Practical Manual of

Biochemistry
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 ADVISORY COMMITTEE

Dr Biswajit Das Dr Devajit Sarmah

Professor and Head, Department of Biochemistry Professor and Head, Department of Biochemistry
Rohilkhand Medical College, Bareilly, UP GMCH, Faizabad, UP

Dr Sohil Takodara Dr Randeep Mukherjee

Assistant Professor, Department of Biochemistry
Associate Professor, Department of Biochemistry
GMCH, Faizabad, UP
Geetanjali Medical College and Hospital, Udaipur, Rajasthan
Dr Shrawan Kumar
Dr Anupama Basvraj Patne Professor and Head, Department of Biochemistry
Associate Professor, Department of Biochemistry Pandit Deendayal Upadhayay Medical College
American International Institute of Medical Sciences Churu Rajasthan
Udaipur, Rajasthan
Dr Manish K Singh
Dr Biswajit Das Assistant Professor and Head, Department of Biochemistry
Professor and Head, Department of Biochemistry Government Medical College
Rohilkhand Medical College, Bareilly, UP Badaun, Rajasthan

Dr Deepa Thadani Dr Dharmveer Yadav

Professor and Head, Department of Biochemistry Associate Professor, Department of Biochemistry
JLN Medical College, Ajmer, Rajasthan AIIMS, Jodhpur, Rajasthan

Dr Tariq Mahmood Dr Shuchi Goyal

Associate Professor and Head, Department of Biochemistry Professor and Head, Department of Biochemistry
SRMSIMS, Bhojipura, Bareilly, UP RNT Medical College, Udaipur, Rajasthan

Dr Sanjay Bhatt Dr Kanchan Sonone

Associate Professor, Department of Biochemistry Associate Professor, Department of Biochemistry
SRMSIMS Bhojipura, Bareilly, UP Lokmanya Tilak Medical College, Sion, Mumbai
Maharashtra
Dr Manoj Gupta
Assistant Professor, Department of Biochemistry Dr Ravi Shankar Chaudhary
SRMSIMS Bhojipura, Bareilly, UP Assistant Professor, Department of Biochemistry
Geetanjali Medical College and Hospital, Udaipur
Dr Shalini Gupta Rajasthan
Professor and Head, Department of Biochemistry
Government Medical College, Bharatpur, Rajasthan Dr Vinay Gehlot
Senior Demonstrator, Department of Biochemistry
Dr Neeta Sahi Geetanjali Medical College and Hospital, Udaipur
Associate Professor and Head, Department of Biochemistry
Rajasthan
PMCH, Udaipur, Rajasthan
Dr Sumeru Samanta Dr Prashant Hisalkar
Assistant Professor, Department of Biochemistry Professor and Head, Department of Biochemistry
Rohilkhand Medical College, Bareilly, UP Government Medical College, Dungarpur, Rajasthan
Practical Manual of
Biochemistry
Second Edition
As per the latest
CBME Guidelines |
Competency Based Undergraduate Curriculum
for the Indian Medical Graduate

GG Kaushik MBBS, MD, PhD, DRIA, FISC

Senior Professor
Department of Biochemistry and
Additional Principal
Jawaharlal Nehru Medical College and Hospital
Ajmer, Rajasthan

Neha Sharma PhD

Professor
Department of Biochemistry
Geetanjali Medical College and Hospital
Udaipur, Rajasthan

Sabira Dabeer MBBS, MD

Assistant Professor
Department of Biochemistry
Geetanjali Medical College and Hospital
Udaipur Rajasthan

Ruchi Jindal BDS, MSc (medical)

Demonstrator
Department of Biochemistry
Geetanjali Medical College and Hospital
Udaipur, Rajasthan

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 Preface to the Second Edition

W e are happy to present the second edition of this book. In this edition we have added one new chapter of
DNA extraction. The author of this chapter is Dr Dharamveer Yadav. The zeal to share our knowledge and
experience has driven us to write this book. It has been written in a simple language for easy understanding of
basic concepts of practical biochemistry. The book caters the need of all the health professionals. We hope students
and teachers will find this book useful.

Salient features of the book

• It covers the entire syllabus of medical biochemistry, core competency based.
• Includes flowcharts and diagrams for easy understanding.
• Presence of colour pictures for clear visualization and understanding.
• Formation of working reagents has also been described in each chapter along with experiments.
• DNA extraction
This is our second edition and in spite of all precautions inadvertent mistakes are likely to happen. We would
appreciate if the same is brought to our notice, so we can improve ourselves in the next edition. We hope this
book will be helpful to the students.

GG Kaushik
Neha Sharma
Sabira Dabeer
Ruchi Jindal
 Acknowledgments

F irst and foremost, we would like to thank God Almighty for his kind blessings on us. Our sincere thanks and
gratitude to Dr FS Mehta, Dean, Geetanjali Medical College, and our colleagues of Geetanjali Medical College
and Hospital, Udaipur.
Our special thanks to Dr Ankita Sharma, Assistant Professor and the entire Department of Biochemistry
of JLN Medical College, Ajmer, Rajasthan. We are thankful to our reviewers and special thanks to
Dr Dharmveer Yadav, Associate Professor, Department of Biochemistry, AIIMS, Jodhpur, for their valuable
suggestions and addition of a new chapter “DNA Extraction and its Applications: An Overview.”
Our humble and sincere thanks to Mrs. Ritu Chawla, Mr. Dinesh S Dheek and CBS Publishers & Distributors
for their trust on us.
We would also like to acknowledge the support of technical staff and postgraduate students for their help. We
wish them good luck in their future endeavour.
Last but not the least, our thanks to our families, we cannot repay the constant support and encouragement
from them.

GG Kaushik
Neha Sharma
Sabira Dabeer
Ruchi Jindal
 Contents

Preface to the Second Edition v

Section 1
Qualitative Analysis
1. Carbohydrate Analysis 3
2. Lipid Analysis 12
3. Protein Analysis 19
4. Urine Analysis 28

Section 2
Quantitative Analysis
5. Colorimeter 43
6. Estimation of Blood Glucose 46
7. Kidney Function Test 50
8. Lipidogram 63
9. Liver Function Test 75
10. Estimation of Blood Calcium 97
11. Estimation of Blood Phosphorus 102

Section 3
Demonstration
12. pH Meter 109
13. Chromatography 110
14. Electrophoresis 112
viii Practical Manual of Biochemistry

Section 4
Miscellaneous
15. Body Fluids 119
16. Reagents Preparation 121
17. Biological Waste Disposal 123
18. Blood Gas Analyzer 126
19. DNA Extraction and its Applications: An Overview 139
 Carbohydrate Analysis ix

Index of Competencies
As per Medical Council of India: Competency Based Undergraduate Curriculum for the Indian Medical Graduate

Topic Competency (core) Page No

Introduction to Practical Laboratory Competency code BI 11.1, 11.24

Section 1: Qualitative Analysis

1. Carbohydrates Analysis BI 11.1, BI 3.1 3
A. Group Test for Carbohydrates
B. Analysis of Polysaccharides, Disaccharides, Monosaccharides
C. Osazone Test
D. Unknown Carbohydrate Identification
E. Viva Voce

2. Lipid Analysis BI 4.1 12

A. Solubility, Saponification, Emulsification, Greasy Spot Test
B. Glycerol Analysis [Dunstan’s, Acrolein Test]
C. Analysis of Cholesterol [Liebermann-Burchard, Salkowski’s Test]
D. Hubl’s Iodine for Unsaturation of Fatty Acids BI 11.24
E. Viva Voce

3. Protein Analysis BI 5.1 19

A. Biuret Test, Ninhydrin Test [Group Color Reaction for Amino Acids]
B. Xanthoproteic, Millon Nasse’s, Hopkins-Cole [Aromatic Amino Acids]
C. Sakaguchi’s [Test for Arginine]
D. Lead Sulphide [Test for Sulphur Group]
E. Neumann’s [Test for Phosphate Group]
F. Coagulation and Precipitation Reaction
G. Identification of Unknown Protein
H. Viva Voce

4. Urine Analysis BI 11.3, BI 11.4, 11.20 28

A. Analysis of Normal Biochemical Constituents of Urine
B. Abnormal Pathological Urine
C. Unknown Urine Sample/Report
D. Viva Voce

Section 2: Quantitative Analysis

5. Colorimeter BI 11.6 43

6. Estimation of Blood Glucose BI 11.21 46

7. Kidney Function Test 50

A. Blood Urea BI 11.21
B. Serum Creatinine BI 11.7
C. Serum Uric Acid BI 11.17
x Practical Manual of Biochemistry

Topic Competency (core) Page No

8. Lipidogram 63
A. Serum Total Cholesterol
B. Serum Triglycerides
C. Serum HDL Cholesterol, Serum LDL, VLDL BI 11.9, BI 11.10, BI 11.9

9. Liver Function Test 75

A. Estimation of Total Bilirubin, Direct Bilirubin BI 11.12
B. Serum Total Protein, Albumin, Globulin, A : G Ratio BI 11.8, 11.22
C. Serum SGOT, SGPT, ALP BI 11.13,11.14

10. Estimation of Blood Calcium BI 11.11 97

11. Estimation of Blood Phosphorus BI 11.11 102

Section 3: Demonstration
12. pH Meter BI 11.2 109

13. Chromatography BI 11.5, 11.16 110

14. Electrophoresis BI 11.16 112

Section 4: Miscellaneous
15. Body Fluids BI 11.15 119

16. Reagents Preparation BI 11.2 121

17. Biological Waste Disposal BI 11.1 123

18. Blood Gas Analyzer BI 11.16 126

19. DNA Extraction and its Applications: An Overview BI 11.16 139

 INTRODUCTION TO PRACTICAL LABORATORY
Equipment and Instruments Used in Biochemistry Laboratory

Colorimeter Spectrophotometer

pH meter Microscope

Autopipettes Urinometer
xii Practical Manual of Biochemistry

Bunsen burner Test tubes with rack

Burette Measuring cylinder Round bottom flask

Glass funnel Glass pipette

 Equipment and Instruments xiii

Beaker Conical Flask

Urine flask Dropper

Spatula Hot air oven

xiv Practical Manual of Biochemistry

Water bath Magnetic stirrer with hot plate

Incubator Electrophoresis chamber and power supply

Electrophoresis buffer Electrophoresis reagents

 Equipment and Instruments xv

Paper chromatography Column chromatography

Amino acids used in chromatography Centrifuge machine

Vortex mixture Desicator

xvi Practical Manual of Biochemistry

Tissue homogenizer Single pan balance

Balance Weights

Digital balance Auto analyzer

 Equipment and Instruments xvii

Write Down the Uses and Principle of Each of the Instruments

xviii Practical Manual of Biochemistry
Equipment and Instruments xix
xx Practical Manual of Biochemistry
Equipment and Instruments xxi
 1
Qualitative Analysis

1. Carbohydrate Analysis
2. Lipids Analysis
3. Protein Analysis
4. Urine Analysis
 1
Carbohydrate Analysis

Definition
• Carbohydrates are aldehyde or ketone derivatives of polyhydroxy alcohols.
• Their main function is to provide energy.
• Glucose is the main form of carbohydrate absorb from the gut.
• Humans can synthesize glucose from non-carbohydrate sources like lactate, glucogenic amino acids, propionic
acid, glycerol, pyruvate by a process known as gluconeogenesis.
Oligosaccharides: Made up of less than ten monosaccharide subunits.

Classification of Carbohydrates

3
4 Practical Manual of Biochemistry

Identification of unknown carbohydrates

 Carbohydrate Analysis 5

Experiment Principle Observation Inference

Molisch‘s test (α
α -naphthol
reaction)
3 ml sugar solution Conc acid dehydrates sugar to Violet/purple ring at the Carbohydrates present in
+ form furfural/furfural junction of two liquids given solution
Molisch reagent (α-naphthol) derivatives which form (Fig. 1.2)
+ complex with molisch reagent
Conc sulfuric acid
Iodine test
3 ml polysaccharide solution Formation of adsorption Starch Colour Polysaccharide is present
+ complex between Glycogen Blue
0.05N iodine soln 2 drops polysaccharide and iodine Dextrin Reddish
(Fig. 1.3) Reddish brown
Benedict’s test
5 ml benedict reagent Reducing sugars have free Colour Conn sugar Reducing sugars present
+ aldehyde or ketone group, Green 0.5 g
8 drops sugar soln which reduces metallic ions. Yellow >0.5–1 g
+ Benedict reagent has cupric Orange >1–2 g
Boil for 2 min ion which is reduced to Red ppt > 2g
Benedict reagent: copper cuprous ion (Fig. 1.4)
sulfate, sodium citrate,
sodium carbonate
Barfoed test
5 ml barfoed reagent In acid medium free aldehyde Red ppt at the bottom of Monosaccharides are present
+ or keto group reduces cupric the test tube (Fig. 1.5)
0.5 ml sugar soln ion into cuprous ion
+
Put in boiling water bath for
2 min
Osazone test
5 ml sugar solution Carbonyl carbon and adjacent Lactose Puff-shaped Confirms the presence of
+ cabon react with phenyl Maltose Sunflower-shaped lactose, maltose, glucose and
300 mg phenyl hydrazine hydrazine to form phenyl Glucose Needle-shaped fructose
mixture hydrazone, phenyl hydrazone Fructose Needle-shaped
+ reacts with two molecules of (Fig. 1.7–1.9)
Heat in boiling water bath phenyl hydrazine to form
(15–45 min depends on type osazones
of sugar)
Cool at room temperature
Avoid rapid cooling
Seliwanoff’s test
3 ml Seliwanoff’s reagent Seliwanoff reagent is resorcinol Cherry red color Fructose is present
+ in dil HCl keto group are readily (Fig. 1.6)
5 drops fructose dehydrated by dil HCl to from
+ hydroxymethylfurfural, which
Heat to just boil condenses with resorcinol to
form red complex
Inversion test
5 ml sucrose soln Sucrose hydrolyze into glucose
+ and fructose when boiled with
2 drops conc HCl conc HCl
+
Boil for 2 mins and divide
into two parts
Part 1: Perform Benedict’s test Part 1: Positive Benedict test Sucrose is present
Part 2: Perform Seliwanoff’s test Part 2: Positive Seliwanoff’s test
6 Practical Manual of Biochemistry

Fig. 1.1: Reagents for carbohydrates identification Fig. 1.2: Molisch’s test

Fig. 1.3: Iodine test Fig. 1.4: Benedict test: Green, yellow, orange, red

Fig. 1.5: Barfoed’s test Fig. 1.6: Seliwanoff’s test

 Carbohydrate Analysis 7

Fig. 1.7: Glucose, fructose-osazone Fig. 1.8: Maltose-osazone

Fig. 1.9: Lactose-osazone

VIVA VOCE
Q1. Name of the group test of carbohydrates.
Ans. Molisch’s test
Q2 Why Benedict test is semiquantative test?
Ans. Color of precipitate indicate quantity of sugar in solution.
Q3. Name of test for polysaccharides.
Ans. Iodine Test.
Q4. How to differentiate monosaccharide’s glucose and fructose?
Ans. By Seliwanoff’s test is positive for fructose.
Q5. Different shape of crystal by osazone test.
Ans. Needle shape—Glucose, fructose
Sunflower shape—Maltose
Cotton ball shape—Lactose
Q.6. Name of common sugar.
Ans. Sucrose
Q7. Carbohydrates preferred in diabetic patients.
Ans. Polysaccharides.
8 Practical Manual of Biochemistry
Carbohydrate Analysis 9
10 Practical Manual of Biochemistry
Carbohydrate Analysis 11
12 Practical Manual of Biochemistry

2
Lipid Analysis

Definition: Lipids are esters of fatty acids with alcohol, which are soluble in organic solvents and insoluble in
water.

Classification of Lipids

12
 Lipid Analysis 13

Test Principle Observation Inference

Solubility test: Reagents This test is based on solubility Oil and water separate quickly in Lipids are insoluble in water
(ether, chloroform, benzene, of lipid in organic solvents and 1st test tube whereas clear and soluble in organic
CCl4) insoluble in water solutions are formed in the case solvents
Procedure: Take 4 test tube of organic solvents
and fill 3 ml of water, ether
chloroform, benzene CCl4 in
each test tube respectively
+
Add 5 drops of oil into each
test tube
+
Shake well and allow to
stand

Saponification test: When oil and fat are boiled A white ppt of insoluble Calcium salt of fatty acid is
Procedure: Take 5 drop oil with alkali than both are calcium soap is formed formed
and 5 ml of alkali in test hydrolyzed and liberated fatty
tube. Keep test tube in acid form salts with alkali called
boiling water bath and boil soap and process is known as
it till the solution become saponification
soapy
+
Cool it take out test tube
and add CaCl2

Emulsification test: When oil and water are shaken • Oil and water separate quickly • Emulsification is unstable
Reagents (0.5% Na2CO3 together the oil is broken down • Separation does not take place • Emulsification is stable with
solution in water + 5% of into small droplets which are with soap, bile salt, Na2CO3 soap, bile salt, Na2CO3
Bile salt solution) dispersed in water. This is
Procedure: Take 5 ml of known as oil in water emulsion.
each water bile salt, The water due to their high
Na2CO3, soap solution in surface tension has a tendency
separate to close together and separate
+ as a layer. Bile salt, Na2CO3,
Add 5 drop of oil each soap solution lowers the surface
+ tension so they are best
In first tube oil and water emulsifying agents
layer separates immediately
indicating. Emulsion is
unstable but in case of
Na2CO3 layer separate
longer time indicating
emulsion is more
stable

Grease spot test reagent: All lipids are grease in nature Ether evaporates and leaving Grease nature of lipid
(Ether) procedure: Take so this test is used as a group behind a translucent spot on
3 ml of ether in test tube test for lipid paper (Fig. 2.3)
+
Add 5 drop of oil to it shake
well and put 1–2 drop of
solution on filter paper
wait 5
14 Practical Manual of Biochemistry

Glycerol Analysis
Test Principle Observation Inference
Dunstan’s test Reagents: Borax hydrolyzed into NaOH Medium is alkaline-pink color Presence of glycerol
(borax solution (strong base) and boric acid disappear. Medium is acidic
+ (weak acid) after addition of than color will reappear
phenolphthalein indicator) glycerol it react with boric acid (Fig. 2.1)
Procedure: 3 ml of borax and forms strong acid
solution in test tube (glyceroboric acid)
+
add drop of
phenolphthalein indicator
pink color produced
indicate medium is alkaline
+
add 20% glycerol drop by
drop until pink color
disappears indicate
medium is acidic. Heat
again until pink color
reappears that indicate
medium has become
alkaline again
Acrolein test: Reagents- On heating with KHSO4 Pungent smell is produced Presence of glycerol
solid KHSO4 glycerol is dehydrated to form
Procedure: Take dry test an unsaturated aldehyde
+ (acrolein)
add 1–2 drop of glycerol
+
add pinch of KHSO4+ heat it

Cholesterol Analysis
Procedure Principle Observation Inference
Salkowski’s test Reagent: When sample is dissolved in Two layer separated chloroform Presence of cholesterol
con H2SO4 chloroform and equal volume layer-cherry red (Fig. 2.2) acid
Procedure: Take 2 ml of of H2SO4 is added. If cholesterol layer–green fluorescence
cholesterol in dry test tube is present solution become blue
+ add 2 ml of conc H2SO4 red and change to violet red and
shake well and allow to H2SO4 become red with green
stand fluorescence
Libermann-Burchard’s Cholesterol is dehydrated by Red color develops followed by Presence of cholesterol
test reagent: Con H2SO4 con H2SO4 and acetic blue and finally whole solution
Acetic anhydride anhydride leading to formation becomes green
Procedure: Take 3 ml of of 2,4 or 3,5 cholestadienes
cholesterol in dry test tube +
add 10 drop of acetic
anhydride + add 1–2 drops
of con H2SO4 mix well

Test for Unsaturation (Hubl’s Iodine Test)

Procedure Principle Observation Inference
Hubl’s iodine test) Unsaturated fatty acid absorb Color of iodine disappears Show degree of unsaturation
Reagents-iodine 26 gm iodine at double bonds until all
Mercuric chloride 30 gm the bonds are saturated with
C2H5OH 1000 ml. Procedure iodine
3 ml of CHCl3 + add
Hubl’s reagent drop
by drop
 Lipid Analysis 15

Fig. 2.1: Dunstan’s test Fig. 2.2: Salkowski’s test Fig. 2.3: Grease spot test

VIVA VOCE
Q1. Name of essential fatty acids.
Ans. Lionlic acid, linoleic acid.
Q2. Name of the test for cholesterol.
Ans. Salkowski, Liberman-Burchard reaction.
Q3. Name of ring present in cholesterol.
Ans. CPPP (cyclopentaperhydrophenenthrene).
Q4. Name of the test for unsaturation of fatty acids.
Ans. Hubl’s iodine test.
Q5. What is the importance of emulsification?
Ans. Digestion of lipids.
16 Practical Manual of Biochemistry
Lipid Analysis 17
18 Practical Manual of Biochemistry
 Protein Analysis 19

3
Protein Analysis

Definition: Polymers of L-amino acids linked by peptide bond.

Classification of Proteins

Color Reactions
A. Biuret Test, Ninhydrin Test [Group Color Reaction for Amino Acids]
Test procedure Principle Observation Inference
Biuret test Violet color formation because Violet color presence (Fig. 3.1) Peptide linkage, presence of
Reagents: 40% of peptide linkage of protein protein
NaOH with CuSO4 in alkali medium
Procedure: 2 ml protein provided by NaOH
solution + 1 ml NaOH mix +
1–2 drops of 0.5% CuSO4
Ninhydrin Heating with ninhydrin Violet—purple color (Fig. 3.2) Presence of α amino acids
Reagents: 0.1% (oxidizing agent) α amino acids Exception: Proline and
Freshly prepared give purple color hydroxyl proline
ninhydrin solution
Procedure: 1 ml protein
solution + 1 ml freshly
prepared 0.1% ninhydrin
solution mix and boil for
1 min, cool

B. Xanthoproteic, Millon Nasse’s, Hopkin’s Cole’s [Aromatic Amino Acids]

Test procedure Principle Observation Inference
Xanthoproteic Reagents: Heating of aromatic amino Yellow to dark yellow convert in Presence of aromatic amino
Con. HNO3, 40% NaOH acids with HNO3 lead to to orange color (Fig. 3.3) acids
Procedure: 3 ml protein nitration of benzene group of
solution + 1 ml HNO3 heat amino acids, by ionization it
for 1 min yellow, cool + give orange color
2 ml 40% NaOH mix-
orange color
Contd…

19
20 Practical Manual of Biochemistry

Test procedure Principle Observation Inference

Millon Nasse’s (tyrosine) Mercuration and nitration of Red-pink color developed in Tyrosine presence
reagents: 15% HgSO4 in phenolic group—development precipitate and solution
6N H2SO4 (Millon Nasse’s of color (Fig. 3.4)
reagent), NaNO2 freshly
prepared
Procedure: 4 ml solution +
1.5 ml Millon’s reagent-
Boil-cool 1 ml NaNO2
Hopkin cole (aldehyde) Indole ring combine with Violet ring at the junction Tryptophan presence
reagents: Conc H2SO4, formaldehyde in presence of (Fig. 3.5)
dilute formaldehyde con H2SO4—violet ring
Procedure: 2 ml protein
solution + 1 ml
formaldehyde mix + 2 ml
H2SO4 along the side wall
of test tube slowly

C. Sakaguchi’s [Test for Arginine]

Test procedure Principle Observation Inference
Sakaguchi reagents: Guanido group react with α Presence of red color (Fig. 3.6) Guanido group (arginine
10% NaOH, α napthol, 10% napthol and sodium presence)
sodium hypobromite hypobromite and give red
Procedure: 3 ml protein color complex
solution + 1 ml 10% NaOH
mix + 2–3 drop of sodium
hypobromite and mix well

D. Lead Sulphide [Test for Sulfur Group]

Test procedure Principle Observation Inference
Lead sulfide (cysteine In alkali medium sulfur release Brown black ppt form (Fig. 3.7) Presence of sulfur con.
and cystine) as sodium sulfide and react amino acids (negative for
Reagents: 40% NaOH, 2% with lead acetate form lead casein and gelatin) lead
acetate sulfide (black-brown color)
Procedure: 3 ml protein +
2.5 ml 40% NaOH boil –
2 min – cool + 3–4 drop
lead acetate

E. Neumann’s [Test for phosphate group]

Test procedure Principle Observation Inference
Neumann’s (casein) Heating with conc H2SO4 + Canary yellow color (Fig. 3.8) Phosphorus group present
Reagents: Ammonium HNO3 casein digested
molybdate, methyl red phosphorus remove, then react
indicator, conc nitric acid, with ammonium molybdate
conc ammonia, conc H2SO4 its form ammonium phoshph-
Procedure: Dry pinch of omolybdate-canary yellow
casein + 6–8 drop of conc color
H2SO4 + 2–3 drop of conc
HNO3– heat untill colorless.
cool + methy indicator
with 3 ml water red +
ammonia-untill change in
yellow + add conc HNO3–
red, boil for 2 min, 2 ml
ammonium molybdate, heat
 Protein Analysis 21

F. Coagulation and precipitation reaction

Test procedure Principle Observation Inference
Heat coagulation 2/3 test Denaturation of protein Dense coagulum formed at upper Presence of albumin and
tube with protein solution part not dissolved adding of globulin (protein)
incline heat upper part + acetic acid (Fig. 3.9)
acetic acid
Precipitation reagents: Shell of hydration, White ppt form Albumin, casein, gelatin
Ammonium sulfate, electrical charge
AgNO3, HgNO3
sulfosalicylic acid
Full saturation procedure:
3 ml protein + solid
ammonium sulfate till
saturation–allow to standing
Half saturation procedure: Shell of hydration, White ppt form Casein, gelatin
3 ml protein solution + electrical charge
saturated 3 ml, mix well-
standing
Heavy metals procedure: Negatively charged protein Ppt form Protein presence
3 ml protein + drop by react with metal and
drop heavy metals salt till neutralize form ppt
ppt formation
Alkaloid reagents Acid base reaction form White turbidity Protein presence
procedure: 3 ml protein + insoluble salt
sulfosalicylic acid mixing

G. Identification of Unknown Protein

22 Practical Manual of Biochemistry

Fig. 3.1: Biuret test: Fig. 3.2: Ninhydrin test: Fig. 3.3: Xanthoproteic test:
Albumin, casein, gelatin, peptone Albumin, casein, gelatin, peptone Albumin, casein, gelatin, peptone

Fig. 3.4: Million Nasse’s test: Fig. 3.5: Hopkin’s Cole’s test: Fig. 3.6: Sakaguchi’s test:
Albumin, casein, gelatin, peptone Albumin, casein, gelatin, peptone Albumin, casein, gelatin, peptone

Fig. 3.7: Lead sulphide test: Fig. 3.8: Neumann’s test: Casein Fig. 3.9: Heat coagulation’s test:
Albumin, casein, gelatin, peptone Albumin
Note: From left to right albumin, casein, gelatin, peptone
 Protein Analysis 23

VIVA VOCE
Q1. Name of the group test for proteins.
Ans. Biuret test.
Q2. Name of the test for aromatic ring containing amino acids.
Ans. Xanthoproteic acids.
Q3. Specific test for tyrosine.
Ans. Millons test.
Q4. Test name for tryptophan.
Ans. Hopkin’s cole’s.
Q5. Specific test for sulphur test.
Ans. Lead acetate.
Q6. Test for casein.
Ans. Neumann’s test.
Q7. How to differentiate albumin and gelatin by saturation test?
Ans. Gelatin gives half saturation, while albumin gives full saturation.
Q8. Name of first class protein.
Ans. Albumin.
24 Practical Manual of Biochemistry
Protein Analysis 25
26 Practical Manual of Biochemistry
Protein Analysis 27
28 Practical Manual of Biochemistry

4
Urine Analysis

NORMAL URINE
Urine is the excretory product of body, formed by kidneys. It is made up of water and water soluble waste
products. Examination of urine gives us idea of renal function.

Normal Constituents of Urine

1. Water 90–95%

2. Urea 25–30 gm/day

3. Creatinine 1–1.2 gm/day

4. Uric acid 0.4–0.7 gm/day

5. Sodium 3–3.5 gm/day

6. Potassium 2–2.5 gm/day

7. Chloride 10–15 gm/day

8. Calcium 0.1–0.3 gm/day

9. Phosphate 0.7–1.2 gm/day

10. Sulfate 1–1.2 gm/day

11. Ammonia 0.6–0.8 gm/day

Physical Examination of Normal Urine

1. Appearance Freshly void urine is clear, it becomes turbid on standing due to precipitation of
phosphates

2. Odor Aromatic odor, which becomes ammonical on standing

3. Color Straw color

4. Volume 1–2 L/day, depends upon the intake of water

5. pH Usually acidic, pH ranges from 4.5 to 8 post meal, pH of urine is alkaline; it is called
alkaline tide

6. Specific gravity 1.010–1.025, measured with the help of urinometer, it tell us about the concentrating
ability of kidneys

28
 Urine Analysis 29

Test Principle Observation Inference

Urea Urease in soybean meal Yellow color Presence of urea
1. Specific urease test reagents: acts on urea and liberate
Soybean meal, phenol red NH3, which makes the
Procedure: 4–5 ml of urine + medium alkaline. In alkaline Red color
pinch of soybean meal + medium phenolphthalein (Due to NH3) (Fig. 4.2a)
1–2 drop of phenol gives red color
indicator mix and wait
10 mins
2. Sodium hypobromite test Breakdown of urea and Effervescence due to Presence of urea
reagents: Alkaline hypobromite release of nitrogen produce production of nitrogen
Procedure: 4–5 ml sample + 2 ml effervescence (Fig. 4.2b)
alkaline hypobromite +
mix

Calcium test reagents: Formation of calcium White ppt of calcium Presence of calcium
Ammonium oxalate oxalate oxalate (Fig. 4.4)
Procedure: Take 2–3 ml
ammonium oxalate +
1–2 ml urine

Phosphate test reagents: Formation of phospho- Canary yellow ppt Presence of phosphate
Ammonium molybdate molybdate (Fig. 4.5)
Procedure: Take 1–2 ml
urine + add con
HNO3 + heat +
ammonium
molybdate

Creatinine test Jaffe’s test Formation of creatinine Orange red color Presence of creatinine
reagents: Alkaline picrate picrate (Fig. 4.3)
Procedure: 3–4 ml urine + 1 ml
alkaline picrate

Uric acid Benedict’s test In alkaline medium, Blue color develop Presence of uric acid
reagents: Sodium tungstste, arsenophosphotungstate of
arsenic pentoxide, phosphoric Benedict’s uric acid regent
acid, HCl, anhydrous sodium is reduced to blue colored
carbonate arsenophosphotungstate
Procedure: 4 ml urine + pinch
of sodium carbonate + mix +
4–5 drop of above reagent
+ mix

Ammonia test reagents: Ammonia is alkaline in Tip of rod turns pink Presence of ammonia
Phenolphthalein indicatior nature which turns
Procedure: 9 ml urine + drop phenolphthalein indicator
of phenolphthalein indicatior + pink
drop of 0.1 N NaOH hold
glass rod at the mouth of
tube
30 Practical Manual of Biochemistry

Fig. 4.1: Reagents

Fig. 4.2a: Test for urea, soybean meal Fig. 4.2b: Sodium hypobromite test Fig. 4.3: Test for creatinine

Fig. 4.4: Test for calcium Fig. 4.5: Test for phosphate
 Urine Analysis 31

PATHOLOGICAL URINE

Physical Examination of Pathological Urine

Normal Pathological
Appearance Clear a. Turbid in UTI
Odor Aromatic a. Fruity in diabetic ketoacidosis, starvation due to presence of ketone bodies
b. Mousy odor in phenylketonuria
c. Foul smell in UTI

Color Straw color a. Red in hematuria

b. Brown to black in alkaptonuria
c. Brown in hemoglobinuria

Volume 1–2 L/day a. Oliguria <300 ml/day

b. Polyuria >2500 ml/day seen in diabetes mellitus and diabetes insipidus
c. Anuria <100 ml/day

pH 4.5–8 a. pH decreases in metabolic acidosis

b. Increase in pH is seen in metabolic alkalosis and UTI
Specific gravity (SG) 1.010–1.025 a. Isothenuria SG is fixed at 1.010 seen in chronic renal failure
b. SG >1.030 hyperosmolar urine, seen in diabetes mellitus, severe dehydration,
adrenal insufficiency and nephrotic syndrome
c. SG <1.008 hypos molar urine, seen in diabetes insipidus, compulsive
polydipsia

Pathological Chemical Constituents Commonly seen in Urine

Abnormal constituents Pathological condition
1. Glucose a. Diabetes mellitus
2. Albumin a. Nephrotic syndrome
b. Eclampsia and pre-eclampsia
c. Heart failure
d. UTI
e. Inflammatory kidney diseases
3. Blood a. UTI
b. Kidney stones
c. Tumors related to renal system
d. Hemoglobinopathies
4. Bile salt a. Obstructive jaundice
5. Bile pigments a. Obstructive jaundice
6. Urobilinogen a. Obstructive jaundice
7. Pentose a. Essential pentosuria
8. Bence-Jones protein a. Multiple myeloma
9. Ketone bodies a. Diabetic ketoacidosis
b. Starvation
c. Hyperemesis gravidarum
32 Practical Manual of Biochemistry

Abnormal Constituents of Urine (Pathological Urine)

Test Principle Observation Inference
Reducing sugar: Benedict test Reducing sugars have free color Con Presence of reducing sugar
Reagents: Sodium citrate + aldehyde or ketone group, Green 0.5–1%
sodium carbonate + copper which reduces metallic ions. yellow 1–1.5%
sulphate Benedict reagent has cupric orange 1.5–2%
Procedure: 5 ml reagent + ion which is reduced to Brick red >2%
7–8 drop of urine sample + boil cuprous ion blue No sugar
2–3 min + cool (Fig. 4.6)

Protein heat coagulation Denaturation of Coagulum is formed at Presence of protein

test-procedure: protein by heat upper part (Fig. 4.8a)
Fill ¾ test tube with sample +
heat upper part + coagulum
formed + add 2–3% CH3COOH
If coagulum is dissolves,
indicate presence of phosphate
Heller’s test-reagents: HNO3
Procedure: 3 ml of sample + add Nitric acid cause White ring formed at Protein present
3–4 ml of HNO3 along the side precipitation of protein junction of two liquid
wall of tube (Fig. 4.8b)
Bence Jones proteins:
Procedure: Heat 40–60°C– Heat–ppt disappear cool– Presence of Bence Jones proteins
ppt + heat 100°C– dissolve reappear
ppt + cool again reapper

Ketone bodies: Rothera’s Ketone bodies form a purple Purple ring formed at the Presence of ketone bodies
nitroprusside test reagents: colored complex with junction of two liquid
Ammonium sulfate, sodium sodium nitroprusside in (Fig. 4.7)
nitroprusside, liquor ammonia alkaline medium.
Procedure: 5 ml urine saturate β-hydroxybutyrate does not
with ammonium sulfate + give this test
sodium nitroprusside + shake +
liquor ammonia side of tube

Bile salt Hay’s sulfur test Bile salt reduces the surface Sulfur powder sinks Presence of bile salts
reagents—sulfur powder tension of water towards bottom
Procedure: 4–5 ml of sample in (Figs 4.9a, and b)
tube + add pinch of sulfur
powder on surface
Bile pigments Fouchet’s test BaCl2 reacts with sulfur to Green blue color appear Presence of bilirubin
reagent: (FeCl3+TCA ) 6 ml form barium sulfate
BaCl2 add 10–11 ml urine
sample + filter Add 1–2 drop
of reagent
Urobilinogen Ehrlich’s test- Urobilinogen in acidic Pink, red color Presence of urobilinogen
reagent: (p-dimethyl- medium react with Ehrlich’s
aminobenzaldehyde) reagent to form colored
Procedure: 5–6 ml urine + 5 ml compound
of reagent + wait 8–10 min +
add 10 ml saturated sodium
acetate
Blood pigment benzidine test- Peroxidase like activity of Blue green color develop Presence of blood pigment
reagents: H2O2 hemoglobin H2O2 →
Procedure: 2–3 drop saturate H2O + O (O) oxidizes the
benzidine solution + add 3 ml benzidine to form blue/
sample + 4 drop H2O2 green colored oxidized
product
 Urine Analysis 33

Fig. 4.6: Benedict’s test Fig. 4.7: Rothera’s test Fig. 4.8a: Heat coagulation test

Fig. 4.8b: Heller’s nitric acid test Fig. 4.9a: Hay sulfur test Fig. 4.9b: Control hay sulfur test

VIVA VOCE
Q1. What is the specific gravity for normal urine?
Ans. 1.010–1.025
Q2. How specific gravity changes in diabetes insipidus?
Ans. It is low, fixed below 1.010.
Q3. In which conditions color of urine sample can be changed?
Ans. Jaundice-deep yellow, red—hemoglobinuria, blood, dark amber—vitamin B complex therapy.
Q4. Name of ketone bodies.
Ans. Acetone, acetoacetate, beta hydroxy butyric acid.
Q5. When ketone bodies appear in urine?
Ans. Uncontrolled diabetes mellitus, severe starvation.
34 Practical Manual of Biochemistry

Q6. Write common causes for proteinuria.

Ans. Nephrotic syndrome, diabetic nephropathy, renal failure, CHD.
Q7. Commonly which protein come into urine?
Ans. Albumin.
Q8. Name of bile salts.
Ans. Sodium and potassium cholic and chenodeoxycholic acid.
Q9. Name of bile pigments.
Ans. Bilirubin, billiverdin.
Q10. Presence of Bence Jones protein in urine indicate…….
Ans. Multiple myeloma.
Q11. Name few conditions where specific gravity of urine increases.
Ans. Diabetes mellitus, proteinuria, presence of ketone bodies in urine.
Urine Analysis 35
36 Practical Manual of Biochemistry
Urine Analysis 37
38 Practical Manual of Biochemistry
Urine Analysis 39
40 Practical Manual of Biochemistry
 2
Quantitative Analysis

5. Colorimeter
6. Estimation of Blood Glucose
7. Kidney Function Test
8. Lipidogram
9. Liver Function Test
10. Estimation of Blood Calcium
11. Estimation of Blood Phosphorus
 5
Colorimeter

The instrument commonly used is colorimetry which uses the basic principle of photometer. Colorless compounds
are converted into colored compounds using chemical reactions. It uses light only in the visible region (400–
760 nm).
Principle of colorimetry: It obeys beer lambert law. It is a combination of two laws.
Beer’s law: Beer’s law states that the absorption of light is directly proportional to the concentration of substance.
AαC
C—concentration of substance
A—absorbance
Lambert’s law: This law states that the absorption of light is directly proportional to the path length travelled
by the light.
Aαt
A = KCt
Log1/T = KCt
K = constant
Log1/T = optical density (OD) or tranmittance
A—Absorbance
C—Concentration of substance

Parts of Colorimeter
1. Source of light: Tungsten lamp
2. Filter: Visible range (400–760 nm)
3. Cuvette: Plain tube
4. Photocell: Convert light energy into electrical energy
Light energy → Electrical energy
5. Measuring device: Galvanometer it can read transmittance and absorbance.

Wavelength Region Color/filter

<380 U.V Not visible
380–440 Visible Violet (green-yellow)
440–500 Visible Blue (yellow)
500–580 Visible Green (red)
580–600 Visible Yellow (blue)
600–620 Visible Orange (green blue)
620–750 Visible Red (green)
>750 IR Not visible

43
44 Practical Manual of Biochemistry

Fig. 5.1: Parts of colorimeter (Courtesy: Dr Sohil)

Fig. 5.2: Instrument

Calculation
OD of test Vol of standard
conc of substance = × × conc of std
OD of std Vol of serum/sample

SPECTROPHOTOMETER
More sensitive and accurate than colorimeter
Works on Beer Lambert’s law.
It covers UV, visible and IR region.

Range Region
200–400 nm UV
400–700 nm Visible
>700 nm IR

Light source: Quartz and deuterium lamp

Cuvette: Quartz
Filter: Prism

Auto Analyzer
• Fully automatic device to measure the absorbance of substance.
• It display results on screen and can also be printed by printer.
 Colorimeter 45

Principle
• It covers Beer Lambert’s law in chemistry.
• In immunochemistry (endocrinology) it follows the principle of chemilumnisence (ECLIA).

Component
• Spectrophotometer/colorimeter component
• Sample tray
• Reagent tray
• Probes
• Cuvette
• Heating and cooling device
• Waste
• Internal and external computer (display)
• Barcode reader
• Printer.

VIVA VOCE
Q1. Difference between colorimeter and spectrophotometer.
Ans.
Spectrophotometer Colorimeter
More accurate Less accurate
More sensitive Less sensitive
Filter: prism Filter: visible (400–700)
Region: UV, visible, IR Region: visible
Cuvette: Quartz Cuvette: plain tube
Light source: Quartz, deuterium lamp Light source: Tungsten lamp

Q2. What is OD?

Ans. 1/T, negative logarithm of transmittance.
Q3. What is the range of OD in colorimeter?
Ans. 0–2.
46 Practical Manual of Biochemistry

6
Estimation of Blood Glucose

Introduction: Estimation of blood glucose use for routine diagnosis of diabetes mellitus hyperglycemia.

Methods of Estimation of Glucose

Enzymatic method: GOD-POD method.

Hexokinase Method
Method based on reducing property: Folin and Wu method.
Nelson and somogyi method.
Asatoor and king method.
Ortho-toludine method.
Aim: Estimation of blood glucose by colorimeter.
Principle: GOD-POD (End point method glucose oxidase-peroxidase method)
Glucose oxidase
Glucose + O 2 ⎯⎯⎯⎯⎯⎯→ Gluconic acid +H 2 O 2
Peroxidase
H 2 O 2 + phenol + 4-aminoantipyrine ⎯⎯⎯⎯⎯
→ Red quinine dye + H 2 O
Procedure: Take 1 ml reagent in tube
Add 10 μl serum
↓
Mix well and incubate for 10 min at 37°C
↓
Read at 520 nm

Observation Table
Blank Standard Test
Reagent 1 ml 1 ml 1 ml
Standard – 10 μl –
Serum – – 10 μl

Calculation
Results: ………………..

Normal range of blood glucose:

Fasting blood glucose—70–110 mg/dl.
Random blood glucose—70–130 mg/dl.
Postprandial (pp) glucose—70–140 mg/dl.
46
 Estimation of Blood Glucose 47

Interpretation
Conditions when blood glucose elevated Hyperglycemia
Fasting >110 mg/dl
Pp >140 mg/dl
1. Hyperactivity of endocrine gland (thyroid, pituitary and adrenal)
2. Emotional stress
3. Disease of pancreas
4. Diabetes mellitus

Conditions when blood glucose decreased Hypoglycemia

Fasting <60 mg/dl

1. Insulinoma
2. Over dose of insulin
3. Prolong starvation
4. Hypoactivity of adrenal gland

VIVA VOCE
Q1. Which vial is used for glucose estimation?
Ans. Sodium fluoride.
Q2. Name of method for glucose estimation.
Ans. GOD-POD.
Q3. What is the range for fasting and PP glucose?
Ans. F-70–110 mg/dl.
PP up to 140 mg/dl.
Q4. What are the causes of hyperglycemia?
Ans. DM, hyperactivity of adrenal and pituitary.
Q5. What are the causes of hypoglycemia?
Ans. Over dose of insulin, prolonged starvation.
48 Practical Manual of Biochemistry
Estimation of Blood Glucose 49
50 Practical Manual of Biochemistry

7
Kidney Function Test

A. ESTIMATION OF BLOOD UREA

INTRODUCTION
Urea is the end product of protein metabolism. Breakdown of protein forms amino acids, deamination of amino
acids form NH3. NH3 is converted into urea in liver and kidney eliminates it in urine.

Methods of Urea Estimation

1. Berthelot method (phenol hypochlorite method).
2. Diacetyl monoxime (DAM) method.
3. Urease method.
4. Nesslerization method.
Aim: Estimation of blood urea by Berthelot method.
Principle: Urease act on urea to liberate NH3, which combines with phenol and hypochlorite to form blue green
color complex. The intensity of color is measured at 580 nm.
Nitroprusside (alk med)
NH 3 + Phenol + Hypochlorite ⎯⎯⎯⎯⎯⎯⎯⎯⎯
→ Indo phenol (Blue green complex)

Procedure
Blank Test Standard
Reagent 1 1 ml 1 ml 1 ml
Distilled water 10 μl – –
Urea standard – – 10 μl
Serum – 10 μl –
Incubate for 5 mins
Reagent 2 1 ml 1 ml 1 ml

Incubate for 10 mins. Read at 580 nm.

Calculation
OD of test Amt of std
× × 100
OD of std Volume of std
Results ……………

Interpretation
Normal blood urea = 10– 40 mg/dl.
Normal urine urea = 23–30 g/day.
Normal blood urea nitrogen (BUN) = 7–25 mg/dl
(1 mg of BUN = 2.14 mg of urea).
50
 Kidney Function Test 51

Elevated Levels of Urea are seen in

Prerenal 1. Congestive heart failure
2. Shock and hemorrhage
3. Salt and water depletion
4. Dehydration due to vomiting and diarrhea
5. Ulcerative colitis
6. Pyloric stenosis with severe vomiting
7. Intestinal obstruction
8. Burns
Renal 1. Acute glomerulonephritis
2. Nephrotic syndrome
3. Malignant nephrosclerosis
4. Chronic pylonephritis
5. Renal tuberculosis
6. Mercurial poisoning

Postrenal 1. Prostate hypertrophy and cancer

2. Stone in urinary tract
3. Stricture of urethra
4. Tumor of bladder

Decrease urea levels are seen in:

1. Sever liver disease.
2. Cancer of liver.

Physiological variations are seen:

1. Pregnancy due to hemodilution.
2. Low in females.
3. Varies with amount of protein in diet.

Urea clearance:
Clearance is defined as volume of plasma cleared of a substance in one minute by kidney.
It is the measure of glomerular function of kidney.
Expressed in ml/min
U×V
C =
P
U = concentration of substance in urine.
V = volume of urine excreted per min.
P = concentration of substance in plasma.
Urea clearance depends on flow of urine.
When flow of urine is >2 ml/minute clearance is 60–95 ml/minute (average = 75 ml/min), it is called maximum
clearance.
When urine flow is <2 ml/minute clearance is 40–65 ml/minute (54 ml/min), it is called standard clearance.

If urea clearance is
Urea clearance Renal impairment
70% Normal
40–70% Mild impairment
20–40% Moderate impairment
<20% Severe impairment

Urea clearance is not a better index of glomerular function as only 10% of urea is excreted.
52 Practical Manual of Biochemistry

VIVA VOCE
Q1. What is normal level of urea in blood?
Ans. 20–40 mg/dl.
Q2. What are the causes of increased blood urea level?
Ans. Prerenal, renal, postrenal.
Q3. Explain few prerenal conditions.
Ans. Fever, CHD, dehydration.
Q4. Explain renal and postrenal conditions.
Ans. See on last page.
Q5. Conditions when blood urea level decreased?
Ans. Cirrhosis of liver.
Kidney Function Test 53
54 Practical Manual of Biochemistry
 Kidney Function Test 55

B. ESTIMATION OF SERUM CREATININE

INTRODUCTION
Creatine is synthesized in liver from glycine, arginine and methionine. It then reaches muscle via blood. Creatine
kinase converts creatine into creatine phosphate. Creatine phosphate acts as a storage form of ATP. Creatinine is
formed spontaneously and non-enzymatically from creatine phosphate at the rate of 2% of total body creatine
per day.
Amount excreted in urine remain constant for a given person and depends only on muscle mass and is
independent of protein in diet. Therefore, serum creatinine is a better index of renal function.

Methods of Creatinine Estimation

1. Jaffe’s reaction.
2. Nitroprusside test.
Aim: Estimation of serum creatinine by Jaffe’s method.
Principle: Creatinine reacts with picric acid in alkaline medium to form orange red creatinine picrate. The intensity
is measured at 520 nm.

Procedure
Blank Standard Test

Jaffe’s reagent 1 ml 1 ml 1 ml

Distilled water 100 μl – –

Standard – 100 μl –

Serum – – 100 μl

Mix well and allow stand for 15 minutes and read at 520 nm.

Calculation

OD of test Amt of std

C= × × 100
OD of std Volume of std

Results ………………………

Interpretation
Normal range
Males: 0.7–1.4 mg/dl.
Females: 0.6–1.2 mg/dl.

Elevated levels are seen in:

1. Renal failure and other renal diseases.
2. Early stages of muscular dystrophy/poliomyelitis.

Creatinine Clearance
Clearance is defined as volume of plasma cleared of creatinine in one minute by kidney.
It is the measure of glomerular function of kidney.
Expressed in ml/min
56 Practical Manual of Biochemistry

U×V
C=
P
U = concentration of substance in urine.
V = volume of urine excreted per minute.
P = concentration of substance in plasma.
Cretinine is negligibly reabsorb and is not secreted by renal tubules; therefore, its clearance is almost similar to
GFR.

Normal creatinine clearance:

Males: 94–140 ml/minute.
Females: 72–110 ml/minute.

VIVA VOCE
Q1. What is the normal serum creatinine level?
Ans. Males: 0.7– 1.5 Mg/dl.
Females: 0.4–1.2 mg/dl.
Q2. Amino acids involved in creatinine synthesis.
Ans. Glycine, arginine, methionine.
Q3. Why is estimation of serum creatinine a better index of KFT than blood urea?
Ans. Its level depends on glomerular filtration not affected by diet and other factors.
Q4. What is the normal daily excretion of creatinine in urine?
Ans. 1–2 gm/day.
Q5. Name of method for creatinine estimation?
Ans. Jaffe’s method.
Kidney Function Test 57
58 Practical Manual of Biochemistry
 Kidney Function Test 59

C. ESTIMATION OF SERUM URIC ACID

INTRODUCTION
Uric acid is the end product of purines metabolism. It is completely reabsorbed in proximal convoluted tubules
and then secreted into distal convoluted tubules. It is partly reabsorbed and partly excreted in urine.

Methods of Uric Acid Estimation

1. Phosphotungstic acid method.
2. Uricase method.
3. High pressure liquid chromatography method.
Aim: Estimation of blood uric acid level by uricase method.
Principle: Uricase enzyme act on uric acid to liberate H2O2. H2O2 reacts with 4-aminophenazone (4AP) and
2,4dichlorophenol sulfonate (DCPS) in presence of peroxidase to form a red colored complex quinoneimine
compound.
Uricase
Uric acid + 2H2O + O2 ⎯⎯⎯⎯ → Allantoin + CO2 + H2O2
POD
2H2O2 + 4AP + DCPS ⎯⎯⎯ → Quinoneimine + 4H2O
Intensity of color is directly proportional to the concentration of uric acid.

Procedure
Blank Standard Test
Working reagent 1 ml 1 ml 1 ml
Standard – 25 μl –
Serum – – 25 μl
Distilled water 25 μl – –

Mix and incubate for 3 minutes at 37°C and read at 650°C.

Calculation
OD of test Vol of standard
conc of uric acid = × × conc of std
OD of std Vol of serum/sample

Results ………………………
60 Practical Manual of Biochemistry

Interpretation
Normal range
Males: 4.5–7.5 mg/dl.
Females: 2.3–6.5 mg/dl.

Causes of Hyperuricemia
Hyporuricemia: When uric acid concentration <2 mg/dl. It is a rare condition seen in:
1. Severe liver disease, purine synthesis decreases.
2. Defective renal tubular reabsorption.

VIVA VOCE
Q1. What is the normal level of uric acid?
Ans. 2–7 mg/dl.
Q2. What are the causes of hyperuricemia?
Ans. Renal failure, leukemia, HGPRTase deficiency.
Q3. Is there any correlation between cancer and uric acid?
Ans. Yes, hyperuricemia, because of increased cell growth, increase breakdown of purines cause to Gout.
Q4. How alcohol affect uric acid level?
Ans. Alcohol increase NADH it is promote formation of monosodium urate, that is less soluble.
Q5. What is tophi?
Ans. Deposition of uric acid crystals in joints or skin or cartilage. Most common site of deposition of crystal in
big toe.
Q6. Why do cancer patients have high levels of uric acid?
Ans. Cancer patients have high cell turnover this causes increase breakdown of purine and hence increase
formation of uric acid.
Kidney Function Test 61
62 Practical Manual of Biochemistry
 Lipidogram 63

8
Lipidogram

A. ESTIMATION OF TOTAL CHOLESTEROL

INTRODUCTION
Cholesterol is one of the causative factors of heart disease, thrombosis, cerebral hemorrhage.
Free 30%
Cholesterol
Estrified 70%
Total plasma lipids are in serum 400–600 mg/dl

Total cholesterol 200–250 mg/dl

Serum triglycerides 25–170 mg/dl
Serum HDL—cholesterol 35–60 mg/dl
Serum LDL—cholesterol Up to 150 mg/dl
Serum VLDL—cholesterol 20–35 mg/dl

Aim: Estimation of total cholesterol by colorimetry.

Methods: CHOD-POD method.
Libermann-Burchard’s method.
ZAK’s method.
Sackets’s method.
Kim and goldberg.
Salkowski’s method.

Principle: (CHOD-POD method)

Cholesterol ester +H2O → Cholesterol + FFA
Cholesterol + O2 → Cholesterol + H2O2
H2O2 + phenol + 4-aminoantipyrine → quinoneimine + H2O
(Red/pink dye)

Procedure
Take 1 ml reagent in tube
↓
Add 20 μl serum
↓
Mix well and incubate at room temperature for 10 minutes AT 37°C
↓
Read at 520 nm

63
64 Practical Manual of Biochemistry

Observation Table
Blank Standard Test
Reagent 1 ml 1 ml 1 ml
Standard – 20 μl –
Serum – – 20 μl

Calculation
OD of test
× conc of standard
OD of standard

Results: …………………..
Normal range of cholesterol in blood 200–250 mg/dl.

Interpretation
Hypocholestremia 1. Hyperthyroidism
2. Abetalipoproteinemia

Hypercholestremia 1. Hypothyroidism
2. von-Gierke’s disease
3. Nephrotic syndrome
4. Alcohol intake
5. Obstructive jaundice
6. Diabetes mellitus

VIVA VOCE
Q1 What is the normal blood cholesterol range?
Ans. 150–250 mg/dl.
Q2. Number of carbon atom present in cholesterol structure.
Ans. 27.
Q3. Name of products formed by the degrading of cholesterol.
Ans. Bile acids, vitamin D, steroid hormones.
Q4. Name of dietary sources for cholesterol.
Ans. Milk, egg, meat.
Q5. Condition for Hypo- and hyper-cholesterolemia
Ans. See on last page.
Lipidogram 65
66 Practical Manual of Biochemistry
 Lipidogram 67

B. ESTIMATION OF TRIGLYCERIDES (TG)

INTRODUCTION
TG estimation is very important for diagnosis and management of hyperlipidemias, nephrosis, diabetes mellitus,
and endocrine disorders.
Aim: Estimation of triglycerides by colorimetric method.
Method: Trinder method.

Principle
Lipase
Triglycerides + H2O ⎯⎯⎯→ Glycerol + FFA
Glycerol kinase
Glycerol + ATP ⎯⎯⎯⎯⎯⎯ → Glycerol 3-PO4 + ADP
Glycerol 3PO4 + O2 ⎯⎯⎯⎯
Oxidase
→ DHAP + H2O2
H2O2 + 4-aminoantipyrine + phenol ⎯⎯⎯⎯⎯
Peroxidase
→ Quinoneimine dye
(Red/pink color)

Procedure
Take 1 ml reagent in tube
↓
Add 10 μl serum
↓
Mix well and incubate at room temperature for 10 minutes (37°C)
↓
Read at 520 nm
Observation Table
Blank Standard Test
Reagent 1 ml 1 ml 1 ml
Standard – 10 μl –
Serum – – 10 μl

Calculation
OD of test
× conc of standard
OD of standard

Results:………………
Normal range of TG in blood 25–170 mg/dl.

Interpretation
TG level ↑ 1. Diabetes mellitus
2. Nephrosis
3. Hypothyroidism
4. Pregnancy
5. Alcoholism
6. Atherosclerosis
7. Ischemic heart disease
8. Intake of OCPs
68 Practical Manual of Biochemistry

VIVA VOCE
Q1. What is the normal range of TG in serum?
Ans. 25–170 mg/dl.
Q2. Conditions when increases serum TG levels ?
Ans. See on last page.
Q3. Name of method for TG estimation.
Ans. Trinder method.
Q4. Which color filter use for TG estimation?
Ans. 520 nm green filter.
Lipidogram 69
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 Lipidogram 71

C. HDL ESTIMATION IN LIPIDOGRAM

INTRODUCTION
HDL cholesterol is good cholesterol its action is cardioprotective because it transport cholesterol from peripheral
tissue to liver (reverse transport of cholesterol).
Aim: Estimation of HDL by colorimetry.
Method: Phosphotungstic acid method.
Principle: Serum phosphotungstate → HDL + (LDL + VLDL + Chylomicrons).
Procedure: Precipitation.
Pipette Volume
Test 250 μl
Precipitating reagent 500 μl

Mixing and stand for 10 minutes at room temperature centrifuge at 4000 rpm for 10 minutes supernatant
↓
Take 1 ml reagent (cholesterol)
↓
Add 50 μl serum
(supernatant)
↓
Mix well and incubate 10 min at 37°C
↓
Read at 505 nm
Observation Table
Blank Standard Test
Reagent 1 ml 1 ml 1 ml
Standard – 50 μl –
Serum (supernatant) – – 50 μl

Calculation
OD of test
× conc of standard
OD of standard

Results: ……………………………
Normal range of HDL in male 30–65 mg/dl.
Normal range of HDL in female 30–80 mg/dl.

Interpretation
HDL level ↓ Atherosclerosis
Smoking
Alcoholism
Myocardial infraction
Coronary heart disease
72 Practical Manual of Biochemistry

LdL-c and VLDL estimation (Friedewald’s equation)

Total cholesterol = VLDL + HDL + LDL
LDL = Total cholesterol-(TG/5-HDL)
VLDL = TG/5

VIVA VOCE
Q1. What is the normal HDL level in serum?
Ans. 30–70 mg/dl.
Q2. Name of the enzyme which take part in reverse cholesterol transport?
Ans. LCAT.
Q3. Why HDL is known as good cholesterol?
Ans. Because of reverse cholesterol transport, it esterifies cholesterol and transfer to liver.
Q4. Name of Apo protein present in HDL.
Ans. Apo A, Apo E, Apo CII.
Q5. How to calculate VLDL?
Ans. TG/5.
Lipidogram 73
74 Practical Manual of Biochemistry
 Liver Function Test 75

9
Liver Function Test

Liver function test is routinely done to assess the function of the liver.

The test routinely done in LFT are:

1. Total bilirubin
2. Direct bilirubin
3. Indirect bilirubin
4. Total protein
5. Albumin
6. Globulin
7. SGOT
8. SGPT
9. Alkaline phosphatase.

ESTIMATION OF BILIRUBIN TOTAL/DIRECT/INDIRECT

INTRODUCTION
Bilirubin is a catabolic product of heme metabolism. Conjugation of bilirubin occurs in liver. Estimation of bilirubin
is very important for diagnosis of liver disease.
Aim: Estimation of bilirubin (total/direct/indirect).
Method: Diazo method.

Principle
Bilirubin + diazotized sulphanilic acid → azobilirubin (pink).
Bilirubin direct—directly react in acidic medium (water soluble).
Indirect bilirubin—Indirect bilirubin solubilised after addition of surfactant.

Procedure
Total Bilirubin Reagent (R1)
Surfactant 1%
HCl 100 mmol/L
Sulphanilic acid 5 mmol/L

Direct Bilirubin Reagent (R2)

Sulphanilic acid 10 mmol/L
HCl 100 mmol/L

Reagent (R3): Sodium nitrite 144 mmol/L.

75
76 Practical Manual of Biochemistry

Reagent Preparation
Test Volume of working reagent Add
R1 R2 R3
Bilirubin total 10 ml 10 ml 0.2 ml
Bilirubin direct 10 ml 10 ml 0.1 ml

Keep the reagent 3 vial plugged after use.

Observation Table

Total Bilirubin/Direct Bilirubin

Pipette in to test tube Blank Standard Test
Working reagent 500 μl 500 μl 500 μl
DW 25 μl
Standard 25 μl
Test 25 μl

Mix well incubate for 5 minutes at 37°C for total bilirubin and direct bilirubin. And read at 540–630 nm.

Calculation
OD of test
× conc of standard
OD of standard
Normal range of total bilirubin—0.2–1 mg/dl.
Indirect bilirubin 0.2–0.6 mg/dl.
Direct bilirubin 0.2–0.4 mg/dl.

Results. ................................

Interpretation
Indirect bilirubin • Hemolytic Jaundice
• Gilbert syndrome
• Neonates
• Crigller—Najjar type I
• Crigller—Najjar type II
Direct bilirubin • Obstructive jaundice
• Hepatocellular jaundice
• Rotor syndrome
• Dubin-Johnson syndrome
Liver Function Test 77
78 Practical Manual of Biochemistry
 Liver Function Test 79

ESTIMATION OF TOTAL PROTEIN

INTRODUCTION
Protein is the polymer of L-α amino acid. Blood contain large number of protein they perform so many functions.
Most of enzymes, hormones, and clotting factors are protein in nature.

Methods of Estimation of Protein

• Biuret method
• Lowry method
• Dye-binding method
• Turbidimetric method
• Nephelometric method
• Kjeldahl’s method.

Aim: Estimation of protein by colorimeter.

Method: Biuret method.
Principle: All compounds contain minimum two peptide linkage in alkaline medium peptide bond react with
biuret reagent. Cupric ion forms chelate with peptide bond and form purple–violet color complex.

Procedure
Take 1 ml reagent in tube
↓
Add 20 μl serum
↓
Mix well and incubate 10 min at 37°C
↓
Read at 540 nm

Observation Table
Blank Standard Test
Reagent 1 ml 1 ml 1 ml
Standard – 20 μl –
Serum – – 20 μl

Calculation
OD of test
× conc of standard
OD of standard

Results. ..........................

Normal range of blood protein:

Total protein: 6–8 g/dl.
Serum albumin: 3.5–5.5 g/dl.
Serum globulin: 2.5–3.5 g/dl.
A/G ratio: 1.4–1.15 g/dl.
80 Practical Manual of Biochemistry

Interpretation
Conditions when serum protein elevated Hyperproteinemia
1. Dehydration

Conditions when serum protein decreased Hyperproteinemia

1. Malnutrition
2. Liver disease
3. Prolong starvation
4. Nephrotic syndrome

VIVA VOCE
Q1. Bilirubin is the catabolic end product of which substance?
Ans. By catabolism of heme.
Q2. Write names of water soluble and insoluble bilirubin.
Ans. Conjugated is water soluble and unconjugated is insoluble in water.
Q3. How to define jaundice?
Ans. Yellow discoloration of skin, conjunctiva, mucosa and increase level of bilirubin is known as jaundice.
Q4. Types of jaundice?
Ans. Prehepatic, hepatic, posthepatic.
Q5. Causes of prehepatic, hepatic and posthepatic jaundice.
Ans. Prehepatic—G6PD deficiency, blood transfusion mismatch.
Hepatic—Alcohol intake, hepatitis A, B, C, E, drug induced.
Posthepatic—Stone, cancer of head of pancrease.
Liver Function Test 81
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 Liver Function Test 83

ESTIMATION OF SERUM ALBUMIN

INTRODUCTION
Albumin is a major plasma protein perform so many function like transport, maintain colloid osmotic pressure,
buffering action and nutritive so albumin act as complete protein. Synthesis of albumin occurs in liver.
Aim: Estimation of serum albumin by colorimeter.
Method: Bromocresol green (BCG).
Principle: Albumin binds with BCG and form blue green color complex.

BCG (undissociated form) −−−→ BCG (dissociated form)

Yellow color blue color

Procedure
Take 1 ml reagent in tube
↓
Add 10 μl serum
↓
Mix well and incubate 10 min at 37°C
↓
Read at 630 nm

Observation Table
Blank Standard Test
Reagent 1 ml 1 ml 1 ml
Standard – 10 μl –
Serum – – 10 μl

Calculation
OD of test Vol of standard
conc of substance = × × conc of std
OD of Std Vol of serum/sample

Results: ……………

Normal Range of Blood Protein

Total protein: 6–8 g/dl.
Serum albumin: 3.5–5.5 g/dl.
Serum globulin: 2.5–3.5 g/dl.
A/G ratio: 1.4–1.15 g/dl.

Interpretation
Conditions when serum albumin elevated Hyperalbuminemia
1. Dehydration
2. Shock
3. Hemoconcentration
84 Practical Manual of Biochemistry

Conditions when serum albumin decreased Hypoalbuminemia

1. Malnutrition
2. Liver disease
3. Prolong starvation
4. Nephrotic syndrome

Serum globulin = Total protein—serum albumin

A/G ratio = Serum albumin/serum globulin
Liver Function Test 85
86 Practical Manual of Biochemistry
 Liver Function Test 87

ESTIMATION OF SGOT/AST
INTRODUCTION
SGOT is a serum enzyme high concentration of SGOT found in liver, heart muscle.
Aim: Estimation of serum glutamate oxalotransaminase/aspartate aminotransferase (SGOT/AST).
Method: IFCC method (international federation of clinical chemistry).

Principle
L-Aspartate + 2-oxoglutrate ⎯⎯⎯
AST
→ Oxaloacetate + L-glutamate
Oxaloacetate + NADH ⎯⎯⎯
MDH
→ L-Lactate + NAD
Sample pyruvate + NADH ⎯⎯⎯
LDH
→ L-Lactate + NAD
AST—Aspartate aminotransferase.
LDH—Lactate dehydrogenase.
MDH—Malate dehydrogenase.

Procedure
Pipette Volumes
Working reagent 1000 μl
Test 100 μl

Mix well and aspirate.

Lagging time 60 sec.
Reading time 60 sec.
Read at 340 nm.

Calculation
3
(ΔA/min) × TV × 10
IU/L =
SV × Absorption × P
P: Cuvette light path = 1 cm.
TV: Total reagent volume in μl.
SV: Sample volume in μ.

Results: ...........................

Interpretation
Increased level of SGOT are seen in:
1. Heart diseases.
2. Myocardial infraction.
3. Liver diseases.

VIVA VOCE
Q1. Parameters included in LFT profile.
Ans. Bilirubin total, direct, indirect, total protein serum albumin, globulin, A/G ratio SGOT, SGPT, ALP.
Q2. Normal value of SGOT.
Ans. 10–45 IU/L.
88 Practical Manual of Biochemistry

Q3. Which enzymes increase in liver disease?

Ans. SGPT, SGOT.
Q4. Very high level of SGPT and SGOT are seen in:
Ans. Acute hepatitis, cardiac disease.
Q5. SGOT and SGPT are dependent on which coenzyme?
Ans. Pyridoxal phosphate (PLP).
Liver Function Test 89
90 Practical Manual of Biochemistry
 Liver Function Test 91

ESTIMATION OF SERUM SGPT/ALT

INTRODUCTION
SGPT is a serum enzyme high concentration of SGPT found in liver.
Aim: Estimation of serum glutamate pyruvate oxalotransaminase/Alanine transaminase (SGPT/ALT).
Method: IFCC method (international federation of clinical chemistry).

Principle

L-alanine + 2-oxoglutrate ⎯⎯⎯

ALT
→ pyruvate + L-glutamate
Pyruvate + NADH ⎯⎯⎯
LDH
→ L-Lactate + NADH

Procedure
Pipette Volumes
Working reagent 1000 μl
Test 100 μl

Mix well and aspirate.

Lagging time—60 sec.
Reading time—60 sec.
Read at—340 nm.

Calculation
3
(ΔA/min) × TV × 10
IU/L =
SV × Absorption × P
P: Cuvette light path = 1 cm.
TV: Total reaction volume in μl.
SV: Sample volume in μ.

Results: .........................

Interpretation
Increased level of SGPT are seen in
• Liver diseases
• Viral hepatitis
• Toxic hepatitis
• Liver cirrhosis
• Dengue.
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Liver Function Test 93
94 Practical Manual of Biochemistry

ESTIMATION OF ALKALINE PHOSPHATASE (ALP)

INTRODUCTION
ALP is produced by osteoblast of bone and associated with calcification of bone. ALP has different type of
isoenzymes which are found in liver, bone, kidney, intestine and placenta.
Aim: Estimation of ALP by colorimeter.
Method: Kind and king method.

Principle
ALP
Phenyl phosphate ⎯⎯⎯
→ phenol + pi
Potassium ferricyanide
Phenol + 4-aminoantipyrine ⎯⎯⎯⎯⎯⎯⎯⎯⎯
→ Orange/red color

Procedure
Blank Standard Control Test

Reagent R1 500 μL 500 μL 500 μL 500 μL

DW 1500 μL 1500 μL 1500 μL 1500 μL

Mix well and incubate at 37°C for 3 minutes

Serum – – – 50 μL
R3 – 50 μL – –

Mix well and incubate at 37°C for 15 minutes

R2 1000 μL 1000 μL 1000 μL 1000 μL
Serum – – 50 μL –

Mix well read at 520 nm.

Calculation
OD of test − OD of control
= × Conc of standard
OD of standard − OD of blank

Results
Normal range of blood ALP: 29–130IU/L.
Increased levels are found in children.

Interpretation
Conditions when ALP elevated Bone disease
Obstructive jaundice
Bone carcinoma
Paget disease
Liver cirrhosis
Disease of intestinal tract
Third trimester of pregnancy
Growing children
Conditions when ALP decreased Severe anemia
Malnutrition
 Liver Function Test 95

VIVA VOCE
Q1. Which enzyme increase in obstructive jaundice?
Ans. ALP.
Q2. What is the normal range of ALP in serum?
Ans. 29–129 IU/L.
Q3. How many isoenzymes of ALP are there?
Ans. Six isoenzymes (bone liver, placenta).
Q4. Which enzyme increase in bone disease?
Ans. ALP.
Q5. Various conditions where increased ALP levels are seen?
Ans. Hyperparathyroidism, disease of intestinal tract.
Q6. What is the normal range of ALP in children?
Ans. 104–345 U/L in 1–3 years.
96 Practical Manual of Biochemistry
 Estimation of Blood Calcium 97

10
Estimation of Blood Calcium

INTRODUCTION
99% of calcium is present in bones. In blood calcium is present in three forms:

Regulation of Blood Calcium

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98 Practical Manual of Biochemistry

Role of Calcium
1. Present in bone along with phosphorus as hydroxyl apatite crystals.
2. Plays important role in blood coagulation cascade.
3. Muscle contraction.
4. Membrane permeability.
5. Neuromuscular transmission.
6. Act as secondary and tertiary messenger for hormone action.
7. Excitation of muscle and nerve tissue.

Methods of Calcium Estimation

1. o-Cresolphthalein complex-one (OCPC) method.
2. Arsenazo-III method.
3. Ion selective electrodes (ISE).
4. Atomic absorption spectrometry.
5. Titration method.
Aim: Estimation of blood calcium level Arsenazo III, end point method.
Principle: Calcium ions form violet complex with o-cresolphthalein complex-one in alkaline medium. Absorbance
is measured at 570 nm. The intensity of color is directly proportional to the concentration of calcium ions.

Procedure
Blank Standard Test
Working reagent 1 ml 1 ml 2.5 ml
Calcium standard – 20 μl –
Distilled water 20 μl – –
Serum – – 20 μl

Mix well then read at 650 nm filter.

Calculation
OD of test Volume of standard
× × 100
OD of std volume of serum

Result: ……………….

Interpretation
Normal range
Total calcium: 8–10 mg/dl.
Ionic calcium: 4.6–5.3 mg/dl.
Hypocalcemia Hypercalcemia
1. Osteomalacia in adult 1. Hyperparathyroidism
2. Rickets in children 2. Hypervitaminosis D
3. Hypothyroidism 3. Bone cancer
4. Steatorrhea 4. Milk alkali syndrome
5. Pregnancy and lactation 5. Thyrotoxicosis
6. Nephritic syndrome 6. Multiple myeloma
7. Hepatocellular disease 7. Polycythemia vera
8. Hypomagnesemia 8. Sarcoidosis
 Estimation of Blood Calcium 99

VIVA VOCE
Q1. What is the normal range of serum calcium?
Ans. 9–11 mg/dl.
Q2. How many forms of calcium present in body?
Ans. Three forms. Ionized calcium, protein bound calcium, complexed calcium.
Q3. Write down regulation of calcium.
Ans. See on last page.
Q4. What is corrected calcium?
Ans. Serum calcium + 0.8 (4-serum albumin in gm%).
Q5. Write sources of calcium.
Ans. Milk, egg, dairy product.
100 Practical Manual of Biochemistry
Estimation of Blood Calcium 101
102 Practical Manual of Biochemistry

11 Estimation of
Blood Phosphorus

INTRODUCTION
90% phosphorus is present in bones. In blood phosphorus is present in two forms:
1. Organic form: Phospholipids, glycerophosphate, nucleoside phosphate.
2. Inorganic form: Phosphates bound to various inorganic cations like Na+, K+, Ca2+, etc.
Parathyroid hormone regulates the level of PO43– in blood. PTH increases the excretion of PO43– in urine and
decreases its level. Vitamin D increases the level of PO43– by increasing its absorption from intestine and reabsorption
from renal tubules.

Methods of Estimation
1. Using reaction of phosphate ions with ammonium molybdate.
2. Vandate—molybdate method.
3. Enzymatic methods by monitoring the formation of NADPH, H2O2 and NADH.
Aim: Estimation of blood phosphate by ammonium molybdate method, end point.
Principle: Phosphate react with ammonium molybdate to form ammonium phospho molybdate which is reduced
by reducer aminonaphthol sulphonic acid (ANSA) to form blue color.

Procedure
Blank Standard Test
Reagent 1000 μl 1000 μl 1000 μl
Distilled water 20 μl – –
Standard – 20 μl –
Test – – 20 μl

Mix well and incubate at room temperature for 10 min, then read absorbance at 340 nm.

Calculations
OD of test Vol. of std
× × 100
OD of std Vol. of serum

Result: ……………….

Interpretation
Normal range
Adults: 2.5–4.5 mg/dl.
Children: 4–7 mg/dl.
102
 Estimation of Blood Phosphorus 103

Hyperphosphatemia Hypophosphatemia
1. Renal failure 1. Hyperparathyroidism
2. Hypoparathyroidism 2. Fanconi’s syndrome
3. Acromegaly 3. X-linked hypophosphatemia
4. Rhabdomyolysis 4. Malabsorption
5. Chemotherapy 5. Acidosis
6. Aggressive phosphate therapy 6. Respiratory alkalosis

VIVA VOCE
Q1. What is the normal range of serum phosphorus?
Ans. 3–5.5 mg/dl
Q2. Causes of decrease phosphorus level.
Ans. Hyperparathyroidism, diabetic coma, decrease renal tubular reabsorption.
Q3. Causes of increase phosphorus level.
Ans. Renal failure, hypoparathyroidism, hypervitaminosis D.
Q4. Is there any role of phosphorus in buffer system?
Ans. Yes, phoshphate buffer is main intracellular buffer.
Q5. Write biochemical functions of phosphorus.
Ans. See on last page.
104 Practical Manual of Biochemistry
Estimation of Blood Phosphorus 105
 3
Demonstration

12. pH Meter
13. Chromatography
14. Electrophoresis
 12
pH Meter

INTRODUCTION
pH meter is an instrument used to determine the pH of a solution.
Principle: pH meter is based on the principle of measurement of ECF (electromotive force), generated between
two electrode. This force occurs due to difference in hydrogen ion concentration.
When metal plate kept in a solution of its own salt, it loses ions into the solution and it becomes negatively
charged.
• Metal plate—negatively charged (zinc).
• Solution—positively charged (zinc sulfate).
If two different metal plate (electrode) are used in which one act as reference electrode (known potential) and
other electrode whose potential we want to measure.

Reference Electrode
• Standard hydrogen electrode.
• Calomel electrode.
• Glass electrode.
• Combined electrode.

Fig. 12.1: pH meter

Procedure
1. Adjust temperature by knob in pH meter.
2. First of all wash electrode with water, wipe with soft tissue paper.
3. Take various standard buffer for pH 4, 7, 9.
4. Calibrate the instruments with 7 pH, then 4 and 9, 10.
5. Then dip unknown solution and read pH.
6. After this wash it again and dip into DW.

Precautions
1. Always wash electrode after use, dip into DW.
2. Followed any specific guideline provided by instrument manual.
3. Temperature correction is also required for assessment of pH.
109
110 Practical Manual of Biochemistry

13
Chromatography

DEFINITION
This technique is used for the separation of closely related compounds, present in a mixture solution like
carbohydrates, lipid, amino acids, vitamins, etc. The term was given by Mikhail Tswett.
Principle: Separation of mixture by chromatography depends on Stationary and Mobile phase. There is
movement of solute mixture in Mobile phase, which move on Stationary phase. This interaction, results in separation
of compounds.
Migration of substance is measured as Rf (ratio of front) value
Distance travelled by substance (solute)
Rf =
Distance travelled by solvent

Classification

Paper Chromatography
Aim: Separation of individual amino acids from a mixture.

Requirements
• Lead pencil.
• Filter paper strip, (Whatman no 1).
• Amino acid mixture (tyrosine, aspartic acid, alanine) (Fig. 13.2).
• Solvent mixture (Butanol: Acetic acid: DW, 12 : 3 : 5).
• Chromatography chamber (Fig. 13.1).
• Capillary tube, pipette, oven, dryer.
• Ninhydrin.

Procedure
• Make 2% solution of amino acid mixture (0.2 gm in 100 ml).
• Cover beaker with aluminum foil to prevent it from oxidation and protection from direct sunlight and air.
• Make buffer solution 12 : 3 : 5.
• Take A clean TLC chamber.
• Take paper and mark two line-one horizontal, one vertical.
110
 Chromatography 111

Fig. 13.1: Chromatography chamber Fig. 13.2: Amino acids for chromatography

• Mark three point on horizontal line.

• With the help of capillary put first solution on Ist mark.
• Marked dot on horizontal line.
• Then dry it with the help of dryer.
• Repeat it with more solutions.
• Keep buffer solution in TLC chamber.
• Put TLC paper into the chamber along the side wall such that the horizontal line get completely emerged into
the solution.
• Then wait for 40 minutes for allowing into stand.
• After 40 minutes take paper out of chamber, and dry.
• Dry for 20 minutes in oven, or help of dryer.
• After that stain will appear on the paper (Fig. 13.3).
• Spry ninhydrin on the stain.
• Mark it with lead pencil.
• Measure the distance from starting of horizontal line to the end point and also measure horizontal-vertical line.
• Calculate Rf value.
• According this substance can be found out.

Result
Calculate Rf value
Distance travelled by substance
Rf =
Distance travelled by solvent

Fig. 13.3: Paper chromatography of amino acids

112 Practical Manual of Biochemistry

14
Electrophoresis

DEFINITION
It is a technique widely used for the separation of biological molecules like plasma proteins, lipoproteins,
immunoglobulin, etc.
Principle: When charged particles are subjected to electric field they move towards the opposite poles, negatively
charged particles move towards the anode and positively charged particle move towards the cathode.
For example, in alkaline medium proteins are negatively charged, when electric field is applied they move
towards the anode.

Migration of particles in electric current depends on following factors:

1. Charge on ions.
2. Strength of electric current.
3. Size and shape of the charge particles.
4. Temperature.
5. Ionic strength buffer.
6. Support medium properties.

Procedure
• Sample is applied on a support media.
• The medium is then placed in a buffer solution, filled in electrophoresis chamber.
• Electric current is passed through the media to carry out electrophoresis.
• Support medium is removed and stained to visualize the separated bands.

Types of Electrophoresis
Free electrophoresis Zone electrophoresis
It is of two types: It has following types:
1. Microelectrophoresis 1. Paper
2. Moving boundary electrophoresis 2. Cellulose acetate
Out dated, mostly used for non-biological experiments 3. Capillary
4. Gel: Further classified into:
a. Agarose gel
b. SDS-PAGE
c. PFGE
d. Two dimensional

Paper electrophoresis: Support medium is a filter paper having adsorptive capacity and uniform pore size.
Filter paper is moistened with buffer and ends of the strip are immersed into buffer reservoirs containing the
electrodes.
112
 Electrophoresis 113

Cellulose acetate electrophoresis: It is a modified version of paper electrophoresis, where instead of filter
paper, cellulose acetate membrane is used.
Capillary electrophoresis: Capillarity of narrow bore tube is employed to separate the samples based on their
size: charge ratio.
Gel electrophoresis: Gel is used as a support media for the separation of DNA, RNA and proteins under the
influence of electric current. It is the most commonly used technique.

Type of gel electrophoresis

Agarose gel Agarose gel
Used for separation of nucleic acid
SDS- sodium dodecyl sulfate polyacrylamide gel Used to separate proteins SDS dissociates proteins into individual
electrophoresis polypeptide subunit and gives a uniform negative charge
Pulse field gel electrophoresis Agarose/polyacrylamide gel molecules are induced to migrate through
the gel under a static electric field. DNA, under the influence of electric
field elongate and align themselves with the field
Two-dimensional electrophoresis Used to purify protein mixture ampholyte solution is incorporated into
the gel. A stable pH gradient is established in the gel after application of
electric field. This is followed by SDS-PAGE

Aim: To perform agarose gel electrophoresis to separate mixture of protein/nucleic acid.

Principle: Molecules are separated based on molecular size. Agarose gel acts as a sieve. Larger and bulky
molecules stay behind whereas the smaller molecules move faster and quickly towards their respective electrodes.

Instruments required:
1. Electrophoretic chamber (Fig. 14.1).
2. Power supply.
3. Trans-illuminator, to visualize the DNA in gel.
4. Agarose gel support media.
5. Buffer: TAE buffer or tris acetate EDTA (Fig. 14.2).
6. Ethidium bromide dye to stain the DNA.

Fig. 14.1: Electrophoretic chamber and power supply

114 Practical Manual of Biochemistry

Fig. 14.2: Reagents required for electrophoresis

Procedure
1. Formation of agarose gel: Dry agarose gel powder is mixed with electrophoresis buffer to the desired concentration
and heated till it completely melts.
2. Ethidium bromide 0.5 mg/ml is added at this point.
3. After cooling at 60°C it is added into the casting tray containing sample comb and allowed to solidify at room
temperature. Once the gel is dry sample comb is removed.
4. The gel is removed and placed in electrophoretic chamber and covered with buffer.
5. Sample containing the mixture of DNA and buffer is applied on sample well.
6. Lid and power leads are placed on the apparatus and current is applied.
7. Current flow is confirmed by the presence of bubbles.
8. DNA being negatively charged move towards the positive electrode.
9. Ethidium bromide gets incorporated into the DNA. Once separation has occurred the fragments of DNA can
be visualized in a UV trans-illuminator.

Preparation of Buffer
Tris buffer: 3 gm tris, 0.39 gm EDTA and 0.23 gm boric acid are dissolved in 250 ml distilled water.
1% agarose: 0.1 gm of dry agarose powder is dissolved in 10 ml tris buffer and boiled till it is completely
dissolved.

DNA Extraction
Procedure of DNA extraction.

Step 1: Source of DNA

a. Cell culture up to 5 × 106 cells should be present.
b. Tissue up to 25 mg.
c. Blood/serum up to 200 μl.

Step 2: Cell lysis—to Remove Proteins, Lipids and RNA

a. Lipids are removed with the help of detergents/surfactants.
b. Proteins are removed with the help of enzyme proteinase.
c. RNA is removed with help of RNAase.
Cell debris consisting of amino acids, lipids, RNAs are clumped together by treating with saline. Centrifugation
removes clumped cell debris from DNA.
 Electrophoresis 115

Step 3: DNA Purification

There are three methods of DNA purification:
a. Ethanol precipitation: DNA being insoluble in ethanol aggregates in it and form pellets on centrifugation.
b. Phenol: Chloroform extraction—protein is denatured by phenol and it remains in the organic phase. DNA
being insoluble in phenol stays in the aqueous phase. DNA is separated from aqueous phase and treated
with chloroform to remove phenol residue.
c. Minicolumn purification: Column chromatography which relies on the principle, nucleic acid binds with
solid phase depending on pH and salt concentration of the solution.
Once the DNA is removed it is kept in alkaline buffer like TE buffer or ultra pure water.

Protein Electrophoresis Interpretation

Normal serum on electrophoresis gets separated into 5 bands
Albumin 55–60%
α1 globulin 3–4%
α2 globulin 5–10%
β globulin 9–10%
γ globulin 10–20%

Pathological conditions identified on serum protein electrophoresis

1. Nephrotic syndrome Loss of albumin in urine, albumin band will reduce
2. Liver cirrhosis Albumin is not synthesized
Albumin band will reduce with wide β globulin band
3. Chronic infection γ globulin increases
4. Multiple myeloma A sharp spike is seen in γ globulin, called M-band
 4
Miscellaneous

15. Body Fluids

16. Reagents Preparation
17. Biological Waste Disposal
18. Blood Gas Analyzer
 15
Body Fluids

Collection, prevention and analysis of body fluid

Fig. 15.1: Various vials used for collection

Types of Fluid
• Milk
• CSF (cerebrospinal fluid)
• AF (ascites fluid)
• PF (pleural fluid)
• Amniotic fluid
• Synovial fluid
• Pericardial fluid.
CSF: Collection of sample by lumbar puncture.
• Fluid is collected in 3 to 4 plain tubes.
• CSF in first tube act as a reserve sample.
• Biochemistry and immunological tests are performed on second tube.
• Culture and other tests related to microbiology are performed on third test tube.
• Tests related to hematology are performed on forth test tube.
If CSF fluid is available in only three test tubes then, biochemistry, immunology and hematology, tests related
to all three are performed on third test tube.
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RBC’s are normally absent in CSF, but it can be present in case of traumatic tap or subarachnoid hemorrhage,
to differentiate between the two conditions RBC count is performed on first tube and third/fourth tube:
In case of traumatic tap, RBC’s will be present in only in tube since first tube is filled first.
In case of subarachnoid hemorrhage RBC’s will be present in both tubes 1 and 3/4

CSF findings in various pathological conditions

Normal Tuberculosis Bacterial Subarachnoid Brain tumor
meningitis meningitis hemorrhage
Color and Clear and colorless Opalescent and Cob Turbid Blood color Clear and colorless
appearance web formed on
standing, yellow
Total cell count 0–5 Increased Markedly increase RBC’s present No change
Protein 15–45 mg/dl Increased Markedly increase Increased Increased
Glucose 45–85 mg/dl Low Markedly low Increased Low

Pleural Fluid
Collection done in 4 mL in lavender top spray coated EDTA tube or dark green top sodium heparin tube.

Biochemical Analysis
• Protein 0–2 gm/dl, sugar 50–70 mg/dl, Cl 95–108 mEqμl/L.
• ADA (adenosine deaminase estimation in pleural fluid is done) 0–10 normal, >10 infection.

AF (Ascites Fluid)
• Sample required > 25 Ml.
• Protein 0–2 gm/dl, sugar 50–70 mg/dl , Cl 95–108 mEq/L, SAAG (serum albumin—ascites albumin).

Synovial Fluid for Crystal

Preferred sample—4 ml dark green top sodium heparin tube, not plasma seperated tube (PST).

Also acceptable
• Sterile container (no additive).
• Red top tube.
• Lavender top spray coated EDTA tube.
Synovial fluid for glucose and protein: 0.5 ml in gold top SST or light green (mint) top PST tube.
Dialysate: 3–5 mL in lavender top spray coated EDTA tube.

Precautions
• Always indicate priority of test orders on the requisition.
• Pleural fluid pH must be collected in a blood gas syringe.
• Do not send other fluids in syringes (with or without needles attached).
• All body fluids must be taken to the laboratory immediately after collection. Hand the specimen directly to the
laboratory personnel (do not leave on counter).
 16
Reagents Preparation

Reagents for Carbohydrates Tests

Reagents Method of preparation
1. Molisch’s reagent 1 gm of α naphthol in 100 ml of ethanol
2. Benedict’s reagent A. 17 gm of sodium citrate and 10 gm of sodium carbonate in 90 ml of distilled water
B. 1.73 gm of copper sulfate in 10 ml of distilled water
3. Barfoed’s reagent 6.6 gm copper acetate and 0.9 ml of glacial acetate in 100 ml of distilled water
4. Seliwanoff’s reagent 50 mg resorcinol in 100 ml of 12% HCl
5. 12% HCl 12 ml of HCl in 88 ml of distilled water
6. Iodine solution 1.27 gm iodine and 3 gm of potassium iodide in 100 of distilled water

Reagents for Test of Lipids

Reagents Method of preparation
1. Emulsification sodium carbonate 0.5% 0.5 gm sodium carbonate in 100 ml of distilled water
2. Saponification
a. KOH/NaOH (10%) 10 ml (KOH/NaOH) in 90 ml of distilled water
b. CaCl2 (5%) 5 gm CaCl2 in 100 ml of ditilled water
3. Dustan test 0.5% boric solution 0.5 gm of boric powder in 100 ml of distilled water
4. Hubel’s iodine 26 gm of iodine and 30 gm of HgCl2 in 1000 ml of ethanol

Reagents for Tests of Proteins

Reagents Method of preparation
1. Biuret test 10% NaOH—10 gm NaOH in 100 ml DW
0.5% copper sulfate—0.5 gm copper sulfate in 100 ml DW
2. Ninhydrin reagent (2%) 2 gm ninhydrin in 100 ml DW
3. Xanthoproteic test 40 gm NaOH in 100 ml DW
4. 40% NaOH
5. Millon’s reagent 10 gm mercuric sulfate in 50 ml DW and 10 ml conc H2SO4, make final volume 100 ml
with DW
6. Sodium nitrate 1% 1 gm in 100 ml of DW
7. Hopkin cole’s 40% formaline reagent 1 ml formaline in 500 ml DW
8. Sakaguchi reagent Molisch’s reagent, sodium hypobromite-1 ml bromine in 100 ml of 10% NaOH
10% NaOH is 10 gm NaOH in 100 ml DW
9. 2% lead acetate 2 gm lead acetate in 100 ml DW
10. Esbach’s reagent 5 gm picric acid and 10 gm citric acid in 500 ml DW

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Reagents for Normal Urine

Reagents for uric acid 0.1 N AgNO3 1.7 GM AgNO3 in 100 ml DW
Reagents for phosphate Conc nitric acid 25 gm ammonium molybdate + 200 ml DW + 300 ml 10 N H2SO4

How to form Normal and Molar Solution

M1V1 = M2V2 (number of solute per mole) W/V
N1V1 = N2V2
5M H2SO4 (98%)
M1V1 = M2V2
5 × 1000 = 18.4 × V2
5000/18.4
271 ml
10 N H2SO4 (98%)
M1 = 10N = 5M
M1V1 = M2V2
5 × 1000 = 18.4 × V2
V2 = 5000/18.4
271.7 ml
9N H2SO4 (98%)
9 × 1000 = 36.8 × V2
9000/36.8 = V2
244.56 ml
.25 N HCL (36%)
.25 × 1000 = 11.65 × V2
V2 = 250/11.65
21.45 ml
N/5 NaOH
N1 = N/5, N2 = 19.4, V1 = 1000
N/5 × 1000/19.4
200/19.4 = 10.30 gm.
Note: Add required volume of DW to make up to 1000 ml of solution.
 Biological Waste Disposal 123

17
Biological Waste Disposal

Any material containing infectious material is called biomedical waste. Biological waste is segregated into
appropriately colored plastic bags.
I. Red category: Recyclable infected waste.
1. Tubes
2. Bottles
3. Soiled gloves
4. Urine bags
5. Syringes
6. IV sets
7. ET tubes
8. All medical plastic equipment.
Disposed by: Autoclaving/microwaving, hydroclaving followed by
shredding or mutilation or combination (Fig. 17.1).
II. Blue category: Recyclable waste (glassware)
1. Broken glass
2. Contaminated glass
3. Medicine vials
4. Ampoules
5. Metallic body parts.
Disposed by: Disinfection by sodium hypochlorite treatment or
through autoclaving, microwaving, hydroclaving (Fig. 17.2).
III. White category: Infected sharp waste
1. Needles
2. Blades
3. Scalpels
4. Burnt needle
5. Syringes with fixed needles
6. All medical used metallic sharps.
Disposed by: Autoclaving or dry heat, sterilization, followed by
shredding, mutilation or combination (Fig. 17.3).
IV. Yellow category: Waste contaminated with blood body fluid and
infected cytotoxic waste.
1. Discarded medicine Fig. 17.1: Autoclaving/microwaving,
hydroclaving followed by shredding or
2. Human anatomical waste multilation or combination

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Fig. 17.2: Disinfection by soldium Fig. 17.3: Autoclaving or

hypochlorite treatment of through dry heat, sterilization, followed by
autoclaving, microwaving, shredding, mutilation or
hydroclaving combination

3. Soiled linen and bedding/mattresses

4. Soiled waste
5. Animal anatomical waste
6. Blood bags
7. Expired medicines
8. Microbiology, clinical and laboratory waste (after pretreatment)
9. Cytotoxic drugs
10. Items contaminated with cytotoxic drugs
11. Infected with cytotoxic secretions.

Disposed by: Incineration or plasma pyrolysis or deep burial (Fig. 17.4).

 Biological Waste Disposal 125

Fig. 17.4: Management incineration or plasma pyrolysis or deep burial

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18
Blood Gas Analyzer

INTRODUCTION
Measurement of blood gases, pH and electrolytes, metabolite,
bicarbonate concentration by blood gas analyzer (Fig. 18.1).
Principle: pH, PCO2, PO2 can be directly measured by analyzer.
Generation of potential difference between two electrodes is
responsible for measurement of pH, PCO2 and PO2. Hb is measured
in percentage saturation. Bicarbonate concentration calculated by
Handerson–Hasselbach equation. For the measurement of electrolytes
Na+, K+, Ca++, Cl–, Li+ ion selective electrode (ISE) method is used.
Sample: Arterial heparinised whole blood sample, volume 70–
100 μl.
Procedure: As per the manual provided with instrument.

Precaution
• Arterial blood sample should be used.
• Blood sample should not be clotted.
• Analyze the sample as soon as possible within half an hour.
• Instrument should be washed after or between uses of samples.

Clinical Applications
• Acid-base disorders.
• Critical care patients.
• Burn cases.

Fig. 18.1

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 DNA Extraction and its Applications: An Overview 139

19 DNA Extraction and its

Applications: An Overview
Dr Dharmveer Yadav

Isolation of nucleic acids (DNA or RNA) is a basic and essential step in molecular biology. The main purpose of
this procedure is to obtain a relatively purified form of DNA for doing the downstreaming techniques like PCR
analysis, mutation or polymorphism studies using restriction fragment length polymorphism (RFLP). Since the
quality and quantity of DNA affect the results of the above downstreaming procedures, procedure of isolation of
DNA plays a crucial role in the molecular biology.
There are several methods for isolation of DNA (Flowchart 19.1). All these methods involve four basic essential
steps:
1. Cell lysis,
2. Digestion/removal of protein,
3. Concentration/precipitation of DNA, and
4. Determination of the purity and quantity of DNA.

1. CELL LYSIS
Cell lysis is the most crucial step in the DNA isolation. Lysis can be done by different methods, i.e. physical
methods (sonication and grinding) or by chemical/enzymatic methods (detergents/lysis buffer). The strong forces
applied in physical methods lead to shearing of DNA. So the best way to do the cell lysis is by chemical/enzymatic
methods

2. DEPROTEINIZATION
The second step in the isolation of DNA is removal of proteins from the cell lysate. This procedure is also called
deproteinization. Depending on the property of solubility differences, different methods were developed. Methods
that exploit solubility differences are: Solubility differences in organic solvents, solubility differences of salts or
detergent complexes and differences in absorption to charged surfaces.

Flowchart 19.1: Methods for isolation of DNA

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Organic solvents: Most commonly used organic solvents are phenol and chloroform with isoamyl
alcohol. Due to the hydrophobic nature of the phenol, it helps in unfolding and precipitation of
proteins.
Detergents: Detergents help in deproteinization by forming insoluble complexes with nucleic acids and proteins
(e.g. CTAB). At high NaCl concentrations, CTAB will form insoluble complexes with protein but not with nucleic
acids. These insoluble complexes are removed from solution by centrifugation and the DNA is collected from the
aqueous phase by ethanol precipitation.
Silica based: The principle of silica matrices purification is based on the high affinity of the negatively charged
DNA backbone towards the positively charged silica particles. In the presence of a high concentration of salts,
DNA will bind to a silica surface, whereas proteins, due to the predominance of hydrophobic characteristics in
high salt, will not. The bound DNA is washed to remove impurities and eluted from the glass beads by lowering
the salt concentration. Besides silica matrices, nitrocellulose and polyamide membranes such as nylon matrices
are also known to bind with nucleic acids, but with less specificity.
Enzyme based: Proteins can be removed from DNA or RNA preparations using an enzyme protease that can
digest all proteins. Proteinase K and pronase are 2 such enzymes which are used DNA purification.

3. CONCENTRATING THE DNA

The main purpose of this step is to: (1) Concentrating the DNA from the deproteinised lysate, and (2) Removing
the impurities (amino acids, nucleotides, etc.) which were present in the lysate. This is achieved by either using
the alcohols or by dialysis. This step can be implemented in two ways: By precipitating DNA with alcohols
(ethanol and isopropanol), or by dialyzing and concentrating the DNA.
Alcohol precipitation is based on the phenomenon of decreasing the solubility of nucleic acids in
water. Whereas, the method dialysis is used if isolation of very high molecular weight DNA (equal or
larger than 200 kb) is desired. Alcohol precipitation can lead to DNA shearing, and indeed, it is difficult
to obtain DNA larger than 150 kb using ethanol precipitation. Dialysis is carried out in pretreated dialysis
tubing, against a large volume of buffer or water (dilution factor, 1000 or more) for a period of at least
24 hours.

4. QUANTIFICATION OF DNA
The last step of any DNA isolation procedure is the determination of concentration and purity of DNA. Method
of choice used for the determination of DNA concentration is ultraviolet (UV) spectrophotometry. Nucleic acids
have maximum absorbance at 260 nm and for proteins at 280 nm. So, the 260/280 is used to measure the purity of
isolated nucleic acids. A 260/280 ratio of 1.8 and 2.0 is generally accepted as pure for DNA and RNA repectively.
Any lower ratio may indicate the presence of protein or other contaminants in the isolated DNA. Similarly other
substances like phenol tend to have absorbance at 230 nm, so the ratio of A260/A230 is also frequently calculated.
A ratio ranging from 2.0 to 2.2 is considered pure. Any contamination from the phenol or guanidine leads to low
260/230 ratio.

DNA Isolation Solutions

Most common obstacles in obtaining a high yield DNA is degradation of DNA by nonspecific DNases. To avoid
these problems, all solutions should contain DNase inhibitors and all glassware and plastic usables should be
sterilized.
Material used for isolation of DNA (cells or tissues) should be suspended in isolation buffer called lysis buffer.
The appropriate buffer concentration, pH, ionic strength and the addition of DNase inhibitors and detergents
fulfil all of these requirements.
Two kinds of DNase inhibitors are in use, ethylenediaminetetraacetic acid (EDTA), and detergents like sodium
dodecyl sulfate (SDS) and cetyltrimethylammonium bromide (CTAB).
EDTA is a chelating agent of divalent cations like Mg2+. Since most of the DNases require Mg2+ ion as a cofactor,
the presence of EDTA will lead to inactivation of nucleases. SDS is a potent anionic detergents used in DNA
purification by density gradient centrifugation. CTAB is a powerful cationic detergent which inhibits DNase by
forming inactive CTAB-protein complexes (Flowchart 19.2).
 DNA Extraction and its Applications: An Overview 141

Flowchart 19.2: Procedure of DNA extraction

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