URINALYSIS

James Knowell E. Chico

Urine

a watery, normally yellowish fluid stored in the bladder and

discharged through the urethra
one of the bodys chief means of eliminating excess water and salt
also contains nitrogen compounds such as urea and other waste
substances removed from the body by the kidneys
95% water, 2.5% urea and the remaining is a combination of salt,
hormones, nutrients, and creatinine
actually sterile in the bladder
can pick up microbes from the urethra and the air as soon as it
leaves the body
pH range: 4.6 to 8.0

Urinalysis

the analysis of urine by physical, chemical, and microscopical

means to test for the presence of disease, drugs, etc.
tests the urine for color, clarity (clear or cloudy), odor,
concentration, and pH (acidity or alkalinity)
purposes:
o General evaluation of health
o Diagnosis of disease or disorders of the kidneys or urinary tract
o Diagnosis of other systemic disease that affect kidney function
o Monitoring of patients with diabetes
o Screening for drug abuse (eg. Sulfonamides or aminoglycosides)

Objectives

Examine and determine the different properties of

urine
Perform qualitative tests for the normal and
pathologic constituents of urine

Materials and Equipment Used

MATERIALS AND REAGENTS
Urine
Concentrated NaOH
Bromine water
20% sodium carbonate
Phosphotungstic acid reagent
Obermayers reagent
Chloroform
Saturated picric acid solution
Dilute acetic acid
Strong ammonia
Lugols solution
Benedicts solution
Orcinol reagent
Concentrated nitric acid
Powdered quaiac
95% alcohol
Hydrogen peroxide
Strong acetic acid
APPARATUS AND EQUIPMENT
Test tubes
Test tube holder
Hot plate
Water bath
Droppers
10mL graduated cylinder

PROPERTIES OF
URINE

Properties of Urine

Procedure:
Examine the
properties of the
urine sample as
to its color,
transparency, and
reaction to red and
blue litmus paper

Properties of Urine

Results
Parameter

Description

Color

Deep yellow

Odor

Putrid, mildly putrescent

Transparency

Clear

Reaction to
litmus

Red litmus remained red; blue litmus

turned to red

Color
Normal urine
pale yellow to
amber yellow
Pigments:
Urochrome
(yellow)
Uroethyrin (red)
Urobilin (orangered)

Odor
Odor

Interpretation

Ammonia-like

(Urea-splitting bacteria)

Aromatic

Normal

Foul, offensive

Old specimen, pus, or inflammation

Sweet

Glucose

Fruity

Ketones

Maple syrup-like

Maple syrup urine disease

Transparency

Normal urine transparency: clear

Abnormal urine transparencies:
o Slightly hazy
o Hazy
o Cloudy
o Very cloudy
o Turbid

Typically cells or crystals, cellular elements

pH

Normal pH range: 4.6 - 8

high (alkaline) pH
o can be caused by severe vomiting, a kidney disease,

some urinary tract infections, and asthma.

low (acidic) pH
o may be caused by severe lung disease (emphysema),

uncontrolled diabetes, aspirin overdose, severe

diarrhea, dehydration, starvation, drinking too much
alcohol, or drinking antifreeze (ethylene glycol).

QUALITATIVE TEST FOR

NORMAL CONSTITUENTS

Test for Urea

Procedure: In a test
tube: 5 drops of
concentrated NaOH
10 drops of urine t4
drops of bromine water

Results: Solution
became lighter in color;
some fumes formed

Test for Urea

Urea major end product in protein metabolism in humans

(mammals).
Ammonia (NH3)product of oxidative deamination (toxic)
Urea cycle conversion reactions of ammonia into urea;
occurs in the liver
NaOH and bromine water was added to form sodium
hypobromite when it is mixed with urea, urea
decomposes to yield off nitrogen, hence the observed fumes

Azotemia

Azotemia
o a medical condition characterized by abnormally high levels

ofnitrogen-containing compounds, such as urea, in the blood

o largely related to insufficient or dysfunctional filtering
ofbloodby the kidneys
o can lead to uremia (the pathological manifestations of severe
azotemia) if not controlled

Urea Cycle

Test for Uric Acid

Procedure:
5 drops of urine 10
drops of 20% NaCO3
mix 5 drops of
phosphotungstic acid
reagent mix
Results: Blue colored
solution

Test for Uric Acid

Uric acid
o
o
o
o

a heterocyclic compound of carbon, nitrogen, oxygen, and hydrogen

with the formula
reducing agent in alkaline medium
a product of the metabolic breakdown ofpurinenucleotides
reduces the phosphotungstic acid into tungsten blue

Sodium carbonate
o

provide the alkaline pH necessary for color development

Hyperuricemia & Gout

Hyperuricemia
is an abnormally high level ofuric acidin theblood
o the amount of urate in the body depends on the balance between the
amount of purines eaten in food, the amount of urate synthesized within
the body, and the amount of urate that is excreted in urine or through
the G.I. tract
o

Gout
High blood concentrations of uric acid can lead togout
o amedical conditionusually characterized by recurrent attacks of
acuteinflammatory arthritisa red, tender, hot,swollen joint
o the uric acid crystallizes, and the crystals deposit in joints,tendons, and
surroundingtissues
o

Indican Test

Procedure: In a test tube:

5 drops of Obermayers
reagent 5 drops of urine
mix 20 drops of
chloroform shake
allow to stand

Results: Formation of 2
layers
o Top - tellow; Bottom faint yellow

Indican and the Indican Test

Indican an indole produced when bacteria in the intestine act

on the amino acid, tryptophan
Indicator of intestinal toxemia and overgrowth of anaerobic
bacteria
Obermayers reagent ferric chloride in hydrochloric acid, and
chloroform
Ferric ions of the Obermayers reagent are oxidized by the indoxyl
from indican)
Clear yellow orange solution with yellow interphase with indigo
blue layer in the chloroform layer (lower layer)

Test for Creatinine

Procedure: In a test tube: 5

drops of urine sample equal
number of drops of saturated
picric acid solution
concentrated NaOH solution
acidify with dilute acetic
acid

Results
o
o

A yellow solution was formed after picric

acid was added to urine
The same yellow solution, after added
with NaOH, turned into a red orange
solution (basic)
The red orange solution turned to yellow

Creatinine and the Test for Creatinine

reaction of creatinine with

alkaline picrate to yield orange
creatinine picrate
adding the acid will destroy
the color madeby creatinine
indicates muscle disorder,
uncontrolled diabetes mellitus,
starvation

How is creatinine synthesized in

the body?
Creatinine comes from the creatine
of the muscles. In the body,
creatine is synthesized from the
amino acids glycine, arginine, and
methionine, primarily in the liver,
kidneys, and pancreas, and it is
transported from there to all the
cells in the body via the
bloodstream.

QUALITATIVE TEST FOR

PATHOLOGIC
CONSTITUENTS

Ketone Bodies

Test performed: Gunnings

test
Procedure: In a test tube:
5mL of urine sample/positive
control 5 drops of strong
ammonia was added
Lugols solution was added
just enough to produce a
black cloud stand for a
few minutes

Results (Positive Control)

o After adding Lugols solution,

black cloud appeared in the dark

red-orange solution.
o After setting it aside, solution
became colorless & no sediments
appeared.

Results (Urine Sample)

o After letting the solution stand for

a few minutes, sediments

appeared.
o Iodoform crystals were seen using
a microscope

Ketone Bodies

Gunnings Test

Gunnings test checks for ketone bodies in the blood or urine

Ketone bodies are three water-soluble compounds that are produced as byproducts when fatty acids are broken down for energy in the body
The excess presence of ketones in urine is associated with diabetes or
altered carbohydrate metabolism
Positive result
o

formation of iodoform crystals; person may be having a starvation, liver damage,

or high fat diet.

Negative result
o

no iodoform crystals; optimum amount of insulin in the body.

Glucose

Test performed:
Benedicts Test

Results (Positive
Control)
o Orange solution with

Procedure: In a test
tube: 5mL of Benedicts
solution 8-20 drops
of urine placed tube
in boiling water for a
span of 2-3 minutes
cool

dark orange precipitate

Results (Urine
Sample)
o Blue solution with no

precipitates

Ketone Bodies

Benedicts Test

It reduces cupric hydroxide to red colour cuprous oxide in an alkaline

medium.
A brick red precipitate indicates the presence of a reducing sugar.
However, the colour of the precipitate may vary from light green (less) to
brick red (more) depending upon the amount of reducing sugar present.
Benedicts reagent sodium carbonate, copper sulfate, and sodium citrate
Sodium carbonate provides basicity of the solution; retains the cupric
hydroxide of copper
Sodium citrate acts as stabilizer; prevent cupric hydroxide precipitation
Glucosauria presence of glucose in the urine

Pentose

Test performed: Bials

orcinol test

Results (Positive
Control)
o Formation of dark blue

Procedure: In a test
tube: 0.5 mL of urine
4.5mL of the
orcinol reagent
place in a boiling
water bath

colored solution

Results (Urine
Sample)
o Rum-like brown solution

with black precipitates

Bials Orcinols Test

Distinguishes between pentoses and hexoses.

Reaction is due to the formation of furfural in the acid medium
which condenses with orcinolin the presence of ferric ions to
give a blue to green colored solution.
Positive result Blue-colored solution
Negative result brown or muddy red precipitate in a brown solution
Pentosuria presence of pentose in urine
Benign pentosuria- inborn error of metabolism, caused by the
defect in the activity of the enzyme l-xylulose dehydrogenase,
which results in high levels of l-xylulose in the urine
o Alimentary pentosuria- normal consequence of excessive
ingestion of some fruits or their juices
o

Albumin

Test performed:
Hellers Test
Procedure: In a test
tube: In a test tube:
5mL of concentrated
HNO3 urine was
slowly added down
the tube

Results (Positive Control)

o Light yellow solution with

white precipitate

Results (Urine Sample)

o Solution had 2 layers- top

- yellow (urine), bottomvery faint light yellow to

colorless
o No precipitate formed

Albumin

Hellers Test

Detects presence of albumin

Nitric acid causes denaturation of proteins with the
formation of a white precipitate.
Positive result formation of white precipitate at the
interface
Albuminuria- presence of albumin in the urine

Why is albumin present in the urine?

o Albumin is smaller than most other proteins and is typically the

first protein that is seen in the urine when kidney dysfunction

begins to develop
o An increase in urinary albumin excretion indicates that there is

Bile

Test performed:
Gmelins Test
Procedure: In a test
tube: 1mL of
concentrated nitric
acid 1mL of urine
sample, superimposed
to the tube

Results (Positive Control)

o Varied colors (reddish-

yellow, greenish-blue, &

violet) observed at the
interface

Results (Urine Sample)

o None observed (changing of

colors at point of contact)

o Solution has 2 layers: top yellow (urine); bottom colorless

Gmelins Test

Detects the presence of bile

Nitric acid oxidizes bilirubin into
biliverdin, thus giving varied colors
(positive result)
Choluria
o presence of bile in urine

Occult Blood

Test performed: Guiaiac

Test
Procedure: In a test tube:
small amount of guaiac
2mL of 95% alcohol
5mL of hydrogen
peroxide acidify with
acetic acid observed
for color changes

Results (Positive Control)

o After adding H2O2 from

dark orange solution to

beige-colored solution
o After adding blood sea
blue green colored solution,
foam produced

Results (Urine Sample)

o Yellow solution with pinkish-

colored precipitates formed

o No blue ring appeared

Occult Blood

Guaiac Test

Detects presence of blood

Hemoglobin decomposes hydrogen peroxide, and the liberated
oxygen from hydrogen peroxide oxidizes a chromogen (guaiac
powder) to yield blue or green colored ring.
Hematuria presence of blood in urine, may be caused by:
o
o
o
o
o

inflammation of the kidney, urethra, bladder, or prostate;

polycystic kidney disease
blood clots
blood clotting disorders, such as hemophilia; or
sickle cell disease

References
Campbell, M.K. and S.O. Farrell. 7th ed. Biochemistry. USA: Brooks/Cole.
Nelson, D.L. and M.M. Cox. 2008. 5th ed. Lehninger Principles of
Biochemistry. USA: W. H. Freeman and Company.
Simerville, J.A., W.C. Maxted, and J.J. Pahira. Urinalysis: A comprehensive
review. American Family Physician, Vol. 71, No. 6, pp. 1153-1162.
Part 2. The Role of pH and Healthy Living.
http://www.chemcraft.net/acidph2.html. Retrieved on 18th November
2015.
Urine test. http://www.webmd.com/a-to-z-guides/urine-test. Retrieved on
18th November 2015.
Urine analysis. https://www.academia.edu/4836857/Urine_analysis.
Retrieved on 18th November 2015

THE END