Biochemistry Practial

1
PRACTICAL NOTE BOOK

OF MEDICAL BIOCHEMISTRY
Authors:
 Prof. Dr. Muhammad Asif Jaleel
MBBS, M.Phil (Head of Department)
 Asst Prof. Dr. Marium Shoukat
MBBS, M.Phil
 Demonstrators
 Dr. Nasreen Aman
MBBS (Senior Demonstrator)
 Dr. Zeemal Iqbal

MBBS (Demonstrator)
 Dr. Sadia Akram

MBBS (Demonstrator)
 Dr. Yasoob Ali Khan

MBBS (Demonstrator)
Biochemistry Department
CMH Multan Institute of Medical Sciences (CIMS)
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DEPARTMENT OF BIOCHEMISTRY
It is certified that
Mr./Ms.________________________________________________
S/D/o_________________________________________________of
_______________________________________ has carried out the
necessary practical work part-1I as per the course according to
the National University of Medical Sciences Rawalpindi and
Pakistan Medical & Dental Council Islamabad, of studies for the
year____________.
University Roll No._____________
___________________________________________
Teacher Incharge Practical Work
___________________________________________
Head of Biochemistry Department
Date: ____________
3
Index
Section-1
Liver function Tests
Ser No Practical Page No

1.1 Estimation of Total Proteins
1.2 Estimation of Serum Albumin
1.3 Estimation of Bilirubin
1.4 Estimation of Serum AST
1.5 Estimation of Serum ALT
1.6 Estimation of Serum ALP
Section-2
Lipid Profile

2.1 Estimation of Cholesterol
2.2 Estimation of Serum Triglycerides
2.3 Estimation of Serum HDL Cholesterol
Section-3
Renal Function Tests

3.1 Estimation of Blood urea
3.2 Estimation of Blood Uric Acid
3.3 Estimation of Serum Creatinine
3.4 Determination of Creatinine Clearance
Section-4
Cardiac Markers

4.1 Estimation of Serum creatine kinase
4.2 Estimation of Serum LDH
Section-5
Enzymes

5.1 Estimation of Serum Amylase
5.2 Estimation of Serum calcium
5.3 Estimation of Serum chloride
4
Section-6
Electrolytes

6.1 Estimation of Serum Sodium
6.2 Estimation of Serum Potassium
6.3 Estimation of Serum Calcium
Section-7
Instrumentation in Clinical Biochemistry

7.1 pH meter
7.2 Laboratory Centrifuge
7.3 Spectrophotometer
7.4 Electrophoresis
7.5 Chromatography
Section-8
Blood Chemistry

8.1 Collection and Prevention of Blood Samples
8.2 Estimation of Blood Glucose
8.3 Glucose Tolerance Test
5
Section-1
Liver function Tests
Ser Practical Page No

No
1.1 Estimation of Total Proteins
1.2 Estimation of Serum Albumin
1.3 Estimation of Bilirubin
1.4 Estimation of Serum AST
1.5 Estimation of Serum ALT
1.6 Estimation of Serum ALP
6
LIVER
Liver is the largest gland in the body. It is concerned with the metabolism with the
metabolism of carbohydrates, lipids and proteins. Several toxins and drugs are
detoxified and excreted by liver. Liver is involved in the activation of vitamin D,
synthesis of many coagulating factors, metabolism of bile pigments, bile salts and
steroid hormones. In the view of the wide range of functions performed by the
liver, assessment of all the functions of liver is not easy. One has to choose
appropriate test depending upon the clinical findings.
Serum markers of liver pathology:
1. Aspartate:
This enzyme released in liver damage in liver disease.
 Amino transferase:
Increase In most alcoholic liver disease, ALT is greater than AST.
 Alanine aminotransferase:
Increase in most liver disease AST is greater than ALT.
Increase of AST in non-alcoholic liver disease suggests progression to
advanced fibrosis or cirrhosis.
2. Alkaline phosphatase:
It increase in cholestasis e.g. biliary obstruction, infiltrative disorders,
liver disease
Functional liver markers:
1. Bilirubin:
It increase in various liver diseases e.g. biliary obstruction, alcoholic or viral
hepatitis, cirrhosis, hemolysis.
2. Albumin:
It decrease in advanced liver diseases.
7
ESTIMATION OF SERUM TOTAL PROTEIN
Experiment # 1.1
Principle:
In alkaline medium CuSO4 reacts with peptide bonds present in protein,

forming a violet coloured complex. Intensity of colour is proportionate to
number of peptide bonds, which in turn depends upon the amount of
proteins present in the given sample.
Reagents:
• Biuret reagent
CuSO4 12 mmol/l
KI 30 mmol/l
Na-K tartarate 32.0 mmol
NaOH
• Standard protein solution (6g/dl)
Procedure:
• Take three test tubes and label them as standard, blank and test.
Sr. No Reagent Test Standard Blank

1. Biuret 5ml 5ml 5ml
reagent
2. Serum 0.2ml - -
3. Standard - 0.2ml -
solution
4. Distilled H2O - - 0.2ml
• Mix the contents of test tubes, by shaking and let them stand for 7
minutes at 37o C. Incubate at 20- 25o C Note the reading at 540 nm of
spectrophotometer and calculate the %age of total protein in 100ml of serum.
8
Calculation:
Plasma Protein concentration/dl=

Absorbance of Unknown x Concentration of protein standard
Absorbance of Standard
Normal Value:
Total plasma protein concentration is 6__8 gms/dl.
PRACTICAL VIVA QUESTIONS

9
Q.1 : write down the causes of hypoproteinemia?
a. Burns
b. Malnutrition
c. Crush wounds
d. Malabsorption
e. Nephrotic syndrome cirrhosis
f. Prokei loosing enteropathies
g. Ceiliac sprue
h. Marasmus
i. Khawiskhor
Q.2: write down the causes of hyperproteinemia?
a. Addison’s disease
b. Dehydration
c. Strenuous exercise
Q.3: what is denaturation of proteins?
When the structure of proteins are disturbed the proteins may lose
their functional and molecular characteristics. The loss of their original
characteristics is called denaturation of proteins.
Q.4: write down methods of determination of protein?
1. Turbidity metric method: In this method proteins are precipitated with

an organic acid, ‘tungstic acid’ and turbidity is measured.
2. Kjeldahl method: In this method concentration of nitrogen is
measured in serum after separation of non-protein nitrogen compound
(urea, uric acid, creatinine, ammonia), 15.1% to 16.8% of nitrogen is
present in proteins.
3. Refractometery: This test is useful when the small amount of serum
is needed.
4. Dye binding method: Bradford had described a dye-binding method
for determining the protein in microgram quantities.
5. Lowry’s method: It is used for the estimation of tyrosine in protein.
10
6. Colorimetric method (biuret): The name biuret is given because of the

reaction of the substance called biuret with cupric ions in alkaline
solution to give it violet color.
Q.5: write down functions of proteins?
a. The maintenance of water distribution between cells and tissues,

interstitial compartments and vascular system of body.
b. Participation as buffer.
c. Involved in hemostasis and coagulation of body.
d. Transportation of metabolic substances.
e. Proteins are biocatalysts (enzymes).
f. They are part of defense system (antibodies).
g. Proteins are also hormones.
Q.6: what are major plasma proteins?
Most of the plasma proteins are synthesized in liver, Albumin,α-

globulin,β globulin and some γ globulins are secreted by hepatocytes into
circulation.
Q.7: What is the normal range of albumin/globulin ration in blood?
1.2 to 1.5
Q.8: What is most commonly used method for determination of serum

proteins?
Ans. Biuret method.
Q.9: What are bence___Jones Proteins?
Ans. These are abnormal proteins which occur in blood and urine of individual
suffering from a disease called “multiple myeloma” (a plasma cell tumor).
Q-8 How can we detect bene ___Jones proteins?

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Ans. Such proteins are identified easily in urine by a simple “heat test”. On heating
the urine upto 50 to 60o C, Bence-Jones proteins are precipitated, but when
heated further they dissolve again. Reserve occurs on cooling.
Q-9 What are the functions of plasma proteins?
Ans. i. Control of body water distribution by plasma colloid osmotic pressure.
ii. glycogenic amino acids provide energy.
iii. Transport of cortisol, thyroxin, fat-soluble vitamins and metals.
iv. Blood coagulation, e.g. fibrinogen.
v. Buffers.
vi. The immunoglobulin’s are antibodies which provide a defense against
infection.
vii. Maintenance of blood viscosity.
Q-10 What are the causes of hypo and hyperproteinemia?
Ans. See clinical importance.
Q-11 Name the major plasma protein which is only synthesized in liver and
Name copper containing protein of plasma?
Ans. a. Albumin.
b. Ceruloplasmin.
Q-12 Which protein principally acts as a storage for iron, name the plasma
protein which binds to extra corpuscular hemoglobin?
Ans. a. Ferritin is principally a storage protein for iron.
b. Haptoglobin binds extra corpuscular hemoglobin.
Q-13 Which immunoglobulin is raised first of all in plasma against infection
and which is hormone directly involved in absorption of dietary
calcium?
Ans. a. 1gM.
b. Calcitriol.
Q-14 Name two agent s that can denature the proteins in the laboratory.
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Ans. Different agent that can denature protein the laboratory are heat, mechanical
mixing, acid or base, detergent Ions of heavy metals.
Q-15 what is the normal range of total protein in serum
Ans. Total: 6.0 to 8.0 gm/dl
Albumin: 3.5 to 5.5gm/dl
Globulin: 2.0 to 3.6gm/dl
13
Results and Calculations

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QUANTITATIVE ESTIMATION OF TOTAL SERUM ALBUMIN

Experiment 1.2
Reagents:
 Test Reagent: Citrate buffer
Bromocresol green solution
 Albumin standard (4gm/dl)
Procedure:
 Take three test tubes and label them as standard, blank and test.
Ser Reagent Test Standard Blank
No
1. Buffer bromocresol 4ml 4ml 4ml
Green Solution
2. Serum 0.02ml - -
3. Standard solution - 0.02ml -
4. H2O - - 0.02ml
 Mix the contents of test tubes, by shaking and
Incubate them for 5 minutes at 37o C. Incubate at 20-25o C.
 Measure the absorbance of sample and standard against reagent blank
within 30 minutes at 456nm.
Calculation
Serum albumin (gm/dl)= Atest x concentration of albumin standard
Atest
Normal value 3.8-5.1 g/d
15

Q-1: write down the causes of hypoalbuminemia?
i. Impaired albumin synthesized in liver

ii. Liver disease
iii. Malnutrition
iv. Malabsorption
v. kidney disease
vi. Intestinal disease
vii. Shock
viii.Burns
Q-2: write down causes of hyperalbuminemia?
i. Dehydration
Q-3: which is most abundant serum protein?
Albumin
Q-4: write down functions of albumin?
1. Maintain water balance in serum and plasma.

2. Transport a wide variety of ligands e.g. Fatty acids, calcium, bilirubin and
hormones such as thyroxin.
3. It also provides endogenous source of amino acids.

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ESTIMATION OF SERUM BILIRUBIN
Experiment 1.3
Description:
a. Unconjugated bilirubin (UCB)

1. Senescent RBCs are phagocytosed by splenic macrophages.
2. UCB is the end product of heme degradation
3. UCB is lipid soluble( indirect bilirubin)
b. UCB combines with albumin in the blood.
1. UCB is taken up by hepatocytes.
2. UCB is conjugated to glucuronic acid to produce conjugated bilirubin
which is water soluble.
c. Conjugated bilirubin is secreted into intrahepatic bile ducts.
1. CB is stored in gall bladder.
2. CB enters duodenum via CBD.
d. Intestinal bacteria convert CB to UBG.
1. UBG is oxidized to urobilin.
2. Urobilin produces brown colour of stool.
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Principle
Bilirubin reacts with diazotized sulphanilic acid to form a colored compound. It is

red in neutral solution and blue in alkaline solution. The water soluble conjugated
bilirubin reacts directly. The absorbance of this blue colored compound can be
determined at 578 nm wavelength. Direct bilirubin is measured as red color
compound at 546 nm wavelength.
Reagents
RI – Sulphanilic acid R3-Caffeine

Hydrochloric Acid Sodium Benzoate
R2- Sodium Nitrite R4-Tartarate
Sodium
Sample: Serum
Procedure: For normal = 578nm

Optical path=1cm
Temperature= 20-25oC.
Sample blank Sample

R1 100ul 100ul
R2 - 25ul
R3 500ul 500ul
R4 500ul 500ul
Sample 100ul 100ul
Read absorbance of sample against sample blank at 578nm.
Total Bilirubin = A x 10.8
For Direct Bilirubin:

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Sample blank Sample

R1 100ul 100ul
R2 - 25ul
NaCl(9g/l) 1000ul 1000ul
Sample 100ul 100ul
Read absorbance at 546nm.
Direct Bilirubin = A x 14.4
Indirect= Total – Direct
Note:
Unconjugated or indirect reacting bilirubin is obtained by the difference of
total and conjugated.
Indirect bilirubin= Total bilirubin – Direct bilirubin
Normal Value of Total Bilirubin in blood
Upto 0.1-1mg/dl

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Q-1 Write down causes of unconjugated hyperbilirubinemia?
1. Hemolytic
2. Physiologic
3. Gilbert syndrome
Q-2 Write down causes of unconjugated hyperbilirubinemia?
1. Biliary tract obstruction.

2. Gall stones.
3. Cholangiocarcinoma.
4. Pancreatic liver Ca
Q-3 Write down causes of mixed hyperbilirubinemia?
1. Hepatitic
2. Cirrhosis
Q-4 What is meant by conjugated or direct Bilirubin?

Ans. When bilirubin is conjugated with glucuronic acid in liver it is called
conjugated bilirubin. It is water soluble and reacts rapidly with reagent to
give direct reaction.
Q-5 What is meant by unconjugated or direct Bilirubin?
It is occurs in blood. It is in soluble in water &U circulates in blood combined with
albumin. It is called indirect bilirubin because it is measures by subtracting direct
bilirubin from total bilirubin.
Q-6Define urobilirubin?
Urobilirubin (in increased amount) which can be found in the urine of those with
conditions causing abnormal haemolysis.
Q-7What are bile acids & bile salts?
Cholic acid, chenodeoxycholic acid are bile acids and glycocholic
acids,Taurochenodeoxycholic acid are bile salts. They are used for the
emulsification of lipid.
20

21
ESTIMATION OF SERUM ASPARTATE TRANSAMINASE (AST) BY KIT

METHOD
Experiment 1.4
Principle
α – ketoglutarate + aspartate GOT glutamate + Oxaloacetate
Oxaloacetate + NADH + H+ MDH malate + NAD
Reagents:
Working Reagent:
 Buffer/Enzyme reagent
i. Tris buffer
ii. Aspadrate
iii. LDH
iv. MDH
v. Sodium azide
 Substrate
i. Oxoglutrate
ii. NADH
iii. Sodium azide
Assay:
Wavelength: 340nm
Optical Path: 1cm
Temperature: 25oC, 30oC or 37oC
Measurement : against air (decreasing absorbance)
Procedure:
1. Take 0.2 ml of sample and 1 ml of working reagent in a cuvette and mix.
2. Read the initial absorbance and at same time start stop watch.
3. read the absorbance again exactly after 1,2 and 3 minutes.
Calculation:
22
AA x Factor (1151)
min
= AA x 1151
3
Note: the constant factors and values used in calculations as specific of
Human Kit (Germany). These factors can vary if other kits are used but the main
principle will remain the same.
Normal value:
M ales: Upto 35U/L
Females: Upto 31 U/L
Q-1 What is pattern of elevation of aspartate transaminase in myocardial

infraction?
Ans. a. It appears and starts to rsie at 6-8 hours.
b. Time aftger infraction fsor peak rise (24-48 hours).
c. Duration of rise (-4-06 days).
Q-2 Write down the clinical significance of AST?
Ans. Aspartate transaminase (AST) also called serum glutamate oxaloacetate
transaminase (SGOT) or aspartate aminotransferase (ASAT/AAT) is similar
to alanine transaminase (ALT) in that it is another enzymes associated with liver
parenchymal cells. It facilitates the conversion of aspartate and alpha ketoglutarate
to oxaloacetate and glutamate and vice versa. It is raised in acute liver damage. It is
also present in red blood cells, cardiac muscle, skeletal muscle, kidney and brain
tissue and may be elevated due to damage to damage to those sources as well.
AST was defined as a biochemical marker for the diagnosis of acute myocardial
infarction. However, AST is commonly measured clinically as a part of diagnostic
liver function tests.

23
ESTIMATION OF SERUM ALANINE TRANSAMINASE (ALT) BY KIT

METHOD
Experiment 1.5
24
Principle:
The enzyme glutamate pyruvate transaminase (GPT) catalyzes an exchange of an
amino group of alanine for a alpha keto group of alpha ketoglutarate. The end
products formed in this reaction are pyruvate and glutamate.
Reaction:
α – ketoglutarate + alanine ALT glutamate + Pyruvate

Pyruvate + NADH + H+ LDH Lactate + NAD+
Reagents:
Working Reagent:
 Buffer/Enzyme reagent
i. Tris buffer
ii. Alanine
iii. LDH
iv. Sodium azide
 Substrate
i. α – ketoglutarate
ii. NADH
iii. Sodium azide
Assay:
Wavelength: 340nm
Optical Path: 1cm
Measurement: against air or distilled water (decreasing absorbance)
Procedure:
2. Read the absorbance after 1 minute and at same time start stop watch.
25
3. Read the absorbance again exactly after 1,2 and 3 minutes.

Calculation:
AA x Factor (1746)
min
= AA x 1746
3
Note: the constant factors and values used in calculations as specific of Human Kit
(Germany). These factors can vary if other kits are used but the main principle will
remain the same.
Q-1 Write down the normal value of ALT?

Males: 10-60 U/L
Females: 8-40 U U/L
Q-2 Write down the clinical significance of ALT?
Alanine transaminase or ALT is a transaminase enzyme. It is also call serum

glutamate pyruvate transaminase (SGPT) or alanine aminotransferase (ALT).
ALT is found in serum and in various body tissues, but is most commonly
associated with the liver (hepatocytes).
When a cell is damage, it leaks this enzymes into the bold, where it is measured.
ALT rise dramatically in acute liver damage, such as viral hepatitis or paracetamol
(acetaminophen) overdose.It is useful diagnosing acute liver disorders and
monitoring the prognosis of liver disease. It is raised in viral hepatitis, cirrhosis,
drug inducted liver damage (opiates, antibiotics; NSAIDs).

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ESTIMATION OF SERUM ALKALINE PHOSPHATE BY KIT METHOD

Experiment 1.6
ALP catalyzes the hydrolysis of 4 nitorphenyl phosphate with the formation of
freed 4-nitrogphenol and inorganic phosphate. Alkaline buffer act as a phosphate
group acceptor. The reaction is monitored kinetically at 405 nm by the rate of
formation of 4 nitorphenol (yellow), proportional to the activity of ALP in the
sampole.
Reaction:
ρ-Nitrophenylphosphate + H2O ALP Phosphate + (ρ-Nitrophenol)
Pyruvate + NADH + H+ LDH Lactate + NAD+
Reagents:
Working Reagent:
• Buffer/Enzyme reagent
Diethanolamine buffer (pH) 10-35+0.2) 1.25 mol/l
Magnesium Chloride 0.625mmol/l
• Substrate
ρ-Nitrophenyl phosphate 55 mmol

Specimen:
Serum,, Hepadrinized or Plasma or EDTA plasma. Avoid Hemolysis.
Assay:
Wavelength: 400-420nm
Optical Path: 1cm
Measurement: against air
Procedure:
27

2. Read the absorbance after 1 minute and at same time start stop watch.
3. Read the absorbance again exactly after 1,2 and 3 minutes.
Calculation:
ALP = AA/3 x Factor (2757)
=AA/3 x 2757
Note: the constant factors and values used in calculations as specific of Human Kit
(Germany). These factors can vary if other kits are used but the main principle will
remain the same.
Normal value: 20-140 IU/L
Q-1 What are the indications of liver function test?
Ans. liver function tests are commonly used.
 To detect hepatocellular damage.

 To assess the impairment of liver function.
 To distinguish between various types of jaundice.
Q-2 Who is ar risk for liver disease and/or damage?
Ans. Some of those who are at risk of liver disease include the following:
 People who have been exposed to hepatitis viruses.

 Heavy drinkers.
 People who take medication that can be toxic to the liver or who are
exposed to other liver toxin.
 People with an inherited disorder affecting the liver such as Wilson
disease or hemochromatosis
Q-3 What is the normal range of AST?

28
Ans. Generally the levels of AST ranges from 5 to 43 units per liter of serum and
of ALT from 5 to 30 or 40 units per liter of serum. The upper level tends to be
lower in females.
Q-4 What does the test result of ALP mean?
Ans. High ALP usually means that either the liver has been damaged or a
condition causing increased bone cell activity is present. If other liver tests such
as bilirubin, aspartate aminotransferase (AST), or alanine arninotransfere (ALT) are
also high, usually the ALP is coming from the liver. If calcium and phosphorus
measurements are abnormal usually the ALP is coming from bone. If a GGT or 5’-
nuclcotidase is also increased, then the high ALP is likely due to liver disease. If
either of these two tests is normal then the high ALP is most likely due to a bone
condition.
Q-5 Is any test preparation needed to ensure the quality of the sample?
Ans. Fasting is preferred but not required for this test. Eating a meal can increase
the ALP level slightly for a few hours in some people. It is usually better to do the
test after fasting overnight. In this case, only water is permitted.
Q-6 Which things affect the values of ALP?
Ans. pregnancy can increase ALP levels. Temporary elevations are also seen with
healing fractures. Children and adolescents normally have higher ALP levels than
adults because their bones are growing and ALP is often very high during a growth
spurt which occurs at different ages in boys and girls. Some drugs may affect ALP
levels. For example, oral contraceptives may decrease levels while anti-epileptics
may increase levels.
Q-7 Write down the clinical significance of Alkaline phosphatase?
 Ans. It is increased in Paget’s disease of bone, hepatitis,

osteomalacia, obstructive liver disease and hepatotoxicity by drugs.
 Physiological increase is seen in exercise, growth and pregnancy
 This enzyme is particularly high in bones, placenta, intestines and kidney,
liver, bile duct.
29
Section-2
Lipid Profile
Ser Practical
No
2.1 Estimation of Cholesterol
2.2 Estimation of Serum Triglycerides
2.3 Estimation of Serum HDL Cholesterol
30
ESTIMATION OF SERUM CHOLESTEROL
Experiment 2.1
Introduction:
1. Cholesterol maintains lipid bilayer of cell membrane and structure of cell

membrane.
2. Cholesterol helps in transfer of molecules and ions across cell membrane.
3. Cholesterol is precursor of steroid hormones secreted by adrenal cortex and
ovary.
e.g. estrogen and progesterone.
It is precursor of catecholamine secreted by adrenal medulla.
It is precursor of vitamin D.
It is precursor of bile acids in bile salts.
4. It causes emulsification of fats and fat soluble vitamins. E.g. A D E K.
Principle:
Cholesterol esters of the sample are hydrolyzed by cholesterol ester hydrolase

(chEH),4 cholesten – 3 one + H2O2 are formed from the release free cholesterol by
cholesterol oxidase. Red quinoneimine complex is formed by reaction of H 2O2 and
4 aminoantipyline in presence of phenol.
chEH
Cholesterol ester + H2O Cholesterol + fatty acid.
Cholesterol oxidase
Cholesterol+ O2 4 cholesterol 3-one + H2O2
2H2O2 + phenol + Quinoenimine (Red) + 4 H2O
Reagents:
R1: Cholesterol standard 200 mg/dl
R2: Phosphate buffer pH – 6.9
Phenol
Sodium cholate
31
4 Aminoantipyline
Cholesterol ester
Cholesterol oxidase
Sample: Serum
Procedure:
Wave lenth 505 nm Temperature 37oC optical path 1cm

No
1. Reagent 1ml 1ml 1ml
2. Standard - 10ul -
3. Sample 10ul - -
4. Distilled water - - 10ul
Calculation:
Absorbance of test
Serum Cholesterol in mg/dl=----------------------- - - x Concentration of Standard
Absorbance of standard
AT
= - - - - - - - - - - - x 200 mg
As
Normal value of serum cholesterol
140-250 mg/100ml.
32

Q-1 What are the symptoms of high cholesterol?
Ans. high cholesterol does not cause symptoms by itself. Instead, it is a risk factor
for the development of atherosclerosis or narrowing of arteries in the body
that can lead to heart attack, stroke, or peripheral artery disease.
Q-2 What can a patient do to prevent high cholesterol?
Ans. Controlling high cholesterol is a lifelong commitment. Important first step

include eating a health diet low in saturated fats, routine exercise, weight
loss, and avoiding or quitting smoking. If these actions fail to lower
cholesterol levels (below 200 mg/dl), most physicians will recommend a
modification to lower cholesterol
Q-3 When would a doctor prescribe a medication to lower cholesterol?
Ans. the main goal of ad treatment program is to lower total cholesterol levels,
LDL (‘bad’) cholesterol levels and triglyceride levels. Treatment may cause a
slight rise in HDL or good cholesterol in the blood. There are two main ways
to control cholesterol;
a. Lifestyle changes.
b. Medication.
Medications may be prescribed by a health care practitioner if attempts at
lifestyle changes fail to make a difference in cholesterol levels (usual goal is
to be under 200 mg/dl). A variety of medications options are available and
the decision as to which medication to use depends upon the individual
situation and other medical conditions that might be present. Usually the
health care practitioner and patient will discuss options and decide together
upon the treatment options. There are many treatment options such statins,
niacin and fibrates.
Q-4 How much cholesterol is synthesized per day in human body?
Ans. For a person of about 68 kg(150 pounds), typical total body cholesterol
synthesis is about 1 g (1,00 mg) per day, and total body content is about 35 g.
Typical daily additional dietary in take in the United States is 200-300 mg.
the body compensates for cholesterol intake by reducing the amount
synthesized.
Q-5 how is cholesterol recycled?
Ans. Cholesterol is recycled. It is excreted by the liver via the bile into the
digestive tract. Typical about 50% of the excreted cholesterol is reabsorbed
by the small bowel back into the bloodstream. Phystosterols can compete
33
with cholesterol reabsorption in the intestinal tract, thus reducing cholesterol

reabsorption.
Q-6 What is the molecular formula of cholesterol?
Ans. C27H45OH.
Q-7 What is the importance of cholesterol in the body?
Ans. It acts as a precursor for the following:
a. Biosynthesis of bile acids
b. Biosynthesis of vitamin D3 and 7 dihydrocholesterol.
c. Biosynthesis of sex hormones
It also transport fats to the liver in the form of cholesterol esters.
Q-8 in which is cholesterol present in the blood?
Ans. Free cholesterol (25-35% of the total cholesterol).
Esterified cholesterol (65-75% of the total cholesterol)
Q-9 What is name of cholesterol nucleus ?
Ans. Cyclopentano perhydro phenanthrene nucleus.
Q-10 write down the causes of hypercholesterolemia?
 Ans. Type 2 familial hypercholesterolemia

 Obstructive Jaundice.
 Hypothyroidism
 Nephritic syndrome
 Uncontrolled diabetes
 Obesity
 Alcohol
 Drugs (thiazide diuretics, cyclosporine, glucocorticoids,
glucocorticoids, beta blockers, retinoic acid)
Q-11 Write down the causes of hypercholesterolemia?
 Hypothyroidism
 Liver disease except obstructive and cholesterol disese.
 Malabsorption
 Stains
 Adrenal insufficiency
 Malnutrition
 Abetalipoproteinemia
34
ESTIMATION OF SERUM TRIGLYCERIDES
Experiment 2.2
35
Principle:
Triglycerides are determined by enzymatic hydrolysis with lipases. Indicator is

quinoneimine formed from H2O2, 4amino antipyrine (or 4-aminophenzone) and 4-
cholrophenol under the catalytic influence of peroxidase.
Contents:-
Reagent
 PIPES buffer (pH 7.5)

 4-chlorophenol
 4-amino phenazone sodium.
 Mg ions
 Sodium Azide.
 ATP, lipases, peroxide, glycerol kinase, glycerol-3 phosphate
oxidase
Triglyceride Standard (200 mg/dl)
Reaction:
Lipases
1. TAGs Glycerol + fatty acids
Glycerol kinase
2. Glycerol + ATP Glycerol-3 Phosphate + ADP
3. Glycerol-3phosphate + O2 Glycerol phosphate oxidase(GPO)

Dihydroxy acetone
phosphate + H2O2.
peroxidase
4. H2O2. + 4-amino antipyrine + 4-chlorophenol
quinonimine+H2O+HCl
Assay:
Wavelength: 456nm
Optical Path: 1cm
Measurement:
against reagent blank.
36
Procedure:
1. Take 3 test tubes and label them blank, standard and sample.
2. Add 1 ml reagent in each test tube, add 0.01 ml sample solution in sample
test tube and 0.01 ml standard solution in standard test tube.
No
2. Serum sample 0.01 ml - -
3. Standard salutation - 0.01 ml -
4. H2O - - 0.01 ml
 Mix the contents for 10 minutes at 20-25 C or for 5 minutes at 37oC.
o
 Note the absorbance of standard and test / or unknown against reagent

blank by placing the cuvettes in spectrophotometer at 546 nm or 500nm.
Calculation
Conc. of TA /100ml = AA Sample x 200mg/dl
AA standard
Q-1 What down the normal value of serum triglycerides?
Normal value: upto 150mg/dl,
F = 40-140 mg/dl
M = 50-165 mg/dl

37
ESTIMATION OF SERUM HDL CHOLESTROL

Experiment 2.3
Introduction:
38
HDL cholesterol or good cholesterol transfers cholesterol from periphery to liver.

Principle
Chylomicrons, LDL’VLDL are predicated by addition of phosphotungstic acid and

magnesium chloride. After centrifuge’ the supernatant fluid contains HDL fraction
Which is assayed for HDL cholesterol.
Reagent:-
Precipitants:- Phosphotungstic acid’ Magnesium Chloride.
Standard 175 mg / dl
Procedure:-
Precipitation:-
 Take test tube add 0.5ml serum and 2ml precipitants.

 Mix well incubate for 10 minutes at room temperature.
 Centrifuge for 10 minutes at 4000 rpm.
 After centrifugation separate the clear supernatants from the
precipitant within one hour and determine the cholesterol
concentration.
Cholesterol Blank Standard Sample

Reagents 1ml 1ml 1ml
Distilled H2O 0.1ml - -
STD - 0.1 ml -
HDL Supernatant - - 0.1 ml
 Mix the incubate for 5 minutes at 37 C or 10 minutes at 20-25oC.
o
 Measure the absorbance of sample and reagent blank with in 60

minutes at 500/546nm.
Calculation:
C= AA Sample x 175 mg/dl
AA standard
LDL (mg/dl)
TC – TG - HDL
39
5
AA standard
Q-1 What down the normal value of serum HDL cholesterol?
Clinical value : In males > 55mg/dl

In female > 65mg/dl
Standard Risk level: In male > 35-55 mg/dl
In female > 45-65 mg/dl
Risk indicator: Male < 35mg/dl
Female <45mg/dl
For LDL:
Loss than 150mg/dl is favorable prognosis
Standard risk level : 190mg/dl is considered as elevated
Q-2 Name the lipoproteins and their functions?
 Chylomicron:
It transport TGs in blood.
 VLDL:
It transport TGs from liver to blood.
 LDL:
It mainly consists of cholesterol and transport cholesterol in blood.
 HDL:
It consists of proteins and transfer protein to liver.
Q-3 What is atherosclerosis?
Endothelial cell damage of muscular and elastic arteries.

40
Renal Function Tests
Ser Practical
No
3.1 Estimation of Blood urea
3.2 Estimation of Blood Uric Acid
3.3 Estimation of Serum Creatinine
3.4 Determination of Creatinine
Clearance
3.5 Estimation of BUN
41
Renal Function Overview:

1. Excretes harmful waste products
 E.g. Urea, Creatinine , Uric acid.
2. Maintain acid-base homeostasis.
 E.g. controls t5he synthesis and excretion of bicarbonate and hydrogen
ions.
3. Reabsorbs essential substances
 E.g. sodium, glucose, amimoacids.
4. Regulates water and sodium metabolism
 E.g. controls water by concentrating and diluting urine.
 Controls sodium reabsorption in this proximal and distal collecting
tubules.
5. Maintain vascular ions.
 E.g. Angiotensin 2 vasocontricts peripheral resistance arterides and
efferent arterioles.
 Renal derived prostaglandins vasodilators afferent arterioles.
6. Produce erythropoietin.
7. Maintain calcium homeostasis.
ESTIMATION OF BLOOD UREA

42
EXPERIMENT #3.1
Principle
This method is based on the following reaction:
Urea + Water urease NH3 = CO2
Salicylate and hypochlorite in the reagent react with ammonium ions to form green
complex
Reagents:
Reagent 1 Urease
Phosphate buffer
Na+ salicylate
Na+ nitroprusside
EDTA
Reagent 2 Na+ hypochlorite
NaOH
Phosphate buffer
Urea Standard: 80 mg
Assay
Wavelength: 578nm
Optical Path: 1cm
Measurement : against reagent blank
Procedure:
 Take e test tubes and label them as standard, test and blank.
Ser Reagent Test Standard Sample
No
1. Working reagent 1 1ml 1ml 1ml
2. Serum 0.01ml - -
4. H2O - - 0.01ml
43
 Shake and incubate for at least 3 minutes at 37oC or 5 minutes at 20-25oC.

 Now add 0.2 ml of reagent 2 in all the above test tube.
 Shake and incubate for at least 5 minutes at 37oC or 10 minutes at 20-25oC.
 Measure the absorbance of standard and test against reagent blank within two
hours at 578 nm.
Calculations:
Urea Concentration = Absorbance of sample x conc. of stanadard
Normal level of blood urea = 10-50 mg/dl
Q-1 What is uremia?

Ans. The term uremia (meaning urea in the blood) is often used as a synonym for
renal failure (both acute and chronic). Azotemia is used in a similar context
and refers to an increase in the blood concentration of nitrogenous
compounds.
Q-2 What is normal level of urea in blood?
Ans. It is 10 to 50 mg/dl.
Q-3 Why the level urea is decreased in pregnancy?
Ans. The blood urea level falls down in pregnancy due to haemodilution and
active utilization of amino acids by the fetus particularly in the third trimester
of pregnancy.
Q-4 What is azotemia?
Ans. Increase of serum blood urea nitrogen (BUN ).
Q-5 write down the reasons of increased BUN?
 Acute glomerulonephritis.
 Acute or chronic renal failure.
 Increase of protein intake.
 Increase of tissue catabolism.
Q-6 write down normal value of BUN?
Ans. 7-18 mg/dl.
44
Q-7 Write down the normal blood urea level?

Ans. 18-45 mg/dl.
Q-8 Write down the causes of increased urea levels?
Ans. Slight increase in urea may occur when there are non renal causes;
 Dehydration (hemo concentration)
 Diuretic therapy (hemo concentration)
 Gastrointestinal blood loss
 Any condition associated with increase protein breakdown such as
pneumonia, malaria, meningitis, typhoid, operations.
 Stress
 Acute myocardial infraction
 Congestive heart failure as result of poor renal perfusion
Q-9 Write down the causes of increased urea levels?
Ans. Low urea levels may be found in:
 Pregnancy (due to increase plasma volume)
 Malnutrition and AIDs
 Severe liver disease
 Water overload or over dehydration
 Syndrome of inappropriate antidiuretic secretion (SIDH)
 Acromegaly

45
ESTIMATION URIC ACID BY ENZYMATIC COLORIMETRIC TEST

(PAP METHOD)
46
Experiment 3.2
Principle:
Uric acid reacts with oxygen and water in the presence of uricase enzyme. Formed
H2O2 reacts under the catalysis of peroxidase with 3,5-dichloro2 hydroxy-benzene-
sulfonic acid (DCHBS) and 4-amino-phenazone (PAP) to give a red violet
quinonimine as indicator.
Reaction
Uricase
Uric acid + O2 + H2O allantion + CO2 + H2O2
Peroxidase
2H2O2 + DCHBS + PAP quinoneimine + HCI + 4 HO
Reagents
Phosphate buffer
4- aminophenazone (PAP)
3,5-dichloro2- hydroxybenzene-sulfonic acid (DCHBS)
Uricase
Peroxidase
Procedure
 Take 3 test tube and label them as standard, test and blank.
Ser Reagent Test Standard Sample
No
1. Working reagent 1ml 1ml 1ml
2. Serum 0.02ml - -
 Shake and incubate for 15 minutes at 20-25 C or for 37 C.
o o
 Measure the absorbance of sample and standard against reagent blank at 546
nm.
Calculations:
Concentration = Absorbance of sample x 8 (mg/dl)
Concentration = Absorbance of sample x 476 (umol/dl)
Reference values:
47
Male 3.4-7 mg/dl or 200-420 umol/l

Female 2.4-5.7 mg/dl or 140-340 umol/l
In Urine 250-750 mg/24 hours or 1.5-4.5 mmol/24 hour

Q-1 What is the normal serum uric acid level?
Ans. See above
Q-2 Where does the synthesis or uric acid occur?
Ans. Inside cells and in intestine (dietary)
Q-3 Which substance yields uric acid as a metabolic end product?
Ans. Uric acid is the end product of purine metabolism.
Q-4 Which disease is caused by increase concentration at uric acid?
Ans. Gout
Q-5 What is Gout?
Ans. Gout is an abnormality of uric acid metabolism that results in the deposition
of sodium urate crystals in joints, soft tissues and urinary tract.
Q-6 What is Xanthinuria?
Ans. An autosomal recessive deficiency of enzyme xanthine oxidase, blocking the
oxidation of xanthine to uric acid and xanthine appears in urine, such conditions is
called xanthinuria.
Q-7 Write down the clinical significance of determination of serum uric
acid?
Ans. Determination of serum uric acid levels are helpful in the diagnosis of
several, pathological conditions. An increase of serum uric acid is seen in case of
gout, and increase metabolism of nucleoproteins of the body e.g leukemia,
polycythemia) . Uric acid levels are elevated in conditions associated with
decreased renal function but the test is rarely used for this purpose because of the
48
great effect of extra renal factors on serum levels. The normal uric acid level of
serum is not significantly affected by the ingestion of a diet rich ion purines unless
there is renal insufficiency.
Indications for ordering this test include suspected gout and suspect uric acid
nephropathy.
Q-8 Write down the conditions in which uric acid levels are increased?
 Burns,
 Crush injuries
 Very severe hemolytic anemia
 Plasma cell myeloma and myeloproliferative disorders may also leads to the
increased uric acid.
 Administration of cortisone or ACTH increases urate excretion. Increased
excretion of uric acid occurs due to accumulation of copper in the kidneys
and lease to damage to proximal renal tables in Wilson’s disease.

49
50
ESTIMATION OF SERUM CREATININE

Experiment 3.3
Principle:
The experiment is based on the reaction of creatinine with sodium picrate and is
called Jaffe’s reaction. Creatinine reacts with sodium picrate in alkaline medium,
forming a colored complex (red). The color formed is directly proportional to the
concentration of creatinine and is measured spectrophotometrically at 500 nm (490-
510 nm).
Reagents:
Picric acid
10% NaOH
Serum sample
Standard solution
Procedure
 Take 3 test tube and label them as blank, standard and test.
No
1. Picric acid 2ml 2ml 2ml
2. 10% NaOH 2ml 2ml 2ml
3. Serum sample 0.25ml - -
5. H2O - - 0.25ml
 Allow all the test tubes to stand for 10 minutes and then take the absorbance
against blank at 500 nm.
Calculations:
Concentration = Absorbance of sample x 2 (concentration of standard)
Normal values:
Male 0.6-1.1 mg/dl or 53-97 umol/l
Female 0.5-0.9 mg/dl or 44-80 umol//l
51
Plasma Creatinine
Decreased Increased
Pregnancy G.I bleeding
Starvation High protein diet
Wasting doses Strenuous exercise
Corticosteroid Dehydration
Low Proteins intake Renal failure
Dialysis Urinary stasis
Diuretics Shock
Surgery
Diabetic nephropathy
Anti-hypertensive drugs
52

Q-1 Which enzyme is responsible to decompose urea to ammonia?
Ans. Urease.
Q-2 What are the sources of enzymes urease?
Ans. Seed of watermelon, Jack beans, Soybeans.
Q-3 What is creatinine coefficient?
Ans. The number of mg of creatinine excreted daily per kg of body weigh is

known as the creatinine coefficient.
Q-4. What are the sources of creatine?
Ans. Muscles, Brain and Blood.
Q-5. In which forms creatine remains present?
Ans. In free or phosphorylated form.
Q-6 what are the precursors of creatine?
Ans. Glycine, Arginine, Methionine.
Q-7 What is the normal excretion of create nine daily?
Ans. 1.0-2.0gm.
Q-8 Name the condition in which urinary excretion of creatinine is

increased?
Ans. Early stages of muscles dystrophy when muscle destruction occurs rapidly.
In any wasting disease involving increased tissues catabolism.
Hyperthyroidism.
Q-9 What do you understand by the words “Hematuria and

‘Hemoglobinuria’?
53
Ans. In hematuria one finds presence of hemoglobin and unruptured RBCs in the
urine. It happens when there is some lesion in the kidney or urinary track.
In hemoglobinuria one finds the presence of hemoglobin only in the urine.
It occurs from haemolysis i,e by the rupturing of the stroma of the RBSs as in
the diseases like malaria, typhoid fever, yellow fever, hemolytic jaundice or
as a result of transfusion with incompatible blood.
Q-10. In which disease excretion of calcium in urine is increased?
Ans. Osteomalacia.
Q-11 In which disease excretion of calcium in urine gets decreased?
Ans. Rickets.
Q-12 In which disease will you find increased amount of pheny pyruvic acid in
urine?
Ans. In the inherited condition known as phenylketonuria. In this disorder

phenylalanine is
Not oxidized to tyrosine due to the absence of an enzyme phenylalanine
hydroxylase.
Q-13 Which vegetables contains high amount of oxalic acid, oxalates or its
precursors?
Ans. Asparagus, Cabbage, Spinach, Tomatoes.
Q-14 Which fruits are rich in oxalic acid, oxalates or its precursors?
Ans. Apple & Grapes.
Q-15 What is percentage of glucose in the urine of patients suffering from
diabetes mellitus?
Ans. 3 to 5% but may reach up to 10% or even more, depending upon the severity.
Q-16 A 66 year old male was sent his GP to casualty because of tight chest
pain that had occurred three days back. The pain had largely resolved
after 6 hours, but he was left feeling weak and breathless, causing him
54
eventually to seek medical attention. His plasma CK was 235 U/L (<250)
and Troponin T was 0.13 igIL (<0.1). What is your diagnosis?
Ans. The plasma CK activity has returned to normal because of the time delay
since myocardial infarction. In normalizes in about 3-5 days. The plasma
troponin I concentration still remains elevated because it remains there for 10
– 14 days. Thus it is useful cardiac marker in both early hours and few days
later.
Q-17 What is creatinine coefficient?
Ans. it is urinary cratin ine expressed in mg/kg body weight.
Q-18 What is normal level of creatinine clearance in an adult male aged less
than 40 years?
Ans. 90-140 ml/min/1.73m2.
Q-19 How many isoenzymes of cratine kinase are found?
Ans. Creatine kinase occurs as three isoenzymes CKMB, CKMM & CKBB.
Q-20 Write down causes of hematuria and enlist the drugs that are given?
Ans. Upper urinary tract, Kidney ureter, Renal stones.
Drugs for the treatment of hematuria are Anticoagulants and
cyclophosphamide..

55
Section-4
Cardiac Markers
Ser Practical
56
No
4.1 Estimation of Serum creatine kinase
4.2 Estimation of Serum LDH
Trop T
Trop I
Risk Factors For Cardiac Diseases:

1. Age
Men >45 years
Women >55 years
2. Family history of premature coronary artery disease or stroke.
3. Lipid abnormalities.
LDL>160mg/dl
HDL<40mg/dl
4. Smoking, tobacco, hypertension, D.M, obesity, physical inactivity, excess
salt, excess salt and alcohol intake.
Estimation of Serum creatine kinase

Experiment 4.1
Reaction Principle
57
ADP = creatine phosphate CK creatinine + ATP
ATP + D-glucose Hexokinase ADP + D-glucose-6 PO4
Glucose-6 PO4 + NADP Glucose 6 –PO54 dehydrogenase 6-phosphogluconolacone
Reagents:
Buffer Imidazole buffer
Glucose
Mg-acetat
EDTA
Sodium azide
Enzyme ADP
AMP
Diadenosine pentaphosphate
NADP
Creastine phosphate
Hexokinase
Glucose 6-PO4 dehydrogen
N-acetylcysteine
Specimen:
Serum: Heparinized plasma or EDTA plasma
Assay
Wavelength: 340nm
Optical Path: 1cm
58

Procedure:
1. Take 0.1 ml of serum in test tube and 1 ml of working reagent, mix them.
2. Read the absorbance after 3 min at 25oC, 2 min at 30oC and 1 min at 37oC.
3. Take a least 3 values of absorbance and multiply it by factor 4127.
Calculation
A x 4127
Note; the constant factors and values used in calculations are specific of human
kit (Germany). These factors can vary if other kits are used but the main principle
will remain the same.
Q-1 Write down clinical significance of CPK?
The major clinical usefulness of the serum CPK measurement is in the
assessment of disease of the skeletal and cardiac muscles.
Q-2 Write down causes of increase CPK level?
 Cardiac disease
 Myocardial infarction
 Skeletal muscle damage (muscular dystrophy)
 Brisk walking and running
 Intramuscular injections
Q-3 Write down isoenzymes and their sources?
MM - Skeletal and cardiac muscle
MB- Cardiac nuscle
BB- brain onle present CSF

59
ESTIMATION OF SERUM LACTATE DEHYDROGENATE (LDH)
Experiment 4.2
Principle
60
LDH catalyzes the reduction of pyruvate by NADH. The rate of of decrease in

NADH is proportional to concentration of LDH present in sample.
Reaction: Pyruvic acid + NADH + H+ LDH Lactate + NAD+
Contents:
Working reagent: Reagent (TRIS buffer, pyruvate, Sodium azide)
Substrate (NADH, Sodium azide)
Specimen:
Serum, hepranized plasma.
Assay
Wavelength: 340nm
Optical Path: 1cm
Temperature: 37oC
Measurement : against air
Procedure:
1. Take 0.1 ml of serum in a cuvette and 1 ml of working reagent, mix them.
2. Read the absorbance after 1 min at the same time start the stop watch.
3. Read the absorbance against exactly after 1,2 and 3 minutes.
Calculations:
Using the absorbance reading calculate the mean absorbance change per minute
(AA/min). Calculate the LDH activity in sample by multiplying A/min with the
factor 16030
Units / liter (37oC) = A/min x 16030
Note: The constant factors and values used in calculations are specific of Human
kit (Germany). These factors can very if other kits are used but the main principle
61
Normal values : 225 – 450 U/l

Q-1 Write down the isoenzymes and sources of LDH?
LDH has 5 isoenzymes i,e.
\LDH 1 Heart & RBCs
LHD 2 RBCs and WBCs
LDH 3 Lungs
LDH 4 Kidney, pancreas, placenta
LDH 5 liver and skeletal muscles
Q-2 Write down causes of increased LDH?
There is marked increase in

 Cirulatory failure
 Circulatory shock
 Hypoxia
 Myocardial infarction
 Megaloblastic anemia
 Acute leukemias
 Renal infarction
There is moderate increase in
 Viral hepatitis
 Pulmonary embolism
 Skeletal muscle disease
 Results and Calculations
62
Section-5
Enzymes
Ser Practical
No
5.1 Estimation of Serum Amylase
5.2 Estimation of Serum Chloride
63
ESTIMATION OF SERUM AMYLASE

Experiment 5.1
Principle:
α-amylase catalyzes the hydrolysis of 3 chloro-4-nitophephenyl maltotrioside
forming 2-chloro-4-nitrophenol and free glycosides.
64
Reactions:
amylase
CNP-G3 G-CNP + CNP-G2+G3+ G
Reagents:
 MES buffer
 CNPG3
 Calcium acetate
 Sodium chloride
 Potassium thiocyanate
 Sodium azide
Assay
Wavelength: 400-410nm
Optical Path: 1cm
Temperature: 25oC or 37oC
Measurement : against water
Procedure:
 Take a cuvette and add 1ml reagent and 0.02 ml sample.
 Mix well and incubate for 1 min at desired temperature.
 Read the absorbance and at the same time start stop watch.
 Read the absorbance again exactly after 1,2 and 3 minutes.
Calculations:
A1min + A2min + A3min x9864 U/L
= A/min x 9864 U/L
Note: The constant factors and values used in calculations are specific of Human
kit (Germany). These factors can very if other kits are used but the main principle
65
Normal values : 21-101 U/L

Q-1 Write down the conditions in which amylase increased?
Increased levels are found in a cute pancreatitis, pancreatic duct obstruction,
intraabdominal diseases, mumps and bacterial prostitis. The activity of enzyme
may fluctuate, rapidly rising during and attack and subsiding to normal shortly after
wards.
Q-2 Write down the conditions in which amylase increased?
 Chronic pancreatitis
 Hepatitis
 Cirrhosis
Q-3 Write down the reaction catalyzed by amylase?
α-amylase breaks starch at alpha 1,4 linkages releasing maltose, maltotriose, and
limit dextrin.
Q-4 Where amylase is synthesized in body?
It is synthesized in salivary glands and pancreas and secreted in saliva and small
intestine. Amylase activity is tested in serum and urine. It is mainly used in
diagnosis of diseases of pancreas and in investigations of pancreatic functions.

66
ESTIMATION OF SERUM CALCIUM

Experiment 5.2
Principle: Calcium ions reacts with o-cresolphthalcin complex one in an alkaline
medium to form purple color complex. The absorbance of this complex is
proportional to the calcium concentration in the sample.
Reagents:
67
 o-cresolphthalcin complexone
 HCi
 8-hydorxy quinoline
 Sodium azide
Standard calcium solution (8 mg/dl)
Specimen:
Assay
Wavelength: 570nm
Optical Path: 1cm
Temperature: 20-25oC or 37oC
Measurement : against reagent blank
Normal value:
8.1-104 mg/dl or 2.02-2.60 mmol/l
PROCEDURE:
Value of calcium in children are greater than in adults.
Ser Reagent Test Stanadard Sample
No
2. Serum Sample 0.02ml - -
4. H2O - - 0.02ml
 Mix and incubate for 5min at room temperature.
 Then read the absorbance against reagent blank.
Calculations:
Concentration = Absorbance of sample x 8mg/dl
Or
Concentration = Absorbance of sample x 2mmol/l
Cl;inical Applications:
Hypocalcaemia
68
Decreased serum level below normal is called hypocalcaemia

Blood Calcium level is decreased.
 Dietary deficiency.
 Vitamin D deficiency
 Hypoproteinemia
 Renal tubular defects
 Acute pancreatitis
 Hyperparathyroidism
 Malnutrition & malabsorption
 Chronic liver disease and liver failure
Hypercalcemia:
Increased serum calcium level above normal is called hypercalcemia Blood Ca12
level is increased in:-
 Increased dietary intake of Ca12 and Vitamin A and D
 Thyroid disorders
 Malignancy
 Acromegaly
 Acute adrenal insufficiency
Q-1 What are the function of serum calcium?
Ans. Ionized calcium helps in coagulation of blood. Calcium along with
phosphours helps in the formation of bones & teeth. It regulates
neuromuscular excitability. It is essential for nerve impulses’ and muscular
contraction. It helps in activation of several enzymes, eg lipases,
dehydrogenases, ATPase etc.
Q-2 in which conditions calcium level of serum falls?
Ans. Blood Ca12 level is decreased in:-
 Dietary deficiency
 Vitamin deficiency
 Hypoproteinemia
 Renal tubular defects
69
 Acute pancreatitis
Q-4 What are the effects of hypocalcaemia?
Ans. 1. Tetany: In this condition nervous system becomes progressively more

and more excitable. It occurs when the blood concentration of calcium
falls to approximately 6 mg/dl and it is usually lethal at about 4 mg/dl.
2. Rickets: Vitamin D deficiency is characterized by faulty calcification

of bones in children.
3. Osteoporosis: In this condition, bones of the adults are decalcified due

to vitamin D deficiency.
Q-5 In which condition calcium level of serum rises?
Ans. Blood Ca+2 level is increased in:-
 Increased dietary n intake of Ca12 and Vitamin A & D
 Thyroid disorders
Q-6 What are the effects of hypercalemia?
Ans. Depression of central and peripheral nervous systems:
 Muscular weakness
 Constipation
 Abdominal pain
70
Section-6
 Electrolytes

Ser Practical
No
6.1 Estimation of Serum Sodium and
Potassium level
6.3 Estimation of Serum Chloride
71
ESTIMATION OF SERUM SODIUM

Experiment 6.1
Principle: The present method is based on reaction of sodium with selective
chromogen (substance providing a chromophore whose absorbance varies inversely
as the concentration of sodium in the test specimen).
Reagents: NA+ at 150 MEV/L (milli equivalent/liter) - standard
Color reagent (ammonium thioglycolate at 140 mmol
Specimen: Freshly drawn non hemolyzed serum
72
Procedure: Wavelength: 623nm((620-640nm)

Optical Path: 1cm
Temperature: 25- 37oC
Reading against blank
Assay type= End point
Blandk Standard Sample
Reagent 1000ul 1000ul 1000ul
Standard - 10 ul -
Sample - - 10ul
 Mix in incubate for 5min at room temperature.
 Read the absorbance of standard and sample.
Calculations:
Na+ in mEq/L = Absorbance of sample / Absorbance of standard x concentration of
standard (i,e.150)
Normal Value:
135- 145mEq/L
CLINICAL APPLICATIONS
Function of Na+
 Maintenance of osmotic pressure of ECF & volume of Na+ ion.

 Transmission of impulse
 Muscle contraction
 Regulation of Acid Base balance.
Hyponatremia:
Serum level below 135 mmol/L is hyponatremia. Hypernatremia, Hyponatremia

usually arises as a complication of other medical illnesses in which excess water
accumulates in the body at a higher rate than can be excreted (for example in
73
congestive heart failure, syndrome of inappropriate anti-diuretic hormone

secretion (SIADH) or polydipsia). Sometimes it may be a result of over-hydration.
Clinical Features:
 Patients present with neurological signs and symptoms such as confusion

and headache. The results from cerebral over-hydration due to hypos
molarity.
 Nausea, vomiting.
 Malaise, lethargy
Biochemical Measurements:
 Serum sodium level

 Serum osmolality
 Urine osmolality
Hypernatremia: serum level more than 145 mmol/L. It is generally not caused by
an excess of sodium, but rather by relative deficit of free water in the body.
Causes:
 Diabetes insipidus
 Conn’s syndrome
 Cushing’s syndro
ESTIMATION OF SERUM POTASSIUM
Experiment 6.2
Principle: The amount pf K+ is determined in a specifically, prepared mixture to
produce a colloidal suspension.
The turbidity of which is proportional to concentration of K+ in the range of 2-7
mEq/L.
Reagents: R1 standard = (5mEq/L)
R2 Color reagent Na+ tetra phenyl boron = 2mmol/L
Specimen: Freshly drawn non hemolyzed serum
74
Procedure: Wavelength: 623nm

Optical Path: 1cm
Temperature: 25- 37oC
Reading against blank
Assay type= End point
Blank Standard Sample
Reagent 1ml 1ml 1ml
Standard - 20 ul -
Sample - - 20 ul
 Mix in incubate for 5min at room temperature.
 Read the absorbance.
Calculations:
K+ (mEq/L) = Absorbance of sample / Absorbance of standard x concentration of
standard (i,e.5)
Function of K+
 Maintenance osmotic pressure of ICF.

 Maintains RMP & plays important role in generation of action potential.
 Regulates acid base balance
 Contraction of muscle
Hyperkalemia: May occur due to:
 Crush injuries of muscle
 Renal failure
 Addison’s disease (loss of aldosterone)
75
 Acidosis
 Aldosterone antagonist
Hypokalemia: May occur due to:
 Vomiting
 Diarrhea
 Surgical fistula
 Hyperaldosteronism
 Renal disease
 Carcinomas that secret ACTH

76
ESTIMATION OF CHLORIDE LEVELS IN SERUM

Experiment 6.3
Principle: Chloride ion in acidic environment in presence of ferric nitrate forms a

colored complex with mercuric thiocyanate.
Intensity of the developed color is proportional to the chloride ion concentration in

the sample.
2Cl + Hg(SCN)2 HgCl + 2 SCN
SCN+ Fe+3 Fe(SCN)++
Reagents:
77
 Mercuric thiocyanate
 Ferric nitrate
 Nitric acid
 Chloride standard (100 mEq/l)
Sample: urine, Serum, CSF
Normal values: 98-107 mEq/l
Procedure:
 Take three test tubes and label them as blank standard and test.
No
1. Buffer bromocresol 1ml 1ml 1ml
Green Solution
2. Serum sample 0.01ml - -
4. H2O - - 0.01ml
 Mix and incubate for 5min at room temperature.
 Then read the absorbance against reagent blank.
Calculations:
Concentration = Absorbance of sample x concentration of standard
Clinical Aspects: Chloride ion represents that anion which is present concentration
in body. It is primarily found as NaCl in extracellular compartment and HCl
Low chloride levels are associated with
 Severe vomiting
 Diarrhea
 Addison’s disease
 Metabolic alkalosis
Increased Chloride levels are associated with
 Dehydration
 Congestive heart failure
 Cushing’s syndrome
 Hyperventilation
78
 Anemia
 Nephritis
 Renal Obstruction
 Primary hyperparathyroidism
79
Q-1 What are the sources of chloride?
Ans. It is present as sodium chloride in milk, water and other elements of diet.
Q-2 What are the functions of chloride?
Ans. 1. As a component of sodium chloride , the element chloride is essential in

water balance, osmotic pressure regulation and in aid base balance.
2. It takes part in the production of hydrochloric acid of gastric juice.

1. Chloride shift is an important phenomenon by which chlorine shifts
alternately from plasma to cell and helps in O2 carriage.
Q-3 What are the daily requirement of chloride?
Ans. Adults : 10-20 gm.

Children: 5-10 gm.
Women during pregnancy and lactation: 10-15 gm.
Q-4 Write down the conditions of low and high chloride level?
Ans. Low chloride levels are associated with
 Severe vomiting
 Diarrhea
 Addison’s disease
 Metabolic alkalosis
Increased Chloride levels are associated with
 Dehydration
 Congestive heart failure
 Cushing’s syndrome
 Hyperventilation
 Anemia
 Nephritis
 Renal Obstruction
 Primary hyperparathyroidism
80
 Section-7
 Instrumentation in Clinical Biochemistry

Ser# Practical
7.1 pH meter
7.2 Laboratory Centrifuge
7.3 Spectrophotometer
7.4 Electrophoresis
7.5 Chromatography
81
PH METER
Experiment 7.1
82
Principle of pH meter:
The term pH is defined by the expression.
pH = log 1 / [H˖] = -log [H˖]
It is defined as “negative log of hydrogen ions concentration expressed in

molarity”. The symbol ‘p’ denotes “negative logarithm”. pH is usually taken by
immersing a glass combination electrode into a solution and reading the pH
directly from a meter. At one time, pH measurement requires two electrodes, a
pH-dependent glass electrode sensitive to H ions and pH-independent calomel
reference electrode. Active electrode will be sensitive to the ion being measured.
Reference electrode will be insensitive to that ion. Reference electrode is filled
with KCL solution. Glass electrode is made up of a very thin glass membrane which
allows passage of H+ ions. The potential difference that develops between the two
electrodes is measured a voltage.
Most pH measurements today are obtained using a single “combination

electrode”. Both the reference and the pH – dependent electrodes are contained
in a single glass or plastic tube. They are much more convenient to use, especially
for smaller volumes of solution. A pH meter is standardized with buffer solution of
known pH before measurement of an unknown solution.
Method: Certain guidelines must be followed while using a pH meter.
a. Before and after use, check the level of standard KCL in the electrode.
b. Turn the temperature control, if available, to the temperature of the
standard calibration buffers and test solution. Be sure the function
dial is set at the pH.
c. Lift the electrode out of the storage solution, rinse it with distilled
water from a wash bottle, and gently clean and dry the electrode with
a tissue.
d. Immerse the electrode in a standard buffer. The standard buffer
should have a pH within two pH units of the expected pH of the test
solution. If not then adjust the meter with the “calibration dial”until
proper pH of the standard buffer is indicated on the dial.
83
e. Remove the electrode and again rinse with distilled water and
carefully blot dry with tissue. Immerse the electrode in a standard
buffer and read pH. It should be within +0.05 pH unit of the known.
f. Clean the electrode and immerse it in the test solution. Record the pH
of the test solution.
VIVA QUESTIONS
Q-1 Write down disadvantages and advantages of pH meter?

Advantages:-
1. It is an accurate method.
2. pH of colored solutions can be measured.
3. pH of semisolids like cheese can be measured.
4. It is affected by factors like slight change in temperature or presence of any
oxidizing or reducing agent.
Disadvantages:-
1. Technical experts are required for proper handling of pH meter.

2. Large sample volume is required.
3. It is time consuming.
4. Cost of this method is more as compared
Q-2 What is pH meter?
A pH meter is an electronics instrument used for measuring the pH ( acidity or

alkalinity of a liquid (though special probes are sometimes used to measure the
pH of semi-solid substances). A typical pH meter consists of a special measuring
probe (a glass electrode) connected to an electronics meter that measures and
displays the pH reading.
84

85
LABORATORY CENTRIFUGE
Experiment 7.2
Defination:
It is a separation technique commonly used in clinical and research laboratory. It is

based on the behavior of particles in an applied centrifuge field.
A laboratory centrifuge is a piece of laboratory equipment, Driven by a motor,

which spins liquid samples at high speed.
Types
There are various types of centrifugation:
 Differential centrifugation often used to separate certain organelles from

whole cells for further analysis of specific parts of cells.
 Isopycnic centrifugation, often used to isolate nucleic acids such as DNA
 Sucrose gradient centrifugation often used to purify enveloped viruses and
ribosome’s and also to separate cell organelles from crude cellular extracts.
Units of measurement: The rate of centrifugation is measured in revolutions per

minute.
Factors affecting sedimentation:
 Their size and shape

 Centrifugal acceleration
 The volume fraction of solids present
 The density difference between the particles
 The liquid and the viscosity
Principle:
Particles which differ in density, size and shape, sediment at different rates in a
centrifugal field. The particle will tend to sediment under the influence of gravity.
If the particles suspended in a liquid are so small or have a density so close to that
of a liquid, then force of gravity fails to sediment the particles in to a separate
86
layer. So the basis of centrifuge technique is to exert a larger force than the
gravitational force to enhance the effective sedimentation force for separating
such particles from liquid.
Working:
Increasing the effective gravitational force will more rapidly and completely cause
the precipitated “pellets” to gather on the bottom of the tube. The remaining
solution is called the “supernate” or “supernatant”.
 The supernatant liquid is then either quickly decanted from the tube
without disturbing the precipitate or withdrawn with a Pasteur pipette.
Precautions:
Centrifuge rotors should never be touched while moving, because a spinning rotor
can cause serious injury.
Q.1: how will you centrifuge the 2ml, of given solution at 2000rpm for 1min?
Ans: a. adjust the centrifuge machine at 2000rpm for 1min
b. pipette out the 2mL of given solution in sterile tube.
c. place the tube in centrifuge machine and balance with anther tube
having equal
Volume.
d. Takes the sample after completing the process.

87

88
SPECTROPHOTOMETER
Experiment 7.3
Components of spectrophotometer:
I. Light source:
Most common light source used is the tungsten iodide lamp for visible
light spectrum and halogen/deuterium lamp for the ultra violet
spectrum. A selector is used to reflect the light from lamp into
monochromator for measurement of light.
II. Monochromators:
Selected filters or prism diffraction gratings are placed in the path of light
to select light of a specific color or wavelength. Glass prisms and lenses
are suitable for this purpose.
III. Sample cell (cuvette):

It can be round or square, the light path must be kept constant in order
to have Lambert-Beer’s law obeyed. The cuvette holds the solution
whose absorbance is to be measured.
IV. Photodetector:
The purpose of photodetector is to convert the transmitted light into an

equivalent amount of electric energy.
V. Slit:
Simple colour reactors have fixed slit, while spectrophotometers have
variable slit width adjustment mechanism.
VI. Wavelength selector:
In spectrophotometer desired wavelength of light can be selected by the
help of wavelength selector.
Digital Display:
The scale reading on the instrument is a measure of and proportional to
optical density of colored solution, and according to Lambert Beer’s law
to concentration of colored substance, so the scale reading appearing on
89
digital display are proportional to concentration of substance under

consideration.
Apparatus & Chemicals:
Cuvettes, test tubes, beaker, spectrophotometer, distilled water, sample,
and standard solution.
Procedure:
For the determination of concentration of substance, the standard
solution which contain known amount of substance is run with unknown
sample.
Both are treated exactly the same way. Readings are taken using
appropriate wavelength and the concentration of unknown sample is
calculated by following formula.
Absorbance of unknown =________________
Absorbance of standard =________________
Concentration of standard =
Concentration of unknown =
Absorbance of unknown x concentration of standard.
Absorbance of standard.
Sometimes a blank test tube is used to set the spectrophotometer at
zero. It contains all things except sample. It is used in many
determinations to avoid false reading. Sometimes blank test tube
contains only distilled water. It is used to set the spectrophotometer at
zero.
Principle
All spectrophotometric measurements are based upon Lambert-Beer’s Law,
which is as follow;
Lambert’s Law:
It states that light absorbed by the sample or any compound is directly
proportional to the thickness of solution.
Beer’s Law:
It states that the amount of light absorbed is directly proportional to the
concentration of solute in the solution.
90
Optical Density:
Optical Density of solution (also known as absorbance or Extinction) is directly
proportional to the concentration of the substance and the depth of the
solution through which light passes. Beer-Lambert’s Law is expressed
mathematically as follows:
Where
I˳ = Intensity of incident light at a given wavelength.
I = intensity of transmitted light.
Å = observed absorbance (optical density, O.D).
ɛ = molar absorptivity of substance under test.
C = concentration of solution in moles/liter.
I = path length of the spectrophotometer cell in centimeters.

91
Q.1: modern spectrophotometer absorbs light in which range?

Ans. It absorbs light in following regions.
700-900 nm_________________infrated region
400-700nm_________________visible region
200-400nm_________________ultraviolet region
Q.2: How enzyme catalyzed reaction can be measured
spectrophotometerically?
Ans: They are measured spectrophotometrically by the appearance
of colored product of disappearance of a substrate.
Q.3: which kind of device is used to disperse white light?
A differential grating, prism or other device is used to disperse
white light into continuous spectrum enabling wavelength of
monochromatic (one color) light to be selected.
Q.4: what are the main uses of spectrophotometer?
Ans: a spectrophotometer is mainly used when tests require
readings at specific wavelength. It is used mainly to study
compounds which give colored product e.g. proteins, amino
acid especially tyrosine and tryptophan.
Q.5: compare spectrophotometer to colorimeter.
Ans: In colorimeter particular band is selected and not the particular
single wavelength. Instead in spectrophotometer single
wavelength is selected, also in colorimeter the filter is used.
Where as in spectrophotometer monochromatic or prism is
used.
Colorimeter uses a coloured light beam to measure ample
concentration. Spectrophotometer uses white light that is
passed through a slit and filter to analyze samples. Another
difference is that colorimeter measures the absorbance of light
in a sample while a spectrophotometer measures the amount
of light that passes through it.
Q.6: Which type of sample cells are used in spectrophotometer?

Ans: Following types of sampe cells are used:
92
Glass Cuvette: They are used in visible range

spectrophotometery.
Quartz Cuvette: They are used in ultraviolet region.
Plastic Cuvette: These are disposable Cuvette used for both
visible and ultraviolet region.
Q.7: You have been provided with three cuvettes having blank,
standard and test, how will you proceed for the
spectrophotometeric analysis of the given sample of bilirubin
at wave length 555nm?
Ans:
a. Adjust the wave length of spectrophotometer at 555nm.
b. Place the blank cuvette into cuvette holder and adjust to
zero.
c. Place the standard cuverre in the cuvette holder and
note the absorbance.
c. Place the test cuvette in cuvette holder and note the
absorbance.
Q.8: In the three labeled tubes, blank, standard and unknown, the
test for triglycerides by end point method has been
performed. Take their absorbance of spectrophotometer.
Ans: a. Adjust the filter at 546 nm, set the instrument at zero
absorbance using blank.
b. Take the absorbance of standard and note.

c. Take the absorbance of unknown and note.
93

94
ELECTROPHORESIS
Experiment 7.4
Principle:
In spite of the many physical arrangements for the apparatus, and regardless of
the medium through which molecules are allowed to migrate, all electrophoretic
separations depend upon the charge distribution of the molecules being
separated.
In electrophoresis, the force moving the macromolecules is the electrical potential;

E. the electtrophoretic mobility ‘u’ of a molecule is ratio of its velocity v, to the
electric potential. Electrophotrtic mobility is also equal to net charge, Z, of the
molecule divided by fractional co-efficient, F which reflect in part a proteins shape.
Thus:
µ= v
Apparatus and chemicals:
Electrophoresis protein buffer. Whatman paper, test tubes, ponceus stain, beaker,
acetic acid, ZnSO4.7H2O, sodium acetate.
Paper Electrophoresis
This technique is useful for the separation of small charged molecules such as
amino acids and small proteins. Paper electrophoresis is a method mostly used for
the separation and quantitative estimation of serum proteins (Albumin, Globulin)
etc).
Methods for paper electrophoresis of serum:
A strip of filter paper is moistened with buffer and the ends of the strip are
immersed into buffer reservoirs containing the electrodes. The samples are
spotted in the center of the paper, high voltage is applied, and the spots migrate
according to their charges.
95
After electrophoresis, the separated components can be detected by a variety of

staining techniques, depending upon their chemical identity.
 This is carried out on whatman paper strips 2-9 x 300cm in a hanging strip
type cell.
 The paper is saturated with protein buffer of pH 6-8 with good ionic
strength and allowed to drain in position) in the cell for 15 min.
 Serum (0.01 ml.) is then applied from a micropipette to the apex of the strip
directly over the silicone-coated glass supporting rod.
 Separation is carried out with a constant current of 8 mA for 8 strips (90-100
v, about 3-3.5 v/cm.) for 16 hr. at room temperature, avoiding any marked
inequalities of temperature from drafts, sunlight, etc.
 The strips were then removed from contact with the feed wick, spread flat,
and dried in an oven at 110-120˚for 30 min.
Staining:
The strips are transferred to a staining rack which holds them apart, and
placed in an aqueous dye bath containing 0.01% ponceus stain, 5% acetic
acid (v/v) and 5% ZnSO4, 7H2O (w/v) for 16 hr at room temperature. They
rinsed 3 times for 5,5 and 10 min, in 2% acetic acid (v/v) without agitation,
and then for 2 min. in a bath of 2% sodium acetate (CH3COONa, 3H2O, w/v)
and 10 % acetic acid (v/v).after being blotted gently on clean filter paper,
the strips are transferred to a drying rack which touches them only on the
ends and dried in the oven for 8-10 min at 110-120.
96
Q.1 Name different types of electrophoresis.
a. Paper electrophoresis.
b. Poly acryl amide gel electrophoresis.
c. Agarose gel electrophoresis.
Q.2. Name the plasma proteins that can be estimated from electrophoresis.
1. Globulin’s which include alpha, beta & gamma globulin.
2. Albumin.
3. Fibrinogen.
Q.3. What is the application of paper electrophoresis technique?
It is mostly used for separation of proteins.
Q-4 Write down uses of electrophoresis?
1. Electrophoretic techniques have also been adapted to other application

such as the determination of protein isoelectric points.
2. It is used to study binding sites and surface features of proteins.
3. Electrophoresis is applied to separations in free solution and has found very
useful application in blood cell separation.
Q-5 Write down precautions of paper electrophoresis?
 Must use a high voltage, otherwise the serum sample diffuses too rapidly.
 Paper must be cooled (usually by water)
97

98
CHROMATOGRAPHY
Experiment 7.5
Principle:
 Chromatography is the technique used to separate a group of similar

substances on the basis of differences in certain physical characteristics.
In every chromatographic separation there is a stationary phase which
remains fixed in a system and it consists of packing within a column. The
stationary phase selectively retards the substances relative to the moving
phase by virtue of the number of attractive forces such as van der Waals
interactions, hydrogen bonds and dipole moments. The other phase is called
mobile phase, which percolates over the surface of the fixed phase. The
stationary phase usually water or another polar solvent and the mobile
phase is relatively non polar. The stationary phase is liquid supported on
silica gel, kieselguhr, cellulose or starch in sheet form. When cellulose is in
the form of paper sheet, it is called as paper chromatography.
All separation by chromatography depend on the fact either the substance
be separated distribute itself between stationary phase and mobile phase in
another. Chromatographic separations are dependent upon multiple
partitions and adsorption process.
Paper chromatography
“The given solute will be partitioned or distributed between two given

immiscible liquids” this is called partition chromatography.
Paper chromatography is an important and useful class of partion

chromatography.
In this case the stationary phase is considered to be made of water

molecules bound molecules bound to cellulose network of paper. The
mobile phase is known as developing solvent consists of other one solvent
or a mixture of different compounds between these two liquid phases. The
99
mobile phase travels by the capillary action through the paper depending on
their way the solvent travel on the paper.
Types of paper chromatography:
i. Ascending chromatography
ii. Descending chromatography
iii. Circular chromatography
Phenomenon of ascending, descending and circular chromatography:
Apparatus:
Ninhydrin, chromotank, filter paper, amino acid solutions.
Procedure:
1. Take a sheet of Whatman chromatography paper of suitable length and

width for the tank in use.
2. Draw a pencil line parallel to and about 8cm from one end. Put the markings
for application of the spots 8cm apart.
3. Take 10 ul of the test solution and load on the spot. Use a hair dryer to dry
after applying the spot. Do not over heat. Make two spots for each sample.
The volume load will vary according to sample and the amount of the
substance present.
4. Reserve on sport for standard, which contains a known amount of all the
expected compounds and their concentration.
5. Hang the paper in the tank; replace the lid run the solvent through the hole
and allow the solvent to flow down. It is desirable to keep the chamber
saturated with the solvent vapour before pouring in the solvent. Hence,
keep a beaker of solvent in the bottom.
6. Remove the paper, mark the solvent front, hang in the fume-cupboard and
wait until the paper is completely dry (blow, if necessary).
7. Spray the spotting compound, with ninhydrin quickly and evenly, over the
entire surface of the paper until the paper becomes damped, work inside
the hood.
100
8. Re-hang the paper to dry. If the chromatogram requires heating for the
development of spot, heat carefully at 95 to 100˚C. Overheating will char
the paper. Keep the oven inside the fume-cupboard.
9. Examine the unknown components, identify them by comparing with the
spot of known compound (Rf calculation will not be necessary) and give a
semi-quantitative report by measuring the area. To do this, outline the spot
on tracing paper, cut out the tracing paper and weigh. The standard spot
with known concentration of test compound will be the reference.
Precautions:
1. Chromo tank should be covered during experiment
2. Do not touch the filter paper before ninhydrin spray.
Descending chromatography:
This basic procedure is similar to ascending chromatography. In descending

chromatography the solvent is placed at the top of the tank. Upper end of filter
paper dips into the solvent and lower end hangs. So the solvent flows down by
capillary action and continuously flow down under the force of gravity. The
distance travelled by the solvent depends upon the length of the paper and height
of the container. Taking out the filter paper, dry it and spray ninhydrin. Measure
the distance travelled by the solvent and amino acids. Calculate the Rf value.
101
Q.1. How does paper chromatography works?
Ans: Chromatography is a process that is used in order to separate various

substances and mixtures into their components e.g. amino acid in a serum
sample.
Q.2. what are the uses of paper chromatography?
Ans: It is used in many scientific studies identify unknown organic and inorganic
compounds.
Q.3 what is circular paper chromatography.
Ans: A paper chromatographic technique in which migration from a spot in the

sheet takes place in 360˚ so that zones separate as a series of concentric
rings.
Q.4 What is Rf value in paper chromatography?
Rf value = Distance traveled by solute (cm) x 100
Distance traveled by solvent (cm)
Rf value represents relative mobility.
Q.5 What are the types of chromatography?

i. Ascending chromatography
ii. Descending chromatography
iii. Circular chromatography
102

103
Section-8
Blood Chemistry
Ser Practical
No
8.1 Collection and Prevention of
Blood Samples
8.2 Estimation of Blood Glucose
8.3 Glucose Tolerance Test
104
COLLECTION & PRESERVATION OF BLOOD SAMPLES

Experiment 8.1
Apparatus
Disposable syringes, spirit, alcoholic swabs, tourniquet, cotton and tube for blood
collection.
Source of Blood: blood can be collected from 3 different sources:-
1. Venous Blood 2. Arterial Blood 3. Capillary

Blood
Types of specimen
1. Random Specimen:
This type of specimen may be collected any time of the day or night to assess
a patient’s condition at the time of collection.
2. Fasting specimen:
The fasting specimen is by far the most common type of specimen requested.
This specimen is drawn after the patient has avoided the intake of food
(fasted) for at least eight hours prior to the drawing of the specimen.
(3) Two-hour postprandial (PP) specimen:
The tow-hour postprandial (2 hours PP) is dawn exactly two hours after a
patient has completed a meal or been given a high carbohydrate drink
(Glucola). This type of specimen is commonly requested in conjunction with
glucose tolerance testing.
105
Procedure:
1. Patient is given as receipt having details for the test to be carried out.
2. Patient is asked to sit on the chair.
3. Take consent of the patient and briefly explain the procedure to the
patient.
4. Wear the gloves. Mostly the blood sample is taken from another anterior
cubital fossa, which is located on the mid of fore arm. This area should be
properly exposed.
5. There should be proper light. Apply the tourniquet in a way that venous
return should be blocked.
6. Sterilize the arm with spirit swab within a range of 5 cm.
7. Make sure the arm is properly positioned.
8. Insect the needle in the vein at an angle of 45o, and slightly putt the
plunger (if the blood appears sin the syringe, it shows that syringe is in the
vein). Collect the required amount of blood.
9. Immediately release the tourniquet.
10.Apply the pressure with thumb on antiseptic swan at puncture site for 2-4
min, till blood stop oozing out.
11.The blood from syringe is distributed to coded-named tubes for various
tests.
12.The syringe is despoiled off by using a cutter.
13.Sample can be temporarily stored by refrigeration. The serum can be
separated and stored at -20oC.
14.Blood samples for serology should not be kept at -20oC but Instead should
be kept at 28oC.
15.Blood from arteries (femoral) is drawn to check acid base balance
(Arterial blood gases). The syringe is inserted at an angle of 90 o in this
case.
Type of anticoagulants:
106
1. EDTA : EDTA is most commonly used in hematology since it preserves the

cellular components of the blood. EDTA cannot be used when calcium
determinations are required as it is a chelating anticoagulant.
2. Citrate: It is used for determination of erythrocyte sedimentation rate.
3. Heparin: It is the most widely used anticoagulant and causes the least
interference.
4. Oxalates: Oxalates form complexes with calcium and also they cause water
to diffuse from RBCs to the plasma. So those tests must not be performed
which are affected by this anticoagulant.
5. Hirudin: it is antithrombin which is extracted from leeches or prepared by

genetic engineering.
Precautions:-
1. Venipuncture area must be thoroughly clean.
2. Tourniquet must not be left in place top avoid prolonged stasis. This alters
concentration of certain constituents specially RBC’s hemoglobin, albumin
and calcium. Blind attempts on puncturing the vein should not be made.
3. Subcutaneous manipulation of the needle to enter a vein should not be done

as it causes a lot of pain.
4. Blood should immediately be transferred to appropriate containers

VIVA QUESTIONS
Q-1 What is plasma and serum?

a. Plasma
It is obtained by removing all formed elements/components from anti
coagulate blood by centrifugation at 5000 rpm for 3-5 minutes.
b. Serum
It is the fluid left behind after blood has clotted. This is the most common
specimen required for chemical and serological tests.
107
ESTIMATION OF BLOOD SUGAR LEVEL IN THE GIVEN SAMPLE

108
Experiment 8.2
Principle: determination of glucose after enzymatic oxidation by glucose oxidase.

The colorimetric indicator is quinoneimine, which is generated from 4-
aminophenazone and phenol by hydrogen peroxide under the catalytic action of
peroxidase.
Reaction:
Glucose + O2 GOD Gluconic acid + H2O2
2H2O2 + Aminoantipyrine + Phenol POD Quinonemine + 4H2O
Sample Material
Serum, Heparinized or EDTA plasma. Avoid hemolysis. Stability after naddition of

a glycolytic inhibitor (NaF or KF).
Reagent
 Reagent contains: Phosphate buffer, phenol, 4-aminoantipyrin,

glucose oxidase, peroxidase.
 Standard (100mg/dl)
Procedure:
Reagent Temperature + 25oC, 30oC or 37oC
Wavelength: 500nm
Light Path: 1cm
Measurement: against reagent blank.
Blank Standard Test
Standard - 0.01 ml -
Sample - - 0.01 ml
Reagent Solution 1 ml 1 ml 1 ml
 Mix. Incubate for 20 minutes at 20-25oC or 10 minutes at 37oC.

 Read Absorbance against the blank within 60 minutes.
Concentration of unknown = Absorbance of unknown x 100 (mg/dl)
109
Calculation:
Glucose concentration = Standard Concentration x AEsa (mg/dl)

AE st
Dilution limit:
If concentration exceed 400 mg/dl (22.2mmol), dilute 1 part of sample mwith 4

parts of 0.9% Nacl solution. Multiply the result by 5.
Normal Range Fasting
75-115 mg/dl (4.2-6.4 mmol/l)
Precautions
The reagent contains sodium azid (0.095%0 as preservative. Do not swallow. Avoid
contact with skin and mucus membranes.
Hyperglycemia
An elevated blood sugar level is known ass hyperglycemia. Fasting hyperglycemia

is highly suggestive of diabetes. On the other hand, diabetes can never be ruled out
by the presence of a normal fasting blood glucose level. So it is advisable to obtain
a blood sugar determination 1 or 2 hour after a meal (postprandial) containing
approximately 100 g carbohydrate.
Other causes of hyperglycemia includes:
1. Hyperthyroidism.
2. Hypopituitarism.
3. Increased secretion of adrenal cortex and medullary hormones.
4. Emotional stress.
5. Infectious disease.
6. Intracranial disease such as meningitis, encephalitis, tumors, hemorrhage.

110
7. Increasing age and pregnancy (gestational hyperglycemia)
hypoglycemia
It is a condition when the blood glucose is below 50 mg/100ml.
Causes
1. Over dosage with insulin in the treatment of diabetes.
2. Insulin secreting tumors of the pancreas.
3. Hypothyroidism (myxedema).
4. Hypopituitarism.
5. Adrenal insufficiency (Addison’s disease).
6. Severe exercise can produce hypoglycaemia due to liver being duple

glycogen from which the blood glucose is maintained.
Q-1 how many methods are used for estimating blood glucose level?
Ans. 1. Folin and Wu’s Method

2. Nelson and Somogy’s Method
3. O-toluidine Method
4. GOD-PAP Method
Q-2 what is the main advantage of O-toluidine test over other method?
Ans. One of the advantages of this method is that it can be directly applied to
plasma or serum without precipitation of proteins.
Q-3 What do you mean by hypoglycemia?
Ans. It is condition when blood glucose is below 50 mg/dl.
Q-4 What do you mean by hyperglycemia?
Ans. An elevated blood sugar level is known as hyperglycemia.
Q-5 What is diabetes mellitus?

Ans. diabetes mellitus is a metabolic disorder which is characterized by
extracellular hyperglycemia and intracellular decreased level of glucose.
Q-6 What is renal threshold for glucose?
111
Ans. Renal threshold for glucose is 180 mg/100 ml of blood.

Q-7 Which hormone is hyperglycemic in nature?
Ans. growth hormone. Cortisol, adrenaline, glucagon thyroid hormone.
Q-8 What is level of true glucose?
Ans. It is 60-80 mg/dl.
Q-9 What is True Glucose?
Ans. True glucose means amount of glucose after excluding other reducing
substances in the blood. It is 30 mg/100 ml less than normal value.
Q-10 What are methods of estimation of blood sugar?

Folin and Wu’s Method: In this method proteins are precipitated by tungstic acid.
Protein free filtrate is heated with alkaline copper tartrate solution. The cupric ions
are reduced to cuprous ions., which in turn reduce phosphomolybdic acid to
phosphomolybdous acid. The intensity of the blue color of phosphomolybdous acid
is measured calorimetrically.
Nelson and Somogy’s Method: In this method proteins are precipitated by zinc
sulphate and brain tartrate. Protein free filtrate is heated with alkaline copper
tartrate solution. The cupric ions are reduced to cuprous ions which in turn reduce
arsenomolybdic acid to arsenomolybdous acid. The intensity of the color is
measured calorimetrically.
O-toluidine Method: This method is commonly used and is simple and specific.
One of the advantages o0f this method is that it can be directly applied to plasma or
serum without precipitation of proteins. Glucose reacts with O-toluidine in glacial
aceticd acid at 100oC to from blue-green colored N-glucosylamine. The intensity of
the color is measured calorimetrically.
112

113
GLUCOSE TOLERANCE TEST
Experiment 8.3
Glucose tolerance test, also referred to as oral glucose tolerance test (OGTT)
measures the body’s ability to metabolize glucose or clear it out of blood stream.
The test can be used to diagnose diabetes, gestational diabetes or pre-diabetes (a
condition characterized by higher than normal blood sugar levels that can lead to
type II diabetes. For patients with or without diabetic symptoms who can not be
specifically classified, an oral glucose tolerance test (OGTT) is helpful for
diagnosis.
In normal subject following a meal, there is a temporary rise in blood sugar level
within 2-3 hours. Glucose tolerance test determines the degree and the duration of
hyperglycemia after an oral or intravenous administration of a known quantity of
glucose.
Indications:
 Gestational diabetes (24-28 weeks of pregnancy).

 If FBG is 6.6-7mmol/l (120-126mg/dl).
 2 hr post parandial glucose is 7.8 – 11.1 mmol/l 140-200mg/dl.
 Evaluation of patients with unexplained neuropathy, nephropathy,
retinopathy along with random blood glucose more than 200 mg/dl.
Preparation of the patient:
1. Patient must be ambulatory and free from fever, acute illness, trauma for
at least two weeks prior to the test.
2. 2. Patient should be on a diet containing at lest 150 g of carbohydrates /
day for least 3 days before the test.
3. Any drug interfering with blood glucose should be discontinued 3 days
prior to this test e.g salicylates, steroids, thiazide diuretics.
4. The hypoglycemia drugs to be discontinued at least on the day of test e.g
insulin and sulfonylureas.
5. The time of test should be 7-9 a.m.
6. Patient to be in fasting state 18-16 hrs before test. 12 hrs fast is ideal.
7. Heavy tea and coffee consumption and smoking to be reduced during the
day proceeding test.
114
8. No physical exercise allowed during the test.

9. Patient should be seated quietly and relaxed for 30 minutes before the test.
Procedure:
1. A zero time (baseline or fasting) blood sample is drawn.
2. Patient is given a measured dose of glucose to drink within a 5 minutes time

frame.
Glucose load:
for non-pregnant adult = 75 gm
for children = 1.75 gm/kg body weight
Lemon can be added to make it palatable and to prevent nausea/vomiting. Time of

oral glucose administration is noted.
4. A total of five specimens of venous blood are collected every ½ hour after
the oral glucose administration i,e ½ hour, 1 hour, 1 and ½ hour, 2 hour and 2 and ½
hour.
5. Glucose contents of all the six (including fasting sample) sample of blood is
estimated.
Sample are tested quantitatively for presence of glucose. A curve is plotted which is
called as “ glucose tolerance curve”.
Interpretation:
The test reveals how quickly glucose is metabolized from blood stream for use by
cells as an energy source. Values given below are for venous plasma glucose. The
following criteria apply to non-pregnant adults.
To consider impaired glucose tolerance three conditions must be fulfilled
1. Fasting plasma glucose above 126 mg/dl (7mmol/l)

2. Intermediate OGTT sample above 200 mg/dl (11.1mmol/l)
3. two hour OGTT sample between 140 and 200 mg/dl (7.8-1.1 mmol/l)
115
Impaired glucose tolerance is often associated with insulin resistance and is often
seen in polycystic ovarian syndrome and obesity.
Individual with impaired glucose tolerance should not be labeled s diabetics and
they should be reviewed regularly and given dietary advice. These individual
should also be encouraged to take exercise.
Gestational diabetes:
For gestational diabetes, a “screening test” of 50 gm over 1 hour is used. If elevated

this is followed with a test of 100 gm over 3 hours (OGTT). Gestational diabetes
can be considered if in pregnancy two or more values exceed the following:
Fasting above 105 mg/dl (5.8 mmol/l)

One hour above 190 mg/dl (10.5 mmol/l)
Two hour Fasting above 165 mg/dl (9.1 mmol/l)
3 hour Fasting above 145 mg/dl (8 mmol/l)
GLUCOSE NORMAL IMPADIRD IMPAIRED DIABETES
LEVEL FASTING GLUCOSE MELLITUS
GLYCEMIA TOLERANCE (DM)
(IFG) (IGT)
Venous Fasting 2 Fasting 2 Fasting 2 Fasting 2
plasma hour hour hour hour
mmol/l <6.1 <7.8 >6.1 & <7.8 <7.0 >7.8 >7.0 >
<7.0 11.1
Mg/dl <110 <140 >110 & < 140 < 126 >140 >126 >200
< 126
Disadvantages:
1. It is more time consuming.
2. It is more complicated than fasting blood glucose test.
3. The patient may feel nauseated, sweaty, light headed or faint after drinking
glucose for test.
4. Hematoma (blood accumulated under the skin) may form.
Q-1 What is the importance of GTT?

116
Ans. It is importance due to following reason:
1. Diagnosing milder and early cases of diabetes mellitus.

2. Investigating a case of symptoms lag type glycosuria.
3. Certain endocrine dysfunction.
Q-2 Name the type of glucose tolerance test.
Ans. This is two types:
2. Standard oral glucose tolerance test.

3. IV (intra-venous) glucose tolerance test.
Q-3 What are the conditions in which hyperglycemia is seen?
Ans. Hyperglycemia is considered when the fasting blood glucose increase above
120 mg/dl and random blood glucose is more than 170 mg/dl. It is raided in :
 Diabetes mellitus (due to insulin deficiency)

 Hyperactivity of thyroid, pituitary and adrenal glands.
 Thyrotoxitivity of thyroid, pituitary and adrenal glands.
 Thyrptpxicpsos (is a hypermetabolic state in which there is
accelerated gastric emptying and increased absorption of glucose
and other carbohydrate from gut).
 Excessive growth hormones
 Excessive glucocorticoid
 Anesthesia (also lead to hyperglycemia depending upon the type,
duration and degree of anesthesia)
 Pheochromocytoma (Tumors of adrenal medulla spontaneously
secrete catecholamine producing hyperglycemia and transient
glycosuria)
 Chronic pancreatitis (it causes a reduction in insulin production
secondary to destruction of islet cells of the pancreas).
 Administration of some drugs (chlorothiazde diuretics causes
suppression of insulin release).
 Post-surgical patients (intravenous infusion of dextrose, which is
often run rapidly to replace the lost fluid volume and depleted
stores of glucose, cause a slight and temporary increase in plasma
glucose concentration).
117
Q-4 What are the complications of diabetes?
Ans. Acute Chronic
Ketoacidosis (DKA) Neuropathy
HONK Neuropathy
Dehydration Retinopathy
Hypoglycemia Vascular insufficiency
MI
Diabetic Foot
Q-5 What are the conditions in which hypoglycemia is observed?
Ans. It is observed when the blood glucose level is below 53 mg/dl. Following are
the conditions in which hypoglycemia is seen
Drug induce hypoglycemia
1. Insulin (common in IDDM, it is related to a missed meal or some other

factor. Insulin requirement in IDDM patients is reduced by exercise).
2. Oral hypoglycemia used in the treatment of type-2 diabetes (NIDDM)
3. Other drugs (hypoglycemia may occur in patients treated with beta-
adrenergic blocking drugs, children poisoned with salicylate, overdoses of
paracetamol, necrosis)
Fasting hypoglycemia
1. Insulin (tumors of insulin secreting beta cells of the pancreatic islets).

2. Endocrine disorders (deficiency of hormones antagonistic to insulin can
cause hypoglycemia, ACTH. Growth hormone and cortisol deficiency).
3. Non pancreatic tumors (tumors like retroperitoneal fibroma, adrenal
carcinoma and hepatoma).
4. Liver disease (inadequate glycogen storage and depressed
gluconeogenesis).
Reactive hypoglycemia
118
1. Glucose induced (post gastrectomy state rapid intestinal absorption of

glucose leases to a large amount of insulin secretion and a consequent
excessive fall in plasma glucose level in 2-3hour).
2. Leucine (it is described in infants and manifests after a high protein meal).
3. Fructosemia (large doses of fructose may lead to hypoglycemia and lactic
acidosis because of saturation of aldolase B, causing accumulation of
fructose-I phosphate and depletion of intracellular ATP and inorganic
phosphate).
4. Galactosemia (galactose can not be metabolized, as a result galactose-I-
phosphate piles up in cell and cannot be converted to glucose resulting in
hypoglycemia).
5. Alcohol
6. Malaria
7. Diarrhea
Q-6 Names some oral hypoglycemic drugs?
Ans. Sulfonylureas
Chlorpropamide
Q-7 how the oral drug produces hypoglycemic effect?
Ans. Increasing pancreatic insulin release.
Decreasing the release of glucose from liver
Increasing sensitivity of peripheral tissues to the action of insulin.

Q-8 How does alcoholism cause hypoglycemia?
Ans. A common cause of fasting hypoglycemia in adults is the consumption of

alcohol in fasting state. The metabolism of alcoholism (ethanol
Acetaldehyde acetate) convers NAD to NADH. The accumulation of
NADH is oxidized back to NAD by the reduction of pyruvate to lactate.
However, the formation of glucose from lactate and alanine (glycogenic
precursors) requires their con version to pyruvate. Thus, the accumulation of
NAD interferes with substrate utilization along the glucogenic pathway.
119
Q-9 What is carbohydrate tolerance?
Ans. The ability of the body utilize carbohydrates may be ascertained by

measuring its carbohydrate tolerance. It is indicate by the nature of blood
glucose curve following the administration of glucose. This “glucose
tolerance” is a valuable diagnostic aid) 70 kg man can ingest approximately
carbohydrate 1500 mg/day.
Q-10 In which disease three is high glucose tolerance d?
Ans. i. Hypopituitarism
ii. Addison’s disease
iii. Hyperinsulinism
iv. In decreased absorption like sprue, celiac disease.
v. In decreased activity of anterior pituitary and adrenal cortex.
vi. In hyperthyroidism.
Q-11. when is intravenous GTT performed?
Ans. It is perfected when there are abnormalities in absorption of glucose. Thus IV

GTT is indicated.
In hypothyroidism
i. In sprue and celiac disease.

ii. When person is unable to ingest.
Q-12. Write the name of different types of glycosuria?
Ans. a. Hyperglycemic glycosuria
b. Renal glycosuria
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1

PRACTICAL NOTE BOOK

 Dr. Zeemal Iqbal

 Dr. Sadia Akram

 Dr. Yasoob Ali Khan

University Roll No._____________

Ser No Practical Page No

Ser No Practical Page No

Ser No Practical Page No

Ser No Practical Page No

Ser No Practical Page No

Ser No Practical Page No

Ser No Practical Page No

Ser No Practical Page No

Ser Practical Page No

Serum markers of liver pathology:

Functional liver markers:

ESTIMATION OF SERUM TOTAL PROTEIN

In alkaline medium CuSO4 reacts with peptide bonds present in protein,

Sr. No Reagent Test Standard Blank

Plasma Protein concentration/dl=

Total plasma protein concentration is 6__8 gms/dl.

PRACTICAL VIVA QUESTIONS

Q.1 : write down the causes of hypoproteinemia?

Q.2: write down the causes of hyperproteinemia?

Q.3: what is denaturation of proteins?

Q.4: write down methods of determination of protein?

1. Turbidity metric method: In this method proteins are precipitated with

6. Colorimetric method (biuret): The name biuret is given because of the

Q.5: write down functions of proteins?

a. The maintenance of water distribution between cells and tissues,

Q.6: what are major plasma proteins?

Most of the plasma proteins are synthesized in liver, Albumin,α-

Q.7: What is the normal range of albumin/globulin ration in blood?

Q.8: What is most commonly used method for determination of serum

Ans. Biuret method.

Q.9: What are bence___Jones Proteins?

Q-8 How can we detect bene ___Jones proteins?

Results and Calculations

QUANTITATIVE ESTIMATION OF TOTAL SERUM ALBUMIN

PRACTICAL VIVA QUESTIONS

i. Impaired albumin synthesized in liver

Q-3: which is most abundant serum protein?

Q-4: write down functions of albumin?

1. Maintain water balance in serum and plasma.

Results and Calculations

ESTIMATION OF SERUM BILIRUBIN

a. Unconjugated bilirubin (UCB)

Bilirubin reacts with diazotized sulphanilic acid to form a colored compound. It is

RI – Sulphanilic acid R3-Caffeine

Procedure: For normal = 578nm

Sample blank Sample

Read absorbance of sample against sample blank at 578nm.

Total Bilirubin = A x 10.8

For Direct Bilirubin:

Sample blank Sample

Indirect= Total – Direct

Normal Value of Total Bilirubin in blood

PRACTICAL VIVA QUESTIONS

Q-1 Write down causes of unconjugated hyperbilirubinemia?

Q-2 Write down causes of unconjugated hyperbilirubinemia?

1. Biliary tract obstruction.

Q-3 Write down causes of mixed hyperbilirubinemia?

Q-4 What is meant by conjugated or direct Bilirubin?