urine, about 250mg in normal
urine in a day. Any damage to the kidney causes an unusual range of albumin, way above the normal
level to enter into the urine. This condition is known as albuminuria. If the albumin level is very little
and persists to stay abnormal, then the condition is referred to as microalbuminuria. Albuminuria can
also be caused by kidney damage caused by heart failure, high blood pressure, diabetes, lupus and
cirrhosis.

A. Heller’s Test

I. Objective
To detect the presence of albumin in the urine.

II. Materials
• Conc. nitric acid, Urine sample, Test tube, Measuring cylinder, Dropper, Test tube holder

III. Procedure
• From the reagent bottle, add 5ml conc. nitric acid accurately using a measuring
cylinder, pour it into a test tube
• Using a dropper, add some drops of the urine sample in a test tube
• Pour some sample of urine along the inner side of the test tube with the help of a
dropper and by inclining the tube.
• The above step is performed so as to form a covering on the nitric acid
• Observe the changes taking place in the test tube.

Reaction:
Nitric acid causes denaturation of proteins with the formation of a white precipitate. As urine
that contains albumin comes in contact with nitric acid, it forms a white ring at the point of
contact.

IV. Observation and result

In the experiment, the changes take place in the test tube when the urine comes in contact
with the nitric acid. Upon taking a small quantity of urine from the urine sample bottle and adding
it to a test tube containing a concentrated nitric acid, it necessary to incline the test tube for the
appearance of the white ring at the junction of two layers will be distinguished clearly. A positive
result is represented by the appearance or formation of a white ring at the junction of the two
distinct layers. This indicates the presence of albumin in the given sample. A negative result is
represented by the absence of a white ring.
 B. Sulphosalicylic Acid Test

I. Objective
To detect the presence of albumin in the urine.

II. Materials
• 30% Sulphosalicylic acid, Urine sample, Measuring cylinder, Burner, Test tube,
• Test tube holder, Dropper

III. Procedure
• From the sample urine bottle, add 2ml sample of the urine accurately using a measuring
cylinder, pour it into a test tube
• Add some drops of the sulphosalicylic acid with the help of a dropper into the tube
holding the urine sample
• Observe the color of the solution.
• Using a test tube holder hold the tube and heat upon the burner gently.
• Observe the changes of the solution

Reaction
When sulphosalicylic acid reacts with albumin, it causes denaturation of protein in the sample
that appears in a form of precipitate.

IV. Observation and result

Upon adding sulphosalicylic acid to the test tube containing a urine sample, a cloudy turbid
solution or precipitate will be observed. The appearance of a cloudy turbid or precipitate indicates
that there is a presence of albumin in the sample urine. The absence of a cloudy turbid or
precipitate indicates the absence of albumin.

3. Detection of urea in urine

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Urea in urine is produced naturally by the body while the proteins are disintegrated during
which amino groups are eliminated from the amino acids that are in the proteins. The eliminated
amino groups are converted into extremely toxic ammonia and furthermore into urea by the liver.
The urea moves into the kidneys, where it is finally eliminated from the body. For detection of urea
in the urine sample, urease tests and sodium hypobromite are performed.

A. Sodium Hypobromite Test

Sodium hypobromite reacts with urea and nitrogen gas is released as brisk effervescence.
When urea is treated with sodium hypobromite it decomposes to give N2

I. Objective
To detect the presence of urea in the given sample of urine.
 II. Materials
• Dropper, Test tube, Urine sample, Measuring cylinder, Sodium hypobromite sol.

III. Procedure
• Take a clean and dried test tube.
• With the help of a measuring cylinder, add 2ml urine sample into the test tube.
• To the same test-tube add 2 to 3 drops of sodium hypobromite solution using a
dropper.
• Mix the solution well.
• Observe the changes.

Reaction:

IV. Observation and result

Upon dropping the sodium hypobromite solution in the urine sample, it had a rapid or
vigorous evolution of gas in the test tube. It is termed as brisk effervescence. The presence of urea
in the given urine sample is indicated by the presence of brisk effervescence of nitrogen around
the test tube. Therefore, the urine sample contains urea and is positive from the Sodium
Hypobromite Test.

B. Urease Test
Urease is an enzyme that carries out the decomposition of urea into carbon dioxide and
ammonia. Addition of an alkaline substance such as ammonium carbonate causes it to turn into
an alkaline solution from slightly acidic urine.

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

II. Materials
• Spatula, Dropper, Urine sample, 1% acetic acid, 2% of Na2CO3, Urease powder.
• Measuring cylinder, Phenol red indicator.

III. Procedure
• Take a clean and dried test tube.
• With the help of a measuring cylinder, measure and add 5ml urine sample into the test
tube.
• To the sample test tube, add 4 to 5 drops of the phenol red indicator using a dropper.
 • With the help of another dropper, add drops of 2% Na2CO3 solution drop-wise until
the pink color starts developing in the test tube.
• With the help of another dropper, add a few drops of 1% acetic acid. Add drop-wise
until the pink color disappears.
• With the help of a spatula, add little urease powder into the urine sample test tube.
• Now shake the test tube slowly until the urease powder get mixed.
• Now observe the changes.

Reaction:

IV. Observation and result

• What alkaline product is produced when urea is broken down?

The alkaline product produced when urea is broken down is ammonia plus carbon
dioxide. Urea can be broken down with the help of the enzyme urease, producing the alkaline
product of ammonia plus carbon dioxide. The existence of urea in the given sample of urine is
indicated when a pink color appears in the solution. If the color remains the same, it shows a
negative result of the Urease Test.

4. Detection of bile salt in urine

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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. Bile salts appear in the urine when there is an obstruction to the biliary
tract, leading to an increase in the blood's bile acids. Serum bile salts may rise even without the
biliary obstruction if liver cell damage usually removes reabsorbed bile salts from the portal blood.
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 is 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.
A. Smith’s Test

I. Objective
To determine the presence of bile salts in the urine

II. Materials
Urine sample, test tube dropper, Smith’s reagent and measuring cylinder

III. 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.
• Observe the formation of green ring that formed at the two junctions of the two
solutions.

Reaction:

The alcoholic iodine mixture or the reagent reacts with the urine, which contains bile
consisting of organic molecules such as bile acids, bilirubin and cholesterol, and water. As urine
was mixed with the reagent, a green ring formation appeared at the layers' intersection.

IV. Observation and result

What indicates the presence of bile salt in urine?

Upon pouring the Smith's reagent on the test tube wall containing the urine sample, a
green ring was observed between the urine and the alcoholic iodine mixture. This green ring
formed at the junction of two layers indicates the presence of bile salts in the urine sample. Thus,
the absence of the green ring formation states the opposite or the absence of bile salts in urine.

C. Pettenkofer’s Test

I. Objective
To determine the presence of bile salts in the urine

II. Materials
Urine sample. Test tube, dropper, measuring cylinder, spatula, Conc. H2SO4, sucrose

III. 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.
• Observe the appearance of red color of the solution.

Reaction:

Bile salt will react with hydroxymethylenephurphural to forms a red solution.

Hydroxymethylenephurphural is formed of sugar that is dehydrated with sulphuric acid.

IV. Observation and result

Sucrose was added to the test tube and was dissolved by shaking. Then 2ml of concentrated
H2SO4 (sulphuric acid) was poured on the wall of the test tube. Seconds later, red color appears in
the solution, indicating that bile salts in the urine sample are present.

Summary of the biochemical tests of urine table:

Name of test Observation Result / inference

1 BENEDICT’S TEST Upon heating the sample, gradually, a For the Benedict’s test, a
yellow precipitate is formed in the test yellow precipitate
tube, which indicates the presence of appeared on the test tube
sugar in the given urine sample. that indicates the presence
Different precipitates are formed of sugar in the given
depending upon the sugar sample of urine. A green
concentration in urine, which can be precipitate appears when
yellow, green, or brick red. there is 0.5 to 1% of sugar
is present, a yellow
precipitate appears when
there is 1 to 2& of sugar is
present, and a brick red
precipitate appears when
there is 2% or more sugar
is present. When a blue
precipitate appears, it
means sugar is absent.
2 FEHLING’S TEST Upon heating the sample, gradually, a With the Fehling’s test, a
green precipitate is formed in the test green precipitate was
tube, which indicates the presence of formed in the test tube
sugar in the given urine that indicates the presence
sample. Different precipitates are of sugar in the given urine
formed depending upon the sugar sample and a green
concentration in urine which can be precipitate appears when
yellow, green, brick red. there is 0.5 to 1% of sugar
is present, a yellow
 precipitate appears when
there is 1 to 2& of sugar is
present, and a brick red
precipitate appears when
there is 2% or more sugar
is present. When a blue
precipitate appears, it
means sugar is absent.
3 HELLER’S TEST In the experiment, the changes take A positive result is
place in the test tube when the urine represented by the
comes in contact with the nitric acid. A appearance or formation of
positive result is represented by the a white ring and a negative
appearance or formation of a white result is represented by the
ring. This indicates the presence of absence of a white ring.
albumin in the given sample. A
negative result is represented by the
absence of a white ring.
4 SULPHOSALICYLIC Sulphosalicylic acid is added to a test A cloudy turbid solution or
ACID TEST tube containing a urine sample. A precipitate will be
cloudy turbid solution or precipitate observed when it is
will be observed. The appearance of positive and an absence of
this indicates that there is a presence it when it is negative.
of albumin. The absence of a cloudy
turbid or precipitate indicates the
absence of albumin.

5 SODIUM Upon dropping the sodium The observed presence of

HYPOBROMITE TEST hypobromite solution in the urine brisk effervescence of
sample, vigorous formation of bubbles nitrogen around the test
occurred as if it is boiling. It had a tube after the interaction
rapid or vigorous evolution of gas in between the urine sample
the test tube, which is termed as brisk and the Sodium
effervescence. Hypobromite solution
indicates that urea is
present in the given urine
sample. Thus, it is positive
from the Sodium
Hypobromite Test.
6 UREASE TEST A light pink liquid was observed in the Through the formation of
test tube containing the urine sample pink colored liquid that was
upon adding drops of the phenol red observed at the end of the
indicator. Thus, when drops of experiment, the presence
Na2CO3 solution was added into the of urea in urine was
test tube, the light pink colored liquid confirmed.
turned into light violet. Then, when
drops of the acetic acid was added
 into the test tube, the liquid turned
colorless/transparent. Lastly, when
the urease powder was added into the
solution, the transparent liquid in the
test tube turned into a pinkish to
reddish liquid.
7 SMITH’S TEST Upon pouring the Smith's reagent on The formation of green-
the test tube wall containing the urine colored ring upon the
sample, a green ring was observed interaction of the Smith’s
between the urine and the alcoholic reagent and the urine
iodine mixture. sample confirms the
presence of bile salt in the
urine.

8 PETTENKOFER’S TEST Upon adding sucrose and The appearance of the red-
concentrated sulfuric acid to the test colored liquid as a result of
tube, containing the urine sample, the adding both the sucrose
appearance of a red-colored liquid and concentrated sulfuric
was observed. acid into the test tube
indicates that there is a
presence of bile salt in the
urine sample.

Questions:

1. What abnormal substances can be detected in urine test?

Many disorders may be detected in their early stages by identifying substances that are
not normally present in the urine and/or by measuring abnormal levels of certain substances.
Some examples include glucose, protein, bilirubin, red blood cells, white blood cells, crystals, and
bacteria. They may be present because:

(1) There is an elevated level of the substance in the blood and the body responds by trying to
eliminate the excess in the urine;
(2) Kidney disease is present; and
(3) There is a urinary tract infection present, as in the case of bacteria and white blood cells.

2. What are the properties of a normal urine?

Normal Urine Characteristics

Characteristic Normal values
Color Pale yellow to deep amber
Odor Odorless
Volume 750–2000 mL/24 hour
 pH 4.5–8.0
Specific gravity 1.003–1.032
Osmolarity 40–1350 mOsmol/kg
Urobilinogen 0.2–1.0 mg/100 mL
White blood cells 0–2 HPF (per high-power field of microscope)
Leukocyte esterase None
Protein None or trace
Bilirubin <0.3 mg/100 mL
Ketones None
Nitrites None
Blood None
Glucose None

3. What is the significance of the biochemical test of urine?

Urine tests can help detect diseases of the urinary system as well as metabolic diseases like
diabetes or liver disease. The color, odor and amount of urine can already indicate whether
something is wrong.

Conclusion:

For the final analysis, urine is a liquid by-product of metabolism in humans and in many other
animals where about 91-96% of urine consists of water. Urine also contains an assortment of inorganic
salts and organic compounds, including proteins, hormones, and a wide range of metabolites, varying by
what is introduced into the body. The color of your urine says about our health, transparent urine is an
indication of over hydration, pale yellow means a person is healthy and hydrated. Transparent and dark
yellow are normal but dark yellow indicates that a person needs more water. If your urine’s color is
brownish yellow, this is an indication of dehydration or a sign of liver disease. Pinkish red is a sign of kidney
disease either UTI or tumor same with the foamy type of urine. Lastly, blue or green is a sign or rare
genetic disease. There are biochemical tests for the urine, we have Benedict’s test and Fehling’s Test
where the objective is to be able to detect the presence of sugar in the given sample of urine. The results
formed different precipitates depending upon the sugar concentration in urine, which can be yellow,
green, or brick red. For the Benedict’s test, a yellow precipitate is formed in the test tube, which indicates
the presence of sugar in the given urine sample. Another test is Fehling’s test, there is a presence of sugar
in the given urine sample where gradually, a green precipitate is formed in the test tube upon heating it
which means both tests that were conducted show a presence of sugar in the given sample urine. Another
biochemical test is to detect the presence of albumin in the urine which was done through Heller’s test
and Sulphosalicylic acid test. Both of the test results show a presence of the albumin in the sample urine
where there is an appearance or formation of a white ring for the Heller’s test and a cloudy turbid solution
or precipitate will be observed for the Sulphosalicylic acid test. The biochemical test for the detection of
urea in the urine, a Sodium Hypobromite test and Urease test were done where the results showed the
presence of urea in the given urine sample. For the Sodium Hypobromite test, there is a presence of brisk
effervescence of nitrogen around the test tube after the interaction between the urine sample and the
Sodium Hypobromite solution and a formation of pink colored liquid that was observed for the Urease
Test. Lastly, Smith’s Test and Pettenkofer’s test was done to determine the presence of bile salts in the
urine. Both of the tests confirm the presence of the bile salt in the urine where Smith’s test shows a
formation of green-colored ring upon the interaction of the Smith’s reagent and an appearance of the
red-colored liquid for the Pettenkofer’s test. As a nursing student, the following tests are essential for
providing information to assist in the diagnosis, monitoring and treatment of a wide range of diseases and
conditions. To be effective, these tests must be performed properly so that the results will be interpreted
correctly to give an overview of the most important aspects of this investigation, highlighting signs to look
forward to and what these results really mean.

References:

https://www.youtube.com/watch?v=JUQExVhugFI

https://byjus.com/biology/test-for-presence-of-urea-in-urine/

https://byjus.com/biology/test-for-presence-of-albumin-in-urine/

https://www.youtube.com/watch?v=i7dZBzhQCaI

https://www.youtube.com/watch?v=8mSzXTF1Q1g

Simulation http://amrita.olabs.edu.in/?sub=79&brch=17&sim=211&cnt=4