Experimen

t 9:
Arcangel
Borlongan
Cayetano
Enriquez
Guinto

URINALYSI
S

INTRODUC
TION

URINE
-

is a liquid waste product secreted by the body and is filtered by

the kidneys. It is composed of urea, dissolved salts and other
organics constituents.

is an important part of the body's disposal process. Its job is to

remove the extra water and water-soluble wastes the kidneys
filter from the blood.

URINALYSIS
A urinalysis is simply an analysis of the urine. It is a very
common test that can be performed in many health-care
settings, including doctor's offices, urgent-care facilities,
laboratories, and hospitals.

OBJECTIVE

Objective
To qualitatively examine the presence of some normal
organic constituents and pathologic organic constituents
of urine

Methodolo
gy

Initial Examination of Urine

Take note the
following:
- Time
- Color
- Turbidity
- Acidity

Qualitative Examination for

Normal Constituents
Test for Urea
Test for Uric Acid
Indican test
Test for Creatinine

Test for
Urea
1mL urine sample

Test for
Uric Acid
1mL urine sample

+ 0.5mL 70% NaOH

+ 5mL 20% Na2CO3

+ 4 drops bromine water

Mix well

Evolution of N2
gasses

+ 5 drops of
phosphotungistic acid
reagent
Mix well
Formation of
blue solution

Indican
Test
5mL urine sample
+ 5mL Obermayers reagent
Mix well
+ 3mL chloroform
Shake and allow the
chloroform layer to settle
Blue color in the
lower (chloroform)
layer

Test for
Creatinine
2mL urine sample
+ 1mL alkaline picrate
solution (5:1, saturated picric
acid-10% NaOH)
Orange colored
solution

Qualitative Examination for

Pathologic Organic
Constituents
Gunnings Test
Benedicts Test
Extons Test
Smiths Test
Test for Occult Blood

Gunnings Test
5mL urine
sample

Positive Control:
5mL urine + 2mL acetone
Basify with 5 drops NH4OH
(red litmus paper)
+ Lugols solution enough
to produce black cloud
which does not disappear
immediately
Stand for 5 mins
Formation of
Iodoform crystals

Benedicts Test
8-10 drops
urine sample

Positive Control:
1mL urine + 1mL 1% glucose solution
+ 5mL Benedicts reagent
Mix well
Boiling water bath for 2-3
mins
Cool via running water

Formation of Precipitate

Smiths Test
5mL urine
sample

Positive Control:
5mL urine + 3-5 drops of
bile from pig gall bladder
Incline the test tube
Overlay with 3mL tincture
alcoholic iodine mixture

Emerald green color at

the point of contact

Test for Occult Blood

Mix 5mL 95% ethanol to a
half spatula guaiac powder
+ 5mL hydrogen peroxide
+ 5mL of the mixture to:
3mL acidified urine
(with acetic acid)

Positive Control:
3mL acidified urine + 3 drops of blood

Presence of blue ring

Discussion of Results

Initial Examination Results of

the Urine Samples
1
Time
Collected

1:07 PM
Mid-day

12:45 PM
Mid-day

12:50 PM
Mid-day

12:27 PM
Mid-day

1:01 PM
Mid-day

Color

Dark
Yellow

Light
Yellow

Light
Yellow

Light
Yellow

Light
Yellow

Turbidity

Clear

Clear

Clear

Clear

Clear

pH of the
urine

pH 6
Acidic

pH 7
Neutral

pH 7
Neutral

pH 7
Neutral

pH 6
Acidic

11/24/16

Initial Examination Results of

the Urine Samples
6

10

1:00 PM
Mid-day

1:00 PM Midday

1:10 PM
Mid-day

1:00 PM
Mid-day

1:08 PM
Mid-day

Color

Yellow

Light Yellow

Light Yellow

Light Yellow

Dark Yellow

Turbidity

Clear

Clear

Clear

Clear

Clear

pH of the
urine

pH 6
Acidic

pH 7
Neutral

pH 7
Neutral

pH 6
Acidic

pH 6
Acidic

Time
Collected
11/24/16

INITIAL EXAMINATION OF THE

URINE SAMPLE
Visual exam. The urine will be looked at for color and
clearness. An infection may make urine look cloudy.
Foamy urine can be a sign of kidney problems.

INITIAL EXAMINATION OF THE

URINE SAMPLE
Acidity (pH) is a measure of the amount of acid in the
urine. A pH that is above normal may be a sign of kidney
stones, urinary infections, kidney problems, or other
disorders.

Results of Qualitative
Examinations for Normal
Constituents

Test for Urea

- Determines the presence of Urea (CH4N2O)

Urea
- is a product of the conversion of ammonia ( and
carbon dioxide (CO2).
- the conversion of urea from ammonia and carbon
dioxide is called the urea cycle or ornithine cycle.
- It is a colorless, odorless solid, highly soluble in water

Test for Urea

Reagents: 70% NaOH, Bromine (Br2) Water
Principle: As Urea is treated with the Sodium Hypobromite, it decomposes
releasing Nitrogen.
Positive Result: Evolution of Nitrogen (N2) gas.

Mechanism
CH4N2O + 3 NaOBr

3 NaBr + N2 + H20 + CO2

Test for Uric Acid

-determines the presence of uric acid

Uric acid
-heterocyclic compound
-chemical produced when the body breaks down food that contains organic
compounds called purines
-purines may also be produced by the natural process of cell breakdown in
the body
-high concentrations can lead to gout and kidney stones

Results

Group number

(+/-) Results

(-)

(-)

(-)

(-)

(-)

(-)

(+)

(-)

(-)

10

(+)

Test for Uric Acid

Reagents: Na2CO3, phosphotungstic acid reagent
Principle: Uric acid is a reducing agent in alkaline medium. It reduces
phosphotungstic acid to tungsten blue
Positive Result: Blue color

Mechanism
H3PW12O40

Tungsten Blue

Indican Test
- Also called Obermeyers Test
- Indicator of intestinal toxemia and overgrowth of anaerobic bacteria

Indican
- an indole produced when bacteria in the intestine act on the amino acid,
tryptophan
- Most indoles are excreted in the feces.
- The remainder is absorbed, metabolized by the liver, and excreted as
indican in the urine.
- increases with high protein diets or inefficient protein digestion

Results

Group number

(+/-) Results

(-)

(-)

(+)

(-)

(+)

(-)

(-)

(-)

(-)

10

(+)

Indican Test
Reagents: Obermayers reagent, chloroform
Principle: Indole is converted to 3-hydroxy indole in the liver.
Positive Result: Blue color in the lower (chloroform) layer

Mechanism
+ H2O2
Indole

Indoxyl

+
Indican

indoxyl
sulfuric
acid K

Test for Creatinine

- Creatinine is a chemical waste product thats normally filtered out of blood
by kidneys.
- When kidneys are not functioning properly, creatinine can accumulate in
the body.
- A creatinine urine test evaluates how well kidneys are working by
measuring the amount of creatinine in the urine.

Test for Creatinine

Test for Creatinine

- Reagents: Alkaline picrate solution (5:1, saturated picric acid- 10% NaOH)
- Positive Result: Formation of orange colored solution
- Principle: Creatinine reacts with Alkaline picric acid (an organic compound
with the formula (O2N)3C6H2OH. Its IUPAC name is 2,4,6-trinitrophenol or
TNP.)

Mechanism

NaOH
Orange colored solution

Results

Group Number

(+/-) Result

(+)

(+)

(+)

(+)

(+)

(+)

(+)

(+)

(+)

10

(+)

Results of the Qualitative test

for Pathologic Organic
Constituents

Gunnings Test
Test for Ketone bodies

Ketones
- Are three water-soluble compounds (acetone, acetoacetic acid, betahydroxybutyric acid) that are produced as byproducts when fatty acids
are broken down for energy in the body.
- are products of incomplete fat metabolism
- may indicate the beginning of diabetic ketoacidosis (DKA)

Gunnings Test
Reagents: Concentrated Ammonium hydroxide, Lugols
solution (Potassium Iodide and iodine), Acetone (for
positive control only)
Principle: Halogenation of the enolate of acetone
Positive Result: Formation of iodoform crystals

Mechanism

Results

Group Number

Results

10

Benedicts Test
Determines the presence of Glucose in the urine.

Glucose (C6H12O6)
- A 6-carbon simple sugar or monosaccharide.
- Can be considered as the most important sugar in human metabolism

Benedicts Test
- Principle: Copper sulphate of Benedicts qualitative solution is reduced
by reducing substances on boiling to form the coloured precipitate of
cuprous oxide.
- Benedict's Reagent(Blue): Copper(II) sulphate, Sodium Bicarbonate
(Alkalinization), Sodium Citrate (Stabilization)
-

Positive: Green ppt (0.5%) , Yellow ppt (1%-1.5%) ,

Orange ppt (1.5%-2%) , Brick red ppt (> 2%)

- Negative:Blue ppt (0%)

Benedicts Test Results for the

Urine Samples
Positive Control Result: Green Solution with Red Brick Precipitate

Group

Results

Negative

Negative

Negative

Negative

Negative

Group

10

Results

Positive

Negative

Negative

Negative

Negative

Mechanism

Extons Test
- Determines the presence of albumin
- Accurate measure of urine protein content

Albumin
- globular proteins
- low molecular weight
- water soluble
- normally found in blood plasma

Extons Test
Reagents: Extons reagent (5% sulfosalicylic acid in sodium sulfate)
Principle: Precipitation of protein as a result of acidification with sulfosalicylic
acid
Positive: Increasing turbidity

Results

Group number

(+/-) Results

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

(-)

10

(-)

Smiths Test
-

Determines the presence of bile pigments (bilirubin)

In humans and most vertebrates, bile is produced by the liver.

Presence of bile pigments in the urine indicate liver dysfunction.

Presence of bile pigments in the urine helps to diagnose increased production of

bilirubin.

Bile Pigments
- Bilins or bile pigments are biological pigments formed in many organisms as a
metabolic product of certain porphyrins
- Coloured compounds breakdown products of the blood pigment
haemoglobin that are excreted in bile. The two most important bile pigments
are bilirubin, which is orange or yellow, and its oxidized form biliverdin,
which is green. Mixed with the intestinal contents, they give the brown colour
to the faeces.

Bilirubin
- In chemical terms, bilins are linear arrangements of four pyrrole rings
(tetrapyrroles). In human metabolism, bilirubin is a breakdown product of
heme. Hydroxymethyl bilane is a major anabolic product, from the
biosynthetic reaction of porphobilinogen (PBG) and uroporphyrinogen I
synthase (known as porphobilinogen deaminase).
- 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.

Formation of Bile Salts

Pathophysiology
-

The main bile pigments are bilirubin and biliverdin and these are the catabolism of hemoglobin.

The main site of the formation of bile pigments is liver.

Ultimately these leave the body in feces and small amount in the urine.

Bile pigments give the urine greenish yellow, yellow or brown color.
- Causes of Hyperbilirubinurea
- Moderate to severe hepatocellular damage and obstruction of bile duct.

Smiths Test
- Reagents: iodine alcoholic tincture (iodine crystals and sodium iodide in
absolute ethanol)
- Principle: bilirubin, bilicyanine, and oholetelin, depend upon oxidation of
the bile pigments resulting in colored derivatives
- Positive Result: formation of emerald green color interphase

Results

Group Number

(+/-) Result

3
4

10

Test for Occult Blood

determines the presence of red blood cells in the urine

Occult Blood
-sometimes referred to as hidden blood
-Heme groups are most commonly recognized in their presence as
components of hemoglobin

Test for Occult Blood

Reagents: 95% ethanol with guaiac powder, hydrogen peroxide and glacial
acetic acid
Principle: The peroxidase activity of hemoglobin which decomposes hydrogen
peroxide and the liberated oxygen oxidizes organic substances such as guaiac
powder forming green or blue color
Positive Result: formation of blue ring

Results

Group Number

Results

10

Conclusion
The qualitative tests can provide information on the normal as well as
pathological constituents of urine. The minimal presence of urea, uric acid,
and indican indicates that the individual is healthy. The presence of other
constituents such as ketone bodies, glucose, albumin, bile pigments, and
blood would indirectly indicate that the individual may suffer from a disease.

References