NORMAL

CONSTITUENTS OF
URINE
 INTRODUCTION

 Urineis the waste product excreted by the

kidneys.

 Crucialfor kidney function evaluation and

disease diagnosis.
 URINE ANALYSIS
 Biochemical analysis

 Qualitative examination:
Physical properties and
presence of abnormal
constituents.

 Quantitative estimation:
Protein, Creatinine, Calcium
etc.
 URINE COLLECTION
 Urine
is collected in a sterile wide-
mouthed container.

Method for urine collection are:-

 Mid stream morning sample

 URINE SAMPLE
‘Mid stream morning sample’
 To collect this sample, the patient is advised to discard the initial
part of the first voided urine in the morning after getting up and
then to collect 15-20 mL of mid stream urine flow in a clean glass
or plastic container.
 It should be immediately analysed or within a few hours of
collection.
 In the meantime it should be refrigerated
 URINE COLLECTION

 24-hour Urinary Sample:- Used for

quantitative estimation e.g. creatinine, proteins,
calcium etc.

 Random Urine Sample:- Anytime of the day

used for detection of glucose, ketone bodies and
proteins.
 URINE PRESERVATIVES

 Concentrated HCI:- Urea, ammonia, protein,

nitrogen and calcium estimations.

 Thymol:- Sodium, Potassium, chloride,

bicarbonate, calcium, phosphorus, creatinine,
amino acids etc.
 PHYSICAL CHARACTERISTICS
OF NORMAL URINE
Appearance Clear, no sediment

Volume 800 mL to 2000 mL/day

pH 4-8;usually acidic (6.0)
Color pale yellow
Specific gravity 1.010-1.025
Odour Aromatic
URINE PH

pH=4-8,
Usually acidic
(6.0)
DETERMINATION OF SPECIFIC
GRAVITY BY URINOMETER
 It is the ratio of the mass of a
solution to that of similar volume
water.

 Measure the concentration of

solutes in urine.
HOW TO MEASURE THE SPECIFIC
GRAVITY OF URINE?
Take a clean 100 ml measuring cylinder
Fill it with urine to about 3/4 of its length.
Gently place the urinometer into the cylinder and allow it
to dip.
Care should be taken that it does not touch the walls of
cylinder.
Note the reading corresponding to the urine level and
calculate the specific gravity.
 The instrument is calibrated at 15˚C

 Temperature correction is applied for room

temperature, which is as follows:

 For every 3˚C rise in room temperature, add

0.001 to the observed specific gravity

 For every 3˚C fall in room temperature, subtract

0.001 from the observed specific gravity
 Calculate change in temperature

 Room temperature=15⁰ C. Divide it by 3

 Multiply it by 0.001
 EXAMPLE
 Let us assume that the temperature is 24⁰ C
 Let us assume that the reading is 1.012

 Temperature difference=24-15= 9⁰ C
 Correction=0.001˟ (9/3)=0.001˟3=0.003

 As the temperature is more we need to add

 Final specific gravity=1.015
 Totalsolids: Last 2 digits of specific gravity are
multiplied by 2.66

 The units for total solids are G/L.

Low specific gravity High specific gravity

Diabetes Insipidus Diabetes mellitus.

Glomerulonephritis Nephrosis
Sever renal damage Fever since urine is conc.
(diminish the concentration Urine preservative
ability of the kidney) substance
Excessive water intake.
PROPERTIES OF NORMAL URINE

Urine

Solids (5- Water (90-

10%) 95%)

Organic Inorganic
 CHEMICAL CONSTITUENTS
Inorganic Organic
Chloride Urea
Sulphate Uric acid
Calcium Creatinine
Inorganic phosphate Hippuric acid
Ammonia
Sodium
Potassium
 CHEMICAL COMPOSITION
Urea 25-30 g/day
Uric acid 0.5-1 g/day
Creatinine 1.0- 2 g/day
Chlorides 5-25 g/day
Sodium 3-5 g/day
Potassium 2-4 g/day
Calcium 0.2 g/day
Phosphate 0.8-1.5 g/day
Sulphate 0.5-1.5 g/day
Ammonia 0.5-0.8 g/day
TEST FOR CREATININE: JAFFE’S
METHOD

 Principle: Creatinine reacts with picric acid in

alkaline medium to form reddish-orange colored
creatinine picrate
Test for creatinine Observation Inference

• 3 mL of urine in a
Indicate the presence
test tube. Reddish orange color of creatinine
is seen
• Add 1 ml saturated
picric acid
solution, and 3-4
drops of 10%
sodium hydroxide
CREATININE IS PRESENT
 urinary creatinine is derived from muscle phosphocreatine.

 Excretion in adults ranges from 1-2 g/day.

 In women and in elderly people the values are lower due to

lesser muscular mass.
• Excretion is increased in:

 Muscular dystrophy

 Hyperthyroidism

 Diabetes mellitus & starvation

 Excretion is decreased in:

 Renal failure

 Paralysis

 Anaemia
 TEST FOR UREA: UREASE TEST

Principle: The enzyme urease under optimum pH and

temperature decomposes urea into ammonia and carbon
dioxide which together form ammonium carbonate (alkaline
substance) which changes the solution to pink color in the
presence of the indicator.
Experiment Observation Inference

1. Take 5 mL of solution A Intense pink-red colour due to Indicate the

in a test tube. the formation of NH3 by the presence of
Urea.
2. Add a drop of phenol action of urease on urea is
red indicator and add, if formed.
necessary, 1% acetic
acid or 2% Na2CO3
solution, till the solution
becomes faint yellow-
red.
3. Add 1.0 mL of urease
solution and shake the
content for a minute and
then allow it to stand for
10 minutes.
UREA POSITIVE TEST
Test For Urea: Sodium Hypobromite Test
 Principle: when urea is treated with sodium hypobromite,
it decomposes to give nitrogen, carbon dioxide and water.
Liberation of Nitrogen Gas produces brisk effervescence.


Experiment Observation Inference

Take 3 mL of urine in a Brisk Indicates the
test tube. effervescence of presence of urea.
Add 5 drops of freshly nitrogen gas is
prepared alkaline seen.
sodium hypobromite
solution and mix.
 Urea is the major nitrogenous constituent of urine.

 Urea is formed in liver as the end product of protein

metabolism and so its excretion depends on protein
intake.

 About 20-40 g of urea is excreted in 24 hours.

Excretion is increased in:
 High protein diet
 Diabetes mellitus
 Fever
Excretion is decreased in:
 Liver diseases
 Acidosis
 Nephritis.
 INORGANIC CONSTITUENTS

Principle: Sulphate is precipitated as barium

sulphate (BaSO₄), forming a white
precipitate.
Test for Observation Inference
Sulphates

2 mL solution +
Curdy white ppt. Inorganic
2mL of Barium sulphates are
precipitated as
chloride solution
BaSO4.
SULPHATES TEST
 CALCIUM AND PHOSPHATE
TEST FOR PHOSPHATES

Principle

Phosphate react with ammonium molybdate to form canary

yellow coloured ammonium phosphomolybdate in the
presence of conc. Nitric acid
 Experiment Observation Inference
Take 5 ml of Canary Yellow Indicate the presence of
urine, add a few precipitate is inorganic phosphates.
drops of conc. formed.
nitric acid and a (ammonium
pinch of phosphomoly
ammonium bdate)
molybdate and
warm.
 POINTS TO REMEMBER

 Urinary Phosphate is derived from the breakdown of

phospholipids, nucleotides and phosphoprotein.
Excretion is decreased in:
 Diarrhoea
 Nephritis
 Pregnancy
 Infections
 Hypoparathyroidism
Excretion is increased in bone diseases like rickets,
osteomalacia and parathyroid dysfunction.
 TEST FOR CALCIUM (SULKOWASKI’S TEST)
Principle: Calcium is precipitated as calcium oxalate with
potassium oxalate in acidic condition.

Experiment Observation Inference

Take 5 ml of urine, White precipitate is Indicate the

add 5 drops of 1% formed. presence of
acetic acid and 5mL Calcium.
of potassium
oxalate solution.
 The vast majority of kidney stone (up to 75%) contain
calcium oxalate and calcium phosphate crystals.
 In such cases, estimating calcium levels in urine help in
evaluating patients suffering from renal calculi.
 Excretion increases in Hyperparathyroidism
Hypervitaminosis D, Multiple myeloma and renal
tubular acidosis.


 TEST FOR AMMONIA
Principle: Ammonia present in urine is liberated by heat. The
evolution of alkaline ammonium vapours changes the color of red
litmus to blue.
Experiment Observat Inferenc
ion e
1. Take 2 mL of urine in a test tube. Red litmus Indicate the
2. Add 1-2 drops of phenolphthalein indicator changes to presence of
and mix.
blue. Ammonia.
3. Add 2% sodium carbonate drop by drop
with constant mixing till the color of the
solution turns faint pink and boil.
4. Hold a piece of red litmus paper at the
mouth of the test tube.
 Urinary ammonia is derived from glutamine and other
amino acids in kidney.
 There is an increase in ammonia excretion when acid
forming foods are taken.
 TEST FOR UROBILINOGEN: EHRLICH’S
TEST

 Principle: Urobilinogen reacts with p-dimethyl amino

benzaldehyde of the reagent to form the red colored complex.

Experiment Observation Inference

1. Take 5 mL of urine in Development of Indicates the
a test tube. Red colour is presence of
2. Add 1 mL of Ehrlich seen. Urobilinogen.
reagent.
3. Mix well and keep for
5 minutes.
POINTS TO REMEMBER

 Urobilinogen in urine oxidizes to urobilin

 Urobilin does not answer the above test.
 Absence of urobilinogen in urine is suggestive of obstructive
jaundice.
 Excess of urobilinogen in urine is indicative of hemolytic
jaundice
TEST FOR URIC ACID:
benedict’s uric acid test or phosphotungstic , schiffs test and
murexide test

a) Principle: Uric acid in alkaline condition reduces

phosphotungstic acid to tungsten blue.
Experiment Observation Inference
1. Take 2 mL of urine Deep blue color Indicates the presence
in a test tube. is formed. of uric acid.
2. Add few drops of
phosphotungstic acid
reagent followed by
a few drops of 20%
sodium carbonate.
POINTS TO REMEMBER
 Uric acid is the end product of purine metabolism.
 The daily output of uric acid varies in the range of 0.6 to 1 g.
 Excretion is increased in:
 Leukemia especially during cytotoxic drug therapy.
 Wilson’s disease
 Administration of Cortisone/ACTH

 Excretion decreases in renal failure.

 Uric acid is the end product of purine metabolism.
 The daily output of uric acid varies in the range of 0.6 to
1g
 Hyperuricemia is mostly associated with gout.
 VIVA QUESTIONS

 What is the volume of normal urine output per day? Describe

the term polyurea, oliguria and anuria.
 What are the common causes of turbidity in a fresh urine
sample?
 What is the pigment responsible for the normal colour of
urine?
 How is the specific gravity of urine measured? Name one
condition where the specific gravity of urine is high.
 What is temperature correction while reading the
urinometer?
 Despite polyuria, specific gravity is high in diabetes
mellites. why?
 Name the condition in which large amounts of calcium
are excreted in urine.
 Name the NPN substance excreted in urine.
 Is urobilinogen normally present in urine ? Name one
condition where excretion of urobilinogen is
increased.name name the test to identify urobilinogen.
 Name the conditions in which excretion of urea in urine
is increased.
 When is the excretion of creatinine increased?