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Phase 1 - Practical Manual - Mbbs 2011
Phase 1 - Practical Manual - Mbbs 2011
MBBS
Phase I
DEPARTMENT OF BIOCHEMISTRY
FACULTY OF MEDICAL SCIENCES
UNIVERSITY OF SRI JAYEWARDENEPURA
2011
1
BIOCHEMISTRY PRACTICALS
2
TABLE OF CONTENTS
1st Term
Foundation Module
Practical 1 - Introduction to Laboratory
Practical 2 - Cell, pH and buffers
Practical 3 - Tests for Carbohydrates and Lipids
Practical 4 - Tests for Amino acids and Proteins
Practical 5 - Enzymology I
Practical 6 - Enzymology II
3rd Term
4th Term
Renal Module
Practical 10 – Analysis of Abnormal Constituents in Urine
3
1st Term
FOUNDATION MODULE
4
Practical - 1
INTRODUCTION TO LABORATORY
When you work in a laboratory, you may be exposed to many accidents and hazards,
which may occur due to specimens (urine/serum), chemicals/reagents, glassware,
electricity/gasses, equipment etc.
Always wear protective clothing (e.g. over (lab) coats, gloves, goggles etc.)
when necessary.
Eating, drinking, smoking, and orating in the lab are hazardous.
Biting fingernails should be strictly avoided.
Any wound, abrasions etc. should be dressed well before entering the lab.
Should not enter the lab with loose hair, high-heeled shoes and fancy dresses and
hanging accessories.
Wash your hands well before leaving the lab.
2. Learn the correct handling of pipettes and never mouth pipette (use fillers/ bulbs)
and consider that all biological materials are hazardous.
3. If any corrosive or infectious materials get spilled on bench, floor or on your
clothes, call a demonstrator or a technician.
4. If anything is splashed on your eyes, quickly wash the eye with running tap
water and report. Always keep the tubes away from the body when
boiling/heating.
5. Do not use any broken glassware or handle them with greatest care. Report any
breakages to the lab staff.
6. When using reagents, read the label carefully and do not move the bottles or
waste and contaminate the reagents. Take care when opening them as some may
exude toxic fumes (e.g. strong acids, NH3 etc.) and close them once used.
7. Do not exceed the specified speed of centrifuges. Allow centrifuge to slow
naturally and wait at least 2 min before opening to prevent contamination
through aerosols.
8. Always switch off the microscope when not in use and never change the field.
9. If there is any fire inside the lab, take actions to stop them. Eg. use sand
baskets/buckets, fire extinguishers, etc.
10. Once you finish working,
Turn off the gas burner.
Wash the glassware used and clean the working bench and sink.
Discard the specimens collected by your selves only (e.g. urine).
5
B. COMMONLY USED LABORATORY EQUIPMENT AND THEIR
APPLICATIONS
10. Filters
6
12. pH Measurement
Used to separate molecules according to the charge / molecular mass ratio (e/m).
E.g. paper / gel electrophoretic apparatus
(ii) Analytical weighing scale – to measure very small amount (Eg g/mg)
(i) Colourimeter
(ii) Spectrophotometer
7
Practical - 2
All biochemical reactions in the body take place in an aqueous environment and most of
these reactions are catalyzed by enzymes. Enzymes are optimally active at a particular
H+ ion concentration.
Concept of pH
HCl H+ + Cl-
Strong acids dissociate to a greater extent and liberate more hydrogen than a weaker
acid. H+ ions can be expressed as mol/L. But a more convenient way to express H +
concentration is in terms of pH.
From the definition: the higher the hydrogen ion concentration the lower the pH and
vice versa.
H2O H+ + OH-
[H+][OH-] = Constant Kw
8
Concentration of 1 x 10-14 1 x 10-7 1x 100
OH- (moles/liter)
pH 0 7 14
2. INDICATORS
Indicators change their colours with a change in the pH of the solution containing
them. They are weak acids or weak bases. Their ionized and unionized forms have
different colours. The actual colour in a solution therefore, depends on the ratio of
these two forms. This ratio in turn depends on the pH of the solution containing the
indicator.
1. Take 1 mL of 0.1 M HCL and 1 mL of 0.1 M NaOH into two separate test tubes
and add 1 drop of red cabbage juice to each tube and observe the colour change.
9
3. MEASUREMENT OF pH
3.1. pH papers
Determine the pH of the solutions A and B, tap water and urine using pH papers
provided.
Question:
a) The blank contains the same volume of sample without the indicator.
b) The tubes are placed in the Lovibond comparator in such a manner that a
window of the disc covers the blank.
c) The disc is rotated so that the colour on the disc matches with the colour in the
test tube.
d) The corresponding pH is read from the disc. This method is superior to the
previous method as the pH of coloured solution (such as urine) can be estimated
without dilution.
4. BUFFERS
Most biological systems will function only within a narrow range of pH and their
activities vary widely within that range. The most important way that the pH of the
blood, urine, extra cellular fluid is kept relatively constant is by buffers dissolved in the
body fluid.
A buffer is a mixture of a weak acid and its conjugate base (respective salt) that resists
change in pH on the addition of small amounts of acids or bases.
A buffer solution has to contain ions, which will remove any hydrogen ions or
hydroxide ions that you might add to it – otherwise the pH will change.
The pH of a buffer solution is determined by the ratio of concentration of the acid to its
salt and pK1 value of the acid (pK1a).
10
Where pH= Pka + log [Salt]
pKa = -log Ka [Acid] of th
(Ka = dissociation constant
a) Take 1 ml of serum and 1ml of tap water to two separate test tubes.
b) Add a drop of phenol red indicator (pH 6.7 – 8.3) to each tube.
c) Then add 0.01 N NaOH to the tube containing water. Counting the number of
drops you add until the colour change.
d) Then add the same number of drops of 0.01M NaOH to the tube containing
serum and observe whether there is any colour change.
e) Then add another few drops of 0.01 M NaOH to the same serum tube and
observe for any colour change upon adding few drops.
Repeat the same procedure with serum and water with 0.01 M HCl.
Questions:
Why did not the serum sample give a colour change even after adding the same
number of acid / base drops to that tube?
b) Wash the beet root pieces until the red colour is no more.
c) Then put a piece of beet root to each tube and observe the tubes after 10-15
minutes.
d) Record your observations and explain the changes.
Exercises
11
1. Calculate the [H+] when
(a) pH = 4 (b) pH = 2.4 (c) pH = 4.4
2. What is the pH value of 0.001 N HCl acid (assume complete dissociation)
3. Calculate pH when,
(a) [H+] = 10-6 (b) [H+] = 4.3 x 10-8 (c) [H+] = 3.2 x 10-10
4. You are given a weak acid HA (pKa = 5.00) using a log table calculate the pH of
the solution.
a) When half the amount of acid has been neutralized what is the pH of the solution?
c) If you are given indicators Bromo Cresol Green (pKa = 4.7) and Phenol Red
(pK = 7.9) which would you choose to determine the pH at
d) When this acid is almost completely neutralized what is the pH of the solution?
12
Practical - 3
Perform with glucose, sucrose and maltose. To 5.0mL Benedict’s reagent add 8 drops of
sugar solution and mix. Boil for 2 min. over a flame (or 5 min in a boiling water bath).
Allow to cool. Note the colour of the solution during heating and the colour of the
precipitate, if any.
Perform with glucose and maltose. Add 1mL of the sugar solution to 3mL of freshly
prepared Barfoed’s reagent. Mix and place the tube in a boiling water bath and boil for 1
min. Allow the tube to stand at room temperature for 5 min.
Barfoed’s reagent contains copper acetate in acetic acid. Only monosaccharides will
answer this test. Disaccharides may answer the test if boiled for a longer time.
Perform with solutions of fructose and Glucose. To 10 drops of solution add 5mL of
Seliwanoff’s reagent and heat for 30 seconds (until it boils). Cherry red colour
indicates the presence of fructose.
(a) Add one drop of dilute solution of iodine to a 6mL of starch solution. Note the
colour produced. Divide the mixture into 3 parts. To one part, add one drop of
dilute HCl. To the second part, add one drop of dilute NaOH. Heat the third
portion gently over a flame. Record your observations.
(b) Warm a starch solution with dilute HCl. Every minute take a drop of the solution
and add to a drop of iodine solution on a white marble. Note the time at which
solution does not give a colour with iodine. Perform Benedict’s test with the
solution. (Not Done)
Questions:
Explain how you would distinguish 2 unknown solutions, one containing glucose
and the other containing fructose?
State the main difference in the principle of Benedict’s and Barfoed’s tests?
13
Summary of the test for carbohydrates.
Dextrin
Glycogen
Test
Lactose
Starch
Glucose
Fructose
Galactose
Pentose
Maltose
Sucrose
Benedict’s Test + + + + + + - - - -
Barfoed’s Test + + + + - - - - - -
Seliwanoff’s - + - - - - - - - -
Test
Iodine Test - - - - - - - + + +
2.1.2 Rancidity
Test (a) rancid coconut oil (b) fresh coconut oil with litmus paper and with 1 drop of
methyl red indicator.
Record your observation.
Questions:
What steps could you take to prevent oil and fats becoming rancid?
14
2.3 Saturation of oils (demonstration)
Perform with coconut oil and sesame oil. Take 2mL of the above in to two test tubes and
keep in the refrigerator for two hours. Comment on your observation and distinguish
between the saturated and unsaturated.
2.4.1 Lecithin
(a) Note its smell
(b) Note its solubility in
(i) Water – only polar part dissolves
(ii) Acetone – only non polar part dissolves
(iii) Chloroform – methanol mixture (4:1v/v)
(c) You are provided with a hydrolysate of lecithin in 40% KOH.
(i) Note the smell of hydrolysate
Acidify 2mL hydrolysate lecithin with 2mL con. HNO3 drop wisely.
Add excess ammonium molybdate (3mL) and heat over a flame
gently. A yellow precipitate indicates the presence of phosphate.
(ii) Perform the acrolein test with hydrolysate.
(a) Dissolve about 5mg cholesterol in 5mL alcohol-ether mixture (1:1). Place a drop
of the solution on a slide and examine the crystal (demonstration).
Questions:
15
Practical - 4
i) To 5 mL of protein solution add an equal amount of saturated (NH 4)2SO4 solution. The
ovoglobulin is precipitated. This test is referred to as the half saturation test.
To 3mL of protein solution, add 5% lead acetate drop by drop and observe the protein
being precipitated. Add more lead acetate. Observe & record your observations. Repeat
the experiment with a solution of AgNO3. Observe the difference.
3) PRECIPITATION BY ACIDS
To 3mL protein solution add a few drops of 20% sulphosalycilic acid. The protein is
precipitated .Repeat the experiment with 1% picric acid and 10% trichloroacetic acid.
4) PRECIPITATION BY ALCOHOL
To 3mL protein solution add 3mL of absolute alcohol. Precipitation occurs. The
mechanism of precipitation in this method is by dehydration, denaturation and removal
of charges.
16
Note: However, if the electrolyte (NaCl) used in dissolving the protein is removed by
dialysis, alcohol is not capable of precipitating the protein.
To 1mL of amino acid solution add 4-5 drops of ninhydrin in alcohol and boil for
one minute. Development of blue colour indicates the presence of amino acid in
the solution.
To 3mL protein solution add an equal amount of 5% NaOH and then 4 drops
of 1% copper sulphate. Mix. A purple (pinkish) colour is produced.
To 3mL protein solution, add 2 mL Conc. HNO3. Boil and cool. What is your
observation?
Place 2mL of concentrated HNO3 in a test tube and incline the test tube. Run a
protein solution down the wall of the test tube until it forms a layer over the
HNO3. Note the protein precipitate at the junction between the two liquids.
You are provided with 3 different solutions containing an Amino acid, Peptide &
Protein (A, B & C). Using above tests try to identify the contents in each test tube.
SUMMARY CHART
17
6) ISOELECTRIC PRECIPITATION
Steps to follow:
a) To 1.0mL of alkaline casein solution add 0.1 M HCl drop by drop until
maximum precipitation occurs.
b) Check the pH using pH papers
c) Continue the addition of HCl, shaking the tube after each addition, until
the precipitate dissolves.
d) At this point add 0.1 M NaOH drop wise until casein precipitates
e) Continue the addition of NaOH until precipitate redissolves.
f) Record your observations
Resources provided:
Questions:
What is the biochemical basis of giving egg white in heavy metal poisoning?
What is the purpose of using ZnSO4 when assaying some serum parameters?
18
Practical - 5
ENZYMOLOGY I
1. Substrate concentration
2. pH
3. Temperature
4. Activators and inhibitors
The rate of the reaction rate of disappearance of substrate / min (iodine test)
rate of appearance of product / min (Benedicts test)
Wash the mouth with some water. Then take 10mL warm distilled water into mouth and
move it for about 2 min. Collect fluid into a clean beaker. Filter if necessary.
Steps to follow:
Note 1: If the amylase is very active, the achromic point may be observed in a minute
or two. If this happens, dilute the saliva (about 1:10) and repeat.
Note 2: Obtain the saliva that has the achromic point at about 5-8 min.
19
2) EFFECT OF SUBSTRATE CONCENTRATION
Dilute the 1% starch solution to obtain 0.8%, 0.4% and 0.2% solutions. To 0.5mL
diluted starch solution, add 0.5mL diluted saliva and 0.5mL buffer (pH 6.0). Incubate at
about 37 C for 5min. Perform iodine test & Benedict’s test with the mixture. Compare
precipitate obtained with each dilution of starch solution. Comment.
3) EFFECT OF pH
To 3 tubes add 1mL phosphate buffer, pH 5.8, 6.6 and 8.0. To each tube add 2mL of 1%
starch solution and 0.5mL diluted saliva. Incubate at 37 C. Determine the time taken by
contents of each tube to reach the achromic point.
4) EFFECT OF TEMPERATURE
Using the buffer of optimum pH (determined in above experiment) add 2mL 1% starch
solution and 0.5mL diluted saliva into 3 labeled test tubes. Place one tube in ice (about 4
C). Keep the other 2 tubes, at 20 C, 37 C. Comment on the optimal temperature.
Demonstration:
Cut the potato / apple into two small equal pieces (1/2 inch) and keep one piece open to
air and immerse the other piece in hot water (40 C – 60 C) and keep there for 1-2 mins
(blanching) and take the potato/ apple out and keep it open to the air. Comment on your
observation.
Blanching
- is a term that describes a process of food preparation wherein the food substance,
usually a vegetable or fruit, is plunged into boiling water (enzyme denaturation),
removed after a brief, timed interval and finally plunged into iced water or placed under
cold running water (shocked) to halt the cooking process.
(Ex: tinned fruits and vegetables)
To 4 tubes, add 2mL of 1% starch solution and 0.5mL of dialysed saliva. Add 2 drops
of 0.1M NaCl, 0.1 M Na2SO4 and mercuric sulphate solution, into each of the 3 tubes.
The 4th one is the control. Test with iodine solution the contents of each of the 4 tubes
after incubating for 5 mins. Comment on your observations.
20
Practical - 6
ENZYMOLOGY II
Introduction
The substrate fibrin is an insoluble clot protein. The dye Congo red (indicator) is
coupled to fibrin. When a protease hydrolyses the protein fibrin, the dye is released into
the solution.
b) To each tube add a shred of Congo red fibrin and incubate the tubes in a bath at 37
0
C for 30 min.
c) Explain the observations you make on each tube.
21
3) OPTIMUM pH FOR TRYPSIN ACTIVITY (Demonstration)
22
6) PRODUCTS OF PEPSIN AND TRYPSIN DIGESTION
Steps as follows:
Questions:
By means of equation indicate the difference between the actions of the two
enzymes.
23
3rd Term
AND
24
Guided Learning Session - 1
25
Guided Learning Session - 2
26
Fixed Learning Module
THYROID HORMONES
At the end of the FLM the student should be able to identify and list the main
features of
27
Guided Learning Session - 3
DIABETES MELLITUS
28
Practical - 7
Note: Every test should be compared with a normal urine sample (control). Note the
colour, smell, and nature of any deposit or any turbidity before testing.
1.1 Benedict’s test (Preliminary test for screening for reducing substances in urine)
To 2.5 mL of Benedict’s reagent, add 4 drops of abnormal urine. Boil for 2 min over a
flame or place the tube in a boiling water bath for 5 min. Allow to cool slowly.
Questions:
What are the simple sugars that will answer for Benedict’s test?
1.2 Seliwanoff’s resorcinol test (Confirmatory test for the presence of fructose)
To 5mL of Seliwanoff’s reagent, add 10 drops of urine and heat for 30 seconds. Cherry
red colour indicates the presence of fructose.
1.3 Clinistix strip test (Confirmatory test for the presence of glucose)
29
Dip the test strip in urine sample. Remove excess urine by touching the side of the
container. Compare the colour developed with the given colour chart in the bottle (read
the instructions).
Theory:
Glucose O2
Gluconic acid H2O2 Chromogen
Glucose oxidase
H2O Peroxidase
Oxidized chromogen
Note: Colour appearing after 2 minutes, does not have any significance.
Questions:
What are the substances impregnated in the strip?
The same principle applied to the clinistix test is used in the reagent kit for the
determination of glucose in serum. Here, the colour intensity of the solution in
measured at 500 nm. The absorbance is directly proportional to the amount of glucose
present in the sample.
Method
Pipette in to labeled test tubes Blank Standard Test
Reagent 1 mL 1 mL 1 mL
Demineralized water 10 μL - -
Standard - 10 μL -
Serum - - 10 μL
Incubate for 10 min at 37oC and measure the absorbance in the spectrophotometer
(Shimadzu – UV 200, Japan), at 500 nm.
30
Guided Learning Session - 4
INTRODUCTION TO ENDOCRINE DISORDERS
Hypothyroidism
Causes and clinical features of hypothyroidism
Biochemical basis for the features
Iodine and goitre
Congenital hypothyroidism (cretinism)
o Causes and clinical features of cretinism
Hyperthyroidism
Causes and clinical features
Growth hormone
Acromegaly
Clinical features of acromegaly
Addison’s disease
Causes and clinical features
Cushing’s syndrome
Causes and clinical features
31
Practical - 8
DIET
Food records, calculation of energy requirement and energy intake by using Food
Composition Tables (FCT).
Preparation:
Keep a one day food record of everything you eat and drink, noting the nature of the
food, method of preparation and portion consumed prior to practical day.
Reference tables:
Please refer the food guide and food comparison tables to calculate RDA values.
(B) Rice
One saucer of boiled rice weight = 225g
Uncooked 100g rice weight = 270g of boiled rice
100g raw rice = 360 kcal
(C) Snack
Cake 1 slice contains 4 teaspoonfuls (tsp) of sugar
Coke (300mL contains 7 tsp sugar)
Biscuit contain ¾ tsp sugar
One tsp (sugar) = 5g (19 kcal)
White sugar contains 387 kcal/ 100g
32
(D)
33
Practical - 9
ESTIMATION OF LIVER ENZYMES AND CONSTITUENTS OF
BILE
1. Constituents of Bile
Sprinkle a little amount of finely powdered sulphur on the surface of the urine in a tube.
Sulphur powder sinks down in the presence of bile salts and floats on normal urine.
Note: Above test depends on the surface tension reducing property of bile salts.
Question:
Give one clinical condition where bile salts can appear in urine.
Slightly acidify 2mL of urine with dil. acetic acid. Add 1mL of 10% BaCl 2, mix and
filter. Spread out precipitate on the filter paper and dry over a Bunsen flame or dry by
placing it on a second dry filter paper. Place a drop of Fouchet’s reagent on the
precipitate. Observe the colour. Green colour indicates the presence of bile pigment
(bilirubin).
Note: When an ion chloride in acid solution is added to a precipitate from urine
containing bilirubin green colour is formed.
Add 8 drops (0.5mL) of Ehrlich’s reagent to 2mL of freshly voided urine. A red colour
indicates the presence of urobilinogen. Warming may intensify the colour.
Serum AST also known as Glutamic Oxaloacetic Transaminase (GOT) is one of several
enzymes that catalyses the exchange of amino and oxo groups between α-amino acids
34
and α-oxo(keto) acids. AST is widely distributed throughout the tissues with significant
amounts in the heart and liver (Normal range: 0-20 U/L at 300C).
Eg. In myocardial infarction (MI), serum AST may begin to rise within 6-8 h after onset
peak within 2 days, and return to normal by the 4th or 5th day post infarction.
Principle:
AST/GOT
2-Oxoglutarate + L-Aspartate Glutamate + Oxaloacetate
Malic dehydrogenase
Oxaloacetate + NADH + H+ L-Malate + NAD+
Materials:
AST reagent
Unhaemolized serum samples
Spectrophotometer
Procedure:
1) Set the wave length of the Spectrophotometer at 340 nm.
2) Zero the Spectrophotmeter with deionized water.
3) For each sample dispense 2.0mL of reconstituted AST reagent in to the cuvettes
or test tubes and warm to the reaction temperature (300C).
4) Add 0.2mL of sample to its respective test tube and mix gently. Incubate for 30
seconds at the reaction temperature.
5) Record the decrease in absorbance at 60 seconds intervals (ΔA/ min). The rate of
change should be constant.
6) If the cuvette is not temperature controlled, incubate the samples at the reaction
temperature between readings.
Calculation:
Note: The micromolar absorptivity extinction coefficient of NADH is 0.0062 at 340 nm.
Serum GPT (Glutamic Pyruvic Transaminase) is known as ALT. ALT is one of several
enzymes that catalyse the exchange of amino and oxo groups between α-amino acids
and α-oxo acids. ALT is widely distributed in human tissues with the largest amount
found in the liver (Normal range: 0-22 U/L at 300C).
35
.
Principle:
ALT/GPT
2-Oxoglutarate + Alanine L-Glutamate + Pyruvate
Lactate dehydrogenase
Pyruvate + NADH + H+ Lactate + NAD+
Materials:
ALT reagent
Unhaemolized serum samples
Spectrophotometer
Procedure:
Calculation:
U/L = ΔA/ min x Total volume (2.2mL)
Absorptivity Sample volume (0.2mL)
Note: The micromolar absorptivity extinction coefficient of NADH is 0.0062 at 340 nm.
ALT is present in very high amounts in liver and kidney, and in smaller amounts in skeletal
muscle and heart. Although serum levels of both AST and ALT become elevated when ever
diseases processes affecting liver cell integrity, ALT is the more liver specific enzyme.
Elevation of ALT persists longer than those of AST activity.
AST is distributed in all body tissues, but greatest activity occurs in liver, heart, skeletal muscles
and in erythrocytes. Although serum levels of both AST and ALT become elevated when ever
disease processes affecting liver cell integrity (viral hepatitis, liver necrosis, cirrhosis),occur,
increased AST activity in serum or plasma appears in more than 97% of cases of myocardial
infarction.
36
Principle:
AST/TGO
2-Oxoglutarate + L-Aspartate Glutamate + Oxaloacetate
ALT/TGP
2-Oxoglutarate + Alanine L-Glutamate + Pyruvate
Then pyruvate or oxaloacetate reacts with colouration reagent, which absorbance at 505
nm in alkaline solution is proportional to AST or ALT activity in the reactional mixture.
Materials:
Procedure:
Tube No 1 2 3 4 5 6
Demineralized water 0.2 0.2 0.2 0.2 0.2 0.2
R1 or R2 1 0.9 0.8 0.7 0.6 0.5
R4 (standard) - 0.1 0.2 0.3 0.4 0.5
R3 (colourant) 1 1 1 1 1 1
Mix. Let stand for 20 minutes at room temperature. Add:
NaOH 0.4 N 10 10 10 10 10 10
Mix. Let stand 5 minutes and read absorbances at 505 nm against water.
TGO units 0 30 70 135 225 350
TGP units 0 40 80 140 225 325
There’s no need to plot a new curve at each determination.
Table 2:
37
Reagent R3 1 mL 1 mL
Mix and let stand 20 minutes at room temperature. Add:
NaOH 0.4 N 10 mL 10 mL
Mix let stand 5 minutes and read absorbances at 505 nm against water.
Note:
Blank reagent: replace serum by demineralized water in table 2.
Calculation:
Expected values:
38
CASE HISTORY I (From Module Book)
A 20 year old student developed flu like illness with loss of appetite, nausea and pain in
the right hypochondrium. On examination the liver was just palpable and was tender.
Two days later he developed jaundice, his urine became darker and his stools became
pale.
Investigations
On presentation One week later Reference range
Serum
Bilirubin 38 μ mol/L 230 μ mol/L 3.4 – 22 μ mol/L
Albumin 40 g/L 38 g/L 38 – 55 g/L
AST 450 IU/L 365 IU/L 5 – 35 IU/L
ALP 70 IU/L 150 IU/L 38 – 126 IU/L
GGT 60 IU/L 135 IU/L 8 – 78 IU/L
Urine
Bilirubin positive positive
Urobilinogen positive negative-
Comments
The first set of results is characteristic of early hepatitis with raised amino-transferase
reflecting cell damage. This usually precedes the rise in bilirubin and the development
of jaundice. Impairment of the hepatic secretion of conjugated bilirubin and of
urobilinogen uptake from the portal blood causes both these substances to be excreted in
the urine.
The second set of results show the expected high serum bilirubin but with a fall in AST
as the phase of maximum cellular damage has passed. An increase in ALP, usually of
not more than three times the ULN (Upper Limit of Normal), is common at this stage.
In hepatitis, the bilirubin in plasma is both conjugated and unconjugated, with the
former predominating. Conjugated bilirubin is excreted in the urine and the pale stool
reflects the decreased biliary excretion. The serum bilirubin has remained normal in this
acute illness.
39
CASE HISTORY 2 (From Module Book)
A middle aged female was admitted to hospital following a haematemesis. Endoscopy
revealed the presence of oesophageal varices. The only biochemical abnormality was an
elevated GGT (245 IU). Her varices were treated by sclerotherapy and no further
bleeding occurred. The patient was told to abstain from alcohol. She was admitted one
year later jaundiced, drowsy and with clinical signs of chromic liver disease.
Comments
The patient had continued to drink and the resulting liver damage eventually affected
hepatic function. The decreased serum albumin, elevated serum bilirubin and enzyme
changes are consistent with cirrhosis and active liver cell damages; the prothrombin time
was also prolonged.
40
4th Term
Renal Module
41
Practical - 10
Introduction
Abnormal Urine may contain reducing substances (hexoses, pentoses, vitamin C),
Proteins (albumin, Bence Jones protein), ketone bodies (acetone, aceto acetic acid), bile
salts & bilirubin (bile pigment).
Every test should be compared with a normal urine sample (control). The colour, smell
and the nature of any deposit or any turbidity should be noticed before testing.
Urine should be sampled immediately after collection for best results. If storage is
essential refrigeration or freezing is required to minimize the microbial degradation
followed by acidification.
Urine samples are preserved by different methods depending on the nature of the
analysis attempted.
Eg: Samples are collected under toluene or oil (determination of pH, amylase activity) or
collected in 10% acetic acid solution (estimation of ascorbic acid)
Sometimes, however, estimation of substances (eg: ascorbic acid) is best carried out as
soon as each specimen is voided. All investigations should be conducted on 24 hours
urine samples.
Note:
Preliminary tests are used to screen for the presence of abnormal groups of substances
in urine.
Confirmatory tests will confirm the presence of a particular abnormal constituent.
Determine the specific gravity by means of the urinometer. Apply the temperature
correction. viz. Urinometer is calibrated at 20 ˚C. Therefore temperature correction is
necessary. The addition of one unit to the third place of decimals in the reading for every
three degrees centigrade rise of temperature above calibration temperature of the
urinometer and vice versa.
42
Eg:
Reading on urinometer = x
Room temperature = 32 ˚C
Temperature correction = 32 ˚C -20 ˚C = 4
3
State
Eg: the importance
If specific of measuring specific gravity
gravity of urine.
= 1020,
Total solids = 2.66 X 20 g/L
Assuming daily excretion = 1500 mL urine
Total solids = 2.66 X 20 X 1.5 g/24 hours
2.1 Benedict’s test (Preliminary test – to screen for the presence of reducing
substances in urine)
To 2.5 ml of Benedict’s reagent, add 4 drops of abnormal urine. Boil for 2 min over
a flame or place the tube in a boiling water bath for 5 min. Allow to cool slowly.
Observe the colour of the precipitate and colour of the supernatant.
Questions:
1. What is the basis of Benedict’s test?
2. What are the simple sugars that will answer for Benedict’s test?
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2.2 Seliwanoff’s resorcinol test (confirmatory test for presence of fructose)
To 5ml Seliwanoff’s reagent, add 10 drops of urine and heat for 30 seconds. Cherry
red colour indicates the presence of fructose.
Note: observe the colour of normal urine, abnormal urine (containing fructose only and
glucose only).
Question:
1. Why does glucose also answer for Seliwanoff’s resorcinol test after prolonged
heating?
Dip the test strip in urine sample. Remove excess urine by touching the side of the
container.
Compare the colour developed with the given colour chart in the bottle (read the
instructions)
Glucose O2
Glucose oxidase
Oxidised Chromogen
H2O
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Globulins
Haemoglobin
Bence-Jones proteins
Note: Normal adult urine contains <150 mg of total protein in 24h urine sample. Of this
15-20 mg is albumin.
Fill ¾ of a test tube with the urine sample. Heat the top 1/3 of the urine in the test tube to
boil. Observe the change.
Note:
Any increase in the turbidity in the upper 1/3 compared to the lower unheated part
indicates the presence of proteins and phosphates. When about 3 drops of acetic acid is
added turbidity due to phosphate disappears. If turbidity remains it indicates the
presence of proteins.
Question:
1. What is the basis for turbidity?
Mix 1ml urine with 2-3 drops of 3% Sulphosalicylic acid. Turbidity indicates the
presence of proteins.
False positive:
Patients on certain drugs (penicillin, tolbutamide)
Presence of high concentration of urates in urine.
3.3 Heat coagulation test [Specific test for Bence-Jones protein (BJP)]
Boil 5ml of faintly acidic urine (check the pH). BJP Precipitate at 40o C, maximally at
60 oC. It disappears at 100 oC and reappears on cooling.
Note: Filter the boiling urine rapidly to remove if any albumin present.
Questions:
1. What are Bench-Jones, Proteins (BJP)?
2. Give one clinical condition where BJP can be present in urine?
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3. Ortho–toludine test (test for haemoglobinuria)
Test sample: Boil 2 ml of urine and cool & Control : Take 2 ml of distilled water.
To both tubes add 4 drops of freshly prepared 4% orthotolidine solution in glacial acetic
acid followed of drops of 10% H2O2 solution. A bluish colour will develop. Observe
within two minutes.
Ketone bodies are detected in urine of persons suffering from diabetic ketoacidosis and
persons on high fat diets or prolonged starvation.
4.1 Rothera , s test (Preliminary test- acetone , acetoacetic acid both will answer)
Add crystals of ammonium sulphate to 5ml of urine and mix in a test tube until
saturation.
Add 8 drops of 5% sodium nitroprusside solution and mix.
Lay (along the test tube wall) 1ml of conc. ammonia solution without shaking.
A purple ring at the junction of liquids indicates the presence of one or both ketone
bodies.
On heating purple colour due to acetoacetate disappears, but colour due to salicylates
persist.
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Sprinkle little amount of finely powdered sulphur on the surface of the urine in a tube.
Sulphur powder sinks in the presence of bile salts, and floats on normal urine.
Note: above test depends on the Surface tension reducing property of bile salts.
Question:
1. State a clinical condition where bile can appear in urine.
Note: when an ion chloride in acid solution is added to a precipitate from urine
containing bilirubin, green colour is formed.
Questions:
1. What is the purpose of doing the Ehrlich’s test?
2. Is it possible to get a positive response to a normal urine sample?
3. How does Urobilinogen from?
4. List a single method for screening the urine for abnormal constituents?
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Purpose of the test, for use by the public is for screening. Complete diagnosis may
require professional experience and judgment followed by further tests.
Reference: http://uristik.com/acatalog/URINE_TESTING_INFORMATION.html
Exercise:
You are provided with an abnormal urine sample containing an abnormal constituent.
Confirm the abnormal constituent using the correct preliminary and confirmatory
tests.
Question:
What condition can give rise to the presence of this abnormal constituent in urine?
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