Biochemical Examination of Urine: Prepared by Jeunne Karmella Madamba, RMT

BIOCHEMICAL
z EXAMINATION
OF URINE
PREPARED BY JEUNNE KARMELLA MADAMBA, RMT
z
A. Inorganic Constituents
 1. Sulfate
 Acidity 5ml of urine with 5 drops of acetic acid and add a few drops
of BaCl2 solution.
 Result : If green (biliverdin) or blue (cholecyanin) color indicates
bilirubin
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 2. Chloride
 Acidity 5ml of urine with 2 drops of dilute nitric acid and add 2
drops of AgNo3 solution.
 Result : Cloudy White precipitate is formed which indicates the
presence of chloride
z
 3. Phosphate
A. Triple Phosphate
 To 5ml of urine add

1ml of NH4OH and
2ml of MgSO4
solution. Examine
crystal under
microscope.
 Result :
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 3. Phosphate
 B. Alkali Phosphate
 Mix 5ml of urine alkaline with 1ml of NH4OH. Warm and note what
happens.
 Result : Formulation of precipitate or crystal which is yellow-brown
in color which is called as ammonium phosphohydroxide. It
became soluble when heated with ammonium hydroxide.
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 4. Iron
 Evaporate 5 ml of dryness under the hood. Cool and supplement 5
drops of 6N HCL to the residue. Dilute 5ml of water. Divide into two
portions.
 A. add 1ml NH4SCN and note the color
 B. treat the second portion with 5 drops of K4Fe(CN)6 and allow to
stand for some time.
 Result: a light reddish brown color is obtained.
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B. Organic Constituents
 1. Urea
 Mix 15ml of urine and 5ml

of baryta mixture. Filter
the precipitate and
evaporate the filtrate to a
thick syrup. Add an equal
volume of 95% ethyl
alcohol and filter. Allow
filtrate to crystallize in a
cool place. Examine
under microscope
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 How does urea become a component of urine?

 It is the principal product of protein metabolism and constitutes
about 80-90% total nitrogen excretion
 Urea is an organic compound which chemical formula is CON2H4
or (NH2)2CO. It is also known as carbamide and one of the most
imp. organic substances in urine which includes uric acid and
creatinine. It is also the major end product in nitrogen metabolism
in humans and mammals.
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 2. Uric Acid
a. 1. to 1ml of urine add 5ml of 20% NaCO3 and mix

2. Add 5 drops of phosphotungstic acid reagent. Mix well
Result: the formation of blue colored solution was observed.

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 2. Uric Acid
b. Acidify 50ml of urine with

5ml conc. HCl and stir.
Examine some crystals
under the microscope.
Result: yellow brown crystals

were formed. These crystals
appear as rhombic plates,
rosettes, prism and are oval
with pointed ends.
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 2. Uric Acid
c. Dissolve some of the crystals in NaCO3 solution. Transfer few

drops of the solution to a strip of filter paper moistened with
AgNO3 solution
Result: the filter paper changes its color to dark brown or black
color due to the precipitation of silver.
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 2. Uric Acid
 What property of uric acid is shown in this procedure?

 Uric acid reduces ammoniacal silver nitrate to metallic silver. The
silver is reduced to metallic silver. This reaction is known as schiffs
test for uric acid.
 How does uric acid became a constituent of urine?

 uric acid is an end product of the oxidation of purines in the body.
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 3. Creatinine
 A. Jaffe’s Reaction
 1. mix 5 volumes of saturated picric

acid and one volume of 10% NaOH
 2. take 2ml of urine and add 1ml of

alkaline picrate solution
 Result: from red colored
solution it forms a yellow to
orange colored solution, which
indicates presence of
creatinine. Creatinine is a
product of muscle metabolism.
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 3. Creatinine
 B. Weyl’s Reaction
 To a 5ml of urine add 5 drops of

sodium nitroprusside. Make this
solution alkaline with dilute
NaOH solution.
 Result: ruby red color is turned

first then to yellow which
indicates the presence of
creatinine in urine.
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 4. Indican (Obermeyer’s test: FeCl3

+ HCl)
 1. To a 5ml of urine add 5ml of

obermeyer’s rgt
 2. mix and add chloroform. Shake

and allow chloroform to settle.
 Result:when indicant is present, the

chloroform layer shows a deep
violet to greenish-blue color.
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 4.Indican (Obermeyer’s test: FeCl3

+ HCl)
 Equation: indole (oxidized) indoxyl +

H202 - indoxyl sulfuric acid K +
indoxyl K sulfate (indicant)
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 4. Indican (Obermeyer’s test: FeCl3

+ HCl)color chart and graded as
follow
Negative (normal) = clear or blue tinge

1+ (slightly positive) = slight blue, yellow, mint green
2+ (Positive) = dark blue, light green, golden brown
3+ (high positive) = violet, indigo, dark brown
4+ (very high positive) = jet black
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 4. Indican
 How does indicant become a constituent of urine?

 The potassium salt of indoxysulfate (indicant) found in
urine is a result of bacterial acyion of tryptophan in the
bowel
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 5. Hippuric Acid
 Mix 10ml of urine and 10ml of

saturated (NH4)2SO4. add 5 drops
of conc. H2SO4. Examine Under
microscope.
 Result: clear to yellow-brown four

sided prism/ rhombic plates
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 5. Hippuric Acid
 Dissolve some crystals in 1ml of conc HNO3. evaporate to

dryness on a waterbath. Transfer the residue to a dry test
tube. Heat and note the odor of nitrobenzene.
 Result: only yellow liquid with a butter almond like odor.
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 6. Oxalic Acid
 20ml of urine in a beaker, add

1ml of a saturated CaCl2.
acidify with HC2H3O2. and
examine under microscope.
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 6. Oxalic Acid
 Calcium oxalate crystals are usually found in acidic urine. They may occur as
either bihydrated or monohydrated coax.
 Bihydrate – appear as bipyramids of various sizes (envelope form)
 Monohydrate – can assume several shapes, including ovoids, biconcave disks,

rod and dumbbells.
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 6. Oxalic Acid
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 7. Pigments
 2ml of urine + dilute NaOH. Stand for a while and then filter. To
the filtrate, supplement with 3 drops of ZnCl3
 Result: formulation of white precipitate
 What pigments are usually present in urine? Urochrome,

urobilinogen and uroerythrin
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 8. Purine Bases
 3ml of urine add 2ml of Magnesium mixture (1:2:4 parts by

weight of MgSO4, NH4Cl, and conc. NH3). Filter then add 1ml
of ammoniacal AgNO3
 Result: Cloudy solution with white precipitate.

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C. Pathologic Constituents
 1. Glucose
 A. Fehling’s Reaction – determination of Glucose

 6 drops of fehling’s rgt (3 drops of fehlings A, mixed with 3 drops of
Fehling’s rgt B) and add 6 drops of urine. Heat until boiling.
 Result: the solution having azure blue color (mixing fehling’s rgt),
turns to red (copper I oxide) when urine having glucose is added.
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 1. Glucose
 B. test portion of the urine sample with combistix strip.
 Result: A. Clinistix/Combistix Rgt strip test
Principle: this is a specific test for glucose based on the

enzyme glucose oxidase, which is impregnated on a distrip.
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 1. Glucose
 A positive reaction is seen as a change of color from red to blue,

depending on the amount of glucose present in the urine
 Over all reaction :
 Contents of the rgt strip: the clinistix, rgt strip contains glucose
oxidase, peroxidase, and O-toluidine
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 2. Albumin (Heller’s Test)
 1ml of conc. HNO3, incline tube and pour a little of urine as to

form a layer over it.
 Result: Appearance of white ring at the junction indicates the

presence of protein.
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 3. Test for Bile Pigments (Smith’s Test)
 A. 5ml of urine in a test tube. Overlay the urine with the tincture
of alcoholic iodine mixture.
 Result: an emerald green ring at the zone of contact shows the

presence of bile pigment.
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 4. Acetone (Legal’s test)
 One small crystal of sodium nitroprusside in a test tube + few

drops of water to dissolve the crystal. Add 5ml of urine, make
alkaline with 10% KOH and acidify with glacial acetic acid.
 Result : A purple or red-violet color indicates the presence of

acetone
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 5. Test for Occult Blood (urine containing hemoglobin appears

brown-gray in color and is usually cloudy)
Result: a green or blue color is a positive test. The chromogen in

this test is gum guaiac. Peroxidase of the hgb molecule liberates
oxygen from the hydrogen peroxide and the liberated oxygen
reacts with an organic rgt or chromogen (gum guaiac or O-
toluidine) to give a colored compound, which is usually blue or
green.
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D. Microscopic Constituents
 2/3 of urine in a tube. Cenrifuge for 5 minutes, then pour off

supernatant liquid. Place a drop of the sediment on a clean dry
slide. Examine under microscope.
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Microscopic
Constituent in
Urine: Crystal
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Microscopic
Constituent in
Urine: Cells
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Microscopic
Constituent in
Urine: Casts
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ABNORMAL ASSOCIATED CAUSES

CONSTITUENT
Protein (Albumin) Albumin is normally too large to pass through
glomerulus. Indicates abnormal Increased permeability
of the glomerulus membrane. Non pathological
causes are: pregnancy, physical exertion, increased
protein consumption. Pathological causes are:
glomerulonephritis, bacterial toxins, chemical poisons.
Glucose Glycosuria. Normally filtered glucose is reabsorbed by
the renal tubules and returned to the blood by carrier
molecules. If blood glucose level exceeds renal
threshold levels, the untransported glucose will spill
over into the urine. Main casue: DM
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CONSTITUENT
Ketones Ketone bodies such as acetoacetic acid, beta
hydroxybutyric acid and acetone can appear in urine in
small amount. Causes: DM, Starvation, diarrhea
Bilirubin Bilirubin comes from the breakdown of hgb in RBC.
Causes: liver disorders, cirrhosis, hepatitis, obstruction
of bile duct.
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CONSTITUENT
Urobilinogen Bile pigment derived from the breakdown of hgb.
Causes: hemolytic anemais, liver dses.
Hemoglobin Hemoglobinuria. Causes: Hemolytic Anemia, blodd
transfucion reactions, massive burns, renal dses.
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CONSTITUENT
RBC Hematuria is the presence of intact erythrocytes.
Almost always pathological. Causes: Kidney stones,
tumors, glomerulonephritis, physical trauma
WBC Pyuria (Pus in the urine) Causes: UTI
Nitrite Presence of bacteria. Causes: UTI

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BIOCHEMICAL

 To 5ml of urine add

 Mix 15ml of urine and 5ml

 How does urea become a component of urine?

a. 1. to 1ml of urine add 5ml of 20% NaCO3 and mix

Result: the formation of blue colored solution was observed.

b. Acidify 50ml of urine with

Result: yellow brown crystals

c. Dissolve some of the crystals in NaCO3 solution. Transfer few

 What property of uric acid is shown in this procedure?

 How does uric acid became a constituent of urine?

 1. mix 5 volumes of saturated picric

 2. take 2ml of urine and add 1ml of

 To a 5ml of urine add 5 drops of

 Result: ruby red color is turned

 4. Indican (Obermeyer’s test: FeCl3

 1. To a 5ml of urine add 5ml of

 2. mix and add chloroform. Shake

 Result:when indicant is present, the

 4.Indican (Obermeyer’s test: FeCl3

 Equation: indole (oxidized) indoxyl +

 4. Indican (Obermeyer’s test: FeCl3

Negative (normal) = clear or blue tinge

 How does indicant become a constituent of urine?

 Mix 10ml of urine and 10ml of

 Result: clear to yellow-brown four

 Dissolve some crystals in 1ml of conc HNO3. evaporate to

 20ml of urine in a beaker, add

 Bihydrate – appear as bipyramids of various sizes (envelope form)

 Monohydrate – can assume several shapes, including ovoids, biconcave disks,

 Result: formulation of white precipitate

 What pigments are usually present in urine? Urochrome,

 3ml of urine add 2ml of Magnesium mixture (1:2:4 parts by

 Result: Cloudy solution with white precipitate.

 A. Fehling’s Reaction – determination of Glucose

 B. test portion of the urine sample with combistix strip.

 Result: A. Clinistix/Combistix Rgt strip test

Principle: this is a specific test for glucose based on the

 A positive reaction is seen as a change of color from red to blue,

 Over all reaction :

 2. Albumin (Heller’s Test)

 1ml of conc. HNO3, incline tube and pour a little of urine as to

 Result: Appearance of white ring at the junction indicates the

 3. Test for Bile Pigments (Smith’s Test)

 Result: an emerald green ring at the zone of contact shows the

 4. Acetone (Legal’s test)

 One small crystal of sodium nitroprusside in a test tube + few

 Result : A purple or red-violet color indicates the presence of

 5. Test for Occult Blood (urine containing hemoglobin appears

Result: a green or blue color is a positive test. The chromogen in

 2/3 of urine in a tube. Cenrifuge for 5 minutes, then pour off

ABNORMAL ASSOCIATED CAUSES

ABNORMAL ASSOCIATED CAUSES

ABNORMAL ASSOCIATED CAUSES

ABNORMAL ASSOCIATED CAUSES

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