Practical Manuel

MBBS

Phase I

DEPARTMENT OF BIOCHEMISTRY

FACULTY OF MEDICAL SCIENCES

UNIVERSITY OF SRI JAYEWARDENEPURA

2011

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BIOCHEMISTRY PRACTICALS

The Biochemistry practical manual is for the undergraduates during the Phase 1 of the MBBS degree course. The manual contains the procedures of laboratory practicals as well as the contents of the guided learning sessions (GLS)/ fixed learning modules (FLM) that will be carried out in the Department of Biochemistry under different modules.

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TABLE OF CONTENTS1st 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

Metabolism, Endocrine and Nutrition Module GLS 1 – Introduction to Disorders of Carbohydrates and Lipid Metabolism GLS 2 - Catabolism and Excretion of Nitrogenous Compounds FLM – Thyroid Hormones GLS 3 – Diabetes Mellitus GLS 4 – Introduction to Endocrine Disorders Practical 7 – Determination of Glucose in Urine and Serum Practical 8 – Diet

Gastro Intestinal Tract Module Practical 9– Estimation of liver enzymes and constituents of bile.

4th Term

Renal Module Practical 10 – Analysis of Abnormal Constituents in Urine

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1st Term

FOUNDATION MODULE

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Practical - 1INTRODUCTION TO LABORATORY

A. Safety in the laboratory.

B. Commonly used laboratory equipment and their applications.

A. SAFETY IN THE 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.

Laboratory Safety Procedures:

1. Each student should take special care about personal hygiene.

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).

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B. COMMONLY USED LABORATORY EQUIPMENT AND THEIR APPLICATIONS Identification and Usage

1. Bunsen Burners – to heat or boil solutions in containers

2. Clamp and Holders – to hold the glassware in a secure manner

3. Porcelain dishes – to evaporate solutions 4. Watch glasses – weigh small quantities 5. Spatulas – to ease the handing of small amount of substances

6. Stirring rods – to mix and make homogeneous solution

7. Water baths – incubate samples at a desired temperature in-vitro e.g. enzyme reactions at 37 C.

8. Urinometer – to measure the specific gravity of urine 9. Pipette filler - to pipette solutions in and out of the pipettes

10. Filters

(i) Filter papers – to filter solutions

(ii) Muslin cloth – to filter solution of great impurities

11. Measuring equipment

(i) Micropipettes – to transfer very small quantities of liquids accurately. Commonly used for serological samples.

(ii) Pasteur pipettes – to transfer solution into another glassware such as measuring cylinder or volumetric flask.

(iii) Graduated pipettes – to transfer a fixed amount of small volumes Eg 1mL, 2mL, 5mL, 10mL.

(iv) Burettes – to transfer varying quantity of solution. Eg in titration

(v) Measuring Cylinders – to measure volumes in larger quantities Eg 10 mL, 100 mL. (vi) Beakers – to measure very large amount of solutions.

Eg 250 mL, 500mL, 1 L, 2L, are available

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12. pH Measurement

(i) pH papers (universal/litmus) - to measure an approximate pH

(ii) Lovibond comparator - to measure pH of a solution by comparison (Eg. urine)

(iii) pH meter - to measure pH accurately

13. Electrophoretic apparatus

Used to separate molecules according to the charge / molecular mass ratio (e/m). E.g. paper / gel electrophoretic apparatus

14. Weighing scale

(i) Electrical weighing scale – to measure substances in gram quantity

(ii) Analytical weighing scale – to measure very small amount (Eg g/mg)

15. Absorbance measurements

(i) Colourimeter

Uses colour filters with fixed wavelengths to measure absorbance of a coloured solution in the visual spectrum.

(ii) Spectrophotometer

Uses a specific wavelength to measure the absorbance of the solution. It can measure absorbance in the visual as well as in the ultra violet range, depending on the instrument.

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Practical - 2

CELL, pH AND BUFFERS1. pH

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.

pH: pH is a measure of the concentration of hydrogen ions (H+ /protons)in a solution.

Numerically it is the negative logarithm of that concentration expressed in moles per liter (M).

From the definition: the higher the hydrogen ion concentration the lower the pH and vice versa.

Water even in its purest form is weakly ionized as follows.

H2O H+ + OH-

According to the law of Mass Action,

K (dissociation constant) = [H+] [OH-] [H2O]

Since H2O is large and remains practically unaltered.

[H+][OH-] = Constant Kw

Kw is referred to as the ionic product of water Kw = 10-14 at 25C (from conductivity measurements)

At neutrality [H+] = [OH-] Then [H+] 2 = 10-14 [H+]= 10-7 (it is assumed that activities of ions are approximately equal to their concentration in dilute solution). When pH < 7, the solution is said to be acidic

PH > 7, that solution is said to be alkaline.

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pH = - log [H+] = log 1 [H+]

Concentration of 1 x 10-14 1 x 10-7 1x 100 OH- (moles/liter)

pH 0 7 14

Concentration of 1 x 100 1 x 10-7 1x10-14 H+ (moles/liter) (as in pure water)

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.

2.1 Natural indicators

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.

Some useful indicators

Indicator pH Range Colour change

Thymol blue (acid range) 1.2-2.8 Red YellowMethyl orange 3.1-4.4 Red OrangeBromophenol blue 3.0-4.6 Yellow PurpleCongo red 3.0-5.0 Violet Red-OrangeBromo cresol green 3.6-5.2 Yellow BlueMethyl red 4.3-6.1 Red YellowBromo cresol purple 5.0-7.6 Yellow PurpleLitmus 5.0-8.0 Red BlueBromothymol blue 6.0-7.6 Yellow BluePhenol red 6.7-8.3 Yellow RedCresol red 7.2-8.8 Yellow VioletThymol blue (alkaline range) 8.0-9.6 Yellow BluePhenolphthalein 8.2-10.0 Colourless Pink

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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:

What is the disadvantage in using pH paper to measure the pH of urine?

3.2. Lovibond Comparator – Demonstration

To 10 ml sample add 0.5ml of appropriate indicator solution.

For the range of pH 4.4 – 6.0 use methyl red For the range pH 6.0 – 7.6 use Bromo thymol blue For the range of pH 6.8 – 8.4 use phenol red.

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.

3.3. Electrometric method (pH meter) – Demonstration

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.

How do buffer solutions work?

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 (pK1

a).

It is expressed in the Henderson – Hasselbalch equation.

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Where pKa = -log Ka (Ka = dissociation constant of th

A buffer has maximum buffering capacity, when [Salt] = 1 [Acid]

i.e. pH = pKa + log 1 pH = pKa

The effective buffering range is pH = pKa ±1

4.1. Physiological buffering action of serum

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:

What is a physiological buffer?

Why did not the serum sample give a colour change even after adding the same number of acid / base drops to that tube?

5. CHANGES IN CELL MEMBRANE INTEGRITY WITH DIFFERENT SOLVENTS

a) Take 3 ml of each of following solutions to separate test tubes. a) Buffer – pH 7.4 b) H2O c) 0.1 M NaOHd) 0.1 M HCl

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

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pH= Pka + log [Salt][Acid]

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.

% Neutralization [A-] log [A-] pH [HA] [HA]

Eg. 1 1/99 -2.00 3.0010 ……. ……. …….20 ……. ……. …….30 ……. ……. …….50 ……. ……. …….70 ……. ……. …….80 ……. ……. …….90 ……. ……. …….99 ……. ……. …….99.9 ……. ……. …….

a) When half the amount of acid has been neutralized what is the pH of the solution?

b) Is this value in anyway related to the pKa of the acid?

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

(i) Half neutralization (ii) Complete neutralization

d) When this acid is almost completely neutralized what is the pH of the solution?

5. Using the Henderson Hasselbalch equation, calculate the pH values of the following mixtures (Pka of acetic acid is 4.74)

a) 22mL of 0.1 M CH3COOH + 78mL of 0.1 M CH3COONa

b) 37mL of 0.1 M CH3COOH + 63mL of 0.1 M CH3COONa

c) 85mL of 0.1 M CH3COOH + 15mL of 0.1 M CH3COONa

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Practical - 3

TESTS FOR CARBOHYDRATES AND LIPIDS

1) TESTS FOR SUGARS

Reduction of copper salts: When cupric hydroxide in alkaline solution is heated it is converted to cupric oxide. If a reducing sugar is present, the cupric oxide is reduced to cuprous oxide.

1.1 Benedict’s Test

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.

1.2 Barfoed’s Test

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.

1.3 Seliwanoff’s Test

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.

1.4 Tests for polysaccharides

(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?

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Summary of the test for carbohydrates.

Test

Glu

cose

Fruc

tose

Gal

acto

se

Pent

ose

Mal

tose

Lac

tose

Sucr

ose

Star

ch

Dex

trin

Gly

coge

n

Benedict’s Test + + + + + + - - - -Barfoed’s Test + + + + - - - - - -Seliwanoff’s Test

- + - - - - - - - -

Iodine Test - - - - - - - + + +

2) TESTS FOR LIPIDS

2.1. Presence of glycerol: (triacylglycerol, phospholipids)

2.1.1 Acrolein Test Place 1 g solid KHSO4 in a dry test tube and add 2 drops of glycerol. Heat slowly at first and then vigorously. Note the smell of acrolein. Repeat the test using 8 drops of coconut oil in place of glycerol.

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:

How does oil become rancid?

What steps could you take to prevent oil and fats becoming rancid?

2.2 Unsaturation of fats and oils

2.2.1 Iodine absorption Dissolve about 0.1 g fat in 5mL CHCl3. Add Hubl’s iodine solution drop wise, shaking the tube with each addition. Continue until the iodine colour persists. Count the number of drops of iodine solution that you had to add before the colour appeared. Compare with a control tube containing only 5mL of CHCl3 and one drop of iodine without the lipid.

Carry out this test with a) Coconut oil b) Butter c) Sesame oil d) Soya oil e) Margarine

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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 Test for phospholipids

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.

2.4.2 Test for cholesterol

(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).

(b) Liberman – Burchard test (use dry test tube) Add 10 drops of acetic anhydride and 2 drops of con. H2SO4 to 2mL of a solution of cholesterol in CHCl3. Note the changes in colour (red to blue to bluish green).

Questions:

What is the relationship between lecithin and respiratory distress syndrome (RDS)?

What foods contain high amounts of cholesterol?

Explain the amphipathic nature of cholesterol.

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Practical - 4

TESTS FOR AMINO ACIDS AND PROTEINS

Physical properties of protein

Precipitation of proteins: Proteins can be precipitated by various methods.

(i) Neutralization of the charges of protein and subsequent dehydration (ii) Proteins may be precipitated by heavy metals: e.g. Zinc proteinate by Zinc

Sulphate, protein tungstate by tungstic acid etc. (iii) Dehydration, denaturation : e.g. alcohol (iv) Flocculation and coagulation (v) Isoelectric pH (vi) Precipitation by neutral salts: e.g. (NH4)2SO4

1) PRECIPITATION BY SALTS (Dehydration)

i) To 5 mL of protein solution add an equal amount of saturated (NH4)2SO4 solution. The ovoglobulin is precipitated. This test is referred to as the half saturation test.

ii) Filter the precipitate iii) To the filtrate add solid ammonium sulphate and saturate it. Ovalbumin is precipitated. This is called the full saturation test.

Know the principle behind: Albumin being more hydrated than globulin requires more salt for dehydration. Serum albumin and globulin may be separated in this manner.

2) PRECIPITATION BY HEAVY METALS

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.

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Note: However, if the electrolyte (NaCl) used in dissolving the protein is removed by dialysis, alcohol is not capable of precipitating the protein.

5) CHEMICAL PROPERTIES: COLOUR REACTIONS

5.1. Test for amino acids (Ninhydrin Test)

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.

5.2. Test for peptide bonds (Biuret Test)

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.

5.3. Test for protein (Xanthoprotein test)

To 3mL protein solution, add 2 mL Conc. HNO3. Boil and cool. What is your observation?

5.4. Test for proteins (Heller’s test)

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.

5.5. Identify the samples

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

Test / Experiment Amino acids Polypeptides Proteins Ninhydrin Test + - -Biuret Test - + +Sulphosalicylic acid - - +Trichloroacetic acid - - +Picric acid - - +Hellers Test - - +

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6) ISOELECTRIC PRECIPITATION

What effect on (a) the charge and (b) the solubility of a protein would you expect if the pH of the protein solution is altered?

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 papersc) 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:

Casein solution (1%) w/v) HCl (0.1 M)NaOH (0.1M) HCl (0.05M) Universal indicator paper Bromocresol green indicator (pH range ≤4.0 yellow≥ 5.6 blue)

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?

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Practical - 5

ENZYMOLOGY I

Using salivary amylase, investigate the effects of,

1. Substrate concentration 2. pH 3. Temperature 4. Activators and inhibitors

Starch Salivary Dextrins / Maltose (Iodine test) amylase

The rate of the reaction rate of disappearance of substrate / min (iodine test) rate of appearance of product / min (Benedicts test)

Collection of dilute saliva

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.

1) FINDING THE ACHROMIC POINT

Setup two tubes containing A. 3mL dilute saliva B. 3mL dilute saliva, boiled for 10 min, and cooled.

Steps to follow:

a) To each tube add 3mL of 1% starch solution. b) Immediately remove a drop from each tube with a glass rod and test with iodine. c) Place tubes in a water bath at about 37 C. Remove drops from each tube at 30

second intervals and test with iodine. d) Observe the colour change from blue to violet, to red and finally becomes

colourless. This is the “Achromic point”. Note the time taken to reach the achromic point.

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.

** Use this dilution of saliva for the tests that follow.

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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.

What is the optimum pH of salivary amylase?

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)

5) EFFECTS OF ACTIVATORS AND INHIBITORS

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.

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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.

1) ACTIVITY OF TRYPSIN – USING CONGO RED FIBRIN AS SUBSTRATE

a) Set up 5 test tubes as follows.

Tube No. Enzyme Add1 2mL Trypsin 2mL of 0.1M HCl2 2mL Trypsin 2mL H2O3 2mL Trypsin 2mL 0.05M Na2CO3

4 2mL Trypsin (boiled and cooled) 2mL 0.05M Na2CO3

5 2mL H2O 2mL 0.05M Na2CO3

b) To each tube add a shred of Congo red fibrin and incubate the tubes in a bath at 37 0C for 30 min.

c) Explain the observations you make on each tube.

2) EFFECT OF pH ON ACTIVITY OF PEPSIN

a) Set up 6 tubes as follows.b)

Tube 1 & 2 Tube 3 & 4 Tube 5 & 60.4% HCl 3mL 1mL 4 dropsDistilled water - 2mL 3mL(pH of the medium) 1.3 1.8 2.51

c) Add to each tube a shred of Congo red fibrin.d) To tube 1, 3 and 5 add 1mL of 0.5% pepsin solution.e) Incubate at 370C for 30 mins.f) Comment on your observations.

Congo red : Blue < pH 4 < Red

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3) OPTIMUM pH FOR TRYPSIN ACTIVITY (Demonstration)

a) Set up tubes as follows;

Tubes Solution Approximate pH1 and 6 1mL water 72 and 7 1mL 0.5% Na2CO3 83 and 8 1mL 10% Na2CO3 94 and 9 1mL 2 % Boric acid 55 and 10 1mL 0.4% HCl 1

b) Add a shred of Congo red fibrin to each tube.c) To tubes 1 -5 add 1mL 0.5% trypsin solution.d) To tubes 6 – 10 add 1mL of 0.5% boiled and cooled trypsin solution.

Explain your observations. What is the optimum pH for trypsin?

4) EFFECT OF TEMPERATURE ON PEPSIN ACTIVITY (Demonstration)

a) Set up tubes as follows;

Solution Tube A Tube BCoagulated egg white 5mL 5mL 0.8% HCL 5mL 5mL 0.5% Pepsin solution 2mL 0 0.5% Pepsin solution 0 2mL

(boiled and cooled) Incubate at 37˚C for 30 minutes .compare the 2 tubes and explain observation.

5) CLOTTING OF MILK (Demonstration)

a) Set up tubes as follows;

Solution Tube 1 Tube 2 Tube 3 Tube 4

Milk 5mL 5mL 5mL 5mLCaCl2 solution 1 drop 1 drop 1 drop 1 dropAcetate buffer, pH 5 5mL 5mL 5mL 5mL Pepsin solution 1mL - - -Pepsin solution(boiled & cooled) - 1mL - -Trypsin solution - - 1mL Trypsin solution (boiled & cooled) - - - 1mL

b) Incubate the tubes at 37 C for 5 min.

c) Explain your observations.

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6) PRODUCTS OF PEPSIN AND TRYPSIN DIGESTION

Solutions of casein in water (0.5% w/v) digested with - 1% Pepsin solution at pH 1.3 (sample A) - 1% Trypsin solution at pH 8 (sample B)

for four days have been provided.

Steps as follows:

a) Take 25mL of sample A, B in boiling tubes and boil.b) Saturate with (NH4)2SO4 crystals.c) Filter the precipitates.d) Boil the filtrate with an equal volume of 0.5% BaCO3 and filter the BaSO4.e) Divide the filtrate in to 3 portions.f) Dissolve the precipitate from step 3 in water and divide in to 3 portions.g) Test each portion obtained in step 5 & 6 with

(i) Ninhydrin(ii) Biuret reagent(iii) 10% Trichloroacetic acid

Questions:

Comment on your observation.

What are the products of peptic and tryptic digestion?

By means of equation indicate the difference between the actions of the two enzymes.

State the differences between an endopeptidase and an exopeptidase.

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3rd Term

METABOLISM, ENDOCRINE AND NUTRITION MODULE

AND

GASTRO-INTESTINAL TRACT MODULE

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Guided Learning Session - 1

INTRODUCTION TO CARBOHYDRATES AND LIPID

METABOLISM

Disorders of Carbohydrate Metabolism:

Defects in Fructose metabolism

Galactosaemia

Glycogen storage diseases

Glucose 6- Phosphate Dehydrogenase (G6PD) Deficiency

Lactose intolerance

Disorders in Lipid Metabolism:

Disorders of fatty acid oxidation

Obesity

o Factors Associated with obesity

o Body Mass Index

Atherosclerosis

o Steps in the formation of atherosclerotic plaque

o Risk factors for atherosclerosis

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Guided Learning Session - 2

CATABOLISM AND EXCRETION OF NITROGENOUS

COMPOUNDS

Catabolism and excretion of haem

Haem metabolism

Major causes of Jaundice

o Hepatic causes

o Pre – hepatic causes

o Post – hepatic causes

Reasons for investigating for Neonatal jaundice

Causes of un-conjugated hyperbilirubinaemia in new born

Van den Bergh test – Direct and Indirect tests for conjugated and

total bilirubin (principle)

Purine nucleotide metabolism and uric acid synthesis

Major inherited metabolic diseases associated with

hyperuricaemia and development of gout

Metabolism and excretion of creatinine

Increased creatinine - report

Biochemical test for Creatinine in urine– Jaffe’s test

Metabolism and excretion of urea

Disorders associated with urea cycle

Biochemical test for urea in urine

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Fixed Learning Module

THYROID HORMONES

At the end of the FLM the student should be able to identify and list the main

features of

Maternal thyroid function during pregnancy

Foetus and thyroid gland

Thyroid deficiency and foetal development

o Isolated maternal hypothyroidism

o Isolated foetal hypothyroidism

o Combined maternal and foetal hypothyroidism (iodine

deficiency)

Role of thyroid hormones in brain development

Hyperthyroidism in pregnancy

Fasting and thyroid hormones

Tests to measure serum thyroid hormones

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Guided Learning Session - 3

DIABETES MELLITUS

Formation of Advanced Glycation End Products (AGE s)

Biochemical mechanisms of development of long term diabetic complications

Eye and eye sight

o Diabetic retinopathy

o Diabetic cataract

Diabetic nephropathy

Diabetic neuropathy

Micro - angiopathy in diabetes mellitus

Formation and structure of Fructosamine

Formation and structure of Haemoglobin A1c

Determination of HbA1c in the laboratory (principle and methods)

Tests for diabetes mellitus (methods and graphs)

Glucose Challenge Test (GCT)

Oral Glucose Tolerance Test (OGTT)

Patterns of OGTT curves

Normal GTT curve

GTT curve in severe diabetes mellitus

GTT curve in mild diabetes mellitus

Flat GTT curve

Lag curve

Curve in renal glucosuria

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Practical - 7

DETERMINATION OF GLUCOSE IN URINE AND SERUM

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. Test for reducing substances in urine

Reducing substances present in abnormal urine could be a. Reducing sugars : hexoses, pentosesb. Other substances : Vitamin C, homogenistic acid, salicylic acid

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.

Observe the colour of the precipitate and colour of the supernatant.

Colour of Precipitate Colour of Solution Approximate Strength

No precipitate Blue with green

opalescence

0.1%

Slight yellow precipitate Blue on standing 0.2%

Slight orange precipitate Green (Blue on standing) 0.3%

Definite orange precipitate Green (Blue on standing) 0.5%

Deep orange precipitate Green (Blue on standing) 1.0%

Bright red precipitate Blue colour almost

disappears

2.0%

Questions:

What is the basis of Benedict’s test?

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.

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1.3 Clinistix strip test (Confirmatory test for the presence of glucose)

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:

Note: Colour appearing after 2 minutes, does not have any significance.

Questions: What are the substances impregnated in the strip?

Why is Clinistix strip test specific for glucose?

2. Determination of glucose in serum

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.

MethodPipette 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.

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Glucose O2

Gluconic acid H2O2 Chromogen

H2O Oxidized chromogen

Glucose oxidase

Peroxidase

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

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Practical - 8

DIETFood 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.

Analyze your food record in terms of nutrients and calorie intake in comparison with the RDA’s (Recommended Daily Allowances).

Calculate your BMI. Do you see ways of improving your diet?

Note: Students are requested to bring their own calculators.

Reference tables: Please refer the food guide and food comparison tables to calculate RDA values.

Eg: Food Comparison Tables of Sri Lanka – MRIFood and Nutrition – T.W.Wickramanayake(Text book)(Annexure 1)

Food Guide Tables: Points to guide (A) One tea cup full of raw provisions (approximate dry weight)

Rice 90gPulses 100gLeafy vegetables 80gOther vegetables 200gCoconut (scraped) 25gLean meat/ fish 50gDried fish 75g

(B) RiceOne saucer of boiled rice weight = 225gUncooked 100g rice weight = 270g of boiled rice100g raw rice = 360 kcal

(C) SnackCake 1 slice contains 4 teaspoonfuls (tsp) of sugarCoke (300mL contains 7 tsp sugar)Biscuit contain ¾ tsp sugar One tsp (sugar) = 5g (19 kcal)White sugar contains 387 kcal/ 100g

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(D)

Food Quantity Protein (g) Energy (Kcal)White bread 1 slice (=1/4 inch) 3 70

MeatLean meat 1 oz (28 g) 7 50

Medium fat 1 oz 7 75

Fat PUFA 1 tsp (5mL) - 45Saturated 1 tsp (5mL) - 45

MilkWhole milk

240mL = glass8 150

Low fat 8 120Skimmed 8 90

Whole egg one 7 160

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Practical - 9

ESTIMATION OF LIVER ENZYMES AND CONSTITUENTS OF BILE

1. Constituents of Bile

1.1 Hay’s test (for bile salts):

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.

1.2 Fouchet’s test (test for bile pigments):

Slightly acidify 2mL of urine with dil. acetic acid. Add 1mL of 10% BaCl2, 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.

1.3 Ehrlich’s test (test for the presence of urobilinogen in urine): 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.

Note: Normal urine also contains urobilinogen.

2. Estimation of Liver Enzymes

Note: Either method 1 or method 2 will be carried out.

Method 1 - Kinetic method

2.1 Test for Aspartate Transaminase (AST)

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

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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:

2-Oxoglutarate + L-Aspartate Glutamate + Oxaloacetate

Oxaloacetate + NADH + H+ L-Malate + NAD+

Materials:AST reagentUnhaemolized serum samplesSpectrophotometer

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:

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.

2.2 Test for Alanine Aminotransferase (ALT)

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).

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AST/GOT

Malic dehydrogenase

.Principle:

2-Oxoglutarate + Alanine L-Glutamate + Pyruvate

Pyruvate + NADH + H+ Lactate + NAD+

Materials:ALT reagentUnhaemolized serum samplesSpectrophotometer

Procedure:

1) Set the wave length of the instrument at 340 nm.2) Zero with deionized water.3) For each sample dispense 2.0mL of reconstituted ALT 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: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.

Method 2 - Colourimetric Method

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.

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Lactate dehydrogenase

ALT/GPT

Principle:

2-Oxoglutarate + L-Aspartate Glutamate + Oxaloacetate

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:

Vial R1: AST/TGO substrateVial R2: ALT/TGP substrateVial R3: Colouration reagentVial R4: Standard solution

Procedure:

Table 1: Standard Curves establishment

Let stand reagents and specimens at room temperature.Pipette into test tubes (mL)

Tube No 1 2 3 4 5 6Demineralized water 0.2 0.2 0.2 0.2 0.2 0.2R1 or R2 1 0.9 0.8 0.7 0.6 0.5R4 (standard) - 0.1 0.2 0.3 0.4 0.5R3 (colourant) 1 1 1 1 1 1Mix. Let stand for 20 minutes at room temperature. Add:NaOH 0.4 N 10 10 10 10 10 10Mix. Let stand 5 minutes and read absorbances at 505 nm against water.TGO units 0 30 70 135 225 350TGP units 0 40 80 140 225 325There’s no need to plot a new curve at each determination.

Table 2:

Let stand reagents and specimens at room temperature.

Pipette into test tubes TGO TGPReagent R1 1 mL -Reagent R2 - 1 mLIncubate for 5 minutes at 37 C. Add:Serum 200 L 200 LMix and incubate at 37 C during Exactly 1hr Exactly 30 mins

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AST/TGO

ALT/TGP

Reagent R3 1 mL 1 mLMix and let stand 20 minutes at room temperature. Add:NaOH 0.4 N 10 mL 10 mLMix let stand 5 minutes and read absorbances at 505 nm against water.

Note:Blank reagent: replace serum by demineralized water in table 2.

Calculation:

Plot standard curve on millimeter paper (absorbances) or semi-log (% of transmission) handling as indicated in table 1.

Abscissa (x axis): Number of units (International Units (IU) / Litre (L))Ordinate (y axis): Absorbances (or % of transmission)

Transfer “Assay” absorbances or % of transmission on standard curve and calculated AST or ALT activity in UL/L.

Expected values:

ALT At 30C At 37CNewborns, Infants 9 -32 13- 45Men 7 - 28 10 - 40Women 5 - 25 7 - 35

AST (IU/L) At 30C At 37CNewborns 25 - 75 39 - 117Infants 15 - 60 23 - 94Adult 8 - 20 13 - 31

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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.

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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.

Investigations Reference range

Serum Albumin 25 g/L 38 – 55 g /L

Bilirubin 260 μ mol/L 3.4 – 22 μ mol/L

ALP 315 IU/ L 38 - 126 IU/ L

AST 134 IU/ L 5 – 35 IU/ L

GGT 360 IU/ L 8 – 78 IU/ L

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.

Hepatic decompensation may be precipitated in chronic liver disease by sepsis, bleeding

in to the gut, for example from varices, erosions and ulcers and by various drugs

including diuretics. Diuretics may be given to treat ascites, a common feature of chronic

liver disease, but must be used with caution. The pathogenesis of ascites is complex.

Portal hypertension due to hepatic venous obstruction causes splanchnic arterial

vasodilation. This leads to under filling of the arterial circulation and stimulation of

renal sodium and water retention. Hypoalbuminaemia may also contribute.

(Abstracted from Clinical Biochemistry, William. J. Marshall, 4th Edition)

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4th Term

Renal Module

41

Practical - 10

ANALYSIS OF ABNORMAL CONSTITUENTS IN URINE

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).

The presence of abnormal constituents in urine is used to diagnose certain diseases. eg; nephritic syndrome.

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.

Storage of urine sample:

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.

1. Measurement of specific gravity and total solids

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.

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Calculate roughly the total solids as follows;

State the importance of measuring specific gravity of urine.

2. Tests for reducing substances in urine Reducing substances present in abnormal urine could be

1. Reducing sugars – hexoses, pentoses2. Other substances – vitamin C, homogentisic acid, salicylic acid.

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.

Colour of precipitate Colour of the solution Approximate strength of reducing sugars (%)

No precipitate Blue with green opalescence 0.1

Slight yellow Blue on standing 0.2

Slight orange Green (Blue on standing) 0.3

Definite orange Green (Blue on standing) 0.5

Deep orange Green (Blue on standing) 1.0

Brick red Blue colour almost disappears 2.0

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Total solids = 2.66 X (last two figures of the four figured number expressing specific gravity)

Eg: If specific gravity = 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

Eg:Reading on urinometer = xRoom temperature = 32 ˚CTemperature correction = 32 ˚C -20 ˚C = 4

3

Specific gravity = 1000 + x + (0.001 * 4) 1000

Note: Urinometer should not touch the walls of the container.

Questions:1. What is the basis of Benedict’s test?2. What are the simple sugars that will answer for Benedict’s test?

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?

2.3 Clinistix strip test (confirmatory test for presence of glucose)

Glucose oxidase method:

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)

3. Test for proteins

Note: Cells, casts in the urine must be removed by centrifugation before performing the test for proteins.

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Glucose O2

ChromogenGluconic acid

Oxidised Chromogen

H2O2

H2O

Glucose oxidase

Peroxidase

Different types of proteins that can appear in urine:

Albumin - in nephritic syndrome, diebetic nephropathy 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.

3.1 Heat precipitation test (Preliminary test)

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.

Add 3 drops of 10% acetic acid and observe.

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?

3.2 Sulphosalicylic acid test (confirmatory test)

Mix 1ml urine with 2-3 drops of 3% Sulphosalicylic acid. Turbidity indicates the presence of proteins.

Note: Proteins are precipitated by Sulphosalicylic acid. The degree of turbidity is an estimation of the quantity of proteins present in the urine sample.

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.

4. Test for ketone bodies (acetone , acetoacetic acid)

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.

4.2 Gerhardt’ s test (Confirmatory test)

To 3ml of urine, add 3% FeCl3 solution drop by drop.Mix while adding. A brown-red precipitate may form, filter and add 3% FeCl3 continuously to the filtrate until a colour (purple) appear. Heat and see whether the colour disappears or not. Purple colour indicates the presence of acetoacetic acid or salicylates.

On heating purple colour due to acetoacetate disappears, but colour due to salicylates persist.

Note: salicylates can interfere with the test.

4.3. Ketostix strips

Dip the strip in fresh urine sample. Remove excess urine by touching the side of the container. Compare the colour developed with the given colour chart after the given time. (Read the instruction).

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5. Test for the presence of the pigment/ bile in urine

5.1 Hay’s test (Preliminary test)

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.

5.2 Fouchet’s test (Test for bile pigment-Confirmatory Test)

Add 1ml of 10% BaCl2 to 2ml of acidified urine. Mix and filter. Spread out the 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.

5.3 Test for Urobilinogen

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?

SCREENING OF URINE FOR THE PRESENCE OF MANY ABNORMAL CONSTITUENTS - Dipstix method

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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.

The urinary constituents which can be identified: Leucocytes, Nitrite, Uroblinogen, Protein, pH, Erythrocytes, Hb, Specifc Gravity, Ketones, Bilirubin and Glucose. Tests are degraded by light. Only remove cap to extract a strip. Replace cap as soon as possible.

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?

Further reading: Varley’s Practical Biochemistry.

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