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Clinical Chemistry of Urine

Urine Formation and Excretion.
Introduction
Urine is composed of unwanted substances that have been filtered from the blood
by nephrons, the functional units of the kidneys. The urine formed in the kidneys
passes through the ureters and is temporarily stored in the bladder.
Urine is a waste by-product formed from excess water and metabolic waste
molecules during the process of renal system filtration. The primary function of the
renal system is to regulate blood volume and plasma osmolarity, and waste
removal via urine is essentially a convenient way in which the body performs
many functions using one process. Urine formation occurs during three processes:
filtration, reabsorption, and secretion.

Filtration
During filtration, blood enters the afferent arteriole and flows into the glomerulus
where filterable blood components such as water and nitrogenous waste will move
towards the inside of the glomerulus, and non-filterable components such as cells
and serum albumins will exit via the efferent arteriole. These filterable components
accumulate in the glomerulus to form the glomerular filtrate. Normally, about 20%
of the total blood pumped by the heart each minute will enter the kidneys to
undergo filtration; this is called the filtration fraction. The remaining 80% of
the blood flows through the rest of the body to facilitate tissue perfusion and gas
exchange.

Reabsorption
The next step is reabsorption, during which molecules and ions will be reabsorbed
back into the circulatory system. The fluid passes through the components of
the nephron (the proximal/distal convoluted tubules, loop of henle, collecting duct)
during which water and ions are removed as fluid osmolarity (ion concentration)
changes. In the collecting duct, secretion will occur before the fluid leaves the
ureter in the form of urine.

Secretion
During secretion, some substances such as hydrogen ions, creatinine, and drugs
will be removed from blood through the peritubular capillary network into the
collecting duct. The end product of all these processes is urine, which is essentially

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a collection of substances that has not been reabsorbed during glomerular filtration
or tubular reabsorption.
Urine is mainly composed of water that has not been reabsorbed, which is the way
in which the body lowers blood volume, by increasing the amount of water that
becomes urine instead of becoming reabsorbed. The other main component of
urine is urea, a highly soluble molecule composed of ammonia and carbon dioxide,
and provides a way for nitrogen (found in ammonia) to be removed from the body.
Urine also contains many salts and other waste components. Red blood cells and
sugar are not normally found in urine but may indicate glomerulus injury and
diabetes mellitus respectively.

Types of urine specimen

There are three types of urine specimens depending upon the time of collection.
i) Random urine sample
ii) Early morning urine sample
iii) 24 hour urine sample
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The three types of urine specimens vary in concentration of solutes as

concentration of urine varies considerably throughout the 24 hour period
depending partly on individual fluid intake, food and activities.
Various solutes appear in greater or lesser amounts at various times of the day.
Eventually, more concentrated urine is preferred for testing rather than a dilute
specimen. Therefore mid-stream sample of the first morning urine is the most
concentrated and therefore suitable for both routine urinalysis and bacterial culture.

Random Samples
These are urine specimens collected at any time of the day for the purpose of
immediate analysis. Random voided urine samples are of less concentration as
compared to the early morning one. However, in most cases it is impractical to
obtain the morning one thus the random sample may be used.
Early morning urine sample
It is the first voided morning urine of the day. A mid-stream sample of this urine is
the most concentrated and the most preferred as it gives a better picture of the
amount of substances in the urine.
24- Hour urine sample
This is a specimen collected over a period of 24 hours. The urine is stored in a
sterile Winchester quart bottle containing a preservative that does not interfere with
the required laboratory investigations.

Preservatives are added to urine to inhibit bacterial activity which leads to the
decomposition of urea to form ammonia and therefore altering the pH.
Tests that require a 24 hour urine specimen include, urinary creatinine, urinary
protein by Esbach’s method.

The preservatives for a 24 hour urine specimen include;

 Concentrated HCl

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Preserves urine for many tests e.g. urea, ammonia and calcium. It must not
be used on urine for protein estimation.

 Thymol
Preserves urine for many tests e.g. protein, calcium, sodium, potassium and
amylase. It prevents aerial bacterial contamination. It does not stop the
multiplication of bacteria already in the urine. Thymol lowers the surface
tension of urine, thus invalidating the detection of bile salts in urine by
Hay’s method.

 Toluene
This is not commonly used. It prevents aerial bacterial contamination.
Unfortunately it contaminates pipettes.

 Chloroform
As a preservative interferes with tests based on reduction for example,
reducing sugars, because it is reducing in nature. Prevents aerial bacterial
contamination.

 Formalin
As a preservative interferes with tests based on reduction for example,
reducing sugars, because it is reducing in nature. Prevents aerial bacterial
contamination.

 10 % Acetic acid
Good for urine ascorbic acid estimation

 5 % metaphosphoric acid
Good for urine ascorbic acid estimation

Changes that take place to urine upon standing

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1. Bacterial multiplication
Occurs in urine specimens left standing at room temperature for several hours.
2. Glucose level changes
Go down as it is utilized by bacteria for growth.
3. Urea level changes
Go down as it is utilized by urease producing bacteria splitting it to ammonia. The
ammonia released dissolves in the urine thus raising the pH of the urine.
4. Casts decomposition
Casts decompose in alkaline urine after hours long of standing
5. Haemolysis
Intact RBCs may haemolyse
6. Phosphates precipitation
Precipitate in alkaline urine but remain in solution when pH is acidic
7. Uric acid crystals precipitation
Precipitates in acidic urine

Urinalysis to reveal abnormal substances in Urine

This is the study performed on urine specimen and involves a number of

procedures, chemical, visual and microscopic leading to the revelation of
substances abnormally present in the urine.
Forms the first line of study that aids in the diagnosis of renal and non-renal
disorders in the physiology of the human body.

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The non-renal disorders are those involving the urinary system as well as disorders
of metabolism i.e. liver diseases.
Urinalysis is divided into two categories; Qualitative and Quantitative analysis.
1. Qualitative analysis
These are methods and procedures that will lead to the detection of substances
abnormally present in the urine. Qualitative procedures performed on urine include
visual, chemical and microscopic examinations of urine specimens.
i) Visual examinations
Include noting of colour, odour and appearance (clarity) of urine samples.
Generally, the procedures involve the noting and reporting of the physical
characteristics of the urine.
ii) Chemical examination
These are tests that will reveal the presence of blood and blood products (Hb),
protein and Bence Jones proteins, reducing substances, bile pigments and bile salts,
among others.
iii) Microscopic examination
These are studies carried out to on urine deposits using low power magnification
lens of a microscope. These are intended to reveal presence of formed elements
(organic), such as RBCs, WBCs and pus cells, casts and parasites such as
Trichomonas vaginalis, yeast cells, ova of schistosoma haematobium, and
inorganic such as calcium oxalate crystals, sodium urate or uric acid crystals. In
alkaline specimens, deposits are more likely to be phosphates, calcium carbonate
or ammonium urate.

2. Quantitative analysis
This is the determination of the actual levels of the substances abnormally present
in the urine. The substances usually quantitated in urine include, specific gravity,
pH, Glucose, proteins, urea and urinary Amylase.

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Testing Urine for Blood by Chemical Method

i. Haemastix for presence of blood
Introduction
Haemastix is a reagent paper strip impregnated with O- toludine
Chromogen system that turns blue in the presence of blood. O – Toludine
test reagent is a buffered mixture of organic peroxide and O – toludine.
Free haemoglobin or haemoglobin derived from Haemolyzed red blood
cells in the urine have peroxide – like activity and catalyze the release of
oxygen from the peroxide so that it oxidizes the Chromogen, o – Tolidine
changing its colour from orange to blue through green depending on the
concentration of haemoglobin. Or to a green – spotting of the orange test
area by non – Haemolyzed erythrocytes in haematuria.
Procedure with strip test
To perform the test,
- Mix the urine by shaking the container
- Dip the strip into the well mixed urine specimen for a second
- Remove and tap the edge of the strip against the container to remove excess
urine
- Compare the colour change visually with the colour chart within 30 – 60
seconds
- Report the findings in the units given by the technique

False positive results are obtained if the urine container is contaminated with
oxidizing cleansing agents, particularly hydrogen peroxide.
ii. Using Spectroscope
Haemoglobinuria can as well be detected by the use of a spectroscope.
Haemoglobin absorbs at a wavelength between α (555 nm) and β (430 nm).

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2. Testing Urine for Bilirubin

Introduction
Bilirubin is not normally passed in urine in detectable levels. When it is
detected in urine, the condition is referred to as bilirubinuria. And occurs
whenever there is an increase of conjugated bilirubin in the blood, bilirubinaemia
as happen in obstructive jaundice and in infectious hepatitis. Tests for the
detection of bilirubinuria are divided into 3 groups:
i. Tests based on the oxidation of bilirubin – which is yellow to Biliverdin –
which is green. Such tests include, Fouchet’s, Gmelin’s Nitric acid ring
test and Iodine test.
ii. Tests based on the diazotization of bilirubin to coloured compound. Such
tests include, Ictostix and Ictotest.
iii. Tests based on the colour of urine froth (the shake test).

Oxidation Tests
I. Fouchet’s Test for Bilirubin
Reagents
i. Fouchet’s reagent – comprising of 25g TCA in 100 ml distilled
water and 10 ml 10 % aqueous ferric chloride (i.e. 10 % FeCl3 in
distilled water)
ii. 10 % BaCl2 in distilled water
Principle of Test
Bilirubin is oxidized by Fouchet’s reagent (10 % FeCl3) to biliverdin –
which is green colour
Reactions:
i. Barium chloride precipitates sulphates present in urine thus; Ba2+ (aq)
+ SO2- (aq) → BaSO4 ↓
ii. Any bilirubin present adheres (i.e. adsorbs) onto the precipitate barium
sulphate.
iii. When Fouchet’s reagent is added to the precipitate the iron III (ferric)
chloride oxidizes the bilirubin (yellow) to biliverdin (green).
Technically, the test can be carried out in two different ways:

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Procedure 1
1. Dispense about 5 ml of urine in a clean centrifuge tube
2. Add 2.5 ml of 10 % BaCl2 solution and mix
3. Spin the mixture at about 2000 rpm for 3 minutes
4. Pour out the supernatant completely and add one drop of Fouchet’s
reagent to the deposit
5. Observe the colour on the top surface of the deposit
Results: A green colour indicates the presence of bilirubin

Procedure II
Instead of centrifuging the mixture (in 3 above) filter and spread the filter paper,
when partly dry add a drop of or two of Fouchet’s reagent onto the deposit on the
filter paper and observe the colour change.
Results: Green colour formation is positive test for bilirubinuria.

II. Gmelin’s Nitric Acid Ring Test for Bilirubin

Reagent – Concentrated nitric acid
Principle of Test
Concentrated nitric acid oxidizes bilirubin in urine to biliverdin whose green
colour appears at the interface of the two fluids.
Procedure
i. Pipette 5.0 ml of concentrated HNO3 to a test tube
ii. Carefully, without shaking add 1.0 ml of urine with a pipette and observe
the colour at the interface.
Results: A green ring at the interface indicates the presence of bilirubin in the
urine.

III. Iodine Test for Bilirubin

Reagent: Tincture of iodine

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Principle of Test:
Iodine oxidizes bilirubin in urine to form biliverdin whose green colour appears
at the interface of the two liquids.
Procedure
i. Dispense 5.0 ml of urine into a test tube
ii. Add very carefully without shaking 1.0 ml of the tincture of iodine with a
pipette and observe the colour at the interface.
Results: A green ring at the interface indicates the presence of bilirubin in the
urine.

Diazotization Tests
1. Ictotest for Bilirubin
Ictotest is a reagent tablet impregnated with the diazo reagent for the
detection of bilirubin in urine as well as in plasma. The method is based on
the principle of the van den berg reaction.
Reaction
Bilirubin couples with diazotized sulphanilic acid to form azobilirubin which is
a purple – coloured compound.
Thus,
Diagram
Procedure
i. Moisten the special test paper mat provided with 5 drops of urine.
ii. Place one tablet in the middle the moistened area
iii. Flow 2 drops of distilled water over the tablet
iv. Observe colour of mat around the tablet exactly after 30 seconds
Results
Purple colour of mat around the tablet within 30 seconds indicates the presence of
bilirubin in urine.

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2. Ictostix for Bilirubin

Ictostix is a reagent test paper strip impregnated with stabilized diazotized 2,
4 – dichloroaniline, a diazo salt for the detection of bilirubin in urine.
Ictostix forms one of the reagent pads on multistix (combur test strips).
Principle of Test: Like Ictotest, Ictostix works on the same principle of van den
berg reaction.
Procedure
i. Dip the whole strip into the a well-mixed urine sample and remove
immediately
ii. Remove excess urine from the strip by tapping the rim of the mouth of the
container
iii. Compare the colour change of the test area with colour chart provided in
30 seconds
Results
Brownish colour indicates the presence of bilirubin whose concentration varies
with intensity of the colour
Tests Based on the Colour of Froth
The Froth Test
This is a crude method of detecting bilirubin in urine. The technique is based
on the production of yellow foam following a vigorous shake of the urine
container.
Procedure
Shake vigorously a bottle containing about 10 ml of urine and observe the
colour of the froth in day light. Compare with that of a normal urine.

Testing Urine for Urobilinogen

Introduction
Urobilinogen, which is synonymous with stercobilinogen is a reduction product
of conjugated bilirubin by the action of intestinal bacteria. Urobilinogen
(colourless) loose two hydrogen atoms on exposure to air or when treated with

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mild oxidizing agents and converted to urobilin (brown coloured). Colourless

urobilinogen show no absorption bands in solution and do not fluoresce on
addition of zinc salts. However, like the urobilin, urobilinogen is precipitated by
saturation with ammonium sulphate, but unlike urobilin, give an immediate red
colour with Ehrlich’s aldehyde reagent, a reaction dependent on the presence of a
central methene group in the molecule.
Qualitative Tests for Urobilinogen in Urine
The traditional test is the red colour formation given by p-
dimethylaminobenzeldehyde of Ehrlich’s test in strongly acid solution, which
utilizes the Wallace and Diamond reaction.
Ehrlich’s Test for Urobilinogen
Reagent: Ehrlich’s Aldehyde reagent (P – dimethylaminobenzeldehyde in
hydrochloric acid solution)
Prepared by dissolving 2 g of P – dimethylaminobenzeldehyde in 48 ml of
concentrated HCl and 50 ml of Water.

Procedure: Add 1 ml of Ehrlich’s reagent to 10 ml of freshly voided urine.

Mix by inversion and stand for 3 – 5 minutes at room temperature

Results: Red or pink colour suggests the presence of increased amounts of

urobilinogen.
NB: Red colour with Ehrlich’s aldehyde reagent is also given by porphobilinogen
and must therefore be distinguished by extraction tests with organic solvents in
which urobilinogen is soluble but not porphobilinogen.
Method: Add 1 – 2 ml of chloroform or amyl alcohol: benzyl alcohol mixture to
the red colour formed with Ehrlich’c reagent and shake thoroughly. Allow the two
layers to separate.
Look for red colour in the chloroform or Amy alcohol layers, Urobilinogen is
soluble in organic layers, so any red remaining in the aqueous layer after extraction

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constitutes a positive test for porphobilinogen which is insoluble in organic

solvents but is in aqueous solvents.
NB: Chloroform layer (red) aqueous layer (clear) – positive for urobilinogen
Chloroform layer (clear) aqueous layer (red) – positive for porphobilinogen

Ehrlich’s Test Strip for Urobilinogen

This is a modification of Ehrlich’s test.
Reagent: makes use of 4 – methoxy-benzene- diazonium tetrafluoroborate (in
BMC products) or p- dimethylaminobenzeldehyde stabilized in an acid buffer on
an absorbent test for pad.
Principle of Test: The biological products rely on the coupling of p- methoxy-
benzene diazonium fluoroborate in an acid solution to give a red azo-dye.
Procedure
1. Dip reagent area of strip in a well-mixed freshly voided uncentrifuged urine.
2. Remove strip from urine, tap the edge of the strip against urine container to
remove excess urine.
3. Allow reaction to continue for exactly 10 seconds.
4. Compare colour change with colour blocks provided by holding the reagent
area of strip closer to the chart.
Results
Strip test gives semi- quantitative results. They detect normal and increased
amounts of urobilinogen corresponding to 1, 4, 8 and 12 mg/100 ml.

Testing Urine for Urobilin

Introduction

Urobilin is the brown coloured oxidation product of the colourless urobilinogen.

Their formation from urobilinogen takes more readily when the urine is exposed to
light. They can be recovered from urine by saturation with ammonium sulphate. In

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solution, they show a distinct absorption band and are converted to zinc salts
possessing a green fluorescence when treated with zinc acetate in neutral solution.

Schlesinger’s Qualitative Test for Urobilin

Reagents
1. Absolute ethanol
2. Powdered zinc acetate
3. Tincture of iodine
Principle of Test
Urobilinogen is oxidized by tincture of iodine and converted to urobilin. The
addition of zinc acetate leads to the formation of a greenish- yellow fluorescent
compound of zinc urobilin.
Procedure
1. To 10 ml of urine in a test tube add 2 -3 drops of tincture of iodine and shake
to mix.
2. To another test tube add 10 ml of absolute ethanol and place about 1 g of
powdered zinc acetate and shake to mix.
3. Mix the two solutions by pouring the contents of one into the other
repeatedly until all the zinc acetate is dissolved
4. Filter the mixture through whatman no.1filter paper into a clean test tube
5. View the filtrate from above.
Results
Greenish – yellow fluorescence is observed if urobilin is present.
Testing Urine for Bile salts
Hay’s Test
Purpose: For the demonstration of bile salts in urine
Principle of Test
Bile salts lower the surface tension of liquids including urine such that when
powdered sulphur is sprinkled on the surfaces of such liquids, it sinks. Hay’s test

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utilizes the reduction of surface tension of bile salts property to demonstrate the
presence of bile salts in urine.
Procedure
Sprinkle a little sulphur powder onto the surface of clear freshly voided urine in a
beaker observe whether the powder will float or sink.
Results
If the urine contains bile salts the sulphur powder will sink. The sulphur powder
will float on urine lacking bile salts. Distilled water is used as a negative test.

Ketone Bodies
Introduction
Ketone bodies are products of the incomplete metabolism of fats that occur in
diabetes mellitus and in carbohydrates starvation during fasting when there occurs
an increased hydrolysis to meet the body’s demand for energy. Under these
conditions the liver yields acetyl-coA at a rate greater than can be metabolized
further to yield energy, water and carbon dioxide as final products. Instead, pairing
up of acetyl-coA molecules occurs resulting to the formation of acetoacetic acid –
a ketone body, some of which is decarboxylated to acetone and some reduced to
Beta hydroxybutyric acid. Ketone bodies accumulate in the blood, giving a
condition known as ketonaemia, some of which is excreted in the urine known as
ketonuria.

Clinical Importance of test

The detection of ketone bodies in the urine is important as part of the routine urine
examination of diabetic patients.
The detection of ketone bodies in urine can be achieved by tests like Rothera’s
nitroprusside and Gerhardt’s 10% ferric chloride tests.
1. Detection of Ketone Bodies by use of the Rothera’s Nitroprusside Test
Reagents

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i. Crystalline sodium nitroprusside

ii. Concentrated ammonia solution ((NH4)2SO4 solution).
Principle of Test
Nitroprusside reacts with a ketone group at an alkaline pH to give a purple
colour.
Procedure
i. Place approximately 1 g of Rothera’s sodium nitroprusside in a test
tube.
ii. Add 5 ml of urine and shake to dissolve the reagent.
iii. Add 1 ml of concentrated ammonia solution and shake to mix.
iv. Observe the colour change.

Results
A purple formation is given by acetoacetic acid and acetone – both of which
contain a ketone group but not β- hydroxybutyric acid as it does not possess a
ketone group but a 2o alcohol group.
2. Gerhardt’s Ferric Chloride Test

Reagents: 10 % Ferric Chloride

Principle of Test
10 % FeCl3 reacts with many substances to give characteristic colour.

Procedure
i. Add the 10 % FeCl3 solution drop by drop to a test tube containing 5
ml of urine.
ii. Observe for colour change.

Results
Purple colour formation is positive test for acetoacetic acid. Acetone does
not react with 10 % FeCl3. Purple colour is also given by salicylates which
are non-ketone bodies. To distinguish purple colour of acetoacetic acid from
that given by salicylates, boil the urine and repeat the test. If acetoacetic acid
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is present the test will be negative whereas if salicylates were present

purplish colour will form after the boiling of the urine. This is because
acetoacetate is volatile while salicylates are not.

Acetest and Ketostix

These are a modification of the Rothera’s and Gerhard’s test in tablet and
paper strip forms respectively for the detection of both acetone and
acetoacetic acid.

Phenylpyruvic acid
Phenylpyruvic acid is a transamination metabolite of phenylalanine amino acid.
Under normal metabolism, phenylalanine is converted to tyrosine by its usual
enzymatic pathways. In phenylketonuria, an inherited disorder of phenylalanine
metabolism, the fundamental defect is the lack of phenylalanine hydroxylase so the
infant is unable to convert phenylalanine into tyrosine. Consequently,
phenylalanine accumulates in the blood and some of it is excreted in urine but a
greater part of it transaminated to phenylpyruvic acid.
(OH)
Phenylalanine Tyrosine

= phenylanine hydroxylase

NH2

Transaminated

Phenylpyruvic acid

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,,,,,
Phenylpyruvic acid is excreted in the urine as early as 2 – 3 weeks after birth. Early
recognition of this disorder is important as dietary control is necessary to prevent
the infant from becoming mentally retarded.
Qualitative Tests for Phenylpyruvic Acid
Phenylpyruvic acid qualitative tests include ferric chloride, phenistix,
microbiological and chromatographic techniques. Chromatographic and culturic
bacterial inhibition assay techniques are far much better than the chemical methods
which are less specific and so have largely been replaced.
Proteins in urine
Proteins in the urine ( proteinuria) generally indicates a renal disease or other
diseases affecting renal function in either case, there are changes in the glomeruli
thereby allowing increased passage of proteins.
In glomerulus damage due to bacterial infections, chemical poisoning or crush
syndromes, the excretion of proteins in the urine is increased.
Tests for urinary proteinuria include,
Boiling test, turbidimetric, nitric acid ring test, protein reagent test paper strip and
electrophoresis

Detection of Proteins in Urine by:

1. Boiling Method – The Heat Coagulation Test
Reagent: 5 % Acetic acid

Principle of Test
Proteins are coagulated and denatured by heat in slightly acid media

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Procedure
i. Pipette about 10 ml clear or centrifuged urine to a boiling tube.
ii. With a test tube holder hold the boiling tube at an angle of 45 o C and
heat the top part of the urine until it boils
iii. Look for cloudness.
iv. Add 3 drops of the 5 % acetic acid and boil again.
Results
i. If turbidity forms before adding 5 % acetic acid and persists after the
addition, then proteins are present.
ii. If turbidity does not appear at first but does after adding 5 % acetic
acid, then proteins are present.
iii. If turbidity appears at first but disappears after the addition of 5 %
acetic acid, then protein is absent.
iv. If no turbidity forms before and after adding the 5 % acetic acid then
there is no proteinuria.
If protein is detected it may be reported semi- quantitatively as;
i. Trace (for very slight turbidity)
ii. + (for slight turbidity)
iii. ++ (for moderate turbidity)
iv. +++ (for heavy turbidity)

2. Turbidimetric Methods of Protein Detection

i. Sulphosalicyclic acid semi- Quantitative Turbidity Method
Principle
Proteins are precipitated by Sulphosalicyclic acid to form (cloudiness)
whose intensity progressively increases with concentration
Procedure: To 5 ml of clear urine in a test tube add 1- 2 drops of reagent and
examine for cloudiness.
Results: In the presence of protein a white precipitate appears whose intensity is
proportional to the amount of protein present. And is reported semi – quantitatively
as for boiling method.

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ii. Procedure with 3 % Trichloracetic Acid

Reagent: 3 % TCA in distilled water
Principle of the Test
Proteins are precipitated by Trichloracetic acid to form turbidity whose
intensity is proportional to the amount of protein present.
Procedure:
i. Mix 6 ml of clear urine with 2 ml of reagent (or 3ml with 1 ml
respectively) and shake to mix.
ii. Observe for turbidity.
iii.
Results: Turbidity is positive test for proteinuria.

3. Detection of Proteinuria by Nitric Acid Ring Test (Heller’s Test)

Reagent: Concentrated Nitric acid
Principle of Test
If urine containing protein is layered carefully on concentrated nitric acid,
proteins are precipitated forming a white ring at the urine – acid junction.
Procedure
i. Carefully layer a little urine on 5 ml of concentrated nitric acid. Do not
mix.
ii. Observe for a white curdy ring at the interface.

Results: white ring at the interface indicates positive test for proteinuria.

4. Protein Testing by Reagent Paper Strip

Urine protein strip tests (detects) mainly albumin. The test strips are
specifically called Albustix. These are cellulose paper strips with the reagent

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are impregnated with the indicator tetrabromophenol blue or

tetrabromophenolphthalein ethyl ester and buffered to an acid pH.

Principle of test
In the presence of protein there is a change in the colour of the indicator
from light yellow to green – blue depending on the amount of protein
present.

Procedure
i. Dip the whole strip into a well-mixed uncentrifuged urine sample.
Results
Are reported semi- quantitatively as follows:
i. Light yellow ---------------------Negative
ii. Light green -----------------------Trace (or 0.3g/l)
iii. Green ------------------------------+ (1 g/l)
iv. Deep green------------------------- ++ ( 3 g/l)
v. Greenish – blue ------------------ +++ (5 g/l)
Other methods for the demonstration of protein in urine include
quantitative test that makes use of the Esbach’s albuminometer.

Testing Urine for Bence Jones Proteins

Three methods of evaluating Bence Jones proteins are available. They are:
i. Heat test
ii. Electrophoresis
iii. Bradshaw’s test

1. Heat Test

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The heat test is based on the peculiar solubility and thermal characteristics of
Bence Jones proteins, which precipitates when heated to temperatures
between 40 and 60 o C but resolubilizes at temperatures below and above
these, whereas ordinary proteins precipitate irreversibly at temperatures
above 60 o C.

Procedure
Read and copy; Refer to handbook for clinical chemistry by V.W. Sitati
page 175 – 176

2. Bradshaw’s Test
This is the most useful screening test and is more reliable than the heating
method.
Reagents: Concentrated hydrochloric acid.

Principle of the test

Bence Jones proteins are precipitated by concentrated hydrochloric acid
forming a white “curdy” precipitate at the interface of the two fluids.

Procedure: read and copy; Refer to handbook for clinical chemistry by

V.W. Sitati page 196 – 197 / Harold varley page 602.

Electrophoresis method
In the electrophoresis of urinary proteins, Bence Jones proteins if present, the
abnormal sharp protein band will, migrate between the β- and ¥- globulin bands
positions. Electrophoresis is used to confirm the screening tests.

Qualitative Tests for reducing substances

Introduction

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A reducing substance is one which will reduce blue cupric sulphate to red cuprous
oxide. The most important reducing substances are sugars (glucose, galactose,
lactose, fructose and ribose, xylose and arabinose).
Alkaline cupric sulphate can also be reduced by substances which are not
carbohydrates such as gluconic acid, salicyluric, uric acid, creatinine and
homogentisic acid if present in urine in sufficiently large concentrations.

1. Benedict’s Qualitative Test for reducing substances

Method (i) Boiling Method
Reagent (qualitative reagent) consist of;
i. Hydrated copper sulphate (CuSO4.5H2O)
ii. Sodium citrate
iii. Anhydrous sodium carbonate (Na2CO3)
iv. Distilled water

Principle of Test
Reducing substances when boiled with Benedict’s qualitative solution,
reduce cupric sulphate (blue) to cuprous oxide which appears green, yellow,
orange or brick red depending on their concentrations.

Procedure
i. To a boiling tube add 0.5 ml of urine followed by the addition of 5.0 ml
of Benedict’s qualitative reagent.
ii. Mix well and place in a boiling water bath for 5 minutes.
iii. Allow to cool and observe for colour change.

Results and their reporting

i. If the solution remain blue, this is interpreted as negative test.
ii. If the solution changes to green, this is reported as trace,
iii. If yellow, +
iv. If orange, ++

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v. If brick red, +++

Method (ii) Clinitest

Alkaline copper reagent in tablet form.
Composition
The tablet contains:
i. Copper sulphate, CuSO4
ii. Caustic soda, NaoH
iii. Sodium bicarbonate, NaHCO3
iv. Citric acid
Reactions
i. NaoH reacts with water (in the urine) and citric acid with the production
of intense heat which boils the mixture.
ii. If glucose or any substance with reducing properties is present, reduce
the copper sulphate in an alkaline medium to cuprous oxide.
Procedure
i. To a boiling test tube add 5 drops of urine followed by 10 drops of
distilled water.
ii. Place one tablet and allow to boil until it stops.
iii. Shake the mixture and observe for colour change.
iv. Compare the colour formed with the colour blocks in the colour chart
provided.
Reporting of Results
The results are reported semi- quantitatively as for the boiling method.

Specific Tests for the Identification of Reducing Substances in Urine

Introduction
The reaction given by both the boiling method and Clinitest is not specific for a
particular reducing substance. Therefore, following a positive Benedict’s
qualitative test it is imperative to perform further tests that are specific for each of

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these substances. And thus lead to their identification. Some specific tests for
individual reducing substances in urine include:
1. Clinistix or Glukotest which are specific for glucose only.
2. Seliwanoff’s test for fructose detection.
3. Bial’s test for Pentoses (xylose, Arabinose and Ribose).
4. Methylamine test for lactose and maltose detection.
5. Osazone test for glucose, fructose, and lactose.
6. Yeast fermentation test for glucose and fructose (Pentoses are non-
fermentable).
7. Chromatography for all sugars.

I. Tests for glucose identification

These include, Glukotest, Clinistix and chromatographic techniques.
Clinistix and Glukotest are reagent test paper strips commercially
available, quick, simple to use and specific for glucose. They are all
based on a glucose oxidase reaction.

Principle of test
i. Glucose (present in the urine) is oxidized by atmospheric oxygen in the
presence of glucose oxidase (on the reagent strip) to gluconic acid with the
release of hydrogen peroxide.
ii. Hydrogen peroxide in the presence of peroxidase enzyme (on the reagent
strip) oxidizes the Chromogen system (on the strip) from colourless state
to shades of a coloured compound.
Thus,

1. Glucose + Oxygen GOD gluconic acid + H2O2

2. H2O2 peroxidase O2 + H2O
3. O2 + Chromogen Coloured compound

Composition
The various glucose reagent strips differ in from one another chemically
as regards the type of Chromogen and buffer system used.
Procedure: similar as for all different strips

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Specificity
Glucose oxidase is a specific enzyme for glucose. Thus no other substance
excreted in the urine gives a positive reaction with these reagent strips. In
particular they do not react with other reducing sugars, for example lactose,
galactose and fructose, or reducing metabolites of some drugs (e.g. salicylates), as
copper reduction methods do. Oxidizing agents such as hydrogen peroxide will
give a false positive reaction.

II. Seliwanoff’s Test for fructose Identification

Reagent
Seliwanoff’s reagent is composed of Resorcinol in concentrated
hydrochloric acid and distilled water.

Principle of Test
Fructose, when boiled in the presence of hydrochloric acid, yields a
derivative furfuraldehyde which condenses with Resorcinol to form a red coloured
compound.

Reaction: C C + HCl . . + 3 H2O

OH O . .CHO

Hydroxyketo group of fructose .

Furfuraldehyde

Then furfuraldehyde condense with Resorcinol to form a red – coloured compound

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Procedure
1. To 5.0 ml of Seliwanoff’s reagent in a boiling test tube add 0.5 ml of urine
and bring it to the boil.
2. Observe for red colour.

Results
Fructose gives a red colour within 30 seconds of boiling.

III. Specific Test for the identification of Pentoses in Urine

Introduction
Pentoses are monosaccharides with 5 carbon atoms in their molecule.
Thus, fall in a class of sugars with the general formular, CnH2nOn. The
most important pentose is Ribose, found in furanose form in the
ribonucleotides and ribonucleic acid (RNA). Other Pentoses include,
Xylose (or wood sugar), Arabinose and Fucose. Pentoses are non-
fermentable sugars. They normally occur in the urine following the
ingestion of large quantities of fruits or fruit products such as plums and
cherries. The presence of Pentoses in urine in detectable levels is termed
pentosuria.

Bial’s Test for Pentoses

Reagent: orcinol in concentrated hydrochloric acid and 10 % ferric chloride
solution.

Principle of Test
Pentoses when boiled with Bial’s reagent, hydrochloric acid acts on Pentoses
to yield aldehydes of furfural type, which in the presence of Orcinol (M-
dihydroxytoluene) condense to form green coloured compounds.

Reaction: CH2OH- CHOH-CHOH-CHOH-CHO HCl HC CH

Pentose ˄

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HC C-HO

O
Furfuraldehyde

Green coloured cmp

Procedure
i. To 5.0 ml of reagent in a boiling test tube add 0.5 urine and shake
to mix
ii. Boil the mixture for 30 seconds

Results
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Green colour appearance within 30 seconds is positive test for

Pentosuria.

Identification Test for Lactose and Maltose

Introduction
Lactose, Maltose and Sucrose are the three important disaccharides –
sugars whose molecules consist of two monosaccharides units
condensed. Both Lactose and Maltose have reducing properties, can be
identified by the methylamine test and chromatographic techniques.

Methylamine Test for Lactose

Reagents
i. 0.2 % aqueous methylamine hydrochloride
ii. 10 % sodium hydroxide

Principle of Test
Lactose and Maltose when warmed with methylamine hydrochloride in
an alkaline medium form a red –
Coloured product.

Procedure
i. To 5.0 ml of urine in a boiling test tube add 1 ml of 0.2 %
methylamine hydrochloride followed by the addition of 0.2 ml of
the 10 % sodium hydroxide and mix by inversion.
ii. Incubate the tube at 56 o C for 30 minutes.
iii. Remove and cool the tube to room temperature.
iv. Observe for red colour

Results

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Red colour is indicative of Lactose or Maltose as they are the only

reducing disaccharides which give a red
Colour with methylamine hydrochloride. However, positive test with
methylamine test is confirmatory for
Lactosuria as maltose is not usually found in urine.

Assignment
Read and note on yeast fermentation test, Osazone test and chromatographic
techniques.

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