TABLE OF CONTENTS

BIOCHEMISTRY STUDENT PRACTICALS........................................................................................................2

INTRODUCTION...........................................................................................................................................2

GENERAL LABORATORY RULES AND SAFETY REGULATIONS........................................................................3

LABORATORY APPARATUS...........................................................................................................................5

LABORATORY REPORT.................................................................................................................................7

EXPERIMENT 1: ESTIMATION OF WHOLE BLOOD GLUCOSE - FOLIN-WU METHOD.....................................9

EXPERIMENT 2: DETERMINATION OF SERUM AMYLASE...........................................................................11

EXPERIMENT 3: DETERMINATION OF TOTAL CHOLESTERAL BY ZAK’S FERRIC CHLORIDE METHOD..........13

EXPERIMENT 4: ESTIMATION OF HIGH DENSITY LIPOPROTEIN (HDL) – CHOLESTEROL.............................15

EXPERIMENT 5: ESTIMATION OF LDL – CHOLESTEROL BY ENZYMATIC METHOD......................................17

EXPERIMENT 6: THIN LAYER CHROMATOGRAPHIC SEPARATION AND IDENTIFICATION OF AMINO ACIDS

..................................................................................................................................................................18

EXPERIMENT 7: BLOOD UREA DETERMINATION BY DIACETYL MONOXIME (DAM) METHOD...................21

EXPERIMENT 8: DETERMINATION OF TOTAL SERUM PROTEIN AND ALBUMIN – GLOBULIN RATIO..........23

EXPERIMENT 9: THE DETERMINATION OF CREATININE IN BLOOD AND URINE.........................................26

EXPERIMENT 10: DETERMINATION OF URIC ACID IN SERUM OR PLASMA (Henry et al Method).............29

EXPERIMENT 11: THE EFFECT OF PH AND TEMPERATURE ON ENZYME ACTIVITY.....................................31

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BIOCHEMISTRY STUDENT PRACTICALS

INTRODUCTION
Biochemistry Laboratory Practical Course, as part of the course curriculum, is the application of
chemistry to the study of biological processes at the cellular and molecular level. It is designed to
supplement the theoretical component of the course. From the outset, the partnership between the
practical of the course with that of the theoretical, should be understood in the right perspective,
and not as an unnecessary extra burden to be loaded on the already-overloaded students.

This is an experimental science that explores the chemistry of living organisms and the
molecular basis for the changes in living cells. It uses the methods of chemistry, physiology,
physics, molecular biology and immunology to study the structure and behaviour of the complex
molecules found in biological material and the ways these molecules interact to form cells,
tissues and whole organisms.

Biochemistry has now become the foundation for understanding all biological processes. It has
provided explanations for the causes of many human and animal diseases.

This practical course in Biochemistry has therefore, been designed to:

(a) Introduce techniques and methods used for the detection, separation and quantitative
estimation of compounds of biological importance.

(b) Develop basic skills used in Biochemistry work

(c) Demonstrate and sometimes amplify material discussed in the lecture room.

The most important aspect of practical work and of all scientific experiments is the keeping of
accurate notes and records. Unless students make a complete record of the experimental details
and results, the time spent in the laboratory is wasted. A full record should be made in the
laboratory, including all items of data, adequately labeled with appropriate units and relevant
notes, graphs, tables and calculations.

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GENERAL LABORATORY RULES AND SAFETY REGULATIONS
1. Be punctual. It is unfair to interrupt your instructor and classmates by coming late for
class.

2. A clean white Laboratory coat and comfortable closed footwear should always be worn
during practical sessions. No hats and caps are allowed.

3. Read your manuals in advance of your practical so that you may be familiar with work
you are about to do.

4. Eating, drinking, smoking and application of cosmetics inside the laboratory is strictly
forbidden.

5. Solid materials (e.g., matches, tissues, filter papers etc.) should not be thrown into sinks.
Use the waste bins provided. When pouring strong acids or alkalis into sinks, always let
the tap run freely but without splashes to dilute the reagents and wash it fully out of the
sink.

6. Do not touch your lips or face with any object that might have been on the bench as this
could be a source of infection or toxicity.

7. Do not bring common reagents bottles to your bench; remove an amount sufficient for
your use from the bottle. Never put pipettes into a common reagent bottle, this is to
ensure that a large quantity is not contaminated by careless use of dirty pipettes.

8. Do not mix up the stoppers on reagent bottles and always replace the stopper on the
reagent bottle as soon as you have finished using the reagent.

9. All glassware should at the end of a practical be thoroughly rinsed in tap water and
placed in the trays provided. Each student should ensure that his bench area is clean and
tidy and ready for use by the next class.

10. All equipment should be left in the condition you hope to find it, i.e. balances should be
spotlessly clean with all weights removed, colorimeters or spectrophotometers should not
have liquid spilled all over them or used cuvette-holders; centrifuges should be washed
and cleaned after use.

11. All breakages and defective operation of apparatus should be reported so that repairs
and/or service could be undertaken.

12. Report all accidents, such as cuts or burns to the instructor.

13. Deposit all waste material in the discard boxes provided and not on the floor.

14. Students ought to know the location of and how to use the following:

(i) Fire extinguishers

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 (ii) Asbestos blankets, emergency eye baths, emergency showers and antidotes for
different poisons.

15. N.B. Never allow an open flame when inflammable liquids are being used. If a spillage of
such a liquid occurs, report it and see to it that it is cleaned up.

16. Restrict the use of organic solvents to the fume hood.

17. Always use the fumehood for spraying of chromatograms

18. Do not pipette volatile poisonous material by mouth. Use pipette filler or a measuring
cylinder or a water pump attached to a pipette

19. Strong acids and alkalis should not be pipette by mouth. If however, you do get acid or
alkali in your mouth, rinse out with plenty of water and for acids, rinse out with carbonate
of soda and then for alkali with dilute acetic or citric acid and report to supervisor.

20. When using corrosive substances such as strong acids or alkalis, ensure that drops of this
do not fall on the floor or bench. Never place pipettes containing traces of these reagents
on the bench without rinsing them. Apart from the danger to the floor, droplets of
corrosive reagents can inflict quite serious burns on cloths and even shoes soon develop
holes.

21. When diluting strong acids, never pour water into acid!!

22. Handle all reagents with care

(a) Always carefully check the label on the reagent bottle before use.

(b) Avoid contamination of reagents by taking too much from the bottle and returning the
unused amount. Take only the amount required because biochemical reagents are
expensive.

(c) Always use a clean spatula to remove a chemical from the bottle.

23. At the end of each laboratory session, leave your bench clean and dry. Check that the sink
is clean and not obstructed and that all the water taps, gas and electric power points are
turned off.

24. Care Of Biochemical Reagents

(a) Buffers, solutions of amino acids, proteins, carbohydrates and nucleic acids and other
special solutions should be stored at 4oC to prevent bacterial of fungal growth.

(b) Protein solutions must be treated carefully.

25. Cleaning of Glassware:

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 (a) Glassware is best cleaned by washing first with detergent solutions approx. 0.5% v/v
teepol or decon, rinsing several times with tap water and finally rinsing with distilled
water.

(b) Pippetes should be soaked (with tips up) in a detergent solution and then rinsed with
tap water and distilled water. Clogged pipettes and burettes can be cleaned with a fine
wire.

(c) Glassware can be dried in an oven, except for volumetric apparatus which can be
distorted on heating.

(d) Plastic cellulose nitrate tubes should never be dried in an oven because cellulose
nitrate is an explosive chemical.

LABORATORY APPARATUS
1. PIPETTES

(a) Two types of graduated pipettes are issued;

i) Calibrated to the tip (serological) – must be blown out or drained.

ii) Calibrated between two marks (Mohr) – do not assume you have a blow/drain
type, check each time- if a Mohr pipette is allowed to drain, it will be greater
than it should be.

(b) Use the correct type of pipette e.g., do not use a 5 ml pipette to deliver 0.1ml or 10 ml
solution.

(c) When delivering small volumes, say 0.1ml, use internal graduations to measure and
not the tip portion of blow out type of pipette.

(d) The outside of a pipette should always be wiped out with tissue before delivery. If
this is not done, the adhering liquid can cause considerable error more especially for
small volumes.

2. BURETTES

A. Always check for leakage with water before filling with the required reagent.
If it leaks, consult technical staff immediately.

B. Do not change taps otherwise leakage will occur. Each tap is ground to fit a
particular burette.

3. CENTRIFUGES

(a) Always balance the load: the minimum requirement is that two tubes opposite to each
other should be filled to the same volume with liquids of the same density. If in doubt
weigh on a balance and match to within 0.3g.

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 (b) Take the speed up slowly. If strange noise emanate, TURN OFF the centrifuge
immediately.

(c) Do not open the lid until the rotor has stopped.

4. SPECIFIC GLASSWARE

Volumetric flasks and spectrophotometer tubes are expensive and should be used only for
specific purposes. Do not distort volumetric flasks by heating or use them for storage. Do
not use spectrophotometric tubes as test tubes.

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LABORATORY REPORT
It cannot be over-emphasized that the practical work is considered to be an important part of
course and the grades obtained for laboratory work are taken into account with the final
examination papers.

The writing up of experimental work reports at the end of every session must be satisfactory and
the students’ actual observed performance of practical work may be included in terms CA tests
and the final examinations. Students should therefore, understand the principles of each
technique or procedure and the manner it may be used to provide information.

Please use the following format to prepare your laboratory report.

1. TITLE: Always write the title of the experiment as the first item on your report.

2. AIM: Always indicate what you want to achieve in your experiment

3. INTRODUCTION:

Introduce the topic; also mention the theory and the principle of the experiment under
study. In the introduction you can include the chemical equations, diagrams etc.. to
support your explanation. Whatever textbooks or published articles you refer to for any
piece of information, ensure that referencing is done orderly. Also in the introduction you
must always include the normal range/values of the parameters you are investigating in
the experiment. This will act as your guide as to whether you have been successful in
your experiment or not.

4. MATERIALA AND METHODS

Always include all the materials that you used for your experiment, and outline a
stepwise methodology of what you did to get your results. Always use past tense in your
report.

5. RESULTS

In this section, you record all your observations and if the results are figurative, please
tabulate all your results well and orderly.

6. DISCUSSION

In this section, you must discuss your results obtained. Always give an explanation as to
why your results may be different from the normal ranges which you quoted in your
introduction. Support your arguments with scientific facts.

7. CONCLUSION

Here you indicate whether you have achieved your goals of your aim or not. You must be
brief and to the point.

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8. REFERENCES

All the textbooks or articles you have referred to your introduction and discussion must
be referenced. Referencing should be presented alphabetically with surname first
followed by initials. The standard format used internationally is as shown below:

“Wada, K., Takai, T and Tanabe, T. (2015). Amino acid sequence of chicken liver
cathepsin L. Euro.J. Biochem.167, 13-18”

So your order of quoting your references must be:

1. Name of the author, followed by

2. Year of publication

3. Title of the textbook or article

4. Name of the journal or publisher

5. Page numbers from where the information was quoted.

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EXPERIMENT 1: ESTIMATION OF WHOLE BLOOD GLUCOSE

PRINCIPLE

When glucose or other reducing agents are treated with alkaline copper solution they reduce the
copper resulting in an insoluble cuprous oxide being formed. The cuprous oxide formed is then
allowed to react with phosphomolybdic acid to form Phosphomolybdous acid (Molybdenum
blue) colored complex. The intensity of this colored solution is a measure of the amount of
glucose present.

A standard containing a known amount of glucose and a blank with distilled water are also
treated simultaneously with the reagent and processed. The Optic Density (O.D) values of all the
three solutions (the blank, standard and the test) are read and recorded colorimetrically at the
wavelength of 490nm. These O.D values are computed to calculate the amount of glucose
present in 100ml of blood.

REAGENTS:
1. 10% of Sodium tungstate
2. 0.66 N H2SO4
3. 10% of NaOH
4. Alkaline copper reagents: Dissolve 40g of anhydrous sodium carbonate in about 400ml of
distilled water and transfer it to 1litre volumetric flask. Dissolve 7.5g of tartaric acid in
this solution and add 4.5g copper sulphate. Mix and make up the volume to 1 litre.
5. Phosphomolybdic acid reagent. To35g Molybdic acid add 5g Sodium Tunstate, 200ml of
10% NaOH and 200ml distilled water. Boil vigorously for 20-40 minutes to remove
nearly the whole of the ammonia present in the molybdic acid. Cool, dilute to about
350ml and add 125ml of orth-phosphoric acid to make up to 500ml.
6. Standard glucose solution 0.1mg/ml. weigh 100mg glucose and to transfer 1000ml
standard volumetric flask; 0.1g/1000ml. Dissolve and make up the volume with distilled
water.

PROCEDURE:
Blood: Venous blood preserved with 2mg potassium oxalate and 1mg sodium fluoride is used.
Fluoride inhibits glycolysis in the blood cells.
Deproteination of Blood: Pipette out 7ml of distilled water and transfer into a 100ml conical
flask and add 1ml of whole blood. Rotate the flask to take the blood. Add 1ml of Sodium
Tungstate solution and mix. Drop by drop with shaking add 1ml of 0.66N H 2SO4. Allow the
mixture to stand for 10 minutes and filter it into a dry test tube. The filtrate should be clear and
colourless, and is taken as Test (T) sample.

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METHODS

1. Standard Calibration Graph

Glucose can be estimated by plotting a standard graph with different concentration of glucose
ranging from 100 to 500mg/dl.

1. Standard glucose (stock) 500mg of glucose is dissolved in 100ml of distilled water. It contains
5mg of glucose per ml.

2. Working standards: Series of working standards of glucose concentrations represented by

S1=100; S2=200; S3=300; S4=400 & S5=500 (mg/dl) are prepared as shown in the table below:

Content Blank S1 S2 S3 S4 S5 Test

Working std (ml) - 0.1 0.1 0.1 0.1 0.1 -
Conc.mg/dl - 100 200 300 400 500 ?
Filtrate (ml) - - - - - - 1
Water (ml) 1 0.9 0.9 0.9 0.9 0.9 -
Alkaline copper 1 1 1 1 1 1 1
Reagent (ml)

Keep in boiling water for 8 minutes. Cool in ice cold water

Phosphomolybdic
Reagent (ml) 1 1 1 1 1 1 1

Make up to 12 ml with distilled water and work out the dilution factor (DF) before
taking absorbance at 490nm

2. Calculations by the formula

2ml of Test sample (Tungastic acid blood filtrate) is transferred to folin-wu tube labeled T and
graduated at 25ml mark.

Similarly, pipette out 2ml of distilled water as blank and 2ml of the standard glucose (100mg/dl)
into folin-wu tubes labeled as B and S, respectively.

To each of the three tubes; T, S and B, add 2ml of alkaline CuSO 4. Rotate to mix the contents
and place the tubes in boiling water bath for 8 minutes. Remove and cool them under ice-cold
water.

Add 2ml of phosphomolybdic acid reagent to each of the tubes. Rotate to mix and make up to the
volume to 25ml mark with distilled water. Mix thoroughly and read the optical densities (O.Ds)

CALCULATIONS
_____mg of glucose in 100ml (or mg/dl) of a given sample
Mg of glucose / 100ml of blood = mg of glucose in standard X (OD of test / OD of Standard) X 100

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Note! Express your answer in mmol/L.

CLINICAL SIGNIFICANCE
The fasting blood glucose value by this method amounts to 80 – 120 mg/100ml in normal
subjects, which is about 20% higher than the true blood glucose level. Blood sugar level is
increased in uncontrolled diabetes mellitus. Increased levels are also found due to hyper
functions of anterior pituitary and adrenal cortex.
In hyperthyroidism, fasting blood sugar level may be normal but there is pronounced
hyperglycemias in the feed state, fear, anger, anxiety and other emotional states that cause a
transient elevation of blood sugar level. This is attributed to the increased secretion of adrenalin,
which has a hyperglycaemic action.

Hyperglycaemia is seen in insulin secretion tumors of the beta cells of pancreas, occationally, it
is encountered in renal diabetes. Insulin overdose also causes hypoglycaemia.

QUESTIONS
1. When and how would you perform an oral glucose tolerance test on an individual?
2. How would you interpret a flat type of glucose tolerance curve?
3. How would you establish an individual as “pre-diabetic”?

EXPERIMENT 2: DETERMINATION OF SERUM AMYLASE

PRINCIPLE
Iodine forms a blue coloured complex with starch. Amylase is the enzyme which breaks down
starch. Serum is incubated with starch substrate at 37oC for 10 minutes. The enzyme reaction is
stopped by the addition of Iodine – EDTA solution. The remaining starch forms a blue colour
with iodine from which the amylase activity is calculated.

REAGENTS

1. Nitric acid 1 mol/L. Dilute 61 ml of concentrated HNO3 to one litre with water.

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 2. Buffered starch solution. Make a paste of 125 mg of starch with about 3 ml of water in a
250 ml beaker. Add about 100 ml of water. Add 2.435 g of tris (hydroxymethyl) amino
methane and 2.875 g of sodium chloride (AR grade). Boil while stirring. Cool to room
temperature. Add 250 mg of sodium azide as preservative. Adjust the pH to 7.4 by the
addition of 1 M HNO3. Transfer to a 250 ml volumetric flask and make to mark.

3. Stock Iodine – EDTA solution: Dissolve 12 g of potassium iodide (KI) in 125 ml of water
taken in a 500 ml volumetric flask. Add 185 mg of disodium EDTA and stir until
dissolved. Add 5.2 g of iodine and stir until dissolved. Make up to 500 ml with water.
Store in a brown bottle.

4. Working iodine – EDTA: Dilute the stock solution 10 in 100 with water every day.

PROCEDURE

1. Label three test tubes “Test (T)”, “Standard (S)” and “Blank (B)”.

2. Add 2 ml of buffered starch solution to T, S and B. Incubate the three tubes at 37 oC for 5
minutes.

3. Pipette 0.05 ml (50µl) of serum to test tube T only. Incubate for 10 minutes.

4. Add 15 ml of Iodine – EDTA working solution to test tubes T, S and Blank.

5. To test tube S add 0.05 ml (50µl) of serum, and to test tube B add 0.05 ml (50µl) of
water. Mix each tube thoroughly and allow them to stand for 3 minutes.

6. Measure and record the absorbance (optical density, O.D.) at 640 nm or red filter.

CALCULATION

Units of enzyme activity: One amylase unit for this method is amount of enzyme that catalyses
hydrolysis of 10 mg of starch in 45 minutes to a stage at which no colour is given by iodine. In
this case

= Conc of starch in 2 ml of buffer x 45 min

10 mg of starch x 10 min

= 1 x 45 = 0.45 units
10 10

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Therefore, under condition of this procedure complete hydrolysis of the substrate per mg
represents 0.45 amylase units per 0.05 ml of serum sample.

For 100 ml of serum sample

= 0.45 100 = 900 units/dl

0.05

Amylase activity in units/100 ml = OD of S – OD of T x 900

OD of S

If the amylase activity is above 1000, 1 in 5 dilution of the sample can be done.

CLINICAL SIGNIFICANCE

Normal serum amylase levels are 80 to 150 Somogyi units/ 100 ml. Certain individuals may
possess up to 300 units/100 ml without any obvious disease. Above this level, however,
pathological processes are likely to have elevated serum amylase activity in a variety of diseases
such as;

1. Carcinoma of the head of the pancreas

2. Intestinal obstruction
3. Hyperthyroidism
4. Kidney disease with impaired renal function
5. Mumps

Increased values are also seen in patients taking certain drugs for other indications, e.g opiates
and chlorothiazide.

Reduced or even absent, serum amylase may occur in patients with diseases, of the liver,
including hepatitis, cirrhosi, abscess and carcinoma. Acute alcoholism has a similar effect.
Elevations have been reported in pregnancy toxaemias.

Lipids: Cholesterol, Triglycerides and Associated Lipoproteins

EXPERIMENT 3: DETERMINATION OF TOTAL CHOLESTERAL BY ZAK’S FERRIC

CHLORIDE METHOD

Background Information

Cholesterol is an unsaturated alcohol of the steroid family of compounds; it is essential for the
normal function of all animal cells and is a fundamental element of their cell membranes. It is
also a precursor of various critical substances such as adrenal and gonadal steroid hormones and

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bile acids. The synthesis and utilization of cholesterol must be tightly regulated in order to
prevent over-accumulation and abnormal deposition within the body.

The estimation of cholesterol in serum helps to detect disorders associated with high cholesterol
levels and treat the subject appropriately.

PRINCIPLE

Cholesterol in acetic acid reacts with ferric chloride and sulphuric acid to produce a red colour.
The absorbance of the red coloured solution is read at 560 nm.

REAGENTS

Analytical grade (AR) chemicals should be used.

1. Glacial acetic acid

2. Ferric Chloride, 0.05 percent reagent: Dissolve 500 mg of ferric chloride in one litre
glacial acetic acid (aldehyde free). Store in brown bottle. It is stable for a month.

3. Conc. Sulphuric acid, AR grade.

4. Stock cholesterol standard solution: 100 mg per 100 ml in acetic acid. Dissolve exactly
100 mg cholesterol in 100 ml glacial acetic acid. Keep in a cool, dark place. Reagent is
stable for one month.

5. Working standard cholesterol solution 0.04 mg/ml: Dilute 4 ml of stock standard

cholesterol solution to 100 ml with ferric chloride reagent (reagent 2). Keep in cool, dark
place.

PROCEDURE

Pipette 9.9 ml ferric chloride reagent into a dry test tube. Add 0.1ml of serum, mix thoroughly
and allow it stand for 10 minutes. Centrifuge the solution and collect 5 ml of the clear
supernatant solution.

1. Set up three test tubes labeled as T for test, S for standard and B for blank as negative
control.

2. Transfer the 5 ml supernatant fluid into T. Pipette out 5 ml of the working standard
cholesterol into S, and 5 ml of ferric chloride reagent into B.

3. To each of the three solutions add 3 ml of conc. sulphuric acid. Mix and incubate for 30
minutes.

4. Read and record the absorbance (optical density OD) at 560nm.

CALCULATION

Mg % of cholesterol per 100 ml serum = OD of T – OD of B x 200

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 OD of S – OD of B

Mg % of cholesterol per 100 ml serum:

= T x 200 = _______mg of cholesterol in 100ml or mg/dl

S

CLINICAL SIGNIFICANCE

Normal serum cholesterol ranges from 150 to 220 mg per 100 ml of serum. This may be slightly
higher in middle age and pregnancy. Increased level of cholesterol in serum is called
hypercholesterolemia and is found in nephrosis, myxoedema, obstructive jaundice and angina
pectoris. Decreased levels called hypocholesterolemia may be associated with hyperthyroidism.

EXPERIMENT 4: ESTIMATION OF HIGH DENSITY LIPOPROTEIN (HDL) –

CHOLESTEROL

Background Information

Atherosclerosis is the condition where low-density-lipoprotein (LDL) – cholesterol is deposited

in the subintimal regions of arteries causing obstruction to the flow of blood. This may lead to
extra burden on the heart, which may be one of the reasons for hypertension. If this condition is
neglected for a long time, it may lead to cardiac diseases and ischemia. HDL Cholesterol is

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inversely related to the risk of atherosclerosis. Hence, it is named as good cholesterol. HDL
values above 60 mg/dl indicate very low-risk for atherosclerosis. LDL Cholesterol is directly
related to risk of atherosclerosis. So LDL is termed as bad cholesterol. LDL values above 160
mg/dl indicate high risk.

PRINCIPLE

Chylomicrons, VLDL, LDL present in serum are precipitated by polyions. Cholesterol esters are
hydrolysed by cholesterol esterase to free cholesterol and fatty acids. Free and liberated
cholesterol are oxidized by cholesterol oxidase to chol-4-ene -3-one and H 2O2 is liberated. The
H2O2 produced couples with 4-aminoantipyrine and phenol in the presence of peroxidase to form
a coloured compound. The intensity of the colour developed is proportional to the concentration
at 500nm (490 – 550) or with green filter.

REAGENTS

1. Phosphotungstic acid reagent: 169 mg of phos-photungstic acid is dissolved in 50 ml of

distilled water. To this 508 mg of MgCl2 is added, mix and the volume is made up to 100
ml with distilled water and mix again.

2. Enzyme reagent. It contains 4-aminoantipyrine, phenol, peroxide and sodium azide in a

powdered form in an amber coloured bottle. The enzyme reagent is reconstituted by
adding 20 ml of diluent reagent.

PROCEDURE

1. Serum 0.2 ml and 0.4 ml phosphotungstic acid reagent containing MgCl 2 is taken in a
centrifuge tube and mixed in a vortex mixer for 10 seconds. Keep it in a room
temperature for 20 minutes.

2. Then it is centrifuged at 1500 rpm for 30 minutes at room temperature. The supernatant is
removed carefully avoiding mixing of the contents. If the supernatant is not clear, the
contents are recentrifuged at high speed and if still slightly turbid the analysis is repeated
on diluting with equal volume of the buffer.

3. Standard 0.2 ml is also treated the test and the supernatant is taken.

4. 25 µl of the supernatant of the test and 25 µl of cholesterol standard are taken in different
tubes using an auto-pipette.

5. 1 ml of each enzyme reagent (chromogen reagent) is added to blank B, standard S and

test T, and contents are mixed well and incubated at room temperature for 20 minutes.

6. Absorbance (OD) for the test, standard and blank are read against distilled water at 546
nm or using green filter.

7. Colour developed is pink and is stable for one hour at room temperature and is protected
from direct light (incubation can also be done for 5 minutes at 37oC).
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CALCULATION

Mg HDL-Cholesterol per 100 ml serum = OD of T – OD of B x conc of std x Dil factor

OD of S – OD of B

HDL-Cholesterol = ______mg/dl

CLINICAL SIGNIFICANCE

The HDL – Cholesterol serves in removing cholesterol from peripheral cells and transporting it
back to the liver where most of the cells of the cholesterol excreted from the body is removed. So
HDL is called as beneficial factor among the other lipoproteins concerned.

Reference:

Shivananda Nayak B. (2207) Manipal Manual of Clinical Biochemistry. 3 rd Edition, Jaypee

Brothers,New Delhi, India 2007:

EXPERIMENT 5: ESTIMATION OF LDL – CHOLESTEROL BY ENZYMATIC

METHOD

PRINCIPLE
LDL – Cholesterol is precipitated by heparin at their iso-electric pH. When the tube is
centrifuged, LDL-cholesterol separates at the bottom. The supernatant contains HDL-cholesterol
and VLDL-cholesterol. The supernatant is tested enzymatically for cholesterol concentration.

Therefore, LDL-cholesterol is calculated as:

LDL-Cholesterol = Total cholesterol minus (-) cholesterol present in the supernatant.

REAGENTS

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 1. Precipitation reagent contains: Heparin-50,000IU/L, Sodium citrate-0.004mol/L pH 5.04.
(The reagent is ready for use).
2. Cholesterol reagent – ready for use.
3. Cholesterol standard – 200mg/dl – ready for use.

PROCEDURE

1. Take 0.1 ml of serum in a centrifuge tube and add 1 ml of precipitation reagent. Mix and
keep it for 10 minutes at room temperature. Then centrifuge at 4000 rpm for 15 minutes.
The supernatant is used for cholesterol estimation.
2. Take 3 test tubes and label them as B, S and T.
3. To B, add 0.05 ml of distilled water and add 0.05 ml of cholesterol standard to tube S. To
tube T add 0.05 ml of the supernatant from step 1.
4. To all the tubes add 1 ml of cholesterol reagent. Mix and keep for 5 minutes at 37 oC or 10
minutes at room temperature.
5. Measure and record the absorbance (optical density) at 505 nm.

CALCULATION

Cholesterol conc in the supernatant = OD of T – OD of B x conc of std

OD of S – OD of B

=_______mg/dl

LDL cholesterol = Total cholesterol – cholesterol in the supernatant.

CLINICAL SIGNIFICANCE

Increased LDL cholesterol increases the risk of coronary heart disease and atherosclerosis.
Normal range: 10 – 160 mg/dl.

EXPERIMENT 6: THIN LAYER CHROMATOGRAPHIC SEPARATION AND

IDENTIFICATION OF AMINO ACIDS

Thin Layer Chromatography (TLC) is an efficient method of separating complex mixtures. It is

sensitive, fast simple and inexpensive analytical technique in carrying out small experiments.

One of the important applications of TLC is the separation of amino acids. Amino acids contain
both the amino groups as well as the carboxylic groups. The most important are the α-amino
acids as these are the units from which proteins are made.

In this experiment you will learn the movement of some simple amino acids on silica gel coated
plates. TLC of amino acids is based on their distribution between a finely divided powder of an
adsorbent and an organic mobile phase.

PRINCIPLE

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TLC is similar to paper chromatography in that the sample is spotted near one end of a plate of
glass or plastic coated with a thin layer of an adsorbent. The TLC plate is placed in a covered jar
containing a shallow layer of developer. The developer rises up by capillary action and the solute
is distributed between the stationary (absorbent) phase and the mobile phase. A solute which is
more strongly adsorbed onto the stationary phase, will spend less time in the mobile phase, and
hence it will migrate more slowly up the TLC plate. The sample is subsequently separated by
development (elution). Treatment with a detector forms the coloured zones of the solutes. The
components of a mixture are identified by the calculation and comparison of Rf values.

REQUIREMENTS

Apparatus Chemicals
TLC jar or tank Propanol-1
(An alternative is a beaker covered Conc. Ammonia solution
By a watch glass or aluminium foil) Any three amino acid :
Spotting capillaries L-alanine
Measuring cylinder 100ml L-leucine
TLC plates (Either arranged from a supplier L-Lysine
Or prepared by the Lab Demonstrator) L-Aspartic acid
Spraying bottle Methionine

Preparation of TLC Plates

TLC plates can be prepared by the spreading method: Take a clean glass and mix about 30g of
Silica Gel G with 60ml of distilled water ( stirring and shaking well to get out any lumps) and
pour on the glass plate. Spread it out with the help of TLC applicator. Let the air dry for ten
minutes and put the plate at 110oC in an oven for at-least 30minutes to activate the adsorbent.

Solutions provided

1. Sample Solution:

Provide two solutions from any of the amino acids above. Label the unknown solutions
as X and Y. Their solutions can be prepared by dissolving 15mg of each amino acid
separately in 1ml of distilled water. Warm if a particular amino acid is not soluble in cold
water.

2. Detector:

Ninhydrin Reagent (0.2%): Take 100ml of 1-butanol and 100ml of water in a separatory
funnel. Shake gently and allow it to form the layers. Remove the lower aqueous layer.
Transfer the upper organic layer to a spraying bottle and to this add 0.2g of Ninhydrin,
shake well and use as the detector for amino acids.

PROCEDURE

19 | P a g e
1. Preparation of Developer: Prepare the developer by mixing 1-propanol and concentrated
ammonia in the proportion of 7:3 respectively by measuring the required volumes with
the help of a measuring cylinder.
2. Take a silica gel coated TLC plate from your demonstrator.
o o
3. Dry the plate in an oven for 30 minutes at 100 C - 110 C, so that they are activated for
adsorption chromatography.
4. Take a plate and make a light pencil line across it, 2cm above the bottom of the plate and
put a short mark at the line centre where known or unknown amino acids will be spotted.

Caution! While marking the plates with pencil, do not press so hard on the pencil that you remove the
silica gel.

5. Label the plate at the top end to indicate known or unknown amino acids.
6. Hold the plate in the left hand cautiously, so that the fingers do not touch the adsorbent
layer. Take a capillary and place in the solution of amino acid to be spotted, let the
solution rise into the capillary, tail out the capillary from the solution and gently touch the
capillary to the layer side of the TLC plate at the marked centre. Allow the solution to
flow on the plate for a short duration so that a spot of the solution is formed but not larger
than 2mm in diameter.

Caution! The spot applied should be kept as small as possible. Application of too much of the solute
should be avoided, as this will result in an elongated zone and may affect the correct calculation of R f
values.

(Note: the lab demonstrator is supposed to demonstrate this technique).

7. Allow the spot to dry. You can blow in order to aid evaporation. Apply more solution at
the same place (if required). The aim is to apply a small but visible and built up spot.
8. Apply the unknown solution on a separate TLC plate in a similar manner.
9. After spotting all the known and unknown solutions, insert the plates into the developing
jars or tanks.
10. Pour the mobile phase into the chamber, with the help of a pipette till the developer level
reaches nearly at 1.5 cm height of the lower edge of the adsorbent layer on the plate
(Remember that the spots should be above this level).
11. Cover the jar and allow the developer to ascend along the plate. The position of the
solvent front can be seen visually as the damp portion of the plate appears darker than the
dry portion.
12. When the developer ascends to a required height on the plate, remove the plate from the
developing chamber, mark the solvent front and dry the plate at 100 oC for about 10
minutes.
13. After the plates have been dried, spray the detector on the plates with the help of a
spraying bottle. The detector is 0.2% ninhydrin solution in butanol saturated with water.
14. Heat the plates at 110oC, either in an oven or on a hot plate, for 5 – 10 minutes till the
zones of amino acids appear as coloured spots on the plates.
15. Mark the periphery of the coloured spots and their centres.
20 | P a g e
 16. Measure the distance of each spot-centre from the starting line and also the distance by
which the solvent front has moved. Calculate the Rf values.
17. From the comparison of Rf values of known and unknown samples you can determine
which amino acids are present in your unknown solutions.

In your report;
(a) Calculate the Rf value of each of the spots on the chromatogram.
(b) Carefully analyze your TLC data by comparing the Rf values and colors of known spots
with those of unknown, deduce the nature of the unknown solutions in X and Y.

EXPERIMENT 7: BLOOD UREA DETERMINATION BY DIACETYL MONOXIME

(DAM) METHOD.

Background Information

Urea is the primary metabolite derived from dietary protein and tissue protein turnover. The
ammonia formed during amino acid metabolism is toxic to the human body. So it should be
converted to a nontoxic substance (urea) and excreted normally. Urea constitutes about 45 % of
NPN substances. Study of their elimination by simultaneous study of blood and urine may help
in understanding of renal disturbances.

Most renal diseases affect urea excretion so that urea levels increase in the blood. Patients with
dehydration or bleeding into the stomach and/ or intestines may also have abnormal urea levels.
Numerous drugs also affect urea by competing with it for elimination by the kidneys.

PRINCIPLE

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Urea reacts with diacetyl monoxime under strongly acidic conditions in presence of ferric ions
and thiosemicarbazide to give a pinkish yellow chromogen which is then quantified by
photometric method to determine the amount of urea.

REAGENTS

1. Diacetyl monoxime: Dissolve 1.56 g diacetyl monoxime in 250 ml distilled water.

2. Ferric Chloride: Dissolve 324 mg of ferric chloride in 10 ml of 56% orthophosphoric

acid. Store in a brown bottle.

3. Thiosemicarbazide: Dissolve 41 mg of thiosemicarbazide in 50 ml of water.

4. Sulphuric acid 20%: Add 200 ml concentrated sulphuric acid to 800ml of distilled water
in a beaker slowly with stirring and cooling.

5. Acid Reagent: Mix 1 litre of 20% sulphuric acid (reagent 4) with 1 ml of Ferric Chloride
(Reagent 2).

6. Trichloroacetic acid, 10%: Dissolve 10 g of TCA in water and make up to 100 ml.
7. Preservative diluents for standard: Boil 250 ml water and add 40 mg of phenyl mercuric
acetate, mix to dissolve. Transfer to 1 litre graduated cylinder. Add 0.3 ml concentrated
sulphuric acid and make up the volume to 1 litre and mix. The use of preservative
diluents is optional.

8. Stock standard urea: 0.5 mg/ml. Dissolve 50mg of urea (GR Grade) in 100 ml of
preservative diluents (Reagent 7) or can be dissolved in de-ionized water it is stable for
week if it is refrigerated.

9. Standard urea for use: 0.01 mg/ml. Dilute 1ml of stock standard (Reagent 8) solution to
50 ml with de-ionized water.

PROCEDURE

Preparation of protein free: Pipette 3.4 ml of distilled water into a dry test tube. Add 0.1ml of
whole blood and mix thoroughly. Pipette 1.5 ml of 10% trichloroacetic acid and mix well. Let it
stand for 10 minutes. Filter into a dry test tube or centrifuge and collect 1 ml of the supernatant.

Development of Colour:
1. Set up three test tubes labeled as T for test, S for standard and B for blank as negative
control.

2. Transfer 1 ml of the filtrate or the supernatant into test tube T.

3. Pipette 1 ml of the standard urea into test tube S, and 1 ml of distilled water into B.

4. To each of the tubes add 1ml of diacetyl monoxime reagent, 1 ml of thiosemicarbazide

solution and 3.0 ml of acid reagent. Mix the solutions thoroughly.

22 | P a g e
 5. Place the tubes in a boiling water bath for 15 minutes. Cool the tubes in a beaker
containing cold water.

6. Read and record the absorbance (optical density OD) at 540nm.

CALCULATIONS

Mg urea in 100 ml of blood = OD of T – OD of B x 5 x 0.01 x 100

OD of S – OD of B 1 0.1

= T – B x 50 = _______mg of urea in 100ml or mg/dl

S–B

Note! -Dilute the sample if the optical density rises above 0.6 and perform the test again.
-Dilution factor should be taken into account during calculations.

CLINICAL SIGNIFICANCE

The normal blood urea level ranges from 12 to 36 mg/100ml. Most of blood urea is excreted via
glomerular filtration in the urine. When plasma volume is diminished as in diarrhea and
vomiting, glomerular filtration decreases and blood urea is elevated.

Blood urea levels are also elevated in advanced age and all other forms of renal diseases such as
acute and chronic glomerulonephritis, later stages of nephrosis, polycystic kidney, malignant
hypertension and hydronephrosis.

Any type of obstruction in the lower urinary tract diminishes glomerular filtration resulting in
elevated blood urea levels. Enlargement of the prostate, stones in bladder are some of the post
renal causes of increased blood urea levels.

EXPERIMENT 8: DETERMINATION OF TOTAL SERUM PROTEIN AND ALBUMIN –

GLOBULIN RATIO

Background Information

Blood is a complex mixture of cells suspended in a fluid medium, plasma. 92 – 93% of this fluid
medium is water and the remaining 8% is dissolved proteins, minerals, glucose, etc. By far, the
largest amounts of the total solutes are the plasma proteins, collectively referred to as TOTAL
PROTEIN. In serum, one of the plasma proteins, FIBRINOGEN, is missing, but it only
constitute 3-6% of the total protein in plasma. To standardize normal values, measurement of
total protein has been confined to serum samples.

On the basis of their solubility properties, the serum proteins can be divided into two major
fractions, ALBUMIN and GLOBULINS which serve a number of different functions.

PRINCIPLE

23 | P a g e
The most widely used method of measuring serum protein is the Biuret reaction method. The
principle of this reaction is that serum proteins react with copper sulphate in sodium hydroxide to
form a violet “biuret” complex. The intensity of the violet color is proportional to the
concentration of protein. The reaction occurs in the peptide bonds of tripeptides or larger.

REAGENTS

1. Sodium Chloride, 0.9%. Dissolve 900mg of NaCl in 100ml of distilled water.

2. Sodium hydroxide, 0.2 N. Dissolve 8g of NaOH in one litre (1L) of distilled water.
3. Biuret reagent: Dissolve 45g of sodium potassium tartarate in about 400ml of 0.2N
sodium hydroxide - NaOH (Reagent 2). Add 15 g of copper sulphate stirring
continuously. Add 5 g of potassium iodide – KI. Dissolve and make up to one litre with
0.2N sodium hydroxide. This is the stock Biuret reagent. Store in a polythene bottle. It is
stable for months.
4. 23% Anhydrous sodium sulphite- Na2SO3 or Sodium Sulphate- NaSO4
5. Standard protein solution. A solution of bovine serum albumin containing 2mg
protein/ml.
6. Ethyl-ether, AR grade.

PROCEDURE

Step 1: Separation of albumin from serum proteins:

Pipette 0.2ml of serum into a test tube. Add 5.8ml of 23% sodium sulphite or sodium sulphate
solution and 2ml ether.

Note! Ethyl-ether is added to help separate the precipitated globulin by centrifugation.

Gently invert the tube 20 times to precipitate the globulins and centrifuge for 5 minutes.

Aspirate and discard the ether layer. Pipette the lower layer for albumin estimation.

Caution! Vigorous mixing may precipitate albumin also.

Alternatively, globulins can also be removed by filtering through the whatman no.44 filter paper,
in which case Ethyl-ether is not required.

Step2: Development of color.

Set up four test tubes as follows:
(a) To the first tube (blank) labeled as ‘B’ add 3ml sodium chloride, 0.9%.
(b) To the second tube (standard) labeled as ‘S’ add 3ml of standard protein solution.
(c) To the third tube (total protein) labeled as ‘TP’ add 0.1ml of serum and 2.9ml of sodium
chloride, 0.9%.
(d) To the fourth tube (albumin) labeled as ‘A’ add 3ml of separated albumin (sodium
sulphite filtrate) from step #1.

24 | P a g e
Step 3: Add 3ml of biuret reagent to each of the above four test tubes. Mix them thoroughly and
allow them to stand for 10 minutes.

Step 4: Record the absorbance (O.D values) of all the four solutions at 540nm using blank to
zero the spectrophotometer.

CALCULATIONS
Total protein in g/100ml = TP – B x 6 x 100 x 1
S–B 0.1 1000 = _________g/dl

Albumin in g/100ml = A – B x 6
S–B

Albumin = ______g/dl

Globulins = total protein – Albumin.

Thus, Albumin/Globulin ratio can be worked out.

CLINICAL SIGNIFICANCE

Total protein concentration in serum is around 7mg/dL in normal subjects. The albumin globulin
ratio is close to 2:1 by this method. Increase in total serum proteins may occur in dehydration
with the ratio remaining unaltered. There are several conditions where increase in total protein is
mainly due to increase of one of the globulins. In many of these conditions, albumin level
remains same or is slightly reduced. Multiple myeloma is a typical example. A decrease in total
proteins is invariably due to a fall in albumin level, which may be accompanied by an increase in
globulin concentration. The ratio is decreased in these cases. Hypoalbuminemia can be due to
either loss of albumin in kidney disease, impaired synthesis in some liver diseases, inadequate
supply of dietary protein or excessive protein catabolism.

Albumin is lost in urine in nephritis and nephrosis. Large amount of albumin is also lost in
severe burns and hemorrhage. In all types of chronic liver diseases particularly cirrhosis,
hypoalbuminemia is seen. Reduced albumin synthesis by the liver also occurs in different
infectious conditions and severe anaemia. Kwashiokor is a typical condition of inadequate
supply of dietary proteins. The serum albumin levels can be as low as 1.0 – 1.5g/100ml in
kwashiorkor. Pancreatic diseases, intestinal fistula, congenital malabsorption syndromes and
severe tuberculosis can also result in low albumin levels in serum. Excessive breakdown of body
proteins together with inadequate supply or defective utilization of proteins is seen in
uncontrolled diabetes, thyrotoxicosis, prolonged febrile disease and trauma.

Questions

1. What are the two major functions of serum proteins?

2. What are the physiological roles of albumin and globulin?

25 | P a g e
 3. Briefly explain why normal saline rather than distilled water is used for protein or
albumin dilutions.

STUDY OF NON-PROTEIN NITROGEN COMPOUNDS

The intermediates or the end products of protein metabolism include Urea, Creatinine, Uric acid, amino
acid and ammonia.

EXPERIMENT 9: THE DETERMINATION OF CREATININE IN BLOOD AND URINE

Background Information
Creatinine is a catabolic product formed from creatine phosphate. Serum creatinine is utilized as
a screening test in the clinical evaluation of renal function [ ]. Serum creatine levels are
influenced by creatinine filtration rate in the kidneys, sex, age, muscle mass and the analytical
method utilized for measurement. Creatinine measurements are useful in the diagnosis and
treatment of renal diseases.

PRINCIPLE
26 | P a g e
The 1886 Jaffe’s reaction, in which creatinine is treated with an alkaline picrate solution to yield
a reddish orange color complex, is still the basis of most commonly used methods for measuring
creatinine in blood and urine. Max Jaffe invented that on reaction with sodium hydroxide and
picric acid solution, creatinine forms a reddish orange colour which can be measured by
spectrophotometer.

Over the years the method has gone through refining phases so as to improve the specificity of
Jaffe’s method. Creatinine is first of all isolated from common interfering chromogens by
adsorption on aluminium silicate such Lloyd’s creatinine, followed by elution and later treated
with alkaline picrate solution. Most of the chromogen is due to creatinine; non-creatinine
chromogen may contribute up to 20% in plasma, but less than 5% in urine.

REAGENTS

1. Picric acid, 0.04M: Dry some picric acid crystals by putting over a pad of filter paper.
Dissolve 9.16 g of dry picric acid in water and make up to 1 litre.
2. Sodium hydroxide, 0.75N: weigh about 7 g of sodium hydroxide and prepare 200 ml
solution in water. Determine the normality by titrating against 0.667 N Sulfuric Acid
(Reagent 1). Adjust the normality of sodium hydroxide to 0.75 by proper dilution.
3. Stock standard creatinine solution, 1mg per ml. Dissolve 100 mg of creatinine in 0.1N
HCl (1ml concentrated HCl diluted 100ml) and make up to 100ml with acid. Store in
refrigerator. Stable for one month.
4. Sulphuric acid, 0.667 N: Dilute 10 ml of concentrated sulphuric acid to 500 ml water.
5. Sodium tungstate, 5%: Dissolve 5 g of sodium tungstate in water and make up to 100ml.
6. Urine working standard, 0.02 mg per ml: Dilute 2 ml of stock standard to 100ml with
water. Urine sample 1 ml diluted to 50 ml in a conical flask.
7. Serum working standard, 0.04 mg per ml: Dilute 4ml of stock standard to 100ml with
water.
8. Lloyd’s Reagent: use Fuller’s earth from BDH Ltd.

Urine Creatinine

Procedure:

Set up the solutions in three test tubes:

1. Add 4ml of water in the 1st tube labeled as B for blank.

2. To the 2nd tube labeled as S add 0.5ml of the standard creatinine solution and 3.5ml of
distilled water.

3. To the 3rd tube labeled as T, add 2ml of diluted urine and 2ml of distilled water.

4. To each of the three test tubes, add 1ml of picric acid and 1ml of sodium hydroxide.

5. Mix the solutions thoroughly and allow them to stand for 15 minutes.

27 | P a g e
 6. Read and record the absorbance at 520nm.

Calculation: = T – B x 0.01 x 50 x100

S–B 2

= T – B x 25 = ______ mg of creatinine in 100ml of urine

S–B

Serum Creatinine

Procedure:

1. Set up three labeled tubes as; T (Test), S (Standard) and B (Blank).

2. Add 2ml of distilled water to Test tube T, another 2ml to test tube S and 4ml of water to
B.

3. Add 2ml of serum to test tube T and 2ml of standard solution to S.

4. To each of the three test tubes add 2ml of 0.667N sulphuric acid and 2ml of 5% sodium
tungstate. Mix the solutions thoroughly and allow them to stand for 10 minutes.

5. Collect 3ml of the supernatant and transfer to three different tubes with the same labels.
To each of these add 1ml of 0.04 M, picric acid and 1ml of 0.75 N sodium hydroxide.

6. Mix the solutions and allow them to stand for 15 minutes.

7. Read and record the optical density at 520nm.

CALCULATIONS = T – B x 0.03 100

S–B 75

= T – B x 4 = _______ (mg of creatinine / 100ml of serum) or mg/dl

S–B

If creatinine is determined in a 24 hour urine sample, and also in the blood serum sample taken
towards the end of the collection period, then the results can be combined to determine
“creatinine clearance” a measure of kidney function. The calculation is as follows:

Creatinine clearance, therefore, can be calculated by applying the formula:

= UxV = ____ml/min where,

P x24 x60

U = Urine creatinine concentration

V = Volume of the urine
P = Plasma creatinine concentration

28 | P a g e
CLINICAL SIGNIFICANCE

The measurement of creatinine concentrations in plasma and urine samples illustrates the
filtration capacity of the glomerulus, also known as the glomerular filtration rate (GFR).
Creatinine is produced endogenously within the blood and is freely filtered by the glomerulus.
These characteristics make creatinine a useful endogenous marker for creatinine clearance. If the
GRF is decreased, as in renal disease, creatinine clearance via the renal system is compromised.
The reduced GRF will then lead to an increase in plasma creatinine concentration. The normal
creatinine excretion is 400 mg to 1800 mg daily, while blood serum ranges from 0.5mg/dl to 1.5
mg/dl.

The measurement of plasma alone should not be used to assess renal function. Plasma creatinine
levels may not be affected until significant renal damage has occurred. In addition, a plasma
creatinine level that is within normal reference range does not equate to a normal functioning
renal system.

Although not as specific as creatinine, blood urea nitrogen (BUN), (to be covered as our next
lab) can also be used as an indicator of renal function. BUN is not the preferred marker for
clearance because it is influenced by factors such as a high protein diet, variables in protein
synthesis, and patient hydration status. Alone BUN is not the ideal marker for GFR. Combined
with plasma creatinine as a creatinine/BUN ratio, BUN can be useful analyte in differentiating
pre or post renal increase of plasma Non protein nitrogen (NPNs).

Reference:

Miltuninovic J, Culter R, Hoover P, Meijsen B, Scribner B. Measurement of residual glomerular

filtration rate in the patient receiving repetitive hemodialysis, Kidney Int. 1985:8:185 – 190.

29 | P a g e
EXPERIMENT 10: DETERMINATION OF URIC ACID IN SERUM OR PLASMA
(Henry et al Method)

PRINCIPLE

Uric acid is oxidized to allantoin and carbon dioxide by a phosphotungstic acid reagent in
alkaline solution, phosphotungstic acid is reduced to tungsten blue which is measured at 710nm.
The uric acid concentration in serum is affected by renal as well as extra-renal factors.

The uric acid determination is helpful in the diagnosis of gout, and when there is severe
impairment in the renal function the uric acid will be varied.

REAGENTS

1. Sulphuric acid 0.667 N: Dilute 10 ml concentrated sulphuric acid to 500ml.

2. Sodium tungstate 10% (w/v): Dissolve 10 g of sodium tungstate in water and make up to
100 ml.

3. Sodium carbonate 14%: Dissolve 14 g of sodium carbonate in about 80 ml of water and

make up to 100 ml.

4. Phosphotungstic acid reagent: dissolve 40 g of sodium tungstate in 300 ml water in a

litre of round bottom flask. Add 32 ml of syrupy phosphoric acid (85%) and several glass
beads or porcelain bits. Attach a reflux condenser and boil gently for 2 hours. Cool to
room temperature. Add 32 g of lithium sulphate and dissolve. Dilute to one litre and store
in brown colored reagent bottle.

5. Stock standard uric acid, 1mg/ml: Dissolve 60 mg of lithium carbonate in 20 ml of water.

Heat the solution to about 60 oC and add exactly 100mg of uric acid. Stir until dissolved.
Cool, transfer to a 100 ml volumetric flask. Add 2 ml of formalin (40%) and 1 ml of 10%
acetic acid solution. Make up to 100 ml mark with water. Keep in a well – stoppered
bottle in the dark or refrigerator. The solution is stable for one month.

6. Working standard: 0.01 mg/ml: dilute 1 ml of the stock solution to 100 ml with water.

PROCEDURE

Preparation of protein free: Pipette 8.0 ml distilled water into a dry test tube. Add 0.1ml of
serum or plasma and mix thoroughly. Pipette 0.5 ml of 0.667 N sulphuric acid and 0.5 ml of 10%
sodium tungstate. Mix well and centrifuge for 10 minutes and collect 3 ml of the supernatant.

Development of Colour:
1. Set up three test tubes labeled as T for the test, S for standard solution and B for blank as
a negative control.

30 | P a g e
 2. Transfer 3 ml of the supernatant into test tube T.

3. Pipette 3 ml of the standard uric acid into test tube S, and 3 ml of distilled water into B.

4. To each of the tubes add 1ml of phosphotungstic reagent, 1 ml of 14% sodium carbonate
solution and 3.0 ml of acid reagent. Mix the solutions thoroughly.

5. Keep the tubes at room temperature for 15 minutes.

6. Read and record the absorbance (optical density OD) at 710nm.

CALCULATION

Mg of uric acid per 100 ml serum = OD of T – OD of B x 0.03 x 100

OD of S – OD of B 0.3

= T – B x 10 = _______mg of uric acid in 100ml or mg/dl

S–B

CLINICAL SIGNIFICANCE

Normal reference range from 2.5 to 7 mg/dL, and hyperuricemia may indicate the risk of acute
gouty arthritis. In patients with gout, the risk of stone formation rises with the level of serum uric
acid. A better correlation exists between stone formation and urinary uric acid excretion. One
study found the prevalence of stones to be 50% in gouty patients excreting greater than 1100 mg
of uric acid per 24 hours. Elevated levels of uric acid serum may also indicate renal failure,
pneumonia, sepsis, leukemia, polycythemia and anemia. Lower than the normal levels may
indicate acromegaly.

31 | P a g e
EXPERIMENT 11: THE EFFECT OF PH AND TEMPERATURE ON ENZYME
ACTIVITY

PRINCIPLE
Enzymes are organic catalysts whose function is to increase the rate of reactions that take place
in the living cells. They are all protein macromolecules and thus their ability to catalyse a
reaction will be affected by any situation which interferes with the structure or conformation of
protein molecules. Two factors which are of special importance in maintaining the correct
conformation of the protein catalyst molecule can be denatured by temperature and hydrogen ion
concentration. The molecule can be denatured by heat and its degree of ionization can be
affected by changes I pH. For each enzyme there is an optimum pH and optimum temperature at
which the catalyst functions most efficiently.

In this experiment, we will study the effect of these two factors on the activity of α-amylase, an
enzyme found in saliva and pancreatic juice, which catalyses the hydrolysis of polysaccharide
such as starch and glycogen. When amylase catalyses the hydrolysis of starch, glucose and other
reducing sugars are released from the originally non-reducing polysaccharide. Observing the rate
at which the reducing sugars appear in the reaction mixture can follow the rate which the enzyme
is breaking down the starch. This can be done by measuring the DNS acid reagent using a
colorimeter.

REAGENTS

1. 0.1 M standard glucose.

2. 0.9 % Starch solution in different buffers to which NaCl has been added (buffers at pH
3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0).

3. Preparation of phosphate buffer: Dissolve 0.2M (2.7218 grams) of KH2PO4 in 100ml of

distilled water. To this solution add 0.5M (2.8053 g) KOH drop by drop till the pH is set
to 6.8. Then make it to 200 ml with distilled water. So the final concentration is 0.1M of
200 ml phosphate buffer.

4. Diluted Saliva (Enzyme source): Saliva is the best and easily available source of amylase.
Collect some saliva in a beaker and dilute it to 1:20 dilution with distilled water.

5. 1% Sodium Chloride (NaCl): Dissolve 1.0 g of NaCl in 100 ml of distilled water.

6. Dinitrosalcylic acid: (DNS reagent) Dissolve 1.6 g of NaOH in 20 ml of distilled water.

Take 1 gram of 3,5-DNS in NaOH solution. In another beaker take 30 g of sodium
potassium tartarate. Dissolve in 5o ml of distilled water. Mix the volume up to 100 ml
with distilled water.

32 | P a g e
PROCEDURE

PART 1: Calibration Curve for estimation of amount of reducing sugar.

A calibration curve for the Optical Density (O.D) of colour produced by the reaction of DNS
acid reagent with a series of glucose solutions of known concentration must first be constructed
as follows:

1. Dilute the glucose solution provided (1ml + 9ml water) to give a solution of 0.01M strength.
Prepare four (4) test tubes as follows:

Tube 1 Tube 2 Tube 3 Tube 4 Tube 5 Tube 6

Blank 2? µmole 4? µmole 6? µmole 8? µmole 10? µmole

Gluc 0.01 M 0ml 0.2ml 0.4ml 0.6ml 0.8ml 1ml

Water 3ml 2.8ml 2.6ml 2.4ml 2.2ml 2.0ml

DNS acid 2ml 2ml 2ml 2ml 2ml 2ml

(Blank contains no sugar, while the amount of sugar in the other tubes is listed above each
column.)

2. Shake these tubes, place in a boiling water bath for exactly 5 minutes. Cool and make up
to 20 ml with water.

3. Read the absorbance (O.D) on colorimeter using the blank tube to set it to zero. Draw
calibration curve, plotting O.D against concentration of glucose.

Part 2: To find the effect of different pH on amylase activity

Prepare a solution of salivary amylase diluted in 1ml to 20ml ratio. Then prepare 4 sets of test
tubes in duplicates as follows:-

Tubes: 1&2 3&4 5&6 7&8 9&10 11& 12

pH: 4.0 5.0 6.0 7.0 8.0 9.0

1. Place 0.4 ml of the buffered starch solutions at the pH indicated in each pair of tubes.
Add 0.5ml of the diluted saliva to each tube. Stand each tube on the bench for exactly 15
minutes.

2. Add 2.0ml of DNS acid reagent to each tube. Develop colour as described in part 1 and
read the O.D values on the colorimeter.

33 | P a g e
 3. Using the already-prepared calibration curve to find the amount of reducing sugar
(glucose equivalent) produced in each tube by the enzyme. Note the optimum pH for the
reaction.

Part 3: To study the effect of different temperatures on amylase action

Set up 10 test tubes as follows:

Tubes 1 – 5: 2ml of 0.9% starch solution in the optimum buffer.

Tubes 5 – 10: 0.5 ml of diluted saliva.

Stand pairs of test tubes as follows:-

(a) 1 & 6 in ice bath

(b) 2 & 7 at room temperature

(c) 3 & 8 in 37oC water bath

(d) 4 & 9 in 60oC water bath

(e) 5 &10 in boiling water

Leave the tubes to equilibrate for 2 min, the transfer 1 ml of starch solution to the saliva tube in
each pair. Put each tube containing the mixture of enzyme + starch back into its own bath and
leave for exactly 15 minutes.

Add 2.0 ml of the DNS acid reagent and develop colour as described above. Using calibration
curve find the amount of reducing sugar produced in each tube.

Report all your findings. Comment on the levels of enzyme activity you found at the different
temperatures.

QUESTIONS

1. Explain what happens when salivary amylase acts on starch (very briefly).

2. Would β-amylase have the same effect?

3. What is the purpose of adding NaCl to the starch solutions in the different buffers?

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