CHEMICAL EXAMINATION OF URINE
Dr. K.Indumathi. MD,DCP, DNB (Path)
Assistant Professor of Pathology
Madras Medical College
Chennai.
The routine urinalysis includes chemical testing for
(1) Protein
(2) Glucose
(3) Ketone bodies
(4) Occult blood
(5) Bile pigments
(6) Bile salts and
(7) Urobilinogen.
PRESERVATIVES
• Urine should be examined within 2 hrs.
• If there is delay specimen should be refrigerated.
• For 24 hrs urine sample (or) transport - one of
the following preservative should be added- to
prevent decomposition and growth of
contaminating organisms.
Toluene, Boric acid, Concentrated HCL,
Formalin, Thymol
For urobilinogn- 2.5ml of sodium carbonate placed.
Requirements:
Glassware :
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Centrifuge tubes,
Pasteur pipettes,
test tubes (10x75, 15x125 and 20x150mm),
Beakers (250 or 500 ml),
Graduated pipettes (5 ml) and
Test tube racks
Reagents and chemicals required for chemical examination of urine.
Benedict’s qualitative reagent
Sulfosalicylic acid, Glacial acetic acid
Rotheras test
Fouchet’s reagent
Ehrlich reagent
Sulfur powder
: Sugar Determination
: Protein Determination
: Ketone estimation
: Bile pigment Determination
: Determination of urobilinogen
: Bile salts detection
DETERMINATION OF PROTEIN
General consideration:
In the normal kidney only a small amount of low molecular weight
protein is filtered at the glomerulus.
Glomerular membrane prevents the passage of high molecular weight
proteins such as albumin (mol. wt.69000) and gamma globulin (mol.
wt. 180000).
After filtration most of the protein is reabsorbed in the tubules.
About <150 mg protein / 24 hrs is generally excreted which is not
detected by chemical methods.
‘Tamm-Horsfallprotein’ - This protein is normally excreted in urine.
There are two main mechanisms by which proteinuria can occur
a) glomerular damage or
b) defect in the reabsorption process of the tubules.
In glomerular damage the capillary walls become more permeable.
• Glomerular proteinuria : Glomerulonephritis, hypertension
and lipoid nephrosis.
• Tubular proteinuria: a) pyelonephritis b) renal tubular
acidosis c) cystinosis d) interstitial nephritis and e) rejection of
kidney allografts.
• Pre-renal conditions which produce it because of secondary
effects on the kidneys. The proteinuria generally disappears
when the primary disease is cured. Examples are dehydration,
intestinal obnstruction, myocardial infarction, intra-abdominal
tumors (since there is intra-abdominal pressure on ascites)
and in fevers.
• Post-renal conditions, proteins may be added in urine as it
passes along the urinary tract. Lesions of the renal pelvis,
bladder, prostate and urethra can all lead to such a condition.
HEAT AND ACETIC ACID TEST
(PROTEIN)
Principle of tests
All the methods are based on the principle of
precipitation of protein by chemical agents (acids) or
coagulation by heat.
Note :
•The Urine should be clear. If it is turbid, it is
necessary, to centrifuge it. In that case supernatant
is tested for protein and sediment for the
microscopic examination. Even after centrifugation
if the urine is turbid then filter it.
•If urine is alkaline, add few drops of glacial acetic
acid and make it slightly acidic.
Reagents:
• 3 g/dl sulfosalicylic acid
2) Glacial acetic acid
Procedure (A):
• Transfer 3 to 4 ml of centrifuged (or filtered) urine to a
small test tube (10x75 mm).
• Add 2 to 3 drops of sulfosalicylic acid on the top of the
specimen.
• Observe for turbidity after 5 minutes.
Observations:
• No formation of turbidity at the upper portion of urine :
protein absent.
• Formation of turbidity : Protein present
• If proteins are present grade the result according to degree
of turbidity as trace, +, ++, +++ and ++++.
Procedure (B): Heat test: Place 5 to 10 ml of clear urine in a
test tube (20x125mm). Boil the upper portion over a flame.
If turbidity develops, add 1 to 2 drops of glacial acetic acid. If
the turbidity is due to phosphate precipitation, it will clear.
Reboil the specimen.
Observation after reboiling the specimen
No turbidity : Proteins absent
Presence of turbidity : Proteins present.
Grade the degree of turbidity as mentioned above.
• Additional Information
• Bence-Jones Protein : It consists of dimmers of either  or 
light chains from immunoglobulins. The molecular weight is
very small, about 44,000, hence it is easily filtered through the
normal glomerulus. It was first detected by Henry Bence Jones
in 1847.
• It has unusual solubility properties. It precipitates when
heated to 40-60oC, but becomes soluble again when boiled. It
reappears after cooling.
• There is malignant proliferation of plasma cells in multiple
myeloma, usually in the bone marrow. This disease is
associated with Bence Jones Proteins.
• Nearly 50-80 percent patients will multiple myeloma will have
Bence Jones Proteins in their urine. The remaining cases can
be diagnosed by serum electrophoresis.
TEST FOR SUGAR
(BENEDICT’S TEST)
ESTIMATION OF GLUCOSE
Glycosuria- Presence of detectable amounts of
glucose in urine called as glucosuria (or) glycosuria
Glucose filtered by the glomeruli is reabsorbed by
the proximal renal tubules and returned to circulation.
Normal renal threshold for glucose is 180mg/dl
CAUSES OF GLYCOSURIA
1.GLYCOSURIA WITH HYPERGLYCEMIA:
A) Endocrine disorders- DM, Pancreatic disease,
hyperthyroidism, cushings syndrome, acromegaly.
B)Non-endocrine disorders- CNS disease, liver disorders
C)Drugs- ACTH, corticosteroids, thiazides
D)Alimentary glycosuria
2.GLYCOSURIA WITHOUT HYPERGLYCEMIA:
Renal glycosuria- renal threshold < 180mg/dl
Renal tubular disease- decreased glucose reabsorptionFanconis syndrome, toxic renal tubular damaage.
During pregnancy
TESTS FOR DETECTION OF GLUCOSE IN URINE
1. Copper reduction methods:
A) Benedicts qualitative test
B) Fehlings test
C) Copper reduction tablet test
2. Reagent strip method
3. Quantitative test for glucose
BENEDICTS TEST
Benedicts qualitative reagent composition–
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Sodium carbonate-100gm,
Copper sulphate – 17.3 gm
Sodium citrate – 173gm,
Distilled water – 1000ml
Take 5ml reagent
Add 8 drops of urine
Boil over a flame for 2 mts
Allow to cool at room temperature, note the color
change, if any.
Contd…
• Cupric ions (blue)+sugar → cuprous oxide(red)
+cuprous hydroxide
Results:
Nil- no change from blue color.
Trace – green without precipitate.
1+: green with precipitate
2+:brown precipitate
3+:yellow – orange precipitate
4+:brick –red preciptate
Also used for screening test for inborn errors of carbohydrate
metabolism.
FALSE POSITIVE RESULTS:
Apart from glucose other substances may
reduce benedicts solution
Carbohydrates – lactose, fructose, pentose and
glucoranates.
Non carbohydrates- salicylic acid, homogentisic
acid, melanogen, formalin
Fehlings test
Take equal volume of fehling solution A and B
mix and boil , to this add few drops of urine
and boil again
If sugar is present precipitate of cuprous
oxide and color changes as in benedicts test
Reagent strip method
• More sensitive than benedicts test.
Specific only for glucose.
• Principle: Based on glucose oxidase-peroxidase
reaction. Reagent area of the strip impregnated
with above 2 enzymes and a chromogen
Glucose+oxygen→ gluconic acid+H2O2
H2O2+chromogen→oxidized chromogen+H2O.
Compared with the color chart provided
• TEST FOR SPECIFIC SUGARS:
Lactose – rubners test , osazone test.
Fructose – sellwanoffs test
Pentoses - Bials test.
ESTIMATION OF KETONES
Excretion of ketone bodies in urine called ketonuria.
(Acetoacetic acid, β-hydroxybutric acid, and acetone)
Causes
1. Decreased utilization of carbohydrate- uncontrolled DM,
Glycogen storage disease.
2.Decreased availability of carbohydrate in the dietStarvation, persistent vomiting, weight reduction program
3.Increased metabolic needs- Fever in children, severe
thyrotoxicosis, PEM, Pregnancy
TESTS FOR DETECTION OF KETONES IN
URINE
• ROTHERAS TEST- Detect acetoacetic acid and
acetone.
• ACETEST TABLET TEST- This is Rotheras test in
the form of tablet.
• Ferric chloride test- Detect acetoacetic acid
only.
• Reagent strip test
• Harls method – betahydroxy butyric acid
ROTHERAS TEST
Take 5ml of urine in a test tube
saturate it with ammonium
sulphate.
Add a small crystal of sodium
nitroprusside. Mix well.
Slowly run along the side of the
test tube liquor ammonia to
form a layer
Immediate formation of purple
permanganate colored ring at
the junction of the two fluids
indicates a positive test.
Ferric chloride test (Gerhardt test )
• To 5ml of urine add 10% aqueous solution of
ferric chloride drop by drop until no further
precipitate of brown ferric phosphate occurs.
filter and then add 2 more drops of ferric
chloride.
If diacetic acid present the filtrate will
develop deep red color.
TEST FOR BILIRUBIN
BILE IN URINE
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Bilirubin
Bile salts
Bile pigments
Urobilin
Urobilinogen
BILE SALTS
• Bile acid synthesis – cholesterol + cytochromep450 forms primary bile acids
• Taurocholic acid, glycocolic acid,
taurochenodeoxycholic acid and
chenodeoxycholic acide
HAY’S TEST
• Hay’s sulfur flower test
• 3ml urine + sulfur powder sprinkled
• Positive – sulfur powder sinks as the bile salts
reduces the surface tension
HAY’S TEST
BILE PIGMENTS
• Breakdown products of blood hemoglobin
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Bilirubin – orange or yellow
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Biliverdin – green
• Mixed with intestinal contents – brown color
to feces
GMELIN’S TEST
• Named after Leopold Gmelin
• Procedure:
• 5ml of urine + 5ml of conc nitric acid
• Different color rings between the two layers
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Nitric acid – oxidising agent
Blue, Green and Violet
Not sensitive
Positive – presence
Negative – not means its absent
GMELIN’S TEST
FOAM TEST
Principle:
Bilirubin if present colors the foam yellow to green.
Procedure:
– Place 5ml urine in a test tube. Place cover.
– Shake the urine vigorously for 3 mins.
– If Bilirubin is present, the foam produced will
have a yellow to light green color.
 In patients with proteinuria, bilirubin bound
to albumin can also appear in urine.*
FOAM TEST
FOUCHET’S TEST/ HARRISON SPOT TEST
• Bariumchloride reacts with sulfates in urine to
form barium sulfate.
• Bilirubin adheres to precipitate
• Yellow bilirubin is oxidized to biliverdin (green)
with Fecl3 in the presence of trichloro acetic
acid.
FOUCHET’S TEST
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PROCEDURE:
10 ml urine + 2.5ml Bacl2
Filter
Unfold the filter paper
Spread it of dry filter paper
Allow 1 drop of fauchet’s reagent on the
precipitate
• Green to blue - positive
TEST FOR BLOOD
(BENZIDINE TEST)
TESTS FOR BLOOD IN URINE
• MICROSCOPY EXAMINATION
• CHEMICAL TEST
BENZIDINE TEST
ORTHOTOLUIDINE TEST
REAGENT STRIP TEST
BENZIDINE TEST
Principle:
The peroxide activity of the blood decomposes hydrogen
peroxide and the liberated oxygen oxidizes the benzidine.
Procedure:
– Place 1cc of Benzidine solution in a test tube.
– Add 0.5cc of urine which was previously filtered.
– Add 0.3cc of H2O2 to the mixture.
– Mix and observe for a change in color.
 Positive result: Green or blue color. (Hematuria)
• ORTHOTOLUIDINE TEST
• More sensitive test than Benzidine test.In
this test, instead of Benzidine
Orthotoluidine is used.
• REAGENT STRIP TEST
• Various reagents strips available use
different chromogens.
EVALUATION OF POSITIVE CHEMICAL TEST
URINE MICROSCOPY
RED CELL PRESENT
RBC CASTS
RBC
RED CELL ABSENT
WBC CASTS
WBC
HEMOGLOBINURIA
GLOMERULONEPHRITIS
MYOGLOBINURIA
PYELONEPHRITIS
LYSIS OF RBC IN
LOWER UTI ALKALINE/
STONE,TUMOUR
TUMOUR HYPOTONIC URINE
COAGULATION DISORDER
FALSE POSITIVE TEST
• Contamination of urine by menstrual blood
• Contamination of urine by oxidizing agent.
FALSE NEGATIVE TEST
• Presence of reducing agent(ascorbic acid)
• Use presertive (FORMALIN)