Chemical examination of

urine
DR. DEVANGI DESAI
Chemical examination
Proteins
Sugars
Ketone bodies
Bilirubin/Bile pigments
Bile salts
Urobilinogen
Porphrins
Blood
Myoglobin
PROTIENS:
NORMAL:<150 mg/day of total protein
Renal tubules secrete TAMM-HORSFALL PROTEIN
which is normally excreted by kidney.
PROTEINURIA:>150 mg/day urinary protein
excretion
The following types of chief proteins can be
present in urine:

# Albumin: Commonest urinary protein, hence

albuminuria term is used
# Globulin: usually found together with albumin
# Mucin: may be present in traces
# Hemoglobin: appears after hemolysis
# Bences Jones Protein: found in myeloma
Causes of Albuminuria
 Asymptomatic/functional causes:
# Exercise
#Orthostatic
#Pregnancy
 Massive renal Albuminuria:
# Post streptococcal glomerulonephritis
# Nephrotic Syndromes: Glomerular lesions associated with
nephrotic sundrome:-
$ Primary renal diseases- MCGN, MGN, MPGN, FSGN
$ Associated systemic disease – SLE, Amyloidosis,
Diabetes, Rhematism, Polyarteritis
Tests for proteins
Test – HEAT & ACETIC ACID TEST
Principle-proteins are denatured & coagulated on
heating to give white cloud precipitate.
Method-take 2/3 of test tube with urine, heat only the
upper part keeping lower part as control.
Presence of phosphates, carbonates, proteins gives a
white cloud formation. Add acetic acid 1-2 drops, if
the cloud persists, it indicates it is protein(acetic acid
dissolves the carbonates/phosphates)
Other tests
Sulphosalicylic acid test:Proteins are
denatured by organic acids and precipitate
out of solution.
Dipsticks
Esbach’s albuminometer- for quantitative
24 hours estimation of proteins
Micro-albuminuria
Defination- urinary excretion of 30-300 mg/24 hrs
of albumin in urine.
The level of albumin protein produced by micro-
albuminuria cannot be detected by urine dipstick
methods.
In a properly functioning body, albumin is not
normally present in urine because it is retained in
the bloodstream by the kidneys.
Significance of microalbuminuria
Earliest sign of renal damage in DM
an indicator of subclinical cardiovascular disease
an important prognostic marker for kidney disease
increasing microalbuminuria during the first 48
hours after admission to an intensive care unit
predicts elevated risk for acute respiratory failure ,
multiple organ failure , and overall mortality
Bence Jones proteins
These are light chain globulins seen in multiple
myeloma, macroglobulimias, lymphoma.
Test- Thermal method(waterbath):
Proteins has unusual property of precipitating at
400 -600c & then dissolving when the urine is
brought to boiling(1000c) & reappears when the
urine is cooled.
SUGAR
A small amount of glucose (2-20mg/dl) may be
present in fasting urine which is not detectable by
chemical methods.
Presence of chemically detectable glucose in urine is
called GLYCOSURIA.
Normal RENAL threshold for glucose is 160-180
mg/dl.
Test for sugar
Test-BENEDICT’S TEST (semi-quantitative)
Principle-benedict’s reagent contains cuso4.In the
presence of reducing sugars cupric ions are converted
to cuprous oxide which is hastened by heating, to give
the color.
Method- take 5ml of benedict’s reagent in a test tube,
add 8drops of urine. Boil the mixture.
 Blue-green = negative
 Yellow-green = + (<0.5%)
 Greenish yellow = ++ (0.5-1%)
 Yellow = +++ (1-2%)
 Brick red = ++++ (>2%)
Benedict’s test
False positive:
# Carbohydrates: fructose, lactose,
galactose, L xylulose
# Non carbohydrates: Uric acid,
creatinine, homogentisic acid, salicylic
acid, melanogen
To confirm it is glucose, dipsticks can
be used (glucose oxidase)
Causes of Glycosuria
Glycosuria with hyperglycaemia-
# Diabetes
# acromegaly
# cushing’s disease
# hyperthyroidism
# drugs like corticosteroids
# Intracranial injury
# functioning Alpha and beta cell pancreatic
tumors
Glycosuria without hyperglycaemia-
# Renal glycosuria
# Alimentary glycosuria
# Fanconi syndrome
Ketone bodies
3 types
Acetone
Acetoacetic acid
β-hydroxy butyric acid
They are products of fat metabolism
Rothera’s test
Principle-acetone & acetoacetic acid react with
sodium nitroprusside in the presence of alkali to
produce purple color.

Method- take 5ml of urine in a test tube & saturate

it with ammonium sulphate. Then add one crystal
of sodium nitroprusside. Then gently add 0.5ml of
liquor ammonia along the sides of the test tube.
Change in color/Purple color ring at junction
indicates + test
Causes of ketonuria
Uncontrolled Diabetes
Chronic starvation
 Severe vomiting/diarrhoea
Glycogen storage disease
Pregnancy toxaemia with vomiting
Bile Pigments
Unconjugated bilirubin of blood cannot pass through
the glomerulus in urine but conjugated bilirubin (water
soluble) is liable to pass through glomerulus in urine.
Increased:
- Extrahepatic obstructive jaundice
- Hepatic jaundice caused by cholestasis
Fouchet’s Test:
-5 ml of urine in TT. Add 2-5 ml of 10% barium
chloride, mix well.
- It is taken on filter paper on which fouchet’s reagent is
added.
- Blue green color indicates positivity.
Bile Salts
Bile salts are the products of cholesterol
metabolism.
Primary bile salts are cholate and
chenodeoxycholate and secondary bile salts
are lithocholate, deoxycholate and
ursodeoxycholate.
Increased: Obstruction of common bile duct
and drug induced cholestasis
Hay’s Sulfur Flower Test
 Principle:
Bile salts when present lower the surface tension of urine.
When sulfur powder is added on the surface of urine, sulfur
particles sink to the bottom of the beaker. In case of a normal
urine sample; sulfur particles float on the surface of urine.
 Procedure:
Take about 10 ml urine in a beaker. Sprinkle dry sulfur powder
on the surface of urine. Observe the sulfurs particles.
 Observations:
Sulfur particles sink to bottom : Bile salts present.
Sulfur particles remain floating: Bile salts absent.
Urobilinogen
Urobilinogen is a colorless by-product
of bilirubin reduction. It is formed in the intestines
by bacterial action on bilirubin. About half of the
urobilinogen formed is reabsorbed and taken up
via the portal vein to the liver, enters circulation
and is excreted by the kidney.
Freshly voided urine should be examined for
urobilinogen in urine as urobilinogen is converted
to urobilin within short time when exposed to air.
Normal:0.5- 2.5 mg/24 hrs
Increase: hemolytic anemia,
Decrease or absent: obstruction to bile flow
(obstructive jaundice), reduction in bacterial flora
of intestine
Variable: hepatocellular jaundice
Porphyrins
Porphyrins are synthesised in the bone marrow and
liver
The porphrins mainly comprise of inherited autosomal
disorders characterised by aberrations in the activities
of the specific enzymes of haem biosynthetic pathway
Porphyrins in the urine may be found in the form of
delta aminolevulinic acid(ALA), porphobilinogen,
uroporphyrin or coproporphrin
Porphobilinogen
Urine bilirubin and urobilinogen in
jaundice
Urine test Hemolytic Hepatocellular obstructive

Bilirubin Absent Present Present

Urobilinogen Inc Inc Absent

Blood in urine
Test- BENZIDINE TEST
Principle-The peroxidase activity of hemoglobin
decomposes hydrogen peroxide releasing nascent
oxygen which in turn oxidizes benzidine to give
blue color.
Method-
 Place pinch of benzidine powder in a test tube.
 Add 2-3 drops of glacial acetic acid and mix well.
 Add about 2 ml of hydrogen peroxide solution and mix well. Transfer 1
ml of supernatant to a test tube.
 Add 5 ml of urine and mix well.
 Observe the color of the mixture after 5 minutes.
 Also observe microscopic examination at the same time.
Causes of hematuria
Pre renal- bleeding diathesis(hemophilia, purpura,
leukemias), hemoglobinopathies, malignant
hypertension, SABE, anticoagulant therapy, drugs
Renal- trauma, calculi, acute & chronic
glomerulonephritis, renal TB, renal tumors
Post renal – severe UTI, calculi, trauma, tumors
of urinary tract
Causes of hemoglobinuria
Paroxysmal nocturnal hemoglobinuria
Mismatched blood transfusion
Autoimmune hemolytic anemia
Black water fever
Snake venom poisoning
Extensive burns
Drug sensitivity with quinine, sulfonamides
 Myoglobin
Presence of myoglobin in urine is called
myoglobinuria.
During acute destruction of muscle fibres
(rhabdomyolysis) myoglobin is released.
It is red brown pigment
It can be detected by spectroscopic and electrophoretic
study.
It is difficult to distinguish between hemoglobin and
myoglobin
Myoglobinuria
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