BIOCHEMISTRY I

Lecture 5
The Kidney and Urinalysis Part III

Test for Renal Function

Urinalysis
3 parts examination (physical, chemical &
microscopic)

Creatinine and blood urea nitrogen (BUN)

Creatinine clearance test

Chemical Examination of Urine

Several biochemical tests can be performed
quickly and easily on urine using reagent strips
Chemical testing by reagent strip = chemical,
biochemical or analyte analysis of urine
Reagent strips are used by dipping it in the
urine and observing chemical reactions on
each reagent pad within a specified time

Principle of urine Chemical Tests by

Reagent Strip
They are sometimes called dipstick
The parameters commonly tested are:

Glucose
Bilirubin
Ketones
Blood
pH protein

- Urobilinogen
- Nitrite
- Leukocyte esterase
- Specific gravity

It is best to prefer to manufacturers instructions

since use of biochemical detection methods may
differ

Glucose
Presence of detectable glucose in urine =
glycosuria (indicates blood glucose level has
exceeded the renal threshold for glucose)
Can occur in diabetes mellitus and gestational
diabetes
Reagent strip is specific for ONLY glucose
It contains enzymes glucose oxidase and
peroxidase with a chromogen (a substance
that produces a colour in chemical reaction)

Glucose
These enzymes react with glucose in the urine
- change colour of the reagent pad
Intensity of the colour formed - proportional
to the glucose concentration
Normal urine is negative for glucose by this
method

Bilirubin
This is the primary bile pigment, formed from
the breakdown of haemoglobin
When present, it indicates:
Liver disease
Bile duct obstruction
Hepatitis

Bilirubin
Test for bilirubin - based on coupling bilirubin
with a diazonium salt in the reagent pad to
form a purple - brown colour
Direct light causes decomposition of bilirubin
Thus, specimens should be protected from
light - prevent false - negative results
Normal urine contains no detectable bilirubin
by this method

Ketones
Ketones are formed when body burns fat
rather than sugar for energy
Ketones include:
Acetone
Acetoacetic acid
Beta-hydroxybutyric acid

Ketones
Ketonuria (presence of ketones in urine) may
be caused by:
Uncontrolled diabetes
Starvation
Prolonged dieting or fasting

Ketone test - based on reaction of ketones

with sodium nitroferricyanide in ketone
reagent pad - cause formation of dark pink to
maroon colour

Ketones
Ketones evaporate at room temperature - thus
must be tightly capped and refrigerated, if
cannot be tested promptly
Normal urine - negative for ketones

Blood
Presence of blood in urine = haematuria
Can be due to infection, trauma to the urinary tract,
bleeding in the kidneys, glomerular damage or
tumour
May be due to menstruation, trauma from catheter
(a hollow flexible tube for insertion into a body cavity, duct,
or vessel to allow the passage of fluids or distend a
passageway. Its uses include the drainage of urine from the
bladder through the urethra or insertion through a blood
vessel into the heart for diagnostic purposes) insertion

Blood
Blood reagent pads detects haemoglobin and
intact RBC
When either is present, a colour forms
because of the peroxidase - like action of
haemoglobin in the RBC reacting with
chromogen and peroxide in the reagent pad
Resulting colour range form orange to green
to dark blue

Blood
Intact RBC can cause a spotty appearance on
the reagent pad
Myoglobin - also cause a positive reaction on
the reagent strip
Normal urine - negative

pH
pH of urine changes with:
Diet
Medications
Kidney disease

Metabolic diseases (e.g. diabetes mellitus)

Indicator dyes (e.g. methyl red and bromothymol
blue) in the pH reagent pad forms colour from
yellow - orange for acid urine to green - blue for
alkaline urine
Normal urine = pH 4.5 - 8

Protein
Proteinuria - increased amount of protein in
urine
Proteinuria is important indicator of renal
disease but can be caused by vigorous
exercise or conditions such as urinary tract
infection (UTI)
Positive tests - caused by presence of albumin

Protein
Protein reagent pad - not a good indicator of
globulins
Protein sulfosalicylic acid turbidity test should
be used if proteins can alter colour of some
acid - base indicator dyes without changing
the pH
The pad is kept at pH 3 with a buffering dye
(e.g. tetrabromophenol blue)

Protein
At the constant acid pH, development of any green
colour on the reagent pad is due to presence of
protein (albumin) and is usually reported using a
plus system (Neg, 1+, 2+, 3+ & 4+)
Colour range form yellow for negative to yellowgreen or green for positive, depending on the
amount of protein present
Normal urine - negative or just a trace of protein

Urobilinogen
Is a bilirubin degradation product - formed by
action of intestinal bacteria
It can be increased in hepatic disease or
haemolytic disease
Urobilinogen reagent pad contains chemicals react with urobilinogen to form a pink - red
colour, based on Ehrlich aldehyde reaction
Normal urine - 0.1 - 1.0 mg/dL (0.1-1.0 EU/dL)

Urobilinogen
Since urobilinogen - unstable in light and in
acidic urine, negative results - not considered
significant
Reagent strip can detect urobilinogen in
concentration as low as 0.1 mg/dL (0.1 EU,
Ehrlich unit)
Normal urine - 0.1 - 1.0 mg/dL (0.1 - 1.0 EU)

Nitrite
Gram-negative bacteria produce enzymes that
convert urinary nitrate (a normal urine constituent)
to nitrite
Nitrite reacts with chemicals on reagent pad to
form a pink colour
Positive nitrite test is an indication of possible
bacterial UTI
Since not all bacteria can convert nitrates, a
negative result is possible in the presence of
infection

Nitrite
Organisms which normally cause UTI and
cause a positive nitrite test are:
Escherichia coli
Klebsiella
Proteus
Pseudomonas

Normal urine - negative for nitrite by reagent

strip method

Leukocytes Esterase
Granular leukocytes (primarily neutrophils)
contain an enzyme called leukocyte esterase
Presence of this enzyme indicates presence of
leukocytes in urine
This may due to infection or inflammation in
the urinary tract

Leukocytes Esterase
Esterase enzyme reacts with esterase
substrates in leukocyte esterase reagent pad form a purple colour
Colour intensity is proportional to number of
leukocytes present
Normal urine - negative result for this test

Specific Gravity
Specific gravity reflects kidneys ability to
concentrate urine
The pad contains indicator which change from
blue-green to yellow-green with relevance to
urine ion concentration
Normal specific gravity for urine is 1.005 to
1.030 by reagent strip method

Reference Values by Reagent Strip

Normal urine:
Negative for glucose, ketone, bilirubin, bacteria
(nitrite), leukocyte esterase and blood
May be negative or contain trace amount of
protein
Usually have pH of 4.5 8.0 and specific gravity of
1.005 - 1.030

 Positive or abnormal results should be

confirmed according to lab policy
Positive leukocyte esterase or nitrite tests
should be confirmed by microscopic
examination of urine sediment

Single Analyte and Special Reagent Strips

Reagent strips that test for only 1 or 2
constituents (e.g. glucose and ketones) useful for monitoring purposes
Examples:
Diastix and Clinistix - used at home to monitor
urine glucose
Ketostix - only test for ketones
Keto-Diastix - for ketones and glucose

Single Analyte and Special Reagent Strips

Multistix PRO - to detect or monitor kidney
disease and diabetes mellitus, also measure
creatinine and protein
Clinitek Microalbumin - detect microalbumin
(protein in the urine in amounts too small to be
detected by routine strips)
Microalbuminuria = condition in which very small
amounts of albumin are present in the urine
This indicates early renal disease and can occur as a
complication of diabetes

Performing Chemical Tests by Reagent Strips

Directions for use and colour comparison

chart are included with each vial
Instructions must be followed for accurate
results

Safety Precautions
All specimens must be considered potential
biological hazards
Gloves and face shields are best to be worn
when handling these specimens
Avoid splashes, spills and creation of aerosols

Quality Assessment
Specimen Collection and Storage
Specimens must be collected and labeled
correctly and tested within required time
limits
Midstream specimen is preferred
Testing should be done within 1 hour of
collection and can be refrigerated up to 4
hours otherwise

Quality Assessment
Specimens should be allowed to reach room
temperature before testing and should be
tightly covered in the dark because urine
components are volatile, labile or light
sensitive

Quality Assessment
1. Reagent Strips
Manufacturers directions for proper storage, use, timing of
reactions must be followed to ensure validity of results
Strips must be stored protected from light, heat and
moisture
They must not be used after expiration date and used only
once
Specimens must be mixed well ( make sure all solid
components will be exposed to the pads
Test are based on colour comparison - technicians must pass
colour blindness test

Quality Assessment
2. Urine Control
Reliability of urine reagent strips validated by
urine chemistry controls ensure the integrity of
strips
Urine controls should be run at least once each shift
(or day)
Commercial normal (-ve), low abnormal and high
abnormal controls should be used
Patients results should not be reported if the strip
does not show valid results based on the
manufacturers stated ranges

Manual Method
Test is performed by quickly
dipping a reagent strip into
recently collected, well mixed
urine
Timing of reactions must start
when the strip is withdrawn from
the urine
Colour changes on pads must be
visually compared to the colour
chart provided by the
manufacturer

Manual Method

Reagent Strip Readers

Are used to interpret the results of urine reagent
strip tests
The readers contain reflectance photometers detect colour formed on the reagent pads
Results may be read out from the display screen
or print out
Reduces errors caused by
incorrect timing of reaction
or interpretation of colours

Performing Copper Reduction Test - Clinitest

Copper reduction test - detects reducing
sugars (glucose, fructose, lactose, galactose)
Substances which may give false-positive
reactions with this test are:
Penicillin
Salicylates
Ascorbic acid
Cephalosporins

Performing Copper Reduction Test - Clinitest

Previously, Clinitest was used to estimate
glucose in urine of individuals with diabetes
But with glucose-specific reagent strips today,
this method is no longer used for glucose
Copper reduction test - now used as a simple
way to screen urine for reducing sugars other
than glucose (e.g. screen newborns for
galactosuria = excretion of galactose in urine)

Performing Clinitest
5 drops of urine and 10 drops of water are added
into heat - resistant test tube
Clinitest tablet is placed in the diluted urine colour is observed while it effervesces
If a reducing sugar is present, the colour changes
from blue to green and then to orange depending on amount of reducing substance
present
When the reaction is complete, colour of the
liquid is compared to the colour chart on the vial

Performing Confirmatory Tests

Methods other than reagent strips - needed to
confirm a positive (or negative) result
Confirmatory tests - more time consuming,
require more reagents and equipments than
reagent strip method
Commonly performed tests:
Proteins
Ketones
Bilirubin

Protein
Most simple confirmatory tests for urine
involve treating a portion of the urine with an
acid - cause protein to precipitate and become
visible
Amount of precipitate proportional to
concentration of proteins present

Protein
Precipitate is graded as:
Negative
Trace (slightly cloudy)
1+ (turbid)
2+ (turbid with granulation)
3+ (granulation and flocculation - lumps / fluff)
4+ (clumps)

Dilute acetic or sulfosalicylic acids can be used

to precipitate protein

Ketones
Acetest - test for ketones and is available in
tablet form
If ketones are present, a drop of urine will
produce a purple colour
Serum or plasma - used to test for ketones
using Acetest tablet
Ketostix and Keto-Diastix - also used to
confirm presence of ketones in urine

Acetest

Bilirubin
Ictotest - specific test for bilirubin and 4x more
sensitive as reagent strip method
A tablet and absorbent mat /paper is used - a
few drops of urine is placed on the mat, tablet
is positioned on the moist area and water is
dropped on the tablet
Purple colour will develop within 60 seconds,
if bilirubin is present

Ictotest