Urinalysis and Renal Function Tests
Collection of Urine
Many urine estimations are carried out on timed specimens (
24hrs ) . The accuracy of the result depends largely on that of
urine collection . Therefore , more care should be taken to
ensure that the collection of urine for 24 hours is done properly .
For example when a 24 hours collection is required between 10
AM on Monday and 10 AM on Tuesday , the following procedure
should be adapted. On Monday 10 AM, empty bladder
completely and discard the specimen. Then collect all the urine
passed in bottle containing the preservative till 10 AM on
Tuesday . The bladder is emptied at 10AM on Tuesday and this is
also added to the above pooled urine .
Preservation for Urine
A preservation must be added to the urine to prevent bacteria
growth and destruction of the substance being estimated .
Hydrochloric acid
Acidification of the specimen is very satisfactory . 10cc of conc.
HCl is adequate for 24 hour specimen . This is suitable for the
determination of urea , ammonia , total nitrogen , calcium and
phosphorus .Toluene is a commonly used preservation . It is
convenient for sodium , potassium , uric acid and protein
analysis . This only prevent further surface contamination with
bacteria . Thymol is also used as a preservative ( a few crystals or
5 ml of 10% solution ) . This is a satisfactory preservative for a
wide range of substances . However , it cannot be used for the
estimation of 17-oxosteroidsusing Zimmermann reaction .
Composition of Urine
Colour
Normal urine is clear and straw coloured . The colour is due to
the pigment urochrome . The colour is lighter when large
amount of water is consumed and darker in fevers and after
excessive perspiration . In jaundice , the urine is yellow due to
excretion of bile pigments . In alkaptonuria and
methaemoglobinaaemia urine is dark brown in colour .
Odour
Freshly voided urine has a mild aromatic odour . It develops
ammoniacal odour on standing , due to the formation of
ammonia by bacterial decomposition of urea . Urine in ketosis
emits the smell of acetone .
Volume
The daily urinary output varies widely and dependents on the
fluid intake and environmental conditions. The volume excreted
varies between 1000-2000 ml . Polyuria or increase in the
volume of urine is seen in diabetes mellitus . A fall in the urinary
output is termed as oliguria which is found in renal failure ,
some diseases of the heart feand lungs , fevers and diarrhea .
Anuria , a complete cessation of urine output is seen in the
terminal stages of renal failure .
Appearance
Normal urine is ordinarily clear and transparent when freshly
voided . It may become cloudy on standing because of the
precipitation of phosphates . Urine may be turbid when RBCs or
pus cells are present .
Specific Gravity
Specific gravity varies from 1.010 to 1.030 depending upon
water and food intake . It is determined in clinical practice by
means of a urinometer . It consists of a weighted cylinder which
floats in urine and a stem caliberated in degrees of specific
gravity usually from 1.000 to 1.060 . The depth to which the
urinometer sinks depends on the density of the urine . The
instrument is usually caliberated for use at 15 .
Procedure
1.Fill the cylinder with urine without producing bubbles.
2. Float the urinometer so that it does not touch the sides.
3. Make the reading from the bottom meniscus .
Temperature Correction
The urinometer is usually caliberated for use at 15 and specific
gravity should be corrected to room temperature by adding .001
for every 3 above the temperature at which the urinometer is
caliberated (15 ) .
Room temperature = 30
Specific gravity read = 1.009
Temperature correction = 1.009 + 5 x .001
= 1.014
Total Solids
Under normal conditions , the total solids present is about 1500
ml of urine varies 60 to 70 gm . The output of urinary solids is
influenced by the intake of fluid diet. The total solids per litre of
urine is obtained by multiplying the last two digits of the specific
gravity by a factor of 2.6 which is called the Longs Coefficient . If
the specific is 1.020 , the total solids would be 52 g / litre .
Reaction
Normally urine is acidic (pH=6) in reaction due to monobasic
salts of phosphoric acid plus small amounts of organic acids . The
acidity of urine is influenced by diet , fluid intake and also by
various drugs . Meat diet will increase acidity whereas fruit
decreases . Fasting and starvation in which the body proteins are
metabolized also tend to increase titrable acidity . Urine on
standing turns alkaline due to the formation of ammonia by the
bacterial decomposition of urea .
Constituents of Normal Urine
Normal urine is composed of : 1. Water , 2. Inorganic salts , 3.
Organic compounds .
Major Inorganic Ions
Anions = Cl_ , PO4---- , SO4---Cations = Na+ , K+ , Ca++ , Mg++ , [NH4]+
Organic Constituents
NPN Nonprotein nitrogen is nitrogen in the blood that is not a
,acid , creatine , creatinine and polypetitides . NPN substances in
urine are urea , creatine , creatinine , uric acid , amino acids ,
allantion and hippuric acid .
Urea About 25 to 30 gm of urea is excreted daily in urine . As
urea is the end product of protein metabolism , urea excretion in
urine is an index of protein intake in diet . Urea in urine is
increased in exaggerated protein metabolism , fevers and
increased adrenocortical activity . It is decreased in the last
stages of severe hepatic diseases and in acidosis .
Creatinine
It is derived from the creatin of the muscle and the amount
excreted ( 1.0 to 1.8 g per day ) depends on the muscle mass
Analysis of Normal Urine
Physical Examination
Note the colour , appearance and odour . Test the acidity with
blue or red litmus paper . Determine the specific gravity using
urinometer . Apply the correction for temperature variation .
Calculate the total solids present and perform the following tests
with the sample .
Test for chlorides
Place 5 ml of albumin free test tube and add 3 drops of Con
HNO3 to prevent precipitation of protein . Add 5 drops of AgCl
indicates presence of chlorides .
Test for phosphates
To 3 ml of urine add 1ml of Con HNO3 and 5 ml of ammonium
molebdate and warm . A canary yellow precipitate indicates the
presence of phosphate.
Test for inorganic sulphates
To 3ml of urine add 1 ml of Con HCl followed by 10% BaCl2
solution .A white precipitate of BaSO4 indicates inorganic
sulphate .Filter and set aside the filtrate for the next
experiment.
Test for ethereal sulphate
Take the above filtrate and boil . A white precipitate is formed
again indicating the presence of ethereal sulphate .
Test for ammonia
Boil urine with equal volume of 10% NaOH. Smell of ammonia
gas indicates the presence of ammonium ion in the urine
sample.
Analysis of Pathological Urine
In the case of abnormal urine , glucose , ketone bodies , proteins
, blood , bile salts and bile pigments are tested in the clinical
biochemical laboratory .
Test for Reducing Sugar. Benedicts Test
Reducing carbohydrates like glucose reduces CuSO4 in alkaline
media to insoluble Cu2O . Colour of the solution depends upon
the particle size of Cu2O formed .
Procedure
Take 5ml of Benedicts qualitative reagent . Boil for a minute and
add 8 drops (0.5ml) of urine , boil for 2 minutes , cool and note
the colour change .
Green
0.5 % sugar
Yellow
1.0%
Reddish yellow
1.5%
Brick red
2.0 % and above .
Test for Proteins (Albumins)
Heat and acetic acid test .Proteins are coagulated by boiling ,
CO2 driven out of the solution , pH of the urine is increased
which may precipitate phosphates . Thus when a sample of urine
is heated precipitate may be due to protein or phosphate
addition of a few drops of acetic will dissolve phosphate , if
present .
Procedure
Fill up 2/3 of a test tube with urine . Boil the upper portion of
the test tube . Add a drop or two % acetic acid . A white cloud in
the heated portion shows the presence of albumin .
Heller's Nitric Acid Test (Cold test )
This sensitive test is based on the fact that protein is converted
into a meta protein which is insoluble in concentrated mineral
acids .
Procedure
Take 3 ml of Con HNO3 in a test tube and pour 2 ml of urine
along the sides . A white ring at the junction of the two layers
shows the presence of proteins .
Sulposalicylic Acid Test
Place 1 ml urine in a test tube , add 3%bsulposalicylic acid and
allow to stand for 10 min . Presence of cloudiness is due to
albumin .
Benzidine Test for Blood in Urine
Prepare a saturated benzidine solution in glacial acetic acid ; mix
well equal parts of benzidine solution and H2O2 . Take 2 ml of
urine and add 2 ml of this mixture . A green or deep blue colour
indicates presence of blood in urine . Any catalyst or enzyme
which splits up H2O2 and liberates nascent oxygen will give this
test . The pus cells ( WBCs in urine ) contain an enzyme called
peroxidase , which can spilt H2O2 and liberate nascent oxygen .
To test haemoglobin in presence of pus cells , the urine must be
boiled to destroy the pus cells enzyme
Normally bile salts and bile pigments do not enter in the general
circulation , and therefore , they are absent in normal urine . But
if there is intrahepatic or extrahepatic obstruction to the flow of
bile ,bile regurgitates into the general circulation and appears in
urine as in obstructive jaundice . Bile pigments give urine a
greenish yellow or brown colour .
a. Hays test for salts
When powder of sulphur is sprinkled on the urine containing bile
salt, the sulphur powder sinks due to lowering of surface
tension. A positive test indicates the liver damage or obstruction
of bile duct.
b. Gmelin's test for bile pigments
When Con HNO3 is added to urine, there is a play of colour.
Green indicates presence of bile pigments. Nitric acid oxidisses
bilirubin to form a series of coloured compounds, biliverdin
(green), billicyanin (blue), choletelin (Yellow), etc.
c. Fouchet's test for bile pigments
Add 5ml of 10% BaCl2 solution to about 10 ml of urine . Mix and
filter . Pour a few drops of Faucets' reagent ( FeCl3 the oxidizing
agent ) on the precipitate in the air dried filter paper . In the
presence of bile pigments , bilirubin turns green and blue due to
oxidation (UBG and SBG ) .
d. Ehrlichs aldehyde test for urobilinogen
Take 10 ml of urine in a test tube . Add 2.5 ml of BaCl2 solution
and filter . Take 3 ml of filtrate add Ehrlich's aldehyde reagent
(para dimethyl aminobenaldehyde) shake well and allow to
stand for 3 minutes. Pink colour indicates a positive test.