Urinalysis- Methods, observations and clinical significance

Urine examination- physical and chemical examination, normal and abnormal urine, methods, observation and interpretations

1. Urinalysis - Methods, Observations
and Interpretations
(Macroscopic Analysis)
By- Professor(Dr.) Namrata Chhabra
Biochemistry for medics- Lecture Notes

2. Introduction
•Urine is an excretory
product of the body
•It is formed in the
kidney
•Urine examination
helps in the diagnosis
of various renal as well
as systemic diseases
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3. Specimen collection
•For most of the routine
investigations fresh mid
stream specimen of 10-20 ml
urine is collected in a clean
dry vial
•In some cases 24 hour urine
sample is also collected
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4. Sample preservation
•There is no single all purpose preservative
•For determination of urea, ammonia, nitrogen and
calcium- Hydrochloric acid is used (2 N or concentrated
HCL)
•For determination of sodium, potassium, chloride,
bicarbonate, calcium, phosphorus, urea, ammonia,
amino acids, creatinine, proteins, reducing substances
and ketone bodies- Thymol is used
•For determination of Ascorbic acid- Acetic acid is used
•Toluene is a very satisfactory preservative for urine
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5. Composition of normal urine
Composition depends
on Kidney functions
• Glomerular filtration
• Tubular reabsorption
and
•Tubular secretion
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6. Composition of normal urine
•Normal urine contains 90-
95 % water and about 60
G/day of solid
constituents which may
be organic or inorganic in
nature
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7. Organic constituents of urine
S.No. Constituent Concentration
(G/day)
1. Urea 25-30
2. Uric acid 0.5-0.8
3. Creatinine 1-1.8
4. Hippuric acid0.7-0.8
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8. Inorganic constituents of urine
S.No. Constituent Concentration
(G/day)
1 Chlorides 10-15
2 Sodium 3-5
3 Potassium 2-2.5
4 Calcium 0.1-0.3
5 Phosphates 0.8-1.3
6 Sulphates 1.0-1.2
7 Ammonia 0.7-0.8
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9. Physical Examination of urine
I) Colour- Freshly excreted urine
is colorless to straw colored
•The normal color of urine is
due to the presence of
pigment urochrome
•Trances of other substances,
such as- Uroerythrin, urobilin,
uroporphyrin and
coproporphyrins also
contribute to the color of urine
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10. Variations in urinary color
S.No.Color Interpretation
1 Dark
yellow
• Concentrated
urine- Mild
dehydration
• Vitamin B complex
therapy
2 Orange Drug induced
3 PinkishExcessive beet root
intake
A) Physiological Variations
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11. Variations in urinary color
B) Pathological Variations
S.No. Color Interpretation
1 Deep yellow Jaundice
2 Reddish Haematuria
3 Brownish Hemoglobinuria, myoglobinuria and
porphyrias
4 Brown to blackAlkaptonuria
5 Cloudy Pus cells and bacteria in infected cells
6 Smoky Red blood cells
7 Black Iron therapy
8 Pinkish brownPresence of urobilin – Hemolytic anemias
9 Milky white Chyluria(Presence of fat globules)
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12. Variations in urinary color
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13. Physical Examination of urine
II) Volume of urine- Normal volume-800-2,500 ml/day
with an average of 1500 ml/day.
Approximately 500 ml/day is the minimum volume of
urine needed in normal health to remove waste products
The volume of urine is affected by-
•Fluid intake
•Fluid loss
•Type of diet
•Cardio-vascular status and
•Renal functions
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14. Variations in volume of urine excreted
A) Polyuria- Polyuria implies an increased volume
of urine excreted per day, generally volume of urine
exceeding 2,500 ml/day is termed as Polyuria.
Conditions causing Polyuria
• Diabetes mellitus
• Diabetes Insipidus
• Late stage of chronic glomerulonephritis
•Drug induced- Diuretics
• Alcohol
• Compulsive polydipsia
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15. Variations in volume of urine excreted
B) Oliguria- Volume of urine less than 500 ml/day is
termed oliguria.
Conditions causing oliguria
•Fever
•Diarrhea (loss of fluid from extra renal sites)
•Severe edema
•Acute nephritis
•Early stage of acute glomerulonephritis
•Cardiac failure and hypertension (reduced
circulatory volume)
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16. Variations in volume of urine excreted
C) Anuria- Complete cessation of urine or volume
of excreted urine less than 100 ml/day.
Conditions causing Anuria
•Acute tubular necrosis
•Blood transfusion reaction
•Surgical shock
•Bilateral renal stones
•Sulphonamide therapy
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17. Physical Examination of urine
III) Specific Gravity- The specific gravity
indicates the concentrating ability of the
kidneys.
In normal health the urinary specific gravity
ranges between 1.016-1.025, the average being
1.020. The specific gravity is affected by-
• Volume of urine excreted and
• The amount of solids present in the urine
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18. Variations in specific gravity of urine
A) Low specific gravity- 1.016 or less
Conditions
•Compulsive polydipsia
•Diabetes Insipidus
•Glomerulonephritis
•Pyelonephritis
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19. Variations in specific gravity of urine
B) High specific gravity of urine- 1.025 or more
Conditions causing high specific gravity of urine
•Severe dehydration
•Nephrotic syndrome (Due to proteinuria)
•Diabetes mellitus ( Due to glycosuria)
•Adrenal insufficiency(Excess of sodium in urine)
•Congestive heart failure
•Hepatic diseases
•Extra renal water losses (fever, vomiting and
diarrhea)
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20. Variations in specific gravity of urine
Fixed specific gravity (Isosthenuria)
• Fixed specific gravity is seen in chronic renal
failure.
• Specific gravity of urine is based on tubular
function
• In the late stages of chronic renal failure,
kidneys fail to concentrate or dilute urine,
which has a constant specific gravity ranging
between 1.008-1.012(average 1.010) same as
that of plasma.
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21. Measurement of specific gravity
•The specific gravity is measured by Urinometer
•The instrument floats in the urine
•The calibration mark that corresponds to the surface
level of urine is read
•It is calibrated at 15
o
C, temperature correction is
done by adding 0.001 for every 3 degree above 15
o
C
or subtracting 0.001 for every 3 degree below15
o
C .
•Proteinuria increases specific gravity, 0.003 is
subtracted for every G/L of urinary protein
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22. Measurement of specific gravity
Urinometer
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23. Physical Examination of urine
IV- Odor of urine
•Normal urine has an aromatic odor
•Variations
•Ammoniacal Odor- On keeping sample for a
long time
•Acetone like Odor- Ketonuria such as Diabetic
ketoacidosis or starvation
•Foul smell due to bacterial infections
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24. Physical Examination of urine
V) pH
Normal urine is acidic, pH ranges between 4.5-8.0 with
a mean of 6.0 in 24 hours
Variations of urinary pH
A)Acidic urine-
Physiologically, It is found after
•A protein rich diet
•Heavy exercise
Pathologically , It is found in conditions of acidosis,
such as diabetic ketoacidosis, respiratory acidosis,
and high fever (break down of tissue proteins)
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25. Physical Examination of urine
Variations of urinary pH
B) Alkaline pH
Physiologically it is found after
•Heavy meals
•Diet rich in citrus fruits
•Excessive intake of milk and antacids
Pathologically, it is found in-
•Urinary tract infections
•Conditions of alkalosis
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26. Measurement of urinary pH
Urinary pH is measured
by-
• pH papers
•Litmus papers
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27. Analysis of normal constituents
A)Inorganic constituents
i) Test for Urinary chlorides(silver nitrate test)
Principle- Silver chloride is precipitated in the
presence of nitric acid and silver nitrate.
Procedure-Take 2 ml of urine and add 0.5 ml of
concentrated nitric acid and 1 ml of silver
nitrate. A white precipitate of silver chloride
appears.
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28. i) Test for urinary chlorides
Interpretation:
a) Increased Urinary chlorides:
Polydipsia, use of diuretics and
Addison's disease.
b) Decreased urinary chlorides:
Excessive sweating, fasting,
diarrhea, excessive vomiting,
edema, diabetes Insipidus,
infections and adrenocortical
hyper function (Cushing's
syndrome).
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29. ii) Test for urinary sulphates
Barium chloride test (test for
sulphates)
Principle: Urinary sulphate is
precipitated as barium
sulphate on reaction with
barium chloride solution.
Procedure: Take 3 ml of urine
and add 1 ml of conc. HCl and
2 ml of 10% barium chloride.
White precipitate indicates
the presence of sulphates.
08/16/14 29Biochemistry for medics- Lecture Notes

30. ii) Test for urinary sulphates
Interpretation
a) Increased urinary sulphate: Cystinuria,
Homocystinuria, melanuria, obstructive jaundice,
hepatocellular jaundice, cyanide poisoning and
high protein diet .
b) Decreases urinary sulphates are observed in
conditions of renal functional impairment.
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31. iii) Test for Urinary phosphates
Ammonium molybdate test (Test for
Urinary phosphates)
Principle: Upon warming with
ammonium molybdate in the presence
of nitric acid, inorganic phosphates are
precipitated as canary yellow
ammonium phospho molybdate.
Procedure: To 3 ml of urine, add a few
drops of concentrated nitric acid and a
pinch of ammonium molybdate. Warm
it. Observe the yellow color of the
precipitate.

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32. iii) Test for Urinary phosphates
Interpretation:
a) Increased urinary phosphates: Rickets,
osteomalacia, hyperparathyroidism, acidosis.
b) Decreased urinary phosphates: Diarrhea,
nephritis, parathyroid hypofunction,
pregnancy, hereditary fructose intolerance
and galactosemia.
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33. IV) Test for Urinary Calcium
Potassium oxalate test (Test for urinary calcium)
Principle: With potassium oxalate in acidic medium,
calcium is precipitated as calcium oxalate.
Procedure: To 2 ml of urine, add 5 drops of 1%
acetic acid and 5 ml of potassium oxalate. White
precipitate of calcium oxalate is formed.
08/16/14 33Biochemistry for medics- Lecture Notes

34. IV) Test for Urinary Calcium
Interpretation:
Increased urinary calcium:
• Hyperparathyroidism,
• Hyperthyroidism,
• Hypervitaminosis D,
• Multiple myeloma,
• Renal stones
• Renal tubular acidosis,
• Steroids and diuretic therapy
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35. V) Test for Ammonia
Principle: Ammonia is evaporated when urine is
made alkaline. Urinary ammonia is derived from
glutamine and other amino acids.
Procedure: To 5 ml of urine add; add 2% sodium
carbonate till the solution is alkaline to litmus. Boil
the solution. Place a piece of moistened red litmus
paper at the mouth of the test tube. Note the
change in color to blue due to evolution of
ammonia.
08/16/14 35Biochemistry for medics- Lecture Notes

36. V) Test for Ammonia
Interpretation:
a) Increased urinary ammonia:
Diabetic keto acidosis, ingestion of
acid forming foods, excessive
water ingestion, urinary tract
infections.
b) Decreased urinary ammonia:
Alkalosis, nephritis.
Damp red litmus paper turns blue
on exposure to fumes of ammonia.
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37. B) Tests for Organic Constituents
1)Test for Urea
(Sodium hypobromite test)
Principle: When urea is treated
with sodium hypobromite, it
decomposes to give
nitrogen.
Procedure: To 2 ml of urine in a
test tube, add 4-5 drops of
sodium hypobromite.
Observe the effervescence
of nitrogen gas.
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38. B) Tests for Organic Constituents
Urease test for urea
Principle: Soyabean powder contains the
enzyme urease. This enzyme under pH 7-8 and
temperature 55
0
C decomposes urea in to
ammonia and carbon dioxide which together
form ammonium carbonate (alkaline
component) which changes the slightly acidic
reaction(yellow color) to alkaline
reaction(pink color).
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39. B) Tests for Organic Constituents
Biuret test for urea
Principle : Urea when heated decomposes with
the liberation of ammonia and the formation
of biuret. Biuret is dissolved in water and
develops a violet color forming a complex
with alkaline copper sulphate solution.
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40. i) Tests for Urea
Interpretation:
a) Increased urinary urea: Fever, diabetes
mellitus, excess of adrenocortical activity
b) Decreased urinary urea: Liver diseases,
metabolic or respiratory acidosis, nephritis
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41. ii) Tests for Creatinine
a) Jaffe’s Reaction (Test for creatinine)
Principle: Creatinine reacts with picric
acid in the alkaline medium to form a
reddish colored complex of creatinine
picrate
Procedure: Take 5 ml of urine and add an
aqueous solution of picric acid. Make
the mixture alkaline with NaOH
solution. A red color is produced.
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42. ii) Tests for Creatinine
b) Nitroprusside test
To 5 ml of urine add a few drops of sodium
nitroprusside and make the solution alkaline
with sodium hydroxide (NaOH).
A ruby red color is formed that turns yellow.
This test is also called Wey’s test.
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43. ii) Tests for Creatinine
c) Nitroprusside -Acetic acid test (Salkowaski
test)
Procedure : Take 5 ml of urine, add a few drops
of sodium nitroprusside and then make the
solution alkaline with NaOH. A ruby red color
is formed that turns yellow.
To the yellow precipitate, add an excess of
acetic acid and heat the solution.
A green color is obtained that turns blue upon
standing.
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44. ii) Test for Creatinine
Interpretation:
a) Creatinuria- Creatinuria occurs in uncontrolled
diabetes mellitus, thyrotoxicosis, myasthenia gravis,
starvation, infancy, pregnancy, muscular disorders
and in growing period.
b) Increased urinary creatinine: Muscular disorders
c) Decreased urinary creatinine: Renal failure
08/16/14 44Biochemistry for medics- Lecture Notes

45. iii) Tests for Uric acid
a) Phospho tungstic acid test (For uric
acid)
Principle: Uric acid is a reducing agent in
alkaline medium. It reduces phospho
tungstic acid to tungsten blue.
Procedure: Take 2 ml of urine, add a few
drops of phospho tungstic acid
reagent followed by a few drops 20%
sodium carbonate. Observe the
appearance of blue color
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46. iii)Tests for Uric acid
b) Benedict’s test
Principle- Uric acid is soluble in alkali. The blue
color is developed due to the reduction of
phospho tungstic acid by uric acid.
Procedure : To 2 ml of urine , add a few drops of
Benedict’s uric acid reagent and add a pinch
of anhydrous sodium carbonate and mix. A
deep blue color indicates the presence of uric
acid.
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47. iii) Tests for Uric acid
Interpretation:
a) Increased urinary uric acid: Cancers,
leukemia, administration of ACTH,
Wilson's disease.
b) Decreased urinary uric acid: Purine free
diet, gout.
08/16/14 47Biochemistry for medics- Lecture Notes

48. Tests for abnormal constituents of
urine
Abnormal urine
Substances which are not present in easily
detectable amounts in urine of normal healthy
individuals but are present in urine under
certain diseased condition are said to be
“Abnormal” or “pathological” constituents of
urine.
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49. Abnormal constituents of urine
•Reducing sugars
•Ketone bodies
•Proteins
•Blood
•Bile salt
•Bile pigments and
•Urobilinogen
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50. Tests for abnormal constituents of
urine
•These constituents are present in normal
health but in very small amount and are not
detected by less sensitive laboratory methods.
•Under certain pathological conditions their
concentration is increased and these are get
detected . The urine is said to be ‘Abnormal ’,
under such conditions.
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51. Tests for reducing sugars
1) Benedict’s Test
Principle :Benedict's reagent contains sodium
carbonate, copper sulphate and sodium citrate.
In the alkaline medium provided by Sodium
carbonate, the copper remains as cupric
hydroxide. Sodium citrate acts as a stabilizing
agent to prevent precipitation of cupric
hydroxide. In alkaline medium, sugars form
enediols, cupric ions are reduced, and
corresponding sugar is oxidized to sugar acid.
08/16/14 51Biochemistry for medics- Lecture Notes

52. Tests for reducing sugars
Benedict’s test
Procedure:
Take 5 ml of Benedict's reagent,
add 8 drops of urine. Boil for 2
minutes or keep it in the boiling
water bath for 5 minutes. A
light green, yellow or brick red
color is produced depending on
concentration of urinary
glucose.
Negative
test
Positive
test
08/16/14 52Biochemistry for medics- Lecture Notes

53. Tests for reducing sugars
Observations
Benedict‘s test is a semi quantitative test. The
color of the precipitate gives a rough estimate of
the reducing sugars present in the given sample.
Green color - Up to 0.5 g%
Green precipitate - 0.5-1.0 g%(+)
Yellow precipitate -1.0-1.5 g% (++)
Orange precipitate- 1.5-2.0 g% (+++)
Brick red precipitate- >2.0 g% (++++)
08/16/14 53Biochemistry for medics- Lecture Notes

54. Tests for reducing sugars
2) Fehling Test
•Another reduction test
•Contains KOH and Sodium potassium Tartrate
in place of Sodium carbonate and sodium
citrate in Benedict’s reagent
•Not used any more, since it is less sensitive,
less specific and the strong alkali causes
caramelisation of the sugars present in the
given sample.
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55. Tests for reducing sugars
Interpretation:
•Positive Benedict's test signifies Glycosuria.
•Glycosuria is a non-specific term. Any reducing
sugar found in urine is denoted by glycosuria
•Lactosuria - in lactose intolerance
•Galactosuria - in galactosemia
•Fructosuria - in hereditary fructose
intolerance
•Pentosuria - in essential Pentosuria
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56. Glycosuria
Causes of Glucosuria are: (Glucosuria and Glycosuria
are used synonymously)
a. Renal glycosuria- pregnancy, hereditary, diseases of
renal tubules, heavy metal poisoning .
b. Diabetes mellitus
c. Alimentary glucosuria
d. Hyperthyroidism, hyperpituitarism and
hyperadrenalism
e. Stress, severe infections, increased intracranial
pressure
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57. Glycosuria
Examples of non-carbohydrate substances
which give a positive Benedict's reaction are:
a) Creatinine
b) Ascorbic acid
c) Glucuronates
d) Drugs: Salicylates, PAS and Isoniazid
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58. Tests for proteins
1)Heat Coagulation test
Principle: Albumin is coagulated when heated,
which is precipitated at the iso-electric point,
when acetic acid is added.
Procedure : Fill 3/4th of the test tube with
acidified urine (add few drops of dilute acetic
acid) and heat the top half of it. Lower part
serves as a control. Note the appearance of
turbidity.
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59. Tests for proteins
Heat Coagulation test- Acidification is
necessary because-
1) In alkaline medium heating may
precipitate carbonates and
phosphates.
2)False negative results may be
obtained since the proteins are
coagulated by heat at a pH near
Isoelectric p H.
3)In alkaline medium proteins may
not be precipitated owing to the
formation of alkaline meta
proteins that are not precipitated
upon heating.
The heated upper half shows turbidity
due to the precipitation of proteins, the
lower half serves as a control for
comparison.
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60. Bence Jone’s proteins
•Bence Jone’s proteins are light chain
immunoglobulins
•Excreted in urine of a patient suffering from
multiple myeloma
•These proteins precipitate between 40-60 degree
centigrade
•Upon further heating, turbidity disappears to
reappear on cooling
•These proteins redissolve on boiling unlike
albumin
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61. Tests for proteins
2) Sulphosalicylic acid test
Principle: Negatively charged sulpho salicylic acid
neutralizes the positive charge on proteins
causing denaturation, and hence precipitation of
proteins.
Procedure: To 1 ml of urine add 3 drops of 20%
Sulphosalicylic acid. A turbidity or precipitate
indicates the presence of proteins.
Absence of cloudiness means absence of proteins.
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62. Test for proteins
S.No. Observation Inference
(Approximate
protein
concentration)
mg/100 ml
1) Barely visible turbidity5
2) Distinct turbidity10-30
3) Moderate turbidity40-100
4) Heavy Turbidity 200-500
5) Heavy
flocculent/precipitation
500
08/16/14 62Biochemistry for medics- Lecture Notes

63. Test for proteins
3) Heller’s Nitric acid ring test
Principle: Concentrated HNO
3
causes denaturation
and hence precipitation of proteins.
Procedure: Take 3-5 ml of concentrated nitric acid.
Incline the tube and to it add carefully, 2-3 ml of
urine, so that it forms the upper layer without
disturbing the lower HNO
3
layer. In a positive
reaction, a white zone of precipitate protein will
appear at the junction of two liquids.
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64. Tests for proteins
Interpretation- Insignificant amounts of
proteins are excreted in urine in normal health
not exceeding 20-80 mg/dl. This small amount is
not detectable by routine methods.
Under certain conditions, as much as 20 G or
more proteins may be excreted per day in urine.
The most common type of proteinuria is
albuminuria; hence proteinuria and albuminuria
are used synonymously.
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65. Proteinuria
When proteins appear in urine in detectable
amounts, it is called proteinuria. It can be
caused by-
a) Increased glomerular permeability
b) Reduced tubular reabsorption
c) Increased secretion of proteins
d) Increased concentration of low molecular
weight proteins in the plasma
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66. Proteinuria
Proteinuria may be- Physiological or
Pathological
I)Physiological Proteinuria
Causes include-
•Violent exercise
•Pregnancy
•Postural
•Alimentary
•Exposure to cold
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67. Proteinuria
II) Pathological proteinuria
I. Pre Renal:
•Severe dehydration
•Heart diseases
•Ascites (due to increased intra-abdominal pressure)
•Severe anemia, and
•Fever
•Collagen diseases
•Toxemia of pregnancy
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68. Proteinuria
II. Renal: All inflammatory, degenerative or
destructive diseases of kidney; the most
common ones are:
•Nephrotic syndrome,
•Pyelonephritis
•Acute and Chronic glomerulonephritis
•Nephrosclerosis
•Tuberculosis of kidney
•Renal failure.
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69. Proteinuria
III. Post Renal – Also called false proteinuria because in
these conditions proteins do not pass through the
kidneys.
Causes include-
•Severe urinary tract infections
•Inflammatory, degenerative or traumatic lesions of
pelvis, ureters, bladder, prostate or urethra
•Bleeding genito urinary tract
•Pus in urine
•Contamination of urine by semen or vaginal secretions
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70. Tests for Ketone bodies
Rothera’s Test
Principle: Nitroprusside in
alkaline medium reacts with
a ketone group to form a
purple ring. It is given by
acetone and acetoacetate,
but not by Beta hydroxy
butyric acid.
08/16/14 70Biochemistry for medics- Lecture Notes

71. Tests for Ketone bodies
Procedure:
• Saturate 5 ml of urine with solid ammonium
sulphate and add 0.5 ml of freshly prepared
sodium nitroprusside (5%).
• Mix well and add liquor ammonia from the
side of tube.
• A purple ring at the junction of the liquid
indicates the presence of ketone bodies.
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72. Tests for Ketone bodies
2) Gerhardt’s ferric chloride test
Principle: A purplish color is given by aceto
acetate. On boiling acetoacetate is converted
to acetone and does not give this test positive.
This test is only given by acetoacetate and not
by beta hydroxy butyric acid directly.
08/16/14 72Biochemistry for medics- Lecture Notes

73. Tests for Ketone bodies
2) Gerhardt’s ferric chloride test
Procedure- Add 10% ferric chloride solution
drop by drop to 5 ml of urine in a test tube. If
phosphates are present, precipitates of ferric
phosphates may form, that should be filtered
off and the ferric chloride is added.
False positive Gerhardt’s test may be obtained
with Salicylic acid and Salicylates.
08/16/14 73Biochemistry for medics- Lecture Notes

74. Tests for Ketone bodies
3) Test for β- OH butyric acid
•No direct test for β- OH butyric acid
•Indirect test is performed
Procedure : Add a few drops of Acetic acid to
urine diluted 1:1 with distilled water. Boil for
a few minutes to remove acetone and aceto
acetic acid. Add about 1.0 ml of H
2
O
2
, warm
gently, cool, and perform Rothera’s test .
08/16/14 74Biochemistry for medics- Lecture Notes

75. Tests for Ketone bodies
Acetone, acetoacetate and beta hydroxy butyrate are
the ketone bodies. Ketonemia and hence ketonuria
occurs mostly in conditions of glucose deprivation.
Causes of Ketonuria:
1) Uncontrolled diabetes mellitus
2) Starvation
3) High fat feeding
4) Heavy exercise
5) Toxemia of pregnancy
08/16/14 75Biochemistry for medics- Lecture Notes

76. Tests for bile pigments
1)Fouchet’s test
Principle: BaCl
2
reacts with sulphate in urine to
form barium sulphate. If bilirubin is present
in urine, it adheres to precipitate and is
detected by oxidation to form biliverdin
(Green) with FeCl3 in the presence of
trichloro acetic acid. Nitric acid oxidizes
bilirubin to biliverdin giving different colors
from green to violet.
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77. Tests for bile pigments
1) Fouchet’s test
Procedure:
Take 5 ml of 10% BaCl2 to 10 ml of
urine and filter. Dry the filter
paper and add a few drops of
Fouchet's reagent (Prepared by
adding 10 mg of 10% FeCl3 to
100 ml of 25% TCA). A green
color is obtained due to oxidation
of bilirubin to biliverdin.
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78. Tests for bile pigments
2) Gmelin’s test
Principle: Nitric acid oxidizes Bilirubin to
Biliverdin giving different colors from green to
violet.
Procedure: To about 5 ml of concentrated HNO
3
in a test tube, add an equal volume of urine
carefully so that the two liquids do not mix. At
the junction of two liquids various colored rings
(Green, blue, red, violet etc.) will be formed.
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79. Tests for bile pigments
3) Iodine test
Procedure : Dilute some tincture of iodine with
one to two volumes of water and layer it
carefully on to some urine in a test tube, a
green ring at the junction of two fluids
indicates the presence of Bilirubin.
It is not a sensitive test, can not detect small
amount of bilirubin present in the given
sample.
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80. Tests for bile pigments
Interpretation
Bilirubin in urine means increased amount of
conjugated bilirubin because unconjugated bilirubin
is water insoluble and is also bound to albumin,
hence cannot cross the glomerular membrane.
Causes of bilirubinuria are:
1) Moderate to severe hepatocellular damage
2) Obstruction of bile duct- Intra or extra hepatic
In prehepatic jaundice, bilirubin is absent in urine.
08/16/14 80Biochemistry for medics- Lecture Notes

81. Test for Bile salts
Hay’s Sulphur test
Principle: Bile salts lower the surface tension
allowing the sulphur powder to sink
Procedure: Sprinkle a little dry sulphur powder
on the surface of fresh urine in a test tube
taking distilled water as control. Sulphur
powder sinks in the presence of bile salts.
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82. Test for Bile salts
Control for
comparison
Positive test
In the control, sulphur
powder remains immiscible
with the underlying liquid.
In the positive test, the
sulphur powder sinks to
the bottom.
Interpretation: Bile salts
and bile pigments are
present in urine in
obstructive jaundice.
08/16/14 82Biochemistry for medics- Lecture Notes

83. Test for Urobilinogen
Ehrlich’s test
Principle: The test for urobilinogen is based on
the Ehrlich Aldehyde Reaction.
P-dimethylaminobenzaldehyde in an acid
medium with a color enhancer reacts with
urobilinogen to form a pink-red color. The
optimum temperature for testing is 22° - 26°C.
A freshly voided sample is best for optimal
results.
08/16/14 83Biochemistry for medics- Lecture Notes

84. Test for Urobilinogen
Ehrlich’s test
Procedure:
Take 5 ml of fresh urine in a test tube and add 5
ml of Ehrlich's reagent to it. Wait for 10
minutes and add 10 ml of saturated sodium
acetate solution. A pinkish color indicates the
presence of urobilinogen. Porphobilinogen is
also detected by Ehrlich's test. The color
intensifies upon addition of sodium acetate if
Porphobilinogen is there.

08/16/14 84Biochemistry for medics- Lecture Notes

85. Test for Urobilinogen
Interpretation:
Urobilinogen is found in urine in hepatic and
prehepatic jaundice. It is present in excessive
amount in prehepatic jaundice and is
completely absent in post hepatic jaundice.
An increased urobilinogen concentration in
urine is a sensitive index of liver dysfunction
or hemolytic disorders.
08/16/14 85Biochemistry for medics- Lecture Notes

86. Test for blood
Benzidine Test
Principle: Hydrogen peroxide liberated from Hb
oxidizes Benzidine to form a colored derivative.
Procedure:
To 3 ml of saturated Benzidine solution in glacial
acetic acid, add 2 ml of urine and add 1 ml of 3%
H2O2. A blue or green color develops within 10
minutes indicating the presence of blood. Color
developing after 10 minutes is not a positive test
but it is due to oxidation of Benzidine by
atmospheric oxygen.
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87. Test for blood
Interpretation:
Presence of blood in urine is called hematuria.
a. Gross hematuria:
Urine appears reddish in gross hematuria and
this is observed in renal stones, malignancies,
trauma, tuberculosis and acute
glomerulonephritis.
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88. Test for blood
b. Microscopic hematuria: Blood is not visible to
naked eyes. It is observed in:
• Malignant hypertension,
•Sickle cell anemia,
•Coagulation disorders,
•Polycystic kidney disease,
•Incompatible blood transfusion,
•Auto immune hemolytic anemia.
08/16/14 88Biochemistry for medics- Lecture Notes

89. Urine test strips
•10 different
substances in urine
can be detected.
•Easy, quick and
bed side procedure
08/16/14 89Biochemistry for medics- Lecture Notes

90. Further reading
1)
http://www.lulu.com/shop/namrata-chhabra-and-kavish-ramessur/clinical-approach-to-practical-biochemistry/ebook/product-21016914.html
2)
http://www.namrata.co/category/practical-biochemistry/normal-urine-analysis/
3)
http://www.namrata.co/category/practical-biochemistry/abnormal-urine/
08/16/14 Biochemistry for medics- Lecture Notes 90