ANALYSIS OF NORMAL URINE

Competency 11.3

Describe the chemical components of Urine.

Objectives:
1. To enumerate the physical characteristics of Normal Urine.
2. To discuss the chemical composition of Normal Urine.
Competency 11.4

Perform urine analysis to estimate normal constituents of urine.

Objectives:
 To perform tests for inorganic and organic constituents of Urine.
 To report the analysis of normal urine.

Clinical laboratory urine examination can provide a variety of useful information regarding
renal and systemic diseases.

Careful laboratory examination of urine often narrows the clinical differential diagnosis of
numerous renal diseases.

Normal constituents of urine in 24-hour sample

Constituents In Grams/24hours
Water 1,300-1600ml
Total Nitrogen 10-17
Urea 20-35
Uric Acid 0.1-2
Creatinine 1-1.5
Amino Acid(Free and Combined) 1.5
Glucose 0.1
Non-Glucose Reducing Substances 1
Citric Acid 0.3
Ascorbic Acid 0.025
Oxalate 0.015
Acetone Bodies 0.01
Total Sulfur 1
Phosphate 0.4-2.2
Sodium 3-6
Potassium 2-4
Calcium 0.2
Urine collection and preservation
Proper urine collection and preservation is important for timely and valid laboratory report
Types of collection
1. Random specimen
2. Mid stream first morning specimen
3. 24-hour collection specimen

1. Random specimen:
The most commonly obtained sample for biochemical and microscopic analysis. These
samples can be collected any time on the spot, therefore, readily available and easy to
obtain.It is commonly used for glucose, ketone bodies, bile and blood pigments etc

2. Mid stream first morning specimen

The specimen of choice for urinalysis as it contains concentrated amount of analytes during
its overnight collection in the bladder. The patient is asked to discard the initial part of first
voided urine in the morning and then collect 15-20ml of urine in a clean glass or plastic
container. Urine sample should be analyzed immediately or stored in a refrigerator if
analysis is delayed by 1 or 2 days

3. 24-hour urine collection

24 hour urine collection is required for estimation of urinary creatinine, urea nitrogen,
calcium, proteins, glulcose, sodium,potassium and hormones like catecholamines,17-
hydroxysteroids etc as these have diurnal variations.

Start collection at 6am. Let the patient empty the bladder and discard the first sampe. From
the second sample onwards, collect into the container all samples of urine passed till next
morning 6 AM sample, which should be the last sample collected.

Preservatives
The commonly used preservatives for 24-hours collection of urine are 50ml of 2N HCl or
10ml of conc. HCl per 24hours collection, or thymol crystals-5ml of 100gms/L solution in
isopropanol

Physical examination of urine

1. Volume
2. Appearance
3. Odour
4. Colour
 5. Specific Gravity
6. pH

Volume
Normally an adult excretes 1 to 2.5 litres per day
1. Polyuria
a. Volume > 2.5 L/day
b. Causes:
i. Diabetes mellitus
ii. Diabetes insipidus
iii. Later stages of chronic renal failure
iv. Drugs like diuretics
2. Oliguria
a. Volume is less than 300ml/day
b. Causes:
i. Fever
ii. Acute nephritis
iii. Early stages of chronic glomerulonephritis
iv. Diarrhea
v. Cardiac failure
3. Anuria
a. Urine output less than 50ml
b. Causes
i. Shock
ii. Acute Tubular Necrosis
iii. Mercury Poisoning
iv. Incompatible Blood Transfusions

Appearance
Usually freshly voided urine is clear
On standing, phosphate/urate/oxalate crystals may form and make appearance turbid
Abnormally urine may appear turbid due to presence of WBC, RBC or bacteria

Odour
Normal urine is slightly ammoniacal in odour
Other variations seen are
 Fruity odour – ketoacidosis due to acetone
 Foul smell – Seen in bacterial infection
 Mousy smell – Seen in Phenylketonuria
Colour
Colour of urine is determined to a large extent by its degree of concentration. Normal urine
is pale yellow in color

Conditions imparting abnormal colour to urine

COLOUR METABOLITE CLINICAL CONDITION

Red RBC Hemoglobin Hematuria, Hemoglobinuria

Red - brown Myoglobin, Porphyrin Myoglobinuria,

Porphyrinuria, Menstrual
contamination

Yellow Urochrome Healthy

Yellow-Orange Urobilin Dehydration, Jaundice

Yellow- Green Bilirubin Jaundice

Biliverdin

Brown- Black Homogentisic acid Alkaptonuria

Methemoglobin
Porphyrin

Milky white Chyle Chyluria

Specific Gravity
 Specific gravity is directly proportional to
concentration of solutes excreted. Normal urine
specific gravity lies between 1. 012 to 1.024
 Specific gravity is determined by urinometer
 High specific gravity conditions
o Restricted water intake, dehydration
o Release of glucose in urine(Diabetes
mellitus)
o Presence of protein in urine(Proteinuria)
o Adrenal insufficiency
 Low specific gravity conditions
o Polyuria
o High fluid intake
o Diabetes insipidus
o Hypothermia
Correction factor

For every 1 gm/dL of glucose, 0.004 is subtracted

For every 1gm/dL of protein, 0.003 is deducted

Temperature correction

Urinometer is graduated at 15 or 20 degrees Centigrade.

For every 3 degrees difference, 0.001 must be added if above or subtracted if below the
calibration temperature

pH
Freshly voided urine is usually acidic to litmus but may be neutral or faintly Alkaline.

pH is influenced by diet

● High Protein or low carbohydrate diet produces acidic urine

● Diet rich in vegetables and fruits produces Alkaline urine

Acidic urine

 High protein diet

 Starvation
 Dehydration and diarrhea
 Diabetic ketoacidosis
 Metabolic and respiratory acidosis

Alkaline urine

 Vegetables and fruits

 Vomiting
 Renal tubular acidosis
 Respiratory and metabolic alkalosis
 Ammonia producing, urea splitting bacteria.
 Chronic renal failure
Analysis of Normal Urine – Organic Constituents
The chief Organic non - protein nitrogenous constituents (NPN) are Urea, uric acid,
creatinine, Urobilinogen

Urea

Urea is the chief end product of protein metabolism and is found in liver.

Tests for Urea

1. Sodium Hypobromite test

 Principle:Urea is decomposed by sodium Hypobromite forming nitrogen gas

Experiment Observation Inference

Take 5ml of urine in a test Marked effervescence is seen Urea is present

tube. Add 1ml of Alkaline
sodium Hypobromite. Mix
gently.

2. Specific Urease test

 Principle:Urease decomposes urea to ammonia and carbon dioxide which
together form ammonium carbonate an alkaline substance which changes
slightly acid reaction (yellow colour) to Alkaline reaction (pink colour). Since
urease is specific for urea, positive test indicates presence of urea

Experiment Observation Inference

Take 2 test tubes C and T. Pink colour is observed in T Confirms the presence of
Add 5ml of urine in both tune urea
tubes. Add 2ml of inactivated
urease in C. No colour change in C tube
Add 2ml of activated urease
in T.
To both tubes, add 1ml of
phenolphthalein indicator
 Clinical interpretation
o Normal level: 25-30gm/day
 Increased urinary urea
o High protein diet
o Hematemesis
o Excess tissue breakdown as in high fever and severe wasting diseases
  Decreased Urinary urea
o Renal failure
o Severe hepatic insufficiency
o Low protein diet
o Severe acidosis

Uric acid
Uric acid ( 2, 6, 8 trihydroxy purine) is the catabolic product of purines. It is synthesized in
liver and excreted through kidney

1. Phosphotungstic acid test

 Principle:Uric acid is a reducing agent. In Alkaline conditions, it reduces
phosphotungstic acid to tungsten blue in the presence of sodium carbonate

Experiment Observation Inference

To 2 ml of urine, add few Deep blue colour is observed Presence of uric acid
drops of phosphotungstic
acid reagent and few drops of
20 % sodium carbonate

2. Schiff's test
 Principle: Uric acid is a reducing agent. Under Alkaline conditions, uric acid
reduces silver nitrate to black colour metallic silver

Experiment Observation Inference

Wet the filter paper with Filter paper turns to black in Presence of uric acid
sample and add few drops of colour
ammoniacal silver nitrate

 Clinical interpretation
o Normal levels: 250- 750 mgms/day
 Increased urinary uric acid
o High protein diet( meat, legumes
o Attacks of gout
o Leukemia
o Administration of cortisone or ACTH
 Decreased Urinary uric acid
o Chronic renal failure
Creatinine
Creatine is a normal constituent of muscles, present in the form of creatine phosphate.
Creatine undergoes spontaneous dehydration to form creatinine.

 Jaffe's test
o Principle:Creatinine in Alkaline medium reacts with picric acid to form
creatinine picrate which is reddish orange in colour

Experiment Observation Inference

Take 2 test tubes, label them as C Reddish orange colour Presence of creatinine
and T is observed in T tube
To C, add 3ml of distilled water.
To T, add 3ml of urine sample.
To both tubes, add 2ml of saturated
picric acid and 2ml of 10% NaOH

 Clinical interpretation
o Normal levels 1 -2 gms/ day , higher in males than females
 Increased urinary creatinine
o Fever
o Myasthenia gravis
o Muscular atrophy, myositis
o Hyperthyroidism
o Starvation

Urobilinogen
 Urobilinogen is formed from bile pigments in intestine and is absorbed and excreted
in urine
 Urobilinogen normally is present in the urine in amounts sufficient to give positive
test in dilution 1:10 to 1:20.
 Its absence indicates complete biliary obstruction.
 Increased amounts are associated with excessive blood destruction/ hemolytic
jaundice
 Ehrlich’s test for Urobilinogen
o Principle: Urobilinogen reacts with Ehrlich’s reagent to form red colour which
is intensified with the addition of sodium acetate.
Analysis of Normal Urine – Inorganic Constituents
Chlorides, sulfates, phosphates, sodium, potassium, Calcium, and Magnesium are chief
inorganic constituents of Urine

Chlorides
Chlorides form chief anion of urine.

 Test for chlorides

o Principle: Chlorine present in urine is precipitated as silver chloride with
silver nitrate in the presence of nitric acid

Experiment Observation Inference

Acidify 2ml of urine with 2 White precipitate of silver Chlorides are present
drops of concentrated nitric chloride is formed
acid and 1ml of 3% silver
nitrate solution

 Clinical interpretation
o Normal levels 10- 12gm/day
 Increased urinary chloride excretion
o Addison's disease
o Polyuria of advanced chronic nephritis and diabetes
 Decreased Urinary Chloride excretion
o Excessive sweating
o Diarrhea and vomiting
o Diabetes insipidus
o Cushing's syndrome
o Extensive burns
o Pneumonia

Phosphates
Phosphates exist in urine as salts of sodium, potassium, ammonium, Calcium and
Magnesium. In Alkaline urine, phosphates crystallize out in characteristic shapes

 Test for phosphates

 o Principle: Upon warming with ammonium molybdate in the presence of
concentrated nitric acid, inorganic phosphates are precipitated as canary
yellow ammonium phosphomolybdate

Experiment Observation Inference

To 5ml of urine, add few drops of Canary yellow precipitate Phosphates are present
concentrated nitric acid and a pinch of is seen
ammonium molybdate and warm it

 Clinical interpretation
o Normal levels: 0.8- 1.2gm/day

 Increased phosphates excretion

o Hyperparathyroidism
o Administration of parathyroid Hormone

 Decreased phosphate excretion

o Hypo-parathyroidism
o Rickets
o Diarrhea
o Acute infection and nephritis due to impaired kidney function
o Pregnancy

Calcium
 Test for Calcium
o Principle: With potassium oxalate in acidic conditions, Calcium is precipitated
as Calcium oxalate

Experiment Observation Inference

To 2ml of urine, add 5 drops White precipitate of calcium Calcium is present

of 1% acetic acid , 5ml of 2% oxalate is formed
potassium oxalate

 Clinical interpretation
o Normal levels: 0.1 -0.3 gm/day
 Increased urinary Calcium
o Hyperparathyroidism
o Hyperthyroidism
 o Hypervitaminosis D
o Multiple myeloma

Sulfates
Sulfates are formed from oxidation of proteins

 Test for sulfates

o Principle: Sulfates are precipitated as barium sulfate with barium chloride

Experiment Observation Inference

To 3ml of urine, add few A white precipitate is seen Sulfates are present
drops of concentrated HCL
and 1 ml of 10% barium
chloride solution.

 Clinical interpretation
o Normal levels : 0.7- 1gm/day
 Increased sulfates
o High protein diet
 Decreased sulfates
o Renal dysfunction

Ammonia
Ammonia is present as ammonium salts in normal urine.

 Test for Ammonia

o Principle:Ammonia is liberated when urine is boiled in Alakaline conditions

Experiment Observation Inference

To 5ml of urine add 2% sodium Red litmus turns to blue Ammonia is present
carbonate till the solution is Alkaline
to litmus. Boil the solution. Place a
piece of red litmus paper at the
mouth of the tube

 Clinical interpretation
o Normal level: 0.4 - 1.0 gm/day
 Increased urinary Ammonia
o Acidosis
 o Severe diabetes mellitus
o Starvation
o Delayed chloroform poisoning
o Hepatic diseases
 Decreased Urinary Ammonia
o Alkalosis
o Damaged distal renal tubules in renal failure
o Glomerulonephritis
o Addison's disease