Laboratory Manual - PSAU · Biochemistry Laboratory Manual PHL ... Estimation of SGOT & SGPT...

Laboratory Manual

Biochemistry II

(PHL 224)

Dr/ Gamal A Gabr

Department of Pharmacology College of Pharmacy

Salman bin Abdulaziz University

1435-1436 / 2014 – 2015

Biochemistry Laboratory Manual PHL 224

Dr. Gamal Gabr 1

TABLE OF CONTENTS

i. Development of Skills 2 ii. Laboratory Safety 2-3 iii. General Laboratory Rules 3 iv. In Case of an Accident 3-4 v. Laboratory Reports 4 vi. Use of Micropipettes & Volumetric and Serological Pipettes 5 vii. Units, Amounts and Concentrations 6-10

S. No. Experiment Page No.

1. To perform Molisch test, Fehling’s test, Benedict’s test for identification of sugars.

11-12

2. A. To measure pH of tap water and distilled water using pH paper strips and a pH meter.

13-14

B. To prepare phosphate, citrate and carbonate buffer & measure their pH.

15-16

3. To detect the presence of amino acids qualitatively by Ninhydrin test, Xanthoproteic test, Hopkins-Cole Test, Millon’s test, Pauly’s test, Ehrlich test, Nitroprusside test, lead acetate test and Sakaguchi test.

17-19

4. To carry out qualitative tests to detect lipids in the given sample using Sudan III reagent.

20-20

5. Estimation of glucose content by kit method and glucometer in blood & urine.

21-22

6. Enzymatic hydrolysis of starch by α & β amylase. 23-24 7. Effects of temperature & pH on the activity of enzymes (catalase

from fresh potato). 25-26

8. Estimation of SGOT & SGPT activity by kit method in blood & urine.

27-28

9. Estimation of LDH activity by kit method in blood & urine. 29-30 10. Estimation of alkaline phosphatase activity by kit method in blood

& urine. 31-33

11. Estimation of total cholesterol, HDL-Cholesterol & Triglycerides content by kit method in blood & urine.

34-37

12. To estimate protein in the given sample by Folin--Lowry’s method.

38-39

13. To estimate the Urea in blood. 40-41

14. To test the given urine sample for the presence of ketone bodies. 42-42


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DEVELOPMENT OF SKILLS

Following is the broad perspective of acquisition of intellectual and motor skills. Due care is to be taken, that a student systematically studying the subject will acquire the skills enlisted below. A) Intellectual skills

1. To understand the concepts. 2. To understand the procedure for performance of experiments. 3. To interpret results of experiments. 4. To investigate and discriminate the various situations. 5. To acquire ability to plan and design the experiment.

B) Motor skills:

1. Handling and using correctly the instruments and equipments. 2. Measuring and recording accurately with the help of instruments/equipments 3. To follow systematic, hygienic and safe procedure of working. 4. To collect sample of blood & Urine.

LABORATORY SAFETY For the welfare of fellow students, and for your own well-being, each student is expected to follow a set of accepted laboratory precautions. In the lab:

1. Do not eat, drink, or apply cosmetics at any time. 2. Mouth pipetting is not permitted. 3. Wear protective eyeware at all times; chemical splash goggles must be worn

when working with solutions of strong acids or bases. 4. Do not sit on the lab benches. 5. Use chemicals with high vapor pressure only in the hood. 6. Handle and dispose of hazardous chemicals properly. Disposal containers are

provided. 7. When chemicals are spilled they should be wiped or swept up or both as soon as

possible. If the spillage is large, immediately notify an instructor. Sponge and brushes are provided and should be used to keep your work area clean.

8. Be aware of objects that can burn or give electrical shocks. 9. Do not use open flames near flammable chemicals. 10. In general, be alert to possible hazards from chemicals, glassware, electrical

connections, and flammable solvents. Read labels and observe suggested precautions.

11. Sweep up broken glassware as soon as possible and deposit it in the labeled container set aside for this purpose.

12. Never work alone.


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13. All solutions that you prepare must be labeled with date, your last name, and the type and concentration of the reagent. Unmarked solutions will be disposed.

14. Wipe your lab bench with a damp sponge at the end of each lab period. 15. Be familiar with location and use of safety items — location of safety showers,

eyewash stations, fire blankets, fire extinguishers, medical kit.

GENERAL LABORATORY RULES 1. Always wear a lab coat before enter the lab. 2. The instructors will make every effort to keep equipment in good working order.

It is your responsibility to read instructions for the use of equipment. Do not turn on an instrument until you have done this. Do not hesitate to ask questions of your instructor after you have read the instructions. If any equipment malfunction is noted, report this immediately to an instructor.

3. Used glassware should be rinsed with tap water and deposited carefully on the designated cart after all tape has been removed. Ink writing on glassware need not be removed.

4. Items stored in the cold room or deep freeze should be removed and disposed of when they are no longer needed.

5. Clean up after yourself after using any piece of equipment. Be considerate of others. All equipment should be left ready for immediate use by another student.

6. Please clean the lab after each class. Your instructor will advise you for saving or disposing your solutions.

7. Keep your work area clean. Your grades may be deducted if you keep littering your bench.

8. You must have a lab notebook for writing everything in black or blue ink (no pencils). When correcting your writing, scratch the error with a single line and write the correct one above or below the line.

9. Pay attention to the due date for each experiment. You will be penalized for late submitting your lab report.

10. You should finish your own lab report. Do not copy old lab reports, or copy your classmate’s report. Plagiarism is illegal and unethical. Any evidence of plagiarism in this class will result in your failure in the class. Further disciplinary actions will be taken upon your instructor’s decision.

IN CASE OF AN ACCIDENT 1. Report all injuries, even minor ones, to an instructor immediately. 2. In case of even minor laboratory accidents, you should go to Health Center for

treatment. 3. For any chemicals splashed in the eye, hold the eye open and flush immediately

with cold water by using the eye wash or the rubber hose attached to the water faucet at the end of each laboratory bench. Flush for at least 5 minutes and call for help from an instructor.


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4. For chemicals spilled on the skin or splashed into the mouth, again, flush with large amounts of cold water for 5 or more minutes. Call for an instructor.

5. For severe bleeding, apply pressure and call for help from an instructor. 6. For burns, flush with cold water and contact an instructor.

Note: Injury to students in the laboratory is not covered by University insurance. Students must provide their own insurance coverage.

LABORATORY REPORTS You are required for submitting a lab report for each experiment. Reports are due after one week from the date you have performed your experiment. There will be a 10% grade deduction upon your late submission. I also strongly recommend you use a graphic program to plot your graphs, in which all axes should be labeled and the proper units are displayed. Your lab reports should consist of the following sections:

1. The title of the laboratory report: this includes the title of your report, the course name and number, and your name and the date.

2. Objective section: the object will be a brief description of your experiment. 3. Principle section: Describe basic principle of the experiment. 4. Materials required: this section should describe the materials used, their strength. 5. Experimental Procedure: Describe the method used. 6. Calculation section: Calculations are required to be shown and explained. 7. Results section: this section presents your results that were obtained in your

experiments. 8. Clinical significance: in this section, you should discuss your findings and

compare your results with the normal values and write clinical relevance.


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USE OF MICROPIPETTES, VOLUMETRIC AND SEROLOGICAL

PIPETTES

Micropipettes The first portion of this lab involves becoming familiar with the use of micropipettes. Micropipettes accurately measure volumes ranging from less than 1 µl to greater than 1 ml, the types that you will use measure from 0.1-2.5 µl (P-2.5), 1-20 µl (P-20), 20-200 µl (P-200) and 100-1000 µl (P-1000). The disposable tips that fit on the end of the micropipettes are sometimes colored yellow and blue. The yellow tips (which are white for our experiments but are sometimes colored yellow) are for volumes of 1- 200 µl and fit on the ends of the P-20 and P-200 micropipettes but not the P-1000 micropipette. The blue tips (which are white for our experiments but are sometimes colored blue) are for volumes of 100-1000 µl and fit on the end of the P-1000 micropipette.

The following steps describe the use of the micropipette. Adjust the micropipette volume to the required setting. Press the tip on firmly with a slight twisting motion to ensure an airtight seal. The plunger on the top of the micropipette is then depressed to the first point of resistance and while holding the micropipette vertical the tip is placed just below the surface of the appropriate solution (1-2 mm for yellow and 2-4 mm for blue). The plunger is then slowly released and the solution is sucked up into the pipette tip, wait 2 seconds to ensure that the full volume has been drawn up into the tip. Once the plunger has been released the tip is removed from the solution (any excess liquid should be wiped from the side of the tip but keep the cloth away from the end) and transferred to the receptacle where the tip is placed on the side of the receptacle and the plunger is once again depressed to the point of first resistance, wait, then continue on past the point of first resistance (overshoot). With the plunger fully depressed withdraw the micropipette from the vessel allowing the tip to slide along the wall of the vessel. Allow the plunger to return to the top position.

Normally the tip would then be ejected by pressing the tip ejector button to prevent cross contamination of samples but for our purposes in this exercise it should only be necessary a few times. Attention should be given to the speed and smoothness during depression and release of the button, pressure on the button at the first stop, immersion depths (if the depth is incorrect bubbles may get into the sample), and minimal angle from the vertical axis.

Please try the above procedure a few times with each of the micropipettes using water as the solution to gain practice. Once the technique has been mastered, take your solution of water over to the balances and pipette out five times each of the following volumes in any order. Change the tip only when you change the volume.

i. 5.0 µl with the 1-20 µl micropipette ii. 20.0 µl with the 1-20 µl micropipette

iii. 20.0 µl with the 20-200 µl micropipette iv. 100.0 µl with the 20-200 µl micropipette


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Volumetric and serological pipettes

Volumetric pipettes are calibrated to deliver a specific volume of solution at a certain temperature and have a single line with no scale. With a rubber bulb (DO NOT USE YOUR MOUTH) draw liquid to a level 2-3 cm above the line, wipe the tip with a kimwipe (not necessary for your practices), touch the tip of the pipette on the inside wall of the container from which it was filled. Release liquid until the meniscus is directly on the fill line. Transfer the pipette to the inside of the second container and release the liquid, when the flow stops wait 5-10 seconds then touch the tip to the side of the vesicle. The pipette should then be removed from the container. The pipette is TD (“to deliver”) and therefore any remaining liquid should not be blown out of the pipette.

Fractional volumes are transferred with graduated pipettes, one example being a serological pipette. The procedure is the same as the volumetric pipette except that the solution starts at the desired amount and the pipette is blown out at the end.

Please try the above procedure a number of times with each of the pipettes using water as the solution to gain practice. Once the technique has been mastered, take your solution of water over to the balances and pipette out five times:

i. 10.000 ml with the volumetric pipette ii. 10.000 ml with the serological pipette.

UNITS, AMOUNTS AND CONCENTRATIONS Units and abbreviations The common units, and their respective abbreviations, used in biomedical sciences are based on the International System of Measurements (SI units): grams (g), meters (m) etc. Often it is necessary to deal with large quantities e.g. 1500 g or, more commonly, very small quantities, e.g. 0.0000015 g. In such cases, such numbers are either expressed by use of powers of 10 (positive or negative) or by use of the appropriate prefix.

Thus: 1500 g = 1.5 x 103 g = 1.5 kg (kilograms) And 0.0000015 g = 1.5 x 10-6 g = 1.5 µg (micrograms)

Note also that biomedical scientists normally express volumes and concentrations in terms of liters rather than in cubic measurements:

e.g. 1 liter, rather than 1 dm3 1 milliliter (1 ml) rather than 1 cm3

A concentration of 1 milligram per liter is also still commonly expressed in the form 1 mg/l rather than 1 mg.l-1 or 1 mg.dm-3.


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The commonly used prefixes are:

Prefix Name Which modifies an amount by Examples

M milli 1/1000th, i.e. by 10-3 mmol, mg, ml

µ micro 1/1000 000th, i.e. by 10-6 µmol, µg, µl

N nano by 10-9 nmol, ng

P Pico by 10-12 pmol, pg

F Femto by 10-15 fmol, fg

K Kilo by 1000 times, i.e. by103 kg

M mega by 106 MPa

The prefixes centi (10-2) and deci (10-1) are only commonly used in specific cases e.g. cm

Concentrations

The determination of the concentration of a substance in a biological fluid is central to many areas of medical and dental practice (e.g. electrolytes in serum or glucose in urine). It is important, therefore, that concentrations are expressed in clear unambiguous terms. There are several ways of doing this. The simplest is to express the concentration as the weight or mass of the substance per unit volume:

e.g. 10 g/l or 20 mg/ml or 2 µg/ml

Another way is to express the concentration of a solution or mixture in terms of per cent (%). This is a somewhat outdated method but you may still come across it, particularly if you read the older medical literature, so you should know what it means. Note that there are different types of % concentration:

% (v/v) (volume by volume) % (w/v) (weight by volume) % (w/w) (weight by weight)

A 1% (v/v) concentration is obtained by diluting 1 volume of a substance into 100 volumes (total) of solution, e.g. 1 ml ethanol diluted with water to a final volume of 100 ml gives a 1% (v/v) ethanol solution.

A 1% (w/v) concentration is obtained by dissolving 1 g of substance in a final volume of 100 ml solution, e.g. 1 g glucose dissolved in water to a final volume of 100 ml solution gives a 1% (w/v) glucose solution.

A 1% (w/w) concentration is obtained by mixing 1 g of substance with something else in a total weight of 100 g, e.g. 1% (w/w) salt in sand.


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Another old method of expressing concentration that you may still see (just look at the side of a tube of toothpaste) is "parts per million (ppm)". One ppm is one part of anything in one million parts of total material, e.g. 1 g of compound X in a million g total, or 1 liter of Y in a million liters total.

Moles and molarity By far the most important unit defining an amount of a biological substance is the mole, with the corresponding concentration being the molarity. As far as possible, the concentrations of specific compounds in serum, urine etc, are now expressed as molarities in clinical laboratories.

One mole of a substance is the molecular weight of that substance expressed in grams. Thus, the molecular weight of glucose is 180, so:

1 mole of glucose = 180 g 1 mmol glucose = 180 mg 1 µmol glucose = 180 µg 100 µmol glucose = 18,000 µg = 18 mg

Note the abbreviations: 1 mmol = 1 millimole; 5 µmol = 5 micromoles.

Concentrations in molarities are given by expressing the number of moles of the substance present in a defined volume of solution:

A 1 molar (1 M) solution contains 1 mole per liter (1 mol/l) A 1 millimolar (1 mM) solution contains 1 millimole per litre (1 mmol/l)

Note: mol and moles mean the same thing (an amount) and moles/litre (long winded

but correct) is a concentration and can be expressed as mol/l, or mol.l-1, or (best and simplest of all) — M. Ensure you know the distinction between concentrations and amounts.

So, if you dissolve 0.5 mol of a compound in one liter of solvent the concentration of the compound is 0.5 mol/l, or 0.5 M. 100ml of the solution contains 0.05 mol.

Prefix notation The value of the prefix notation can now be seen as it allows rapid mental calculations to be performed (after much practice!). The following concentrations are all the same:

0.5 M, 0.5 mol/l, 0.5 mmol/ml, 0.5 µmol/µl, 0.5 pmol/pl

You see that by scaling both the units (amount and volume) in the concentration up or down by a factor of 1000, the value of the concentration remains the same. If you only scale one of the terms (amount or volume), then you can express the same concentration in yet more ways:

0.5 mol/l = 0.5 mmol/ml = 500 µmol/ml = 500,000 pmol/µl


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Now, let’s say you wanted to determine the amount of cholesterol in the blood of a newborn infant. You know it will be around 5 mM. The detection limit of the method you are going to use is about 20 nmol and you can only take 20 µl of blood. Will this be enough?

5 mM cholesterol contains 5 mmol/l, or 5 µmol/ml, or 5 nmol/µl. It is easy to see now that 20µl contains 100 nmol of cholesterol - ENOUGH.

Examples How many µmol are dissolved in 2 l of a 20 mM solution?

20 mM = 20 mmol/l, so 2 l contain 40 mmol. 40 mmol = 40,000 µmol.

The molecular weight of NaCl is 58. How many mg are in 50 µmol of NaCl? 1 mol of NaCl is 58 g, so 1 µmol is 58 µg, so 50 µmol is 2,900 µg = 2.9 mg.

What is the molarity of a 1% (w/v) solution of glucose? (molecular weight = 180) 1% (w/v) contains 1 g in 100 ml and, therefore, 10 g in 1 liter. A 1 M solution of glucose contains 180 g/l, so 10 g/l represents a molarity of 10/180 = 0.056 M (or 56 mM).

Dilutions

This is another source of great confusion. Most experiments require you to make dilutions of reagents, either before use or as a consequence of the actual assay. When you are asked to make a ten-fold dilution of a reagent, the objective is to produce a solution that has a reagent concentration one-tenth of the original. It follows that the molecules of reagent that occupied a volume of "x" before must now occupy a volume of “10x". This is obtained by adding to one volume of reagent to nine volumes of diluent (sometimes referred to as a “1 plus 9” dilution or, more commonly, a "1 in 10" dilution).

When a solution of known concentration is diluted, it is obvious that the concentration will fall. Less obvious is the amount by which it falls! Take a typical example: A solution of a compound is maintained as a stock solution at 5 mM. What is the final concentration of the compond in which 0.15 ml of stock solution is mixed with 0.4 ml of buffer and 0.2 ml of water?

There are many ways to derive this answer. Here are a few different approaches — all equally acceptable. Take the one that seems most logical to you! i. The final volume is 0.75 ml, therefore the concentration of the substrate will

be 0.15/ 0.75 x 5 mM = 1 mM ii. 5 mM is 5 mmol/l, or 5 µmol/ml. Therefore 0.15 ml of stock solution contains

0.15 x 5 = 0.75 µmol. This is expanded into a volume of 0.75 ml and therefore there are now 0.75 µmol/0.75 ml, or 1 µmol/ml, or 1 mmol/l, or 1 mM

iii. The formula for a dilution is v1 x c1 = v2 x c2 (where c1 and c2 are the concentrations before and after dilution, and v1 and v2 are the volumes


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before and after dilution). Therefore, c2 = v1/v2 x c1, which works out to be c2 = 0.15 / 0.75 x 5 mM , i.e. 1 mM

Note that the last and first are identical, except that the latter defines the problem as a formula. It may be valuable for dilutions when a solution of known concentration must be diluted to a new concentration. The formula will give the new volume, but remember that part of that volume will come from the sample before dilution! Method 2 seems tortuous with this example, but it suits some to work this way.


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Experiment No. 1

Objective: To perform Molisch test, Fehling’s test, Benedict’s test for identification of

sugars.

Principle: Carbohydrates undergo dehydration reactions (loss of water) in the

presence of concentrated sulfuric acid. Pentoses and hexoses form five member oxygen containing rings on dehydration. The five member ring, known as furfural, further reacts with Molisch reagent to form colored compounds. Molisch test is used to detect carbohydrates in several substances.

All monosaccharides and many disaccharides with free aldehyde groups can reduce weak oxidizing agents like cupric ions (Cu2+ ion) in alkaline medium (Fehling's or Benedict's reagents) to produce red or orange colored precipitate of cuprous ions. These carbohydrates are called reducing sugars. Heating in a boiling water bath is necessary for these reactions.

Materials required: Glassware: Beaker, water bath, test tubes, graduated pipettes. Chemicals: Molisch reagent (1% α-naphthol in alcohol), concentrated sulphuric acid, distilled water, Fehling's solution A (7.93% copper sulphate in water), Fehling's solution B (250g sodium hydroxide and 320g sodium potassium tartarate in 500 ml water), Benedict's reagent (dissolve 173g sodium citrate and 100g anhydrous sodium carbonate in about 800 ml water, separately dissolve 17.3g copper sulphate in 100ml water, mix both the solution and make volume to 1000 ml with water).

Experimental Procedure: S. No. Test Observation Inference

1. Molisch test: Mix 2ml of sugar sample with 5 drops of Molisch's Reagent in a test tube. Add gently through the side by tilting the tube, about 2 ml of concentrated H2SO4 so as to form a bottom layer.

Violet / purple ring at the junction of two liquids

Sugar present.

2. Solubility: Compound + water

Soluble Insoluble

Mono and disaccharides present Polysaccharides present

3. Fehling’s test: 2 ml of Fehling's solution A + 2ml of Fehling's solution B+ 2 ml of Sugar solution Boil.

Yellow or brick red ppt is observed.

Reducing sugar present


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4. Benedict’s test: Take 5ml of Benedict's qualitative reagent, add 8 drops of sugar solution. Boil over a flame for 2 minutes or place in boiling water bath for 3 minutes. Allow to cool.

Green, yellow, orange or brick red ppt is observed

Reducing sugar present

Results: On the basis of above observations the given sample was found to be sugar.


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Experiment No. 2 (A)

Objective: To measure pH of tap water and distilled water using pH paper strips and

a pH meter.

Principle: pH is the acidity or alkalinity of a substance. A pH of seven is neutral. Any

number lower than seven represents an acidic substance while any number higher than seven represents an alkaline substance. There are substances which have the property of changing their color when they come in contact with an acidic or basic environment. These substances are called pH indicators. Usually, they are used as dissolved substances, as for instance phenolphthalein and bromothymol blue. Often, to measure the pH, special papers which have been soaked with indicators are used. These papers change color when they are immersed in acidic or basic liquids. This is the case of the well-known litmus paper. More recently, it has become possible to measure the pH with electrical instruments like the pH meter.

The pH meter is an electronic instrument supplied with a special bulb which is sensitive to the hydrogen ions which are present in the solution being tested. The signal produced by the bulb is amplified and sent to a liquid-crystal or an analog meter display. These instruments are much more precise and convenient to use than the indicating papers.

Materials required: 1. Tap water 2. Distilled water 3. pH meter 4. pH paper strips 5. Glassware

Experimental Procedure:

1. Measurement of pH by pH meter: The pH meter is put on and allowed to

warm and calibrate the probe using two standard solutions (pH 4, 7, and 10 buffers are recommended, dependant on the range). Calibration procedures vary by instrument, so following the manufacturer's instructions is highly recommended. BE SURE TO RINSE THE PROBE THOROUGHLY BETWEEN BUFFERS USING DEIONIZED WATER AND CAREFULLY BLOT THE PROBE DRY USING A KIM WIPE. pH meters should be calibrated before each use (before each series of samples, not between each sample itself) or when measuring a large range of pH. The standard buffer solution is taken in a beaker. The temperature of buffer solution is noted and electrodes are dipped in the solution. The selector switch is now turned to proper range 0-7 or 7-14. The meter will show the pH of buffer. Set the pointer to the exact value of pH of buffer. Bring the selector switch again to zero, clean the electrodes with water. Collect sample water in a glass or


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plastic container. Collect enough so the probe tip can be submerged in sample; either rinse the probe with deionized water (and blot dry) or with sample before inserting the probe into the collection vessel. Submerge the probe into the sample and wait until the pH reading on the meter stabilizes. Many meters have automatic temperature correction (ATC), which calculates the pH taking into account temperature, if your meter does not have this feature, you may need to adjust a knob on the meter to correct the pH for temperature. Record the measurement when the pH reading is stable.

2. Measurement of pH by pH paper strips: Using Litmus paper is simple. First

of all, it is necessary to immerse an end of it in the liquid you wish to examine and to remove it immediately. The pH of the liquid is determined by comparing the color of the paper to the scale of colors which is printed on its packet

Precautions: 1. The pH meter must be calibrated by checking against a standard buffer of know pH. 2. Keep the electrodes immersed in water when not in use.

Results: The pH of tap water & distilled water by pH meter was found to be ______ &

____ respectively and by pH paper strip was found to be ____ & ____ respectively.


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Experiment No. 2 (B)

Objective: To prepare standard buffer solution (phosphate, citrate and carbonate

buffer) and measurement of pH.

Principle: Buffer is the solutions that resist changes in pH when small amounts of acid

or base are added. There are two types of buffer. a. Weak acid and the salt of the same weak acid, (for example a solution containing

ethanoic acid and sodium ethanoate). This gives a buffer solution with a pH less than 7

b. Weak base and the salt of the same weak base (for example ammonia and ammonium chloride solution). This gives a buffer with a pH greater than 7

The first (acidic) buffer works in the following way. If an acid is added it combines its free hydrogen ions with the ions from the salt of the weak acid making molecular weak acid that cannot affect the pH. If a base is added the OH- ions from the base react with the H+ ions that are present from the weak acid dissociation. Having been removed from the solution this stimulates the weak acid to produce more H+ ions (Le Chatelier's Principle) and the original pH is re-established.

Materials required: 1. Potassium dihydrogen phosphate (KH2PO4) 2. Disodium hydrogen phosphate (Na2HPO4) 3. Sodium carbonate (Na2CO3) 4. Sodium Bicarbonate (NaHCO3) 5. Citric acid 6. Sodium citrate 7. Distilled water 8. pH meter 9. Glassware

Experimental Procedure: 1. Preparation for Phosphate Buffer:

Solution I: 1.36 g KH2PO4 was dissolved in sufficient water to produce 100 ml. Solution II: 3.58 g of Na2HPO4 was dissolved in sufficient water to produce 100 ml. Then, 96.4 ml of solution I was mixed with 3.6 ml of solution II.

2. Preparation for Carbonate Buffer: 0.84 g of NaHCO3 and 1.06 g of Na2CO3 was dissolved in sufficient water to produce 50 ml.


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3. Preparation for Citrate Buffer: Solution I: 0.21 g of citric acid was dissolved in sufficient water to produce 10 ml. Solution II: 0.29 g of sodium citrate was dissolved in sufficient water to produce 10 ml. Then, 2.8 ml of solution I and 2.2 ml of solution II was mixed in sufficient water to produce 100 ml.

4. Measurement of pH: The pH meter is put on and allowed to warm and calibrate

the probe using two standard solutions (pH 4, 7, and 10 buffers are recommended, dependant on the range). Calibration procedures vary by instrument, so following the manufacturer's instructions is highly recommended. BE SURE TO RINSE THE PROBE THOROUGHLY BETWEEN BUFFERS USING DEIONIZED WATER AND CAREFULLY BLOT THE PROBE DRY USING A KIM WIPE. pH meters should be calibrated before each use (before each series of samples, not between each sample itself) or when measuring a large range of pH. The standard buffer solution is taken in a beaker. The temperature of buffer solution is noted and electrodes are dipped in the solution. The selector switch is now turned to proper range 0-7 or 7-14. The meter will show the pH of buffer. Set the pointer to the exact value of pH of buffer. Bring the selector switch again to zero, clean the electrodes with water. Collect sample water in a glass or plastic container. Collect enough so the probe tip can be submerged in sample; either rinse the probe with deionized water (and blot dry) or with sample before inserting the probe into the collection vessel. Submerge the probe into the sample and wait until the pH reading on the meter stabilizes. Many meters have automatic temperature correction (ATC), which calculates the pH taking into account temperature, if your meter does not have this feature, you may need to adjust a knob on the meter to correct the pH for temperature. Record the measurement when the pH reading is stable.

Precautions: 3. The pH meter must be calibrated by checking against a standard buffer of know pH. 4. Keep the electrodes immersed in water when not in use.

Results: The pH of phosphate, citrate and carbonate buffer was found to be ______,

____ & ____ respectively.


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Experiment No. 3

Objective: To detect the presence of amino acids qualitatively by Ninhydrin test,

Xanthoproteic test, Hopkins-Cole Test, Millon’s test, Pauly’s test, Ehrlich test, Nitroprusside test, lead acetate test and Sakaguchi test.

Principle: Amino acids of the general formula RCH(NH2)COOH are amphoteric,

behaving as aliphatic amines in some reactions and as carboxylic acids in others. Ninhydrin test: Amino acids react with ninhydrin (triketohydrindene hydrate)

at pH=4. The reduction product obtained from ninhydrin then reacts with NH3 and excess ninhydrin to yield a blue colored substance.

Xanthoproteic Test: Amino acids containing an aromatic nucleus form yellow nitro derivatives on heating with concentrated HNO3. The salts of these derivatives are orange in color. Apply this test to tyrosine, tryptophan and glutamic acid.

Hopkins-Cole Test: The indole group of tryptophan will react with glyoxylic acid in the presence of concentrated H2SO4 to give a purple color. Glacial acetic acid, which has been exposed to the light, always contains glyoxylic acid CHOCOOH as an impurity. Apply this test to tyrosine, glycine and tryptophan.

Millon’s Test: Phenols only give this reaction; tyrosine is the only common phenolic amino acid. Millon’s reagent is concentrated HNO3, in which mercury has been dissolved. A yellow precipitate of HgO in a test is NOT a positive reaction but usually indicates that the solution is too alkaline. Apply this test to tyrosine, phenylalanine, glycine and β-naphtol.

Pauly Test: The imidazole ring of histidine, in the presence of sodium nitrite, reacts with sulfanilic acid forming a yellow product. This is a diazo coupling reaction.

Ehrlich Test: Aromatic amines and many organic compounds (indole and urea) give a colored complex with this test. Apply this test to urea, glycine and tryptophan.

Nitroprusside Test: Thiol groups give a red color with sodium nitroprusside in the presence of excess ammonia. Apply this test to cystine, cysteine and methionine.

Lead Acetate test: Sulfur containing amino acids, such as cysteine and methionine, are degraded in strongly alkaline media to release sulfide ion (S2-) in the form of H2S (hydrogen sulfide). The sulfide ions can react with lead (II) acetate to form a brownish-black precipitate.

Sakaguchi Test: The only amino acid, which contains a guanidine group, is arginine. Arginine gives a red color with α-naphthol, in the presence of an oxidizing agent like Bromine solution. Apply this test to arginine.

Materials required: Glassware: Beaker, water bath, test tubes, graduated pipettes.

Chemicals: 0.2% ninhydrin solution (triketohydrindene hydrate), NH3, conc HNO3,

NaOH (20%, 40%), glyoxylic acid, glacial acetic acid, conc H2SO4, Millon’s reagent, 5%


Dr. Gamal Gabr 18

sodium nitrite, 1% sulfanilic acid, sodium nitroprusside, lead (II) acetate, α-naphthol, Bromine solution


S. No. Test Observation Inference

1. Ninhydrin test: To 1ml solution add 5 drops of 0.2% ninhydrin solution in acetone. Boil over a water bath for 2 min. Allow to cool

blue color formed Amino acid present

2. Xanthoproteic test: To 2ml of solution in a boiling test tube, add an equal volume of conc. HNO3. Heat over a flame for 2 min and observe the color. Now COOL THOROUGHLY under the tap and CAUTIOSLY run in sufficient 40% NaOH to make the solution strongly alkaline.

Yellow color formed Amino acid present

3. Hopkins-Cole Test: To a few ml of glacial acetic acid containing glyoxylic acid, add 1-2 drops of the amino acid solution. Pour 1-2ml concentrated H2SO4 down the side of the sloping test tube to form a layer underneath the acetic acid.

purple color at the interface

Amino acid present

4. Millon’s test: To 2ml of amino acid solution in a test tube, add 1-2 drops of Millon’s reagent. Warm the tube in a boiling water bath for 10min.

A brick red color is a positive reaction.

Amino acid present

5. Pauly’s test: Into clean test tube, dispense 1mL of 1% sulphanilic acid and 2 drops of 5% sodium nitrite. Mix for 1 min. Add about 0.5mL of amino acid solution.

Yellow product formed

Amino acid present

6. Ehrlich test: To 0.5ml of the amino acid solution, add 2ml Ehrlich reagent

A colored complex formed

Amino acid present

7. Nitroprusside test: Add 2ml of the amino acid solution into test

Red color formed

Amino acid present


Dr. Gamal Gabr 19

tubes. Add 0.5ml fresh sodium nitroprusside solution and shake thoroughly. Add 0.5ml ammonium hydroxide

8. Lead acetate test: Everything needed to carry out this test will be in the hood and should not remove anything from the hood. A toxic, stinky gas will be made (in small, but immensely smelly quantities) and you don’t want to smell it. Dispense about 0.5mL of the amino acid solution only into a clean test tube (found in the hood, where you will leave it when you are done). Add 0.5mL of 20% NaOH and insert the test tube in a boiling water bath for 1 min. Add 2 drops of lead (II) acetate solution.

brownish-black precipitate

Amino acid present

9. Sakaguchi test: 1ml NaOH and 3ml of the arginine solution is mixed and 2 drops of α-naphthol is added. Mix thoroughly and add 4-5 drops Bromine solution

Red color formed

Amino acid present

Results: The amino acids were found to be present in given sample on the basis of

above chemical tests.


Dr. Gamal Gabr 20

Experiment No. 4

Objective: To carry out qualitative tests to detect lipids in the given sample using

Sudan III reagent.

Principle: Sudan red is a fat-soluble dye that stains lipids red.

Materials required: Glassware: test tubes, graduated pipettes.

Chemicals: Sudan III reagent, distilled water


1. To a test tube, add equal parts of test liquid and water to fill about half full.

2. Add 3 drops of Sudan III stain to test tube. Shake gently to mix.

3. A red-stained oil layer will separate out and float on the water surface if fat is present.

Results: The lipids were found to be present in the given sample on the basis of above

qualitative test.


Dr. Gamal Gabr 21

Experiment No. 5

Objective: Estimation of glucose content by kit method and glucometer in blood &

urine.

Principle: the oxidation of glucose is catalysed by glucose oxidase (GOD). The

resultant hydrogen peroxide (H2O2) is oxidatively coupled with 4-aminophenazone and phenol in the presence of peroxidase (POD) to yield a red quinonemine dye, the concentration of which at 546 nm is proportional to the concentration of glucose. Mutarotse α-D-glucose β-D-glucose GOD β-D-glucose + H2O + O2 D-gluconic acid + H2O2

POD H2O2 + 4-aminophenazone + phenol quinonemine + 4 H2O

Materials required: Reagent composition: (Kit supplied by Crescent Diagnostics, Jeddah)

1. Phosphate Buffer (pH 7.5) 0.1 mol/l 4-aminoantipyrine 0.25 mmol/l Phenol 0.75 mmol/l Glucose oxidase 15 KU/l Peroxidase 1.5 KU/l Mutarotase 2 KU/l

2. Glucose standard 100 mg/dl or 5.5 mmol/l


1. Glucose estimation by Kit Method Pipette into cuvettes Micro Macro

Blank standard sample Blank standard sample

Test sample --- --- 0.01 --- --- 0.025

Standard --- 0.01 --- --- 0.025 ---

Distilled water 0.01 --- --- 0.025 --- ---

Reagent 1.0 1.0 1.0 2.5 2.5 2.5

Mix and incubate for 10 min at 20-25C or 5 min at 37C Measure the absorbance of the sample (As) and standard (Astd) against the reagent blank.


Dr. Gamal Gabr 22

Calculation:

As Glucose (mg/dl) = X concentration of standard Astd To convert mg/dl to mmol/l divide by 18

Results: The glucose levels in blood & urine were found to be ____ & ____ by kit method and ____ & ____ by glucometer respectively.


Dr. Gamal Gabr 23

Experiment No. 6

Objective: Enzymatic hydrolysis of starch by α & β amylase.

Principle: The reducing groups released from starch are measured by the reduction of

3,5-dinitrosalicylic acid. One unit releases from soluble starch one micromole of reducing groups (calculated as maltose) per minute at 25°C and pH 6.9 under the specified conditions.

The rate at which maltose is released from starch is measured by its ability to reduce 3,5-dinitrosalicylic acid. One unit releases one micromole of β-maltose per min at 25°C and pH 4.8 under the specified conditions.

Materials required: Reagents α -amylase 0.02 M Sodium phosphate buffer, pH 6.9 with 0.006 M sodium chloride 2 N Sodium hydroxide Dinitrosalicylic acid color reagent. Prepare by dissolving 1.0 gm of 3,5-

dinitrosalicylic acid in 50 ml of reagent grade water. Add slowly 30.0 gms sodium potassium tartrate tetrahydrate. Add 20 ml of 2 N NaOH. Dilute to a final volume of 100 ml with reagent grade water. Protect from carbon dioxide and store no longer than 2 weeks.

1% Starch. Prepare by dissolving 1.0 gm soluble starch, (Merck) in 100 ml 0.02 M sodium phosphate buffer, pH 6.9 with 0.006 M sodium chloride. Bring to a gentle boil to dissolve. Cool and bring volume to 100 ml, with water, if necessary. Incubate at 25°C for 4-5 minutes prior to assay.

Maltose Stock Solution. Prepare by dissolving 180 mg maltose (MW 360.3) in 100 ml reagent grade water in a volumetric flask.

β amylase 0.016 M Sodium acetate, pH 4.8 2 N Sodium hydroxide Dinitrosalicylic acid color reagent: same as above 1% Starch: Prepare by dissolving 1.0 gram of soluble starch (Merck) in 100 ml of

0.016 M sodium acetate buffer pH 4.8. Bring to a gentle boil to dissolve. Cool and, if necessary, dilute to 100 ml with reagent grade water. Incubate at 25°C for 4-5 minutes prior to assay.

Maltose stock solution, 5 micromoles/ml. Prepare by dissolving 180 mg maltose (MW 360.3) in 100 ml reagent grade water. Incubate at 25°C for 4-5 minutes prior to assay.


Dr. Gamal Gabr 24

Enzyme Dilute to a concentration of 1-10 micrograms/ml. A minimum of three different concentrations in this range should be run.

Mg/ml=A280 X 0.38

Experimental Procedure: α -amylase 1. Adjust spectrophotometer to 540 nm and 25°C. 2. Using the maltose stock solution prepare a maltose standard curve as follows: In

numbered tubes, prepare 10 maltose dilutions ranging from 0.3 to 5 micromoles per ml. Include two blank tubes with reagent grade water only. Into a series of corresponding numbered tubes pipette 1 ml of each dilution of maltose. Add 1 ml of dinitrosalicylic acid color reagent. Incubate in boiling water bath for 5 minutes and cool to room temperature. Add 10 ml distilled water to each tube and mix well. Read A540 versus micromoles maltose.

3. Enzyme assay: Pipette 0.5 ml of respective enzyme dilutions into a series of numbered test tubes. Include a blank with 0.5 ml reagent grade water. Incubate tubes at 25°C for 3-4 minutes to achieve temperature equilibration. At timed intervals, add 0.5 ml starch solution (at 25°C). Incubate exactly 3 minutes and at timed intervals add 1 ml dinitrosalicylic acid color reagent to each tube. Incubate all tubes in a boiling water bath for 5 minutes. Cool to room temperature and add 10 ml reagent grade water. Mix well and read A540 versus blank. Determine micromoles maltose released from standard curve.

β amylase 1. Prepare a maltose standard curve as above 2. Enzyme assay: same as above

Calculation:

Results:


Dr. Gamal Gabr 25

Experiment No. 7

Objective: Effects of temperature & pH on the activity of enzymes (catalase from

fresh potato).

Principle: Enzymes are biological catalysts that carry out the thousands of chemical reactions that occur in living cells. They are generally large proteins made up of several hundred amino acids and often contain a nonproteinaceous group called the prosthetic group that is important in the actual catalysis.

Each enzyme is specific for a certain reaction because its amino acid sequence is unique and causes it to have a unique three-dimensional structure (tertiary or quaternary structure). The business end of the enzyme molecule, the active site, also has a specific shape so that only one or a few of the thousands of compounds in the cell can interact with it. If there is a prosthetic group on the enzyme, it will form part of the active site. Any substance that blocks or changes the shape of the active site will interfere with the activity and efficient of the enzyme. If these changes are large enough, the enzyme can no longer act at all and is said to be denatured. There are several factors that are especially important in determining the enzyme’s shape and these are closely regulated both in the living organism and in laboratory experiments to give the optimum or most efficient enzyme activity: salt concentration and temperature.

In this exercise, you will study the enzyme catalase, which accelerates the breakdown of hydrogen peroxide, a common end product of oxidative metabolism, into water and oxygen according to the summary reaction:

Catalase 2H2O2 2H2O + O2 This catalase-mediated reaction is extremely important in the cell because it prevents the accumulation of hydrogen peroxide, a strong oxidizing agent that tends to disrupt the delicate balance of cell chemistry.

Catalase is found in animal and plant tissues and is especially abundant in plant storage organs such as potato tuber, corns and the fleshy parts of fruits. Catalase has been isolated from potato tubers. The activity and efficiency of enzymes are influenced by various factors, including temperature and pH conditions. Temperatures above 60º C/140ºF damage (denature) the intricate structure of enzymes, causing reactions to cease. Each enzyme operates best within a specific pH range, and is denatured by excessive acidity or alkalinity.

The higher the temperature of water, potato and H2O2, the rate at which the Enzyme will work will be faster therefore producing more oxygen. The reaction will be the same without the catalase (potato). Therefore in both experiments the Enzyme will work more rapidly and produce more oxygen. The optimum pH condition for Catalase is pH 7.52.


Dr. Gamal Gabr 26

Materials required: Fresh potato, 1% H2O2, Distilled water, Crushed ice Homogenizer, Thermometer, Measuring cylinder, Beaker, Water bath etc.

Experimental Procedure: Extraction of Catalase: 1. Peel a fresh potato tuber and cut the tissue into small cubes. Weigh out 50 gm of

tissue. 2. Place the tissue, 50 mL of cold water and a small amount of crushed ice into a

prechilled blender. 3. Homogenize for 30 seconds at high speed.

From this point on, the enzyme preparation must be carried out in an ice bath! 4. Filter the potato extract through cheesecloth and pour the filtrate into a 100 mL

graduated cylinder. Add cold distilled water to bring up the final volume to 100 ml. This extract will be arbitrarily labeled 100 units of enzyme per mL (100 units/mL).

Effect of temperature Using 40 mL of a 1% H2O2 solution as the substrate and 5 mL aliquots of the 100-units/ml-enzyme solution, measure the enzyme activity. Run the reactions in the different temperature water baths. The catalase and substrate (H2O2) should be brought to the testing temperature before they are used. Allow 10 minutes for equilibration. Record the exact temperature and your data in a table in your notebook. Also test the activity of the enzyme that has been boiled. DO NOT boil the H2O2. These assays should be run in duplicate.

Effect of pH Obtain five 50-mL beakers and label them as follows: 1. pH 4 2. pH 6 3. pH 7 4. pH 8 5. pH 10 Into each beaker, pour 10 mL of enzyme preparation and 30 mL of buffer solution at the appropriate pH. Using 40 mL of a 1% H2O2 as the substrate, measure the enzyme activity in the usual manner.

Results:


Dr. Gamal Gabr 27

Experiment No. 8

Objective: Estimation of Serum Glutamate Oxaloacetate Transaminase (SGOT/AST)

& Serum Glutamate Pyruvate Transaminase (SGPT/ALT) activity by kit method in blood.

Principle: For AST AST catalyzes the following reaction:

AST L-Aspartate + 2-Oxoglutarate Oxalacetate + L-Glutamate In the present method a diazonium salt is used which selectively reacts with the oxalacetate to produce a color complex that is measured photometrically.

For ALT In this procedure ALT catalyzes L-alanine and a-ketoglutarate to form pyruvate and glutamate. The pyruvate is then reacted with 2 4-dinitrophenylhydrazine (2 4-DNPH-ine) to form 2 4-DNPH-one. The addition of sodium hydroxide dissolves this complex allows 2 4-DNPH-one to be measured at 505 nm. ALT L-Alanine + a-ketoglutarate Pyruvate + Glutamate H+

Pyruvate + 2 4 - DNPH-ine Pyruvate + 2 4-DNPH-one

Materials required: Reagent composition: For AST 1. AST Substrate: 33 mM Aspartic acid 5 mM ketoglutaric acid phosphate buffer pH

7.4. 2. AST Color Reagent: 0.25% w/v Diazonium salt preserved with formalin. 3. AST Standard: A lyophilized serum with AST (SGOT) value provided in each lot

Reconstitute with distilled water let stand until dissolved and swirl to mix. Stable for 5 days at 2 - 8°C after reconstitution. Aliquot into small portions and keep frozen.

For ALT 1. ALT Substrate: 0.2 M L-alanine 2.0 mM a-ketoglutarate 100 mM phosphate buffer at

pH 7.4 + 0.05 0.2% v/v preservatives. 2. ALT Color Reagent: l.0mM 2 4-dinitrophenylhydrazine in 1N Hydrochloric Acid

preservative. 3. ALT Color Developer: 0.5N sodium hydroxide.

4. ALT Standard: Solution of sodium pyruvate in 100 mM phosphate buffer at pH 7.4 .


Dr. Gamal Gabr 28

Experimental Procedure: For AST 1. Place 0.5 ml of AST substrate into test tubes labeled "Blank" "Standard" and "Test".

Warm vials in 37°C heating bath for at least 4 min. 2. At timed intervals add 0.1 ml (100µl) of samples into their respective tubes gently

mix and return to 37°C heating bath for exactly 10 min. Use distilled water for sample blank.

3. After 10 min and in the same timed sequence add 0.5 ml of AST Color Reagent. Mix gently and immediately return to 37°C heating bath for another 10 min.

4. After 10 min add 2.0 ml of 0.1 N Hydrochloric acid and mix by inversion. 5. Set the wavelength of the spectrophotometer at 530 nm and zero the instrument

with the Blank. Read and record the absorbance of all tubes. NOTE: The final color developed in the reaction must be read within 60 minutes.

For ALT 1. Label test tubes "Blank" "Standard" "Test". 2. Transfer 0.5 ml of ALT substrate to each tube and place in a 37°C heating bath for 3-

5 min. 3. At timed intervals (about 15-30 seconds) add 0.1 ml (100µl) of sample to the

correspondingly labeled tube. Mix and immediately return to 37°C healing bath for exactly 30 min.

4. After exactly 30 min add 0.5 ml of ALT Color Reagent to each tube maintaining the timed interval sequence. Mix and return to 37°C heating bath for exactly 10 min.

5. After exactly 10 min add 2.0 ml of ALT Color Developer (maintaining the same timed intervals). Mix and return to 37°C heating bath for 5 min.

6. Zero the spectrophotometer with the reagent "blank" at 505 nm. Read and record absorbance of all tubes.

Calculation:

Abs. of sample AST (IU/L) = X Concentration of standard (IU/L)

Abs. of standard

Abs. of sample

ALT (IU/L) = X Concentration of standard (IU/L) Abs. of standard

Results: The SGOT & SGPT activity in blood sample was found to be ____ & ____ respectively.


Dr. Gamal Gabr 29

Experiment No. 9

Objective: Estimation of LDH activity by kit method in blood & urine.

Principle: Lactate dehydroenase (LDH) catalyses the conversion of Lactate to

Pyruvate accompanied by the simultaneous reduction of NAD to NADH. LDH activity in the serum is directly proportional to the increase in absorbance due to reduction of NAD.

LDH Lactate + NAD Pyruvate + NADH

Materials required: (Diagnostic kit supplied by Reckon diagnostics Ltd.) 1. Micropipettes 2. Cuvette Preparation of the working reagent:

1 LDH (Coenzyme) x14 2 LDH (Buffered substrate) x1

Reconstitute one vial of the 1LDH with 1.1 ml of the 2 LDH. Mix them gently to dissolve the contents. Use within five minutes.

Reaction Parameters: Type of reaction : Kinetics/ increasing OD

Flow cell temperature : 37C Wavelength : 340 nm Interval time : 30 seconds Delay time : 60 seconds Reagent volume : 1.0 ml Factor : 3376

Sample volume : 50 l (0.05 ml) Zero setting with : Distilled water Path length : 1.0 cm


Pipette into test tube Test

Working reagent (ml) 1.0

Sample (ml) 0.05

Mix and read the first absorbance of the test exactly at one minute and thereafter at 30, 60, and 90 seconds at 340nm. Determine the mean change in absorbance per minute.


Dr. Gamal Gabr 30

Calculation: LDH activity = Δ A/minute x F Where F = 1/6.22 X TV/ SV X 1000 = 3376 TV = Total volume (1.05 ml)

SV = Sample volume (0.05 ml) 6.22 = Milimolar extinction coefficient of NADH at 340 nm

Results: The LDH activity in blood & urine was found to be ____ & ____ respectively.


Dr. Gamal Gabr 31

Experiment No. 10

Objective: Estimation of alkaline phosphatase activity by p-nitrophenol method in

blood & urine.

Principle: Phosphatases are enzymes which catalyse the splitting of a phosphate from

mono-phosphoric esters. Alkaline phosphatase (ALP), a mixture of isoenzymes from liver, bone, intestine and placenta, has maximum enzyme activity at about pH 10.5. Serum ALP measurements are of particular interest in the investigation of hepatobillary and bone diseases.

Paranitrophenyl phosphate, which is colourless, is hydrolysed by alkaline phosphatase at pH 10.5 & 370C to form free paranitrophenol, which is coloured yellow. The addition of NaOH stops the enzyme activity and the final colour shows maximum absorbance at 410 nm.

Materials required: 2-amino 2- methyl 1-propanol (AMP) buffer pH 10.5: Add 116 ml of AMP to 600 ml

of distilled water. Mix and adjust the pH to 10.5 with 6 M HCI and then make up to 1 litre with distilled water. Stable for 6 months at 2-80C.

Magnesium chloride (1.5 mmol/l): Dissolve 300 mg of magnesium chloride hexahydrate in distilled water and make up to 1 litre. Stable for 6 months at room temperature (25-350 C)

Substrate: Dissolve 83.5 mg of disodium paranitrophenyl phosphate in 1.0ml magnesium chloride solution. Stable for 24 hours at 2-80C. This solution should be colourless; do not use it if the OD at 410nm > 0.800.

Sodium hydroxide 0.25 M: Dissolve 10 g of NaOH in about 800 ml of distilled water and then make up to 1 litre with distilled water. Store in a polythene bottle at room temperature (25-35°C). Stable for 6 months.

Stock paranitrophenol (PNP) 10.8 mmol/l: Weigh out 150 mg of PNP and dissolve in about 80ml of NaOH (0.25M) and then make up to 100 ml with the same NaOH

solution. Store in a brown glass bottle at room temperature (25-35C). Stable for 3 months.

Working PNP 54 m mol/l: Pipette 0.5 ml of the PNP stock solution into a 100ml volumetric flask and make up to the mark with NaOH solution (0.25 M). Prepare fresh before use.

Equipment, glassware and other accessories

Experimental Procedure: The protocol of the procedure is described below.


Dr. Gamal Gabr 32

Preparation of standards (SI -S6) Pipette the following into appropriately labeled test tubes & Mix well

S1 S2 S3 S4 S5 S6

Working PNP solution (ml) 0.5 1.0 2.0 3.0 4.0 5.0

NaOH solution (ml) 4.5 4.0 3.0 2.0 1.0 -

Activity (U/L) 40 80 160 240 320 400

Set the spectrophotometer to zero absorbance at 410 nm against 0.25M NaOH and measure the absorbance of the above standards. Enzyme measurement in test Pipette the following into another set of appropriately labeled 18 x 150 mm tubes.

Blank Test

AMP buffer (ml) 1.4 1.4

Mix and Incubate at 370C for 5 minutes

Test Sample (ml) - 0.05

Substrate (ml) 0.1 0.1

Mix and Incubate at 370C for 15 minutes

NaOH (ml) 4.0 4.0

(Note: NaOH should be added to each tube in sequence maintaining timed intervals)

Mix and cool the tubes to room temperature (25-35C). Measure the absorbance of test at 410nm, setting the spectrophotometer to zero with the blank.

Calculation: The working PNP concentration is 54 m mol/L. Standard S1 contains 0.5ml PNP

54 Concentration of PNP in SI = ---------------- x 0.5 = 0.027m mol.

1000

ALP activity in U/L = Liberation of 1 m mol of PNP per minute at 37C incubation per litre serum. In the assay protocol, 0.05 ml serum is mixed with reagent and incubated for 15 minutes and the total volume is made up to 5.55ml. But the total volume in the case of each standard (SI to S6) is 5.0 ml.

Test absorbance 0.027 5.55 1000 PNP (umol/L) or ALP activity (U/L) = -------------------- x -------- x --------x-----------

Std absorbance 15 5.0 0.05


Dr. Gamal Gabr 33

Test absorbance = ----------------------- x 40 Std absorbance i.e, ALP activity equivalent for S 1 = 40 U/L. Similarly the ALP activities represented by other standards are: S2 = 80, S3 = 160, S4 = 240, S5 = 320 and S6 = 400 U/L. Construct a calibration graph by plotting the equivalent activity of ALP of the standards against their corresponding absorbance values. The measurable range with this graph is from 10 to 400 U/L. Plot the absorbance values of test on the calibration graph and read off the concentrations. Once linearity is proved, it will be enough if a single standard is set up every time that sample are analysed. Use standard S6 in the assay and calculate the results using the formula:

Test Absorbance ----------------------- x 400 U/L Std Absorbance

Results: The alkaline phosphatase activity in blood & urine was found to be ____ &

____ respectively.


Dr. Gamal Gabr 34

Experiment No. 11

Objective: Estimation of total cholesterol, HDL-Cholesterol & Triglycerides content

by kit method in blood.

Principle: Cholesterol reacts with hot solution of Ferric Perchlorate, Ethyl Acetate and

Sulphuric acid (Cholesterol reagent) and gives a purple colored complex which is measured at 560 nm.

For Total Cholesterol Materials required: Kit contents: Reagent 1: Cholesterol reagent Reagent 2: Working Cholesterol standard (200 mg %) Reagent 3: Precipitating reagent All reagents were ready for use.

Experimental Procedure: Pipette into 3 test tubes labeled blank (B), standard (S) and test (T) as shown below in Table:

Reagent 2 ml Procedure 3 ml Procedure

B S T B S T

Cholesterol reagent 2 ml 2 ml 2 ml 2 ml 2 ml 2 ml

Working Cholesterol standard (200 mg %)

- 10 µl - - 10 µl -

Specimen / serum - - 10 µl - - 10 µl

Distilled water - - - 1 ml 1 ml 1 ml

Mix well and keep the tubes immediately in the boiling water bath exactly for 90 seconds. Cool them immediately to room temperature under running tap water. Measure the OD of S and T against B on a colorimeter with a yellow green filter or on a spectrophotometer at 560 nm.

Calculation: Absorbance of Test Total Cholesterol (mg/dl) = X 200

Absorbance of Standard


Dr. Gamal Gabr 35

For HDL-Cholesterol Principle: High Density Lipoproteins (HDL) was obtained in the supernatant after

centrifugation. The cholesterol in the HDL fraction was also estimated by this method.

Materials required: Reagents: Reagents supplied in kit were used directly. Kit contents: Reagent 1: Cholesterol reagent Reagent 2: Working Cholesterol standard (200 mg %) Reagent 3: Precipitating reagent

Experimental Procedure: Step 1: HDL-cholesterol separation Pipette into a centrifuge tube: Serum 0.2 ml Precipitating reagent 0.2 ml

Mixed well and allowed to stand at room temperature for 10 minutes. Centrifuge at 2000 rpm for 15 minutes to get a clear supernatant. If the supernatant is not clear (high TG level) dilute the sample 1:1 with normal saline and multiply the result with 2.

Step 2: HDL-cholesterol estimation Pipette into 3 test tubes labeled blank (B), standard (S) and test (T) as shown below in Table:

Reagent 2 ml Procedure 3 ml Procedure

B S T B S T

Cholesterol reagent 2 ml 2 ml 2 ml 2 ml 2 ml 2 ml

Working Cholesterol standard (200 mg %)

- 10 µl - - 10 µl -

Specimen from step 1 - - 80 µl - - 80 µl

Distilled water - - - 1 ml 1 ml 1 ml

Mix well and keep the tubes immediately in the boiling water bath exactly for 90 seconds. Cool them immediately to room temperature under running tap water. Measure the OD of S and T against B on a colorimeter with a yellow green filter or on a spectrophotometer at 560 nm.

Calculation:

Absorbance of Test HDL-Cholesterol (mg/dl) = X 50



Dr. Gamal Gabr 36

For Triglycerides Principle: Lipase hydrolyses triglycerides sequentially to Di- and mono-glycerides

and finally to glycerol and free fatty acids. Glycerol kinase (GK) using ATP as PO4 source converts liberated glycerol to glycerol-3-phoshate. Glycerol-3-phoshate oxidase (GPO) oxidizes glycerol-3-phoshate formed to dihydroxy acetone phosphate and hydrogen peroxide is formed. Peroxidase (POD) uses the hydrogen peroxide formed, to oxidize 4-aminoantipyrine and TOOS (N-ethl-N-sulphohydroxy propyl-m Toluidine) to a purple colored complex. The absorbance of the colored complex is measured at 546 nm (530-570 nm or with yellow filter) which is proportional to the triglycerides concentration.

Lipase Triglyceride + H2O Glycerol + fatty acids

GK Glycerol + ATP Glycerol-3-phoshate + ADP

GPO Glycerol-3-phoshate + O2 dihydroxy acetone phosphate + H2O2

POD H2O2 + 4-aminoantipyrine + TOOS Quinoneimine + H2O

Materials required: Kit content: 1 – Triglycerides (Enzyme, Chromogen) 2 – Triglyceride (Buffer) * Triglyceride standard (conc. 200 mg/dl)

Preparation of working reagent: Dissolve the content of one vial labeled (1) containing Triglycerides with 1.1 ml of second vial labeled (2) containing Triglyceride (Buffer). Mix gently to dissolve. Reaction Parameters Type of reaction : End point Wavelength : 546 nm (530-570 nm)

Flowcell temperature : 30C /37C Incubation : 15 minutes at 37oC Standard concentration : 200 mg/dl

Sample volume : 10 l (0.01 ml) Reagent volume : 1.0 ml Zero setting with : Reagent Black Light Path : 1.0 cm


Dr. Gamal Gabr 37

Experimental Procedure: Pipette into 3 test tubes labeled blank (B), standard (S) and test (T) as shown below in Table:

Reagent 1 ml Procedure 0.5 ml Procedure

B S T B S T

Triglycerides reagent (1) 1 ml 1 ml 1 ml 0.5 ml 0.5 ml 0.5 ml

Triglycerides standard (2) - 10 µl - - 5 µl -

Specimen (serum) - - 10 µl - - 5 µl

Mix well and incubate at 37oC for 15 minutes and read the absorbance of S and T against B at 546 nm or with yellow filter (530-570 nm). The final color was brownish purple and stable at room temperature.

Calculation:

Absorbance of Test Triglycerides (mg/dl) = X 200


Results: The total cholesterol, HDL-Cholesterol & Triglycerides levels in blood were

found to be _____, _____ & _____ respectively.


Dr. Gamal Gabr 38

Experiment No. 12

Objective: To estimate protein in the given sample by Folin--Lowry’s method.

Principle: Lowry’s method of protein estimation is the most widely used and

accepted method for accurate determination of protein concentration. The method is a combination of biuret reaction and folin-ciocalteau reaction. In the first step of the reaction protein binds to copper in alkaline medium and produces Cu++ In the second step, Cu++ catalyses oxidation of aromatic amino acid by reducing phosphomolybdotungstate to heteropolymolybdanum blue. This reaction produces strong blue color, which predominantly depends upon tyrosine and tryptophan content of protein and to a lesser extent cysteine and other residues in protein. Materials required: Preparation of the reagents: 1. Alkaline sodium carbonate solution (2 % Na2CO3 in 0.1 N NaOH): 100 ml of 0.1 N

NaOH solution was prepared by dissolving 400 mg of NaOH in distilled water and the volume was made upto 100 ml. Then 2 g of Na2CO3 was dissolved in 100 ml of 0.1 N NaOH solution.

2. Copper sulfate sodium tartarate solution (0.5 % CuSO4 in 1 % Na-K tartarate): Sodium potassium tartarate solution: 0.5 % copper sulfate solution was mixed with 1 % Na-K tartarate.

3. Alkaline solution: Prepared on the day of use by mixing 50 ml of reagent 1 and 1ml of reagent 2.

4. Folin ciocalteau phenol reagent: The commercial reagent was diluted with 2 volumes of distilled water on the day of use.

5. Standard protein: Bovine serum albumin (0.5 mg/ml)- 5 mg of bovine serum albumin was dissolved in 10 ml of distilled water to get a solution of 2.0 mg/ml of protein.

Experimental Procedure: 5 ml of alkaline solution was added to 1 ml of protein sample and allowed to stand for 10 minutes. 0.5 ml of diluted folin reagent was added and the tube was shaken to mix the solution. After 30 min check the absorbance of unknown sample against blank at 750 nm and determine the concentration of the unknown sample using the standard curve. Preparation of Calibration standard curve of protein: 5 ml of bovine serum albumin solution (0.5 mg/ml) was prepared and different volumes were taken in 6 test tubes. To all tubes distilled water was added to make up the volume in each tube to 1 ml. The protein concentration in the above six tubes was estimated in the same way as for the sample. A graph was plotted between


Dr. Gamal Gabr 39

concentration of protein and optical density. The calibration standard plot was used to calculate the concentration of protein in each ml suspension of the sample.

Results: The total protein levels in given sample was found to be _____.


Dr. Gamal Gabr 40

Experiment No. 13

Objective: To estimate the Urea in blood.

Principle: Urea reacts directly with diacetyl monoxime under strong acidic conditions

to give a yellow condensation product. The reaction is intensified by the presence of ferric ions and thiosemicarbazide. The intense red color formed is measured at 540nm/ yellow green filter.

Materials required: Stock acid reagent: Dissolve 1.0g of ferric chloride hexahydrate in 30 ml of distilled

water. Add 20 ml orthophosphoric acid and mix. Store in a brown bottle at room temperature (25-350C).Stable for 6 months.

Mixed acid reagent: Add slowly 100 ml of Conc. H2S04 to 400 ml distilled water taken in a 1-litre flat-bottom conical flask kept in an ice cold water bath. Mix well and add 0.3ml of stock acid reagent. Mix and store in a brown bottle at room temperature (25-350C). Stable for 6 months.

Stock color reagent – A: Dissolve 2g diacetyl monoxime in distilled water and make the volume up to 100 ml in a volumetric flask. Store in a brown bottle at room temperature (25-350C). Stable for 6 months.

Stock color reagent – B: Dissolve 0.5 g thiosemicarbazide in distilled water and make up to 100 ml in a volumetric flask. Store in a brown bottle at room temperature (25-350C). Stable for 6 months.

Mixed color reagent: Mix 35 ml of stock color reagent A with 35 ml of stock color reagent B and make up to 500 ml with distilled water. Store in a brown bottle at room temperature (25-350C). Stable for 6 months.

Stock urea standard: Weigh 1.0g of analytical-grade urea and dissolve in 100ml of benzoic acid (1g/dl). Use a 100ml of volumetric flask for preparing this. Store at room temperature (25-350C). Stable for 6 months.

Working standard 50mg/dl: Dilute 5.0ml of stock urea standard to 100 ml with benzoic acid. Store at room temperature (25-350C). Stable for 6 months.

Equipment, glassware and other accessories: Experimental Procedure: The protocol of the procedure is described below. Dilution of Standards (S1-S3) & Test


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Pipette the following into appropriately labeled test tubes & Mix Well

S1 S2 S3 Test

Distilled Water (ml) 1.9 1.8 1.7 1.9

50 mg/dl Urea (ml) 0.1 0.2 0.3 -

Test sample (ml) - - - 0.1 Color Development The color reagent is prepared fresh at the time of analysis by mixing distilled water, mixed acid reagent and mixed color reagent in the ratio 1:1:1. Pipette the following into another set of appropriately labeled test tubes.

Blank S1 S2 S3 Test

Color reagent (ml) 3.1 3.0 3.0 3.0 3.0

Respective diluted standard ml) - 0.1 0.1 0.1 -

Diluted test (ml) - - - - 0.1

Mix all tubes well. Keep them in a boiling water bath for 15 minutes. Remove from water bath and cool the tubes for 5 minutes. Set the spectrophotometer to zero with blank at 540nm and measure the absorbance of the other tubes.

Calculation: Concentration of standards: S1 = 50 mg/dl S2 = 100 mg/dl S3 = 150 mg/dl Plot the absorbance values of standards against their respective concentrations. The measurable range with this graph is from 10 to 150 mg/dl. A calibration graph should be constructed whenever a new set of reagents is prepared. Plot absorbance values of test on the calibration graph and read off the concentrations. Once linearity is proved, it will be enough if S3 is set up every time that sample are analyzed and the results calculated using the formula:

Absorbance of test Urea in test sample = ----------------------------- x 150 mg/dl


Results: The urea level in blood was found to be _____.


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Experiment No. 14

Objective: To test the given urine sample for the presence of ketone bodies - Rothera's

test.

Principle: The three main ketone bodies are acetone , acetoacetic acid (diacetic acid)

and beta-hydroxybutyric acid. Testing for ketone bodies should be done on fresh urine

or the specimen kept at 4C. Acetone and acetoacetic acid react with sodium nitropruside in the presence of alkali to produce a purple color.

Materials required: Rothera's Reagent (Dry mixture): Pulve210rize 7.5g sodium nitropruside with 200g

ammonium sulphate. Store in a clean amber bottle at 250-35C. Stable for 6 months.

Ammonia concentrated, specific gravity 0.91

Experimental Procedure: To about 5ml of urine taken in an 18 x 150mm glass tube, add about one teaspoon of the mixture, mix well, then add 0.5 to 1.0 ml of concentrated ammonia down to the side of the tube so that it layers on top of the urine. Observe for any colour change within 30-60 seconds. If acetone and diacetic acid are present, then a purple (permanganate calomel red) colour will form at the junction of the two layers within 30-60 seconds. The result can be graded from trace to 3+ based on the intensity of the color formed, as detailed below.

No change in colour - Negative Pinkish ring - + Red ring - ++ Deep purple ring - +++

Results: The ketone bodies were found to be present in given urine sample on the

basis of above chemical tests.


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Carbohydrate Report

Unknown ( ) Student name ………………….. Student No. ………………

Arabic Date…………………….. English Date……………..

No. Test Observation Inference

1. Solubility:

Compound + water

2.

Molisch test: Procedure: Mix 2ml of sugar sample with 5

drops of Molisch's Reagent in a test tube. Add

gently through the side by tilting the tube,

about 2 ml of concentrated H2SO4 so as to form

a bottom layer.

3.

Benedict’s test:

Procedure: Take 5 ml of Benedict's qualitative

reagent, add 8 drops of sugar solution. Boil over

a flame for 2 minutes or place in boiling water

bath for 3 minutes. Allow to cool.

Fehling’s test: Procedure: 2 ml of Fehling's solution A + 2ml of

Fehling's solution B + 2 ml of Sugar solution Boil.

5.

Barfoed’s test Procedure: Sugar solution + Barfoed’s reagent

(Cu Acetate/Acetic acid), heat for 3 min. on

boiling W.B


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6.

Seliwanoff's Test

Procedure:Sugar solution+ few crystals of

Resorcinol + Equal volume of conc.HCl and

warm on W.B.

7.

Bial's test:

Procedure: 2 ml of sample solution is placed in a

test tube and 2 ml of Bial's reagent is added,

solution is heated gently in hot W.B

8.

Osazone test

Procedure:

1- Add 10 drops of glacial acetic acid to 5 ml of

sugar solution in test tube.

2- Then add a knife point of phenyl hydrazine

hydrochloride and double the amount of

sodium acetate crystals.

3- Mix and warm a little to see that the solids

are dissolved.

4- Filter the solution, and keep the filtrate in a

boiling W.B for 20 min.

5- Cool slowly in water bath not under the tap

to have better crystals.

6- Examine the crystal under microscope.

9.

Hydrolysis of sucrose (Inversion test)

Procedure: add 2 drops of HCl and 1 drop of


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thymol blue to 5 ml of sucrose solution.

The development of pink color indicates that the

solution is acidic.

Divide it in to two equal parts

Boil one portion for about one min., cool under

tape water.

Neutralize both portions by adding 2% sodium

carbonate drop by drop.

Iodine test: Procedure: iodine is added to an peeled potato

10.

Iodine/Potassium iodide test:

Procedure: Two ml of a sample solution is placed

in a test tube. Two drops of iodine / potassium

iodide solution and one ml of water are added.

Results:.


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Amino Acids Report

Unknown ( ) Student name ………………….. Student No. ………………

Arabic Date…………………….. English Date……………..

No. Test Observation Inference

1. Ninhydrin test: To 1ml solution add 5 drops of 0.2% ninhydrin solution in acetone. Boil over a water bath for 2 min. Allow to cool

2. Xanthoproteic test: To 2ml of solution in a boiling test tube, add an equal volume of conc. HNO3. Heat over a flame for 2 min and observe the color. Now COOL THOROUGHLY under the tap and CAUTIOSLY run in sufficient 40% NaOH to make the solution strongly alkaline.

3. Hopkins-Cole Test: To a few ml of glacial acetic acid containing glyoxylic acid, add 1-2 drops of the amino acid solution. Pour 1-2ml concentrated H2SO4 down the side of the sloping test tube to form a layer underneath the acetic acid.

4. Millon’s test: To 2ml of amino acid solution in a test tube, add 1-2 drops of Millon’s reagent. Warm the tube in a boiling water bath for 10min.

5. Pauly’s test: Into clean test tube, dispense 1mL of 1% sulphanilic acid and 2 drops of 5% sodium nitrite. Mix for 1 min. Add about 0.5mL of amino acid solution.

6. Ehrlich test: To 0.5ml of the amino acid solution, add 2ml Ehrlich reagent

7. Nitroprusside test: Add 2ml of the amino acid solution into test tubes. Add 0.5ml fresh sodium nitroprusside solution and shake thoroughly. Add 0.5ml ammonium hydroxide


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8. Lead acetate test: Everything needed to carry out this test will be in the hood and should not remove anything from the hood. A toxic, stinky gas will be made (in small, but immensely smelly quantities) and you don’t want to smell it. Dispense about 0.5mL of the amino acid solution only into a clean test tube (found in the hood, where you will leave it when you are done). Add 0.5mL of 20% NaOH and insert the test tube in a boiling water bath for 1 min. Add 2 drops of lead (II) acetate solution.

9. Sakaguchi test: 1ml NaOH and 3ml of the arginine solution is mixed and 2 drops of α-naphthol is added. Mix thoroughly and add 4-5 drops Bromine solution

Results:.


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Attendance Sheet

Group No. ( ) Student Name:

Marks Exp. Date Week

1st

22d

3rd

4th

5th

6th

7th

8th

9th

10th

11th

12th

13th

14th

Total marks

Laboratory Manual - PSAU · Biochemistry Laboratory Manual PHL ... Estimation of SGOT & SGPT...

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Transcript of Laboratory Manual - PSAU · Biochemistry Laboratory Manual PHL ... Estimation of SGOT & SGPT...