This document provides information on chemical examination of urine, including tests to detect various analytes like protein, glucose, ketone bodies, bilirubin, urobilinogen, bile salts, and blood. It describes the principle, requirements, procedure, and interpretation for common tests to identify these substances, which can indicate underlying renal or metabolic conditions. Causes for abnormalities in the various analytes are also outlined.
3. PROTEINURIA
• The presence of detectable protein in the
urine is known as proteinuria.
• It indicates glomerular injury.
• If turbid, filter or centrifuge the urine before
testing.
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4. TESTS FOR PROTEIN
• A. Heat and acetic acid test
• B. Sulfosalicylic acid test
• C. Heller’s test
• D. Dipstick method
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5. Heat and Acetic Acid Test
• Principle :
• Heat induced coagulation of proteins and
precipitation. Coagulation can further
enhanced when drops of acetic acid are
added.
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6. PROCEDURE
Fill three –fourth ¾ of a test tube with clear
urine.
Heat the upper part (1/3) of the urine .
(lower part of the urine acts as a control)
Add a few drops of (3-5) 10% glacial acetic
acid
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8. • Interpretation :
• If turbidity persists it is due to proteins
• Depending upon the amount of precipitate,
results are interpreted below…….
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11. SULFOSALICYLIC ACID TEST
• This test detects all types of proteins
(albumin, globulin, glycoproteins and Bence
Jones Protein).
• Principle : Cold precipitation of proteins by a
strong acid
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12. PROCEDURE
Take 2.5 mL of urine in a small test tube
Slowly pour 2.5 mL of Sulfosalicylic acid
Wait for 5 Min
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13. INTERPRETATION
• Presence of a cloudy precipitate indicates the
presence of proteins in urine.
• However, it also precipitates mucus and Bence
jones protein.
• Caution : Filter urine if turbid, as it interferes
with the final reading
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17. REDUCING SUBSTANCES IN THE URINE
• Reducing substances are those compounds
which reduce cupric ions ( from copper
sulphate in Benedict’s reagent) in an alkaline
solution to cuprous ions ( cuprous oxide) .
• Such substances may be sugar or non sugar.
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18. Sugars : These include :
• 1. Glucose
• 2.Fructose
• 3.Pentose
• 4.Galactose
• 5.Lactose
• 6.Maltose
• 7.Sucrose
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22. PRINCIPLE
• The copper sulphate present in the Benedict’s
reagent reacts with the reducing substances in
the urine which convert cupric sulphates to
cuprous oxide in hot alkaline media.
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24. PROCEDURE
Take 5 mL of Benedict’s qualitative reagent in a test
tube
Boil to exclude presence of reducing substance in
reagent
Add 8 drops (0.5 mL) of protein free urine.
Boil the mixture for 5 min.
Allow to cool ( under running tapwater)
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25. • The ratio of 5 mL Benedict’s reagent and 8
drops (0.5 mL) of urine ratio is important
because it is a Semiquantitative test.
• Interpretation : The change of color from blue
to green, yellow orange / red depends on the
amount of sugar present.
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30. KETONE BODIES
• Ketone bodies are three water soluble
molecules ( Acetoacetic acid , beta-hydroxy
butyric acid and acetone) that are produced
by the liver from fatty acids during low food
intake , carbohydrate restrictive diets ,
starvation.
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31. Tests for Ketone bodies
• 1. Rothera’s test
• 2.ferric chloride test
• 3.Hart’s test
• 4.Dipstick method
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32. ROTHERA’S TEST
• Principle :
– Acetoacetic acid and acetone react with sodium
nitroprusside in presence of an alkali to form a
purple color compound
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33. PROCEDURE
Take 4 mL of urine in a test tube
Add a few crystals of sodium nitroprusside
saturate the urine with ammonium sulphate
By mixing vigorously
Overlay with few drops of liquor ammonia along
the wall of tube
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34. • INTERPRETATION
• Development of purple ring indicates the
presence of Acetoacetic acid / acetone or
both.
• A brown or red colour is of no significance.
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38. BILIRUBIN (Bile Pigment)
• Tests for Bilirubin in urine provides
information concerning metabolic or systemic
disorders, especially liver function.
• Bilirubin is a breakdown product of
hemoglobin and is normally is not present in
urine.
• Bilirubinuria causes yellow-brown to greenish-
brown urine and forms yellow foam on
shaking.
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39. Tests for Bilirubin
• A. Fouchet’s test
• B. dipstick method
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40. Fouchet’s test
• Principle :
• Fouchet’s reagent contains trichloroacetic acid
and ferric chloride. In acetic medium ferric
chloride oxidizes Bilirubin to produce a dark
green colored bileverdin.
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41. • Requirements :
• 1.Test tubes
• 2.pasteur pipettes
• 3.What man No.1 filter paper
• 4. 10% Barium chloride
• 5.Fouchets reagent
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42. PROCEDURE
Take 10mL of urine in a test tube
add 3 mL of 10% barium chloride solution
Mix the two and filter the mixture through filter paper
(Bilirubin along with barium salt remains on filter paper)
Add a few drops of Fouchet’s reagent onto the filter
paper
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43. Interpretation
Green or blue color
Indicates Bilirubinuria
Precaution : test to be done on fresh urine
sample
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44. Causes of Bilirubinuria
• Obstructive Jaundice : Urine shows Bilirubin
with out urobilinogen
• Hepatocellular jaundice.
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45. UROBILINOGEN
• Urobilinogen is a colorless by-product of
bilirubin reduction.
• It is formed in the intestines by bacterial
action on bilirubin.
• Urobilinogen is normally present in urine in
trace amount (1-12 mg/dl) and is insufficient
to cause a significant positive reaction.
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46. UROBILINOGEN
• Whenever the liver is unable to efficiently
remove the reabsorbed urobilinogen from the
portal circulation ( e.g. Liver disease,
hemolytic anaemia )
• more urobilinogen than normal is routed
through the kidney and hence excreted in the
urine.
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48. Ehrlich’s Test
• Principle :
• Ehrlich’s reagent reacts with urobilinogen and
forms a pink colored aldehyde complex.
• Reagent : Paradimethyl amino benzaldehyde
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50. PROCEDURE
Take 2.5 mL urine in a test tube
Add 2.5 mL of Ehrlich’s aldehyde reagent
Mix well by Inversion
Add 10 mL of saturated sodium acetate solution and
mix
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51. Interpretation
• Pink to cherry red color indicates presence of
urobilinogen
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52. Causes of increased urobilinogen in
urine
• Hemolytic anaemias
• A. Thalassaemia
• B.Sickle cell anaemia
• C. hereditary spherocytosis
• Liver Diseases
• A. Drug or toxic hepatitis
• B.cirrhosis
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53. Causes of decreased / absent
urobilinogen in urine
• In obstructive Jaundice.
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54. Bile salts
• Bile salts are composed of mixture of bile
acids and glycine or taurine.
• Two important bile salts are sodium and
potassium salts of glycocholates.
• Normally bile salts are not present in urine
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55. • Requirements :
• 1.Test tubes 10 mL
• 2.Suphur powder
• 3.Pasteur pipette
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56. Tests for bile salts
• Hay’s Sulfur test :
• Principle : Bile salts have unusual property of
lowering the surface tension of urine markedly
even when present in small concentrations.
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57. Procedure
Take 10 mL urine in a wide bore test tube or small
beaker
Sprinkle sulfur powder over its surface, watch for 5
min
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58. Interpretation
• Sulfur powder sinks to the bottom of test tube
in the presence for bile salts in urine
• Causes : Hepatocellular and obstructive
Jaundice
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59. Tests for Blood in Urine
• These tests detects
• hematuria,
• hemoglobinuria or
• myoglobinuria
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60. • Hematuria : presence of red cells in urine E.g.
Renal stones
• Hemoglobinuria : Presence of free
hemoglobin in urine E.g. Intravascular
hemolysis
• Myoglobinuria : presence of myoglobin in
urine . E.g. Crush injury to muscle.
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61. Tests for blood
• A. Benzidine test
• B. Orthotoludine test
• C. Dipstick method
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62. Benzidine test
• Principle :
• The test depends upon the ability of heme
compounds derived from hemoglobin to
catalyze the oxidation of bezidine by hydrogen
peroxide.
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64. Procedure
Dissolve a small amount of Benzidine in 2 mL of
glacial acetic acid and equal volume of 3 %
hydrogen peroxide
From the above, take 2 mL in another test tube and
add 2 mL of previously boiled and cooled urine
and mix
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66. Causes of hematuria
• 1.Renal causes
a. Acute glomerulonephritis
b. Bacterial hypertension
c. Malignant hypertension
2. Non glomerular causes
a. polycystic kidney
b. Renal cell carcinoma
c. Renal stones
3. Blood Disorders
a. Bleeding disorders
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