Experiment 5

Urine Analysis

Introduction
Urine is an important excretory biological fluid that is formed by the kidneys.
Presence of certain metabolites in urine reflects physiological and metabolic status of the
body. Further, the urine specimen can be easily collected for routine examination and
thus is non-invasive in nature. Microscopic and biochemical investigation of urine is an
important tool in diagnosis and in obtaining information of various pathological
conditions. Normally, complete urinary reports includes (i) physical and microscopic
examination, (ii) qualitative and quantitative analysis of urinary constituents.
A normal urine is colorless to straw colored. The color of the urine is due to the
presence of urochrome, which is an oxidized form of urochromogen. Other pigments that
contribute to the color of urine are urobilin, uroerythrin, hematoporyphyrin etc. The
normal color of the urine is influenced by the volume of urinary output. Abnormal urinary
color is seen in various pathological conditions. Brown to black colored is noted in clinical
conditions such as porphyrinuria, methaemoglobinuria, melanotic sarcoma and in
alcaptonuria- an inborn error of phenylalanine metabolism. A red to brown colored urine
is seen in conditions such as haematuria and myoglobinuria. A deep yellow or greenish-
yellow colored urine is observed in jaundice and in severe dehydration.
An aromatic odor is characteristic of freshly voided urine, due to the presence of
certain volatile organic acids. Urine upon standing, develops an ammoniacal odor due to
bacterial action on urinary urea. Similar ammoniacal odor is observed in subjects
suffering from certain urinary tract infections. Infection due to Gram negative bacteria
imparts an unpleasant odor to urine. In diabetics, excretion of ketone bodies (ketonuria)
imparts a fruity odor to urine.
The daily output of urine in normal individuals ranges between 0.6-2.5 L/day.
However, depending upon the physiological and pathological conditions, the 24-hour
urinary volume may vary considerably, the mean output being 1.2 L/day. Various factors
influence urinary output. For example, high dietary intake in proteins has diuretic effects
leading to increased urinary volume, while high ambient temperature reduces output of
urine. Polyuria- a clinical condition wherein there is an increased urinary output (>
3L/day). This condition is observed in Diabetes mellitus and insipidus, chronic renal
failure, and ingestion of diuretic drugs. Olgiuria is a condition which is characterized by a
decreased urinary output (<400 mL/day) and is seen in acute renal failure, severe
dehydration and oedema. Presence of bilateral kidney stones, shock, and acute nephritis
lead to cessation of urine excretion- a condition termed anuria.
Freshly voided urine is clear and transparent. Flocculation of normal urine on
standing is attributed to the presence of mucoproteins, nucleoproteins and ephithelial
cells originating from genito-urinary tract. Turbidity of urine is due to the presence of pus
cells, crystals, and chyle that can be confirmed by microscopic examination. Precipitation
of calcium phosphate, ammonium urate in alkaline urine and uric acid also results in
turbidity.
The specific gravity of normal urine ranges from 1.003 1.030 and is measured by
urinometer. Polyuria results in urine of low specific gravity. On the contrary, in diabetic
polyuria, the specific gravity of urine is high due to the presence of glucose and ketone
bodies. Values greater than 1.030 are observed in clinical conditions such as adrenal
insufficiency (addisons disease), diabetes and nephrotic syndrome. Lowered specific
gravity in urine is seen in Dibetes insipidus and chronic renal failure.
Normal urine is acidic the mean pH value of a 24-hour normal urine is 6.0, with a
range of 4.5-8.5. The acidity of urine increases in fever, metabolic acidosis, respiratory
acidosis, and after a protein rich meal. Alkalinity of the urine upon standing is due to the
formation of ammonia from urea by the action of microorganisms and subsequent loss of
CO2. Excretion of alkaline urine is characteristic of urinary tract infection (UTI).

Objectives

1. To examine the urine sample physically and microscopically.

2. To tests for abnormal urinary constituents in the urine sample.
3. To determine the urinary titratable acidity.

Materials and Reagents

Micro test tube Glacial acetic acid Things to bring:

pH paper Benedicts Reagent: 1N Own Urine Sample
thermometer NaOH Amber colored bottle
microscope slide 1 N NaOH solution
light microscope 3% H2O2
centrifuge 1% (w/v) Sodium acetate
droppers solution
pipettes Sodium nitroprusside (10%
Bunsen Burner w/v)
Test tube holder Chilled ammia
Water bath Ammonium sulfate
Beakers 10% (w/v) Ferric chloride
Iron Stand 10% (w/v) BaCl2
Funnel Fouchets reagent:
Funnel Support 25 g of trichloroacetic
Filter Paper acid
Erlenmeyer flask and 10 mL 10 % (w/v)
Burette ferric
Burette Holder chloride making up the
vol. to
100 mL
Concentrated Nitric Acid
Elemental sulfur powder
Standardized 0.1 N NaOH
Phenolphthalein (1% w/v)
Powdered potassium oxalate
salt
70% NaOH
Bromine water

Procedure

A. Materials and Sample Preparation

1. The urine sample used for the experiment should be preferably collected over a
period of 24 hours in a clean amber bottle.
B. Physical Examination of Urine

1. Place 2 mL of urine sample in a micro test tube observe its color, odor and turbidity.
Write your observations in your data sheet.
2. Determine the pH using a pH paper and report it in your data sheet.
3. Get the temperature of the sample.
4. Measure the weight of an empty 10-mL graduated cylinder.
5. Transfer the 2 mL sample and get the actual volume.
6. Weigh the graduated cylinder with the sample and measure the density of the urine
sample.
7. Get the specific gravity of the urine sample against the density of water at 4 oC (1
g/mL) and record it in your data sheet.

C. Microscopic Examination of Urine

1. Place 10 mL urine in a micro test tube and centrifuge at 3500 x g for 15 min.
2. Discard the supernatant and resuspend the sediment in 3-5 drops of supernatant
urine.
3. Transfer a small aliquot in a microscope slide and observe under a light microscope.
4. Write your observations in your laboratory manual.

D. Qualitative Test for Abnormal Urinary Contents

a. Test for proteins

Coagulation Test

1. Take 5 mL of urine specimen into micro test tube and add 4-5 drops of glacial acetic
acid.
2. Heat the contents of the test tube over a Bunsen burner to boiling.
3. Write your observations in the data sheet.

Test for Bence-Jones protein

1. Take 5 mL of urine specimen into micro test tube and heat the contents in a water
bath, maintained at 60oC. Observe
2. Heat further to boiling. Observe.
3. Write your observations in the data sheet.

b. Test for sugars (Benedicts test)

1. Take 0.5 mL of urine specimen into micro test tube and make it alkaline by adding a
drop of 1N NaOH solution.
2. Add 5 mL of benedicts reagent and heat the contents in boiling water bath for 3-5
minutes
3. Write your observations in the data sheet.

c. Test for Ketone Bodies

Rotheras Test
1. Saturate 5 mL of urine sample with sodium ammonium sulfate in a test tube.
2. Add 2-3 drops of sodium nitroprusside reagent.
3. Add few drops of chilled liquid ammonia and mix.
4. Observe and write observation in the data sheet.

Gehardts Test

1. Transfer 5 mL of the urine specimen into a clean micro test tube.

2. Add 2-3 drops of ferric chloride and mix.
3. Write observation in the data sheet.

d. Test for Bile Pigments and Bile Salts

Fouchets test

1. Place 10 mL urine sample into a clean micro test tube and add 5 mL BaCl2 solution.
2. Mix and filter. Save the precipitate.
3. Directly, add a drop of Fouchets reagent on the precipitate.
4. Observe and write observation in the data sheet.

Gmelins Test

1. Carefully layer 2 mL of urine sample over 2mL of nitric acid in a test tube.
2. Wait for some time, observe and write observation in the data sheet.

Hays Test

1. Place 20 mL urine sample in a beaker.

2. Sprinkle sulfur powder on the surface of urine sample.
3. Repeat step 2 in a 20 mL distilled water.
4. Compare and write observation in the data sheet.

e. Test for Urea

1. Add 0.5 mL 70% NaOH and 4 drops of bromine water to 1 mL urine sample
2. Observe evolution of gas.

f. Test for Creatinine

1. Add 1 mL alkaline picrate solution to 2 mL urine sample

2. Note the change in color.

E. Quantitative Analysis of Urinary Metabolites

a. Determination of Urinary Titratable Acidity (Perform 3 trials)

1. Transfer 25 mL of fresh urine specimen into clean 250 mL Erlenmeyer flask.

2. Add 5 g K2C2O4 salt to the contents and mix.
3. Add 5 drops of phenolphthalein indicator and titrate the contents against the
standardized NaOH solution to pale pink endpoint.
4. Record the volume consumed and solve for the urinary acidity using the equation
below:

Urinary Acidity (mEq) = N NaOH Volume of NaOH ( mL ) Dilution Factor

Data:

A. Physical Examination of Urine

Physical Characteristics Observations Comments

Color

Turbidity

Odor

Physical Characteristics Value Comments

pH

Specific Gravity

B. Microscopic Examination of Urine

Observations:

Drawing of the specimen:

C. Qualitative Test for Abnormal Urinary Contents

Urinary Contents Test Observations Comment

Protein Coagulation Test
 Bence-Jones
Protein

Sugar Benedicts Test

Rotheras Test
Ketone Bodies
Gehardts Test

Fouchets Test

Bile Pigments and

Gmelins Test
Salts

Hays Test

Urea
Creatinine

C. Determination of Urinary Titratable Acidity

Trial 1 Trial 2 Trial 3

Total Volume of
Urine (mL)
Volume of Urine
Aliquot (mL)
Volume of NaOH
used (mL)
Urinary Acidty
(mEq)

Sample Computation:

Guide Questions for Discussion

1. Describe the urea cycle.

2. What is the significance of a high amount of uric acid in urine samples?
3. What are the amino acids involved in creatinine synthesis?
4. What is the function of bile in lipid catabolism?
5. What tests are important in examining several kidney disorders?

References

Rao, B. and Deshpande, V. experimental Biochemistry: A Student Companion, 2005

G.C. Bernas, Ysrael, M.C. and Bernaldez, A.T. Basic Laboratory Studies in Biochemistry. 3 rd
edition. UST Press: Manila, 1997