Qualitative Analysis of Normal and

Abnormal Urine
Urine is an ultrafiltrate formed by the kidneys carrying the
waste and toxic substances from the blood. The
composition of urine is a mirror not only of renal function
but also of many physiological and metabolic processes
occurring in the body. Thus, examination of urine may lead
to the diagnosis of many metabolic and systemic diseases.
EXAMINATION OF URINE
Examination of urine includes:
1. Physical examination
2. Chemical examination
3. Microscopic examination.
SPECIMEN COLLECTION
1. Fresh mid-stream specimen of 10- 20 ml is collected in a
clean dry container.
2. For most of the qualitative tests, a random urine
sample
is satisfactory.
3. Morning specimen is desirable for normal analysis.
4. 24 hours urine is collected for total urinary proteins,
calcium, uric acid, ketosteroids and certain hormonal
assays, as the concentrations vary at different times of
the day. The patient is instructed to collect the urine
Qualitative Analysis of Normal Urine :
PRESERVATION OF URINE SAMPLES
1. Several changes like urinary decomposition,
precipitation of phosphates, crystallization of uric acid
and bacterial action may alter the urinary composition if it
is kept for long periods, especially in the collection of 24
hours urine samples. Also urine may become alkaline, due
to precipitation of uric acid and urates

This requires addition of preservatives (to prevent the

growth of bacteria and moulds such as 2N hydrochloric
acid, conc. sulfuric acid, toluene, liquid petroleum crystals
of thymol or 10% acetic acid, etc. depending on the
analysis of parameters in urine.

GENERAL AND PHYSICAL CHARACTERISTICS:

Volume
Normal adult excretes around 800 to 2500 ml/ day with
an average of 1500 ml/day.
Day output is greater than night output.
Factors influencing the volume are :
Quantity of fluid intake.
Quality of food taken.
Climate- output is low in hot climate due to excessive
sweating.
Physical exercise.
A high protein diet causes a physiological polyuria due
to the diuretic effect of urea, the end product of protein
metabolism.

Polyuria: An increase in urinary output. Occurs in:

Diabetes mellitus
Diabetes insipidus
After administration of drugs like diuretics, digitalis,
salicylate, etc.
Certain nervous disorders
Later stages of chronic renal failure.

Oliguria: A diminished urinary excretion (< 500 ml).

Occurs in:
Acute nephritis
Fever
Diarrhea and vomiting.

Anuria: A total suppression of urine formation.

Occurs in:
Shock
Acute tubular necrosis
Incompatible blood transfusion
Mercury poisoning
Bilateral renal stones.
Appearance
Freshly voided normal urine is clear and transparent.
On standing it may become turbid due to the bacterial
action that converts urea to ammonium carbonate. This
makes urine alkaline and causes precipitation of
phosphates/oxalates/urates.
It may also become turbid due to the precipitation of
nucleoproteins and mucoproteins.
Abnormal urine is turbid due to
Presence of pus cells in urinary tract infections
Increased excretion of phosphates in alkaline urine
Chyluria- milky white urine- presence of fat globulins
due to obstruction in the lymphatics of urinary tract as in
filariasis.
Color
Fresh normal urine is straw or amber yellow due to the
presence of the pigment urochrome, a compound of
urobilin or urobilinogen.
The color may be light or dark depending on the volume
of urine.
Yellow colored urine will be present in people who
consume vitamin B complex.
Odor
Fresh urine has an aromatic odor due to the presence of
volatile organic acids.
On standing urine undergoes decomposition converting
urea into ammonium carbonate giving an unpleasant

ammoniacal odor.
Color Condition

Odor:

Specific Gravity
The specific gravity of normal urine varies in the range
of
1.012 to 1.024.

 Physiologically, the specific gravity may decrease with

high fluid intake where the urine volume is increased and
may rise with restricted water intake where the urine
volume is low.
It can be as low as 1.001 when water intake is high and
as high as 1.04 when water intake is restricted.
The specific gravity is directly proportional to the
concentration of solutes excreted.
Specific gravity is measured with Urinometer.
Specific Gravity in pathological condition :
Increased in acute nephritis and fever.
Decreased in diabetes insipidus.
CHEMICAL CHARACTERISTICS
Reaction
Fresh urine is normally acidic with a mean pH of 6 (4.87.5) pH of urine is influenced by the nature of the diet.
In people on high protein diets the urine is more acidic
because more sulfates and phosphates are eliminated
from the protein catabolism.
Diet rich in fruits and vegetables makes the urine
alkaline.
Urine on standing becomes alkaline by the bacterial
action on urea and formation of ammonia.
After meals, due to hydrochloric acid secretion in the
stomach, the urine becomes alkaline. This is known as
the alkaline tide.
pH changes in pathological condition:

Significantly acidic urine is voided in fever and diabetes.

Alkali therapy and urinary retention make urine alkaline.
Decrease in urinary pH: metabolic acidosis
Increase in urinary pH: metabolic alkalosis

Constituents of Normal Urine

Normal urine contains both inorganic and organic
constituents.
The inorganic constituents include sodium, potassium,
magnesium, chloride, calcium, phosphorus, inorganic
sulfates and ammonia.
The normal organic contents are urea, uric acid,
creatinine, amino acids and ethereal sulfates (also
urobilinogen, hippuric acid, indican).
The normal non-protein nitrogenous contents are urea,
uric acid, creatinine.
The total non-protein nitrogen varies from 10 to 15 mg
per day depending mainly on the protein intake.
In addition to these major organic constituents,
detoxified products like indican and ethereal sulfates are
found in urine.
ANALYSIS OF NORMAL URINE
Physical Examination

Determination of Specific Gravity:

Specific gravity of urine is measured by an apparatus

known as Urinometer. Urinometer consists of a thin stem
graduated from 1000 to 1060 corresponding to Specific
Gravity of 1.0 to 1.06. Urinometer is calibrated at 60F
(15C).
Procedure: Take sufficient urine in a urine Jar. Allow the
urinometer to float in it without touching the sides.
Observe the reading at the meniscus. This gives the
observed specific gravity at the temperature at which the
urinometer is
calibrated. Note the urine temperature (room
temperature).
Calculation: Suppose the meniscus of the urine coincides
with the reading, 1010 and the room temperature is 37C.
Urinometer is calibrated at 15C. Since the room
temperature is higher, a temperature correction has to be
applied. For
every 3C rise over the temperature of calibration (15C),
a correction factor of 0.001 is added to the last digit of the
observed reading.
The difference between 37C and 15C is 21C. This when
divided by 3 gives 7.
Thus, the corrected specific gravity = 1.010 + 0.00 7 =
1.017
If the room temperature is below 15C, one unit should be
subtracted from the last digit for every 3C difference in
temperature.

Inorganic Constituents:
Tests of inorganic constituents are as follows.
Test for Chloride
Principle: A white precipitate of silver chloride is formed
when acidified urine reacts with silver nitrate.

Points to Remember:
Chloride ion is the chief anion in urine.
Excreted as sodium chloride.
On an average diet, 10- 12 gm of chloride is excreted per
day.
Urates and phosphates can interfere with this test by
forming silver urates and silver phosphates. Hence, nitric
acid is added to prevent such interference.
Decreased urinary chloride is seen in:
Excessive sweating
Fasting
Diarrhea and vomiting
Diabetes insipidus
Cushings syndrome
Infections
Increased urinary chloride is seen in:
- Excessive intake of fluids
- Addisons disease
Test for Inorganic Sulfates:
Principle: Urine being acidified with hydrochloric acid
forms a white precipitate of barium sulfate by the reaction
with barium chloride solution.

Points to Remember:
There are three forms of sulfates:
Inorganic sulfates of sodium and potassium
(80-85%)
Organic sulfates- ethereal sulfates (5%)
Neutral sulfur (15-50%)
Sulfates are derived from the metabolism of sulfur
containing amino acids such as cysteine, cystine and
methionine.
The presence of hydrochloric acid prevents the
precipitation of other inorganic salts like phosphates.
On an average diet about 0.7-1 gram of inorganic sulfate
is excreted per day.
Excretion is increased in:
High protein diet
Acute hyperthyroidism
Cystinuria
Decreased in renal dysfunction.
Neutral sulfur increases in poisoning.

Test for Phosphates and Calcium:

Procedure: Take 10 ml of urine in a test tube. Add 3 ml of
ammonium hydroxide boil and cool. A flaky precipitate of

calcium phosphate is formed. Filter and discard the

filtrate.
Add 3 ml of hot 10% acetic acid on to the residue on the
filter paper, through the sides of the paper. Collect the
filtrate and divide into two parts.
Test for phosphates
Principle: Phosphates of calcium and magnesium are
precipitated by ammonium hydroxide on boiling and these
phosphates are dissolved in hot dilute acetic acid. This
forms yellow precipitate of ammonium phosphomolybdate
reacting with ammonium molybdate.

Points to Remember:
Normally 0.8-1 gm of phosphorus as phosphate is
excreted per day.
Phosphates are present in urine as salts of sodium,
potassium, ammonium, calcium and magnesium. These
are crystallized out in alkaline urine.
Excretion is increased in bone diseases like rickets,
osteomalacia, and parathyroid dysfunction.
Excretion is decreased in:
Diarrhea
Infections
Nephritis
Hypoparathyroidism
Pregnancy

Test for calcium:

Principle: Calcium is precipitated as calcium oxalate with
potassium oxalate in acidic condition.

Points to Remember:
The excretion of calcium is 100- 200 mg/day.
Excretion increases in:
Hyperparathyroidism
Hyperthyroidism
Hypervitaminosis D
Multiple myeloma
This test is known as Sulkowaskis test and is useful in
evaluating parathyroid abnormalities and cases of kidney
stones.
Urinary calcium level is related to serum calcium level.
When serum calcium level is less than 7.5 mg/ dl there
may be no detectable calcium in urine.
When serum calcium level is 7.5- 9 mg/ dl, urine shows
slight cloudiness in this test.
A heavy precipitate indicates high serum calcium.
Test for Ammonia:

Principle: Ammonia present in urine is liberated by heat.

The evolution of alkaline ammonium vapors changes the
color of red litmus to blue.

Points to Remember:
Urinary ammonia is derived from glutamine and other
amino acids in kidney.
The average excretion of ammonia is about 0.7 gm/ day.
There is an increase in ammonia excretion when acid
forming foods are taken.
Ammonia is excreted as ammonium salts.
The kidneys manufacture ammonia in proportion to the
amount of acid radicals excreted in urine.
In alkaline urine, ammonium salts are absent.
Excretion of ammonia is increased in acidosis.
Excretion of ammonia is decreased in alkalosis
Impaired protein metabolism increases the output of
ammonia in urine.
To enhance the conversion of NH4 into NH3, the solution
is made alkaline before boiling.
If the solution is made strongly alkaline, urea will
interfere with the reaction.

Organic Constituents:
Tests for organic constituents are as follows:
Test for Urea
Sodium Hypobromite Test:
Principle: When urea is treated with Sodium hypobromite,
it
decomposes to give nitrogen, carbon dioxide and water.
Liberation of nitrogen gas produces brisk effervescence.

Points to Remember:
Urea is the major nitrogenous constituent of urine.
Urea is formed in liver as the end product of protein
metabolism and so its excretion depends on protein
intake.
About 20-40 grams of urea is excreted in 24 hours.
Excretion is increased in:

 High protein diet

Fever
Diabetes mellitus
Excretion is decreased in:
Liver diseases
Nephritis
Acidosis
Test for Uric Acid:
Phosphotungstic Acid Reduction Test/ Benedicts Uric Acid
Test:
Principle: Uric acid in alkaline condition reduces
phosphotungstic
acid to tungsten blue.
Benedicts uric acid reagent: Composed of sodium
tungstate,
orthophosphoric acid, concentrated Sulfuric acid and solid
sodium carbonate.

Points to Remember:
Uric acid is the end product of purine metabolism.
The daily output of uric acid varies in the range of 0.6 to
1 gm.
Excretion is increased in:
Leukemias especially during cytotoxic drug therapy
Wilsons disease

 Administration of cortisone/ ACTH

Excretion decreases in renal failure.

Test for Creatinine (Jaffes Test):

Principle: Creatinine reacts with picric acid in alkaline
medium to form reddish orange colored creatinine picrate.

Points to Remember:
Creatinine is the anhydride of creatine.
Urinary creatinine is derived from muscle creatine.
It is not influenced by the protein intake.
Excretion in adults ranges from 1-2 gm/day.
In women and in elderly people the values are lower due
to lesser muscular mass.
Excretion is increased in:
High intake of meat, fish
Fever
Myopathy/wasting diseases
Excretion is decreased in:
Renal failure
Anemia
Paralysis
Chemical Constituents

The commonly encountered pathological chemical

constituents of urine are:
Proteins (may be albumin, globulin, Bence Jones protein)
Blood (hemoglobin, erythrocytes)
Reducing sugar (usually glucose and in special cases
lactose, galactose, pentose and rarely fructose)
Ketone bodies (acetone, acetoacetic acid)
Bile salts and bile pigments
Porphobilinogen
Urobilinogen (increased or decreased).

Qualitative Analysis of Abnormal Urine

Test for Proteins:
Test for proteins are as follows:
Heat and Acetic Acid Test
Principle: On heating the protein loses its structure and
becomes denatured to form a coagulum. It is precipitated
after the addition of acetic acid, which provides the
suitable
pH to get the maximum precipitate.
Sulfosalicylic Acid Test
Principle: Sulfosalicylic acid is an alkaloidal reagent and so
it neutralizes the positively charged protein to produce
precipitation.

Points to Remember:
The amount of protein excreted normally in 24 hours
urine is insignificant and it is less than 150 mg/day.
When proteins appear in detectable quantities in urine,
it is called proteinuria/albuminuria.
The presence of detectable amount of protein is
characteristic of kidney diseases.
The normal glomeruli of kidneys are not permeable to
substances with molecular weight of 70 kD. The plasma
proteins of molecular weight of more than 70 kD, hence
are absent in normal urine.
When glomeruli are damaged or diseased, they become
more permeable and plasma proteins appear in urine.
The smaller molecules of albumin pass through damaged
glomeruli more readily than the heavier globulin and so,
when the proteins appear in urine, the albumin fraction
predominates.
Bence Jones protein, an immunoglobulin appears in urine
in cases of multiple myeloma. Protein precipitates
between 40- 60C, disappears at 100C and reappears on
cooling.

 Rating of proteinuria: Proteinuria can be rated as +, ++,

+++ depending upon the visibility of newspaper held at
the other side of the test tube after the coagulation test is
performed.

Test for Reducing Sugar (Benedicts Test):

Principle: In mild alkaline medium reducing sugars
undergo tautomerization to form enediols which reduce
cupric ions to cuprous ions. Cuprous hydroxide is formed.
During the process of heating cuprous hydroxide is
converted to
cuprous oxide which gives different shades of color
precipitate depending upon the concentration of the
sugar.

Points to Remember:
The presence of detectable amounts of sugar in urine
iscalled glycosuria.
Positive Benedicts test is usually suggestive of
presence of glucose in urine.
Common causes of glycosuria are:
Diabetes mellitus
Endocrinal disorders such as hyperpituitarism,
hyperthyroidism, hyperadrenalism.
Emotional glycosuria: It is a benign condition seen in
anger, fear, etc. due to hypersecretion of adrenaline in
stress.
Renal glycosuria in which glucose reabsorption by kidney
tubules is defective.
Alimentary glycosuria: It is a benign condition which is
seen after excessive intake of carbohydrate or patient is
on glucose infusion.

 Non-sugars such as ascorbic acid, glutathione,

salicylates, uric acid, glucuronides and homogentisic
acid will also give positive result with Benedicts
reagent.

Test for Ketone Bodies:

Test for ketone bodies are as follows:
Rotheras Test for Acetone and Acetoacetic Acid
Principle: Acetone and acetoacetic acid form
permanganate colored complex with sodium nitroprusside
in presence ofammonia.

Gerhardts Test for Acetoacetic Acid

Principle: Acetoacetic acid gives a red color with ferric
chloride.

Precaution:
A large number of substances such as aspirin,
antipyrin,salicylates, etc. may develop similar port-wine
color. If the urine is boiled, acetoacetic acid is converted
into acetone; but the other substances remain unchanged.
Now, if the urine gives negative test, it indicates the
presence of acetoacetic acid.
Fresh urine is necessary for this test as acetoacetic acid
is quickly decomposed into acetone and carbon dioxide.
Points to Remember:
Ketone bodies are acetone, acetoacetic acid and hydroxy butyric acid.
Ketone bodies do not appear in urine because
acetoacetic acid, which is produced nor mally in the liver,
is completely oxidized in tissues. Ketone bodies are
formed in excess when the glucose metabolism is
impaired as in
diabetes mellitus or when fat is used exclusively to give
energy as in starvation (starvation ketosis). This condition
is called as ketosis.

 The tissues are unable to oxidize the excess amount of

acetoacetic acid with the limited supply of oxygen. A part
of excess acetoacetic is decarboxylated to acetone and
remaining circulates in blood as acetoacetic acid and hydroxy butyric acid.
Rotheras test is very sensitive. It is answered even by
small amounts of acetone and acetoacetic acid.
-hydroxy butyrate does not answer Rotheras or
Gerhardts test because it does not have a ketone group.
It gives positive when converted to acetoacetic acid and
then to acetone by oxidation.
The excretion of ketone bodies in urine is called
ketonuria. This occurs in ketosis where there will be
ketonemia and ketonuria.
Total ketone bodies are found in normal urine to the
extent of about 20 mg/day.
Ketonuria may also be seen in conditions like intake of
high fat and low carbohydrates diet and toxemia of
pregnancy.
Whenever glucosuria is more than 0.5 mg% (++) the
patient should be tested for ketone bodies also.
If Gerhardts test is negative and Rotheras is positive,
acetone is present.
Gerhardts test or ferric chloride test is useful in
detecting a large number of abnormal constituents in
urine, in rare disorders. In addition to metabolites, drugs
excreted can be detected by this test. Some of the
compounds detected
are listed below.

Test for Bile Salts (Hays Test)

Principle: Hays test is based on the fact that bile salts
lower the surface tension of urine allowing the sulfur to
sink.

Points to Remember:
Bile salts are sodium and potassium salts of
glycocholates
and taurocholates.
Normally bile salts and bile pigments do not enter the
general circulation and therefore, they are absent in the
normal urine.
But, if there is intrahepatic or posthepatic obstruction to
the flow of bile, regurgitation occurs in the general
circulation and bile salts appear in urine.
Bile salts are present in urine along with bile pigments
in
obstructive jaundice.
This is not a specific test for bile salts but is usually
done
to detect bile salts.
Alcohol and salicylates give a false positive test.

Test for Bile Pigments

Tests for bile pigments are as follows:
Gmelins Test
Principle: Bile pigments are oxidized by nitric acid to
various colored products, e.g. biliverdin (green), bilicyanin
(blue), bilifuscin (red) and choletelin (yellow)

Fouchets Test
Principle: Bile pigments adsorbed on barium sulfate
precipitate are oxidized to colored products by Fouchets
reagent.
Fouchets reagent: 10% ferric chloride in 25%
trichloroacetic acid.

Filter. Unfold the filter paper. Add a few drops of Fouchets

reagent
on the precipitate.
Points to Remember:
Bile pigments are bilirubin and biliverdin.
They are produced by the breakdown of heme in the
reticuloendothelial system.
Bilirubin is in unconjugated form soon after it is
produced
from heme. It gets conjugated with UDP glucuronic acid
in liver to form mono/di-glucuronide. Bile contains
conjugated bilirubin which is excreted into the intestine.
In normal persons bile pigments are not present in
urine.
Fouchets test is a highly sensitive test for bilirubin.
Ferric chloride, present in the Fouchets reagent acts as
an oxidizing agent. It oxidizes bilirubin to biliverdin
(green) or bilicyanin (blue).

Test for Blood:

Principle: Hemoglobin (peroxidase) of blood decomposes
hydrogen peroxide catalytically and liberates nascent
oxygen. This oxygen oxidizes benzidine to a blue or green

compound. This color changes to brown within a few

minutes on exposure to air.
Benzidine reagent: It contains benzidine and glacial acetic
acid.

Points to Remember:
This is a very sensitive test but not specific for blood.
Presence of blood in urine is called hematuria.
Causes:
Injury to urinary tract or kidney.
Infection of urinary tract.
Benign or malignant carcinoma of kidney or urinary
tract.
Enlargement of prostrate due to rupture of engorged
venous plexus.
Obstruction due to urinary stones.
Nephritis.
Nephrotic syndrome.
Due to trauma, caused by introduction of catheter
through the urethra.
Tuberculosis.
Acute glomerulonephritis.
Hematuria can be frank when urine appears red (due to
blood) or it can be microscopic when it is not visible to
naked eye (occult blood)
Microscopic hematuria may be seen in:
Malignant hypertension
Sickle cell anemia

 Coagulation abnormalities
Polycystic kidney diseases.
Excretion of free hemoglobin in urine is called
Hemoglobinuria.
This occurs in severe burns, chemical poisoning,
incompatible blood transfusion, malaria, typhoid and
hemolytic jaundice.
This test is also positive when pus cells are present in
urine. These cells contain a peroxidase, which is
responsible for the positive reaction. However, if urine is
subjected to heat treatment (95-100C), the enzyme is
inactivated and the test becomes negative.
Heme, is stable to heat.
When high concentration of ascorbic acid is present in
urine, it is oxidized more readily than benzidine by oxygen
liberated from hydrogen peroxide. The benzidine reaction
then becomes negative although sufficient blood is
present in urine.