MEDICINAL BIO CHEMISTRY

PRACTICAL MANUAL

PREPARED BY

Dr. N. RAMALAKSHMI

DEPARTMENT OF PHARMACEUTICAL CHEMISTRY

C.L. BAID METHA COLLEGE OF PHARMACY

(AN ISO 9001:2000 CERTIFIED INSTITUTION)

THORAIPAKKAM,

CHENNAI-600097

1
2
 CONTENTS

S.No Name of experiment P. No Date Signature

QUALITATIVE EXPERIMENTS
1 Qualitative analysis of normal 5
constituents of urine

General procedure for Analysis of 7

abnormal constituents of urine

2 Analysis of abnormal constituents of 9

urine Sample No:1

3 Analysis of abnormal constituents of 11

urine Sample No:2

4 Analysis of normal and abnormal 13

constituents of urine Sample No:3

5 Analysis of normal and abnormal 16

constituents of urine Sample No:4

QUANITATIVE EXPERIMENTS
6 Estimation of urine sugar by 19
Benedict’s method

7 Estimation of urine chlorides by 21

Volhard method

8 Estimation of urine creatinine by 23

Jaffe’s method

9 Estimation of urine calcium by 25

precipitation method

10 Estimation of serum cholesterol by 27

Libermann Burchard’s method

11 Preparation of Folin wu filtrate from 29

blood

3
12 Estimation of blood creatinine by 30
Jaffe’s method

13 Estimation of blood sugar by Folin 33

Wu tube method

14 Estimation of SGOT in serum 35

15 Estimation of SGPT in serum 37

16 Estimation of urea in serum 39

17 Estimation of protein in serum 41

18 Determination of serum bilirubin 43

19 Determination of Glucose by glucose 45

oxidase

20 Hydrolysis of starch by amylase 46

21 Determination of Na+ and K+ in 47

solution by flame photometry

22 Determination of serum calcium 50

23 Preparation of buffer and 53

measurement of pH

24 Determination of Total Cholesterol 55

25 Estimation of HDL Cholesterol 57

26 Estimation of LDL 59

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 EXP NO 1: Qualitative analysis of normal constituents of urine

S.No EXPERIMENT OBSERVATION INFERENCE

1 NATURE OF URINE

Specific Gravity Test: ¾ of It is around 1.018 Normal range is

urine in meter cylinder and gently 1.015 to 1.025
flow through urino meter

2 Test For pH: Test the urine with Blue litmus changes pH of urine varies
pH litmus to red litmus from 5 to 5.8

3 Test for chloride: To 5ml A curdy white Chloride precipitates

urine add 1ml of conc. HNO3 and precipitate as AgCl
1ml AgNO3

4 Test for earthly phosphate or

calcium: To 10ml of urine strong
ammonium solution filter and absorb
the precipitate with the water and
dil.acetic acid is added and filter
it.filterated is dividede into 2 parts

i) conc.HNO3 and 5ml of ammonium Yellow precipitate Presence of

molybdate solution and boil phosphate
White precipitate
ii) Add 5ml of 2% potassium oxolate formed Presence of calcium

5 Test for Ammonium: 10ml of Ammonium gas Presence of

urine and add a drop of turns the ammonia in urine
phenolpthalein, N/10 NaOH till the pink(indicator)
colour becomes pink boil and hold a
glass rod dipped in phenolpthalein

6 Test for organic sulphates: To 5ml White colour Presence of

of urine and 1ml of conc.HCl add turbidity inorganic sulphates
BaCl2 solution and boil

7 Test for urea

Specific urease test: To 5 ml of Orange colour Specific solutions
urine add 2 drops of phenol red in changes to red turn red from orange
another test tube take a spatula of as ph changes due to
horse gram powder add 5ml of water conversion of urea
and 2 drops phenol red. Then adjust to ammonium
to the reaction in both the test tubes
5
 in adding 2% Na2CO3 carbonate by adding
6%CH3COOH. Until the solution is 2% Na2CO3 or 6 %
faint orange,if the original solution is CH3COOH until the
yellow use 20% NaOH until it solution is faint
becomes orange. The pH is orange, enzyme in
7,transfer the test tube with horse the horse gram. The
gram powder and note the colour pH of phenol red is
before and after mixing. 6 to 8.3.presence of
urea.

8 Test for urea-HNO3 test:

To 3ml of urine add 2ml of conc. Crystalline white Presence of urea due
HNO3 and cool it colour has formed to formation of urea
crystals.

9 Sodium hypobromide test:

To 5ml of urine add 1ml of alkaline Effervescence is Presence of urea

hypobromide and 8% NaOH formed

10 Test of oxalic acid:

To To 3ml of urine add 2ml of CaCl2 White precipitate Presence of oxalates

11 Test for creatinine:

Jaffe’s test: Take 5ml of water in Orange red colour is Due to presence of
one test tube add 5ml of urine to formed creatinine colour
same test tube.add 1ml of saturated changes from
picric acid solution and 10drops of yellow to orange.
NaOH.

12 Test for uric acid:

Benedict’s uric acid test: 3ml of Deep blue colour is Presence of uric acid
urine add 1ml of benedict’s reagent formed
and 20% Na2CO3.

6
 General procedure for analysis of abnormal constituents of urine

S.NO Experiment Observation Inference

1 Test for reducing Green/yellow/orange/ Presence of reducing sugar.
sugar brick red precipitate is Reducing sugar gets excreted
To 5 ml of Benedicts obtained in urine during diabetes
reagent add 8 drops of mellitus. Color is suggestive
urine. Boil it for 2 of approximate amount of
minutes. glucose in urine
Green-0.5%
Yellow-1%
Orange-1.5%
Brick red -2%
2 Rotheras test for A permanganate colour Indicates the presence of
acteoacetic acid and ring is formed at the acetone or acetoacetic acid or
acetone junction of 2 layers. both. Acetone,acetoacetic acid
Take 5 ml of urine. and β-hydroxy butyrate are
Saturate with solid known as ketone bodies. It is
ammonium sulphate. excreted in urine during
Add 5 drops of 5% uncontrolled diabetes mellitus
solution of sodium and starvation. They also et
nitroprusside and gently excreated in urine during
shake. Add 2 ml of Ketonemia and diabetic
strong ammonia ketoacidosis.
through the sides of test
tube.
3 Gerhardts test for A brown color is Presence of acetoacetic acid.
acetoacetic acid obtained
To 5 ml of urine add
dilute ferric chloride
drop by drop until a
maximum precipitate of
ferric phosphate is
obtained. Filter and add
ferric chloride in excess
to the filterate.
4 Test for proteins Coagulation is obtained Presence of proteins.
Heat coagulation test Albuminuria is present in
To a clean test tube add kidney diseases like
5 ml of urine. Heat it at glomerulonephritis,
the top nephroscelrosis, tuberculosis,
carcinoma of kidney,
nephritic syndrome.
5 Hellers test White ring is formed at Presence of proteins.
Take 5 ml nitric acid in the junction of 2 layers
a test tube. Add 5 ml
urine.

7
6 Benzidine test for Blue or green color is Indicates the presence of
haemoglobin obtained haemoglobin. Haemoglobin is
Take a knife point of excreted in urine in
benzidine in a very haematuria
clean test tube and add
1 ml of urine and 1 ml
of hydrogen peroxide.

7 Hays test for bile salt Sulphur flower sinks Presence of bile salts. Bile
Take half test tube of into bottom of test tube salts get excreted in urine
diluted urine and half in tube containing urine during obstructive jaundice.
test tube of water in
another test tube as
control. Gently sprinkle
sulphur flowers in to
both

8 Gmelins test for bile Rings of yellow, blue, Presence of bile pigments
pigments green colour is found.
Take 5 ml nitric acid in
a test tube. Add 5 ml
urine.

9 Fouchets test for bile Blue or green colour is Presence of bile pigments.
pigments obtained Bile pigments are found in
Take 5 ml given urine urine in jaundice.
sample in a test tube
and acidify with few
drops of dilute acetic
acid. Add a crustal of
magnesium sulphate.
Shake the test tube and
dissolve. Add 2 ml of
BaCl2. Filter and discard
the filtrate and dry the
paper and add fouchets
reagent

8
 EXP.NO 2 Analysis of abnormal constituents of urine Sample No:1

S.NO Experiment Observation Inference

1 Test for reducing sugar
To 5 ml of Benedicts reagent
add 8 drops of urine. Boil it
for 2 minutes.
2 Rotheras test for acteoacetic
acid and acetone
Take 5 ml of urine. Saturate
with solid ammonium
sulphate. Add 5 drops of 5%
solution of sodium
nitroprusside and gently
shake. Add 2 ml of strong
ammonia through the sides of
test tube.
3 Gerhardts test for
acetoacetic acid
To 5 ml of urine add dilute
ferric chloride drop by drop
until a maximum precipitate
of ferric phosphate is
obtained. Filter and add ferric
chloride in excess to the
filterate.
4 Test for proteins
Heat coagulation test
To a clean test tube add 5 ml
of urine. Heat it at the top
5 Hellers test
Take 5 ml nitric acid in a test
tube. Add 5 ml urine.

6 Benzidine test for

haemoglobin
Take a knife point of
benzidine in a very clean test
tube and add 1 ml of urine
and 1 ml of hydrogen
peroxide.

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7 Hays test for bile salt
Take half test tube of diluted
urine and half test tube of
water in another test tube as
control. Gently sprinkle
sulphur flowers in to both
8 Gmelins test for bile
pigments
Take 5 ml nitric acid in a test
tube. Add 5 ml urine.
9 Fouchets test for bile
pigments
Take 5 ml given urine sample
in a test tube and acidify with
few drops of dilute acetic
acid. Add a crustal of
magnesium sulphate. Shake
the test tube and dissolve.
Add 2 ml of BaCl2. Filter and
discard the filtrate and dry the
paper and add fouchets
reagent

10
 EXP.NO 3 Analysis of abnormal constituents of urine Sample No:2

S.NO Experiment Observation Inference

1 Test for reducing sugar
To 5 ml of Benedicts
reagent add 8 drops of
urine. Boil it for 2
minutes.
2 Rotheras test for
acteoacetic acid and
acetone
Take 5 ml of urine.
Saturate with solid
ammonium sulphate. Add
5 drops of 5% solution of
sodium nitroprusside and
gently shake. Add 2 ml of
strong ammonia through
the sides of test tube.
3 Gerhardts test for
acetoacetic acid
To 5 ml of urine add
dilute ferric chloride drop
by drop until a maximum
precipitate of ferric
phosphate is obtained.
Filter and add ferric
chloride in excess to the
filterate.
4 Test for proteins
Heat coagulation test
To a clean test tube add 5
ml of urine. Heat it at the
top
5 Hellers test
Take 5 ml nitric acid in a
test tube. Add 5 ml urine.

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6 Benzidine test for
haemoglobin
Take a knife point of
benzidine in a very clean
test tube and add 1 ml of
urine and 1 ml of
hydrogen peroxide.

7 Hays test for bile salt

Take half test tube of
diluted urine and half test
tube of water in another
test tube as control.
Gently sprinkle sulphur
flowers in to both

8 Gmelins test for bile

pigments
Take 5 ml nitric acid in a
test tube. Add 5 ml urine.
9 Fouchets test for bile
pigments
Take 5 ml given urine
sample in a test tube and
acidify with few drops of
dilute acetic acid. Add a
crustal of magnesium
sulphate. Shake the test
tube and dissolve. Add 2
ml of BaCl2. Filter and
discard the filtrate and
dry the paper and add
fouchets reagent

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EXP.NO 4 Analysis of normal and abnormal constituents of urine Sample No:3

S.NO Experiment Observation Inference

Analysis of normal constituents
1. Test for urea
Specific urease test: To 5
ml of urine add 2 drops
of phenol red in another
test tube take a spatula of
horse gram powder add
5ml of water and 2 drops
phenol red. Then adjust
to the reaction in both the
test tubes in adding 2%
Na2CO3 6%CH3COOH.
Until the solution is faint
orange,if the original
solution is yellow use
20% NaOH until it
becomes orange. The pH
is 7,transfer the test tube
with horse gram powder
and note the colour
before and after mixing.
2 Test for urea-HNO3
test:

To 3ml of urine add 2ml

of conc. HNO3 and cool
it

3 Test for urea-HNO3

test:

To 3ml of urine add 2ml

of conc. HNO3 and cool
it

4 Test for creatinine:

Jaffe’s test: Take 5ml of

water in one test tube add
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 5ml of urine to same test
tube.add 1ml of saturated
picric acid solution and
10drops of NaOH.

5 Test for uric acid:

Benedict’s uric acid test:

3ml of urine add 1ml of
benedict’s reagent and
20% Na2CO3.

Analysis of abnormal constituents

1 Test for reducing sugar

To 5 ml of Benedicts
reagent add 8 drops of
urine. Boil it for 2
minutes.
2 Rotheras test for
acteoacetic acid and
acetone
Take 5 ml of urine.
Saturate with solid
ammonium sulphate. Add
5 drops of 5% solution of
sodium nitroprusside and
gently shake. Add 2 ml of
strong ammonia through
the sides of test tube.
3 Gerhardts test for
acetoacetic acid
To 5 ml of urine add
dilute ferric chloride drop
by drop until a maximum
precipitate of ferric
phosphate is obtained.
Filter and add ferric
chloride in excess to the
filterate.
4 Test for proteins
Heat coagulation test
To a clean test tube add 5
ml of urine. Heat it at the
top

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5 Hellers test
Take 5 ml nitric acid in a
test tube. Add 5 ml urine.

6 Benzidine test for

haemoglobin
Take a knife point of
benzidine in a very clean
test tube and add 1 ml of
urine and 1 ml of
hydrogen peroxide.

7 Hays test for bile salt

Take half test tube of
diluted urine and half test
tube of water in another
test tube as control.
Gently sprinkle sulphur
flowers in to both

8 Gmelins test for bile

pigments
Take 5 ml nitric acid in a
test tube. Add 5 ml urine.
9 Fouchets test for bile
pigments
Take 5 ml given urine
sample in a test tube and
acidify with few drops of
dilute acetic acid. Add a
crustal of magnesium
sulphate. Shake the test
tube and dissolve. Add 2
ml of BaCl2. Filter and
discard the filtrate and
dry the paper and add
fouchets reagent

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EXP.NO 5 Analysis of normal and abnormal constituents of urine Sample No:4

S.NO Experiment Observation Inference

Analysis of normal constituents
1. Test for urea
Specific urease test: To 5
ml of urine add 2 drops of
phenol red in another test
tube take a spatula of horse
gram powder add 5ml of
water and 2 drops phenol
red. Then adjust to the
reaction in both the test
tubes in adding 2%
Na2CO3 6%CH3COOH.
Until the solution is faint
orange,if the original
solution is yellow use 20%
NaOH until it becomes
orange. The pH is
7,transfer the test tube with
horse gram powder and
note the colour before and
after mixing.
2 Test for urea-HNO3 test:

To 3ml of urine add 2ml of

conc. HNO3 and cool it

3 Test for urea-HNO3 test:

To 3ml of urine add 2ml of

conc. HNO3 and cool it

4 Test for creatinine:

Jaffe’s test: Take 5ml of

water in one test tube add
5ml of urine to same test
tube.add 1ml of saturated
picric acid solution and

16
 10drops of NaOH.

5 Test for uric acid:

Benedict’s uric acid test:

3ml of urine add 1ml of
benedict’s reagent and
20% Na2CO3.

Analysis of abnormal constituents

1 Test for reducing sugar

To 5 ml of Benedicts
reagent add 8 drops of
urine. Boil it for 2 minutes.
2 Rotheras test for
acteoacetic acid and
acetone
Take 5 ml of urine.
Saturate with solid
ammonium sulphate. Add
5 drops of 5% solution of
sodium nitroprusside and
gently shake. Add 2 ml of
strong ammonia through
the sides of test tube.
3 Gerhardts test for
acetoacetic acid
To 5 ml of urine add
dilute ferric chloride drop
by drop until a maximum
precipitate of ferric
phosphate is obtained.
Filter and add ferric
chloride in excess to the
filterate.
4 Test for proteins
Heat coagulation test
To a clean test tube add 5
ml of urine. Heat it at the
top
5 Hellers test
Take 5 ml nitric acid in a
test tube. Add 5 ml urine.

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6 Benzidine test for
haemoglobin
Take a knife point of
benzidine in a very clean
test tube and add 1 ml of
urine and 1 ml of hydrogen
peroxide.

7 Hays test for bile salt

Take half test tube of
diluted urine and half test
tube of water in another
test tube as control. Gently
sprinkle sulphur flowers in
to both

8 Gmelins test for bile

pigments
Take 5 ml nitric acid in a
test tube. Add 5 ml urine.
9 Fouchets test for bile
pigments
Take 5 ml given urine
sample in a test tube and
acidify with few drops of
dilute acetic acid. Add a
crustal of magnesium
sulphate. Shake the test
tube and dissolve. Add 2
ml of BaCl2. Filter and
discard the filtrate and dry
the paper and add fouchets
reagent

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 EXP.NO 6 Quantitative Estimation of urine sugar by Benedict’s method

Aim: To estimate the amount of sugar present in urine by Benedict’s method.

Principle: Benedict’s test is an alkaline copper reagent containing copper sulphate, Sodium
2+
carbonate, Sodium citrate and potassium ferrocyanate. The alkaline Cu in the reagent is
reduced to cuprous oxide which is red in color.

2Cu(OH)2→Cu2O

Coppersulphate: Furnishes cupric ions.

Sodium carbonate: makes the medium alkaline as the reagent is positive in alkaline medium.

Sodium citrate: precipitates cupric ions by forming citrated cupric ion complex which on
dissociation supplies cupric ions.

Potassium ferro cyanate: keep the cuprous oxide as precipitate.

The test is nonspecific. Other sugars like pentoses, fructose, galactose and lactose also react.

Procedure: In a 100ml conical flask pipette out 10ml of Benedict’s quantitative reagent. Add
3g of anhydrous sodium carbonate and 15 ml of distilled water.Mix it well. Place the conical
flask in boiling water bath. Fill the burette with urine solution. Add urine from the burette to
the conical flask while it is boiling. Continue the titration until blue colour disappears.

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 Volume of Weight of Biurette reading Volume Indicator End
reagent Na2CO3 of urine point
Initial Final

Calculation:

Amount of glucose present in 100 ml=20*100/x Where x is titre value.

Clinical significance

Normally glucose is present in urine in negligible amount. Glucose is pathologically seen in

urine in conditions like

-Diabetes mellitus

-Renal glycosuria

-Endocrine hyper activity (hyper thyroidism, hyper pitutirism and hyper adrenalism)

-liver and pancreatic diesese

-severe infections

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 EXP.NO 7 Quantitative Estimation of urine chlorides by Volhard method

Aim:

To estimate the amount of chlorides by Volhard method.

Principle:

The chloride present in urine reacts with silver nitrate. The excess unreacted chloride is back
titrated with ammonium thiocyanate using ferric alum as indicator.

Procedure:

Measure 10 ml urine into 100ml conical flask. Add approximately 25 ml of distill water and
exactly 20 ml of silver nitrate and few drops of ferric alum. Make up to the mark. Filter the
mixture and transfer 50 ml of solution to clean conical flask. This contains 5 ml of urine and
10 ml of silver nitrate.titrate this with ammonium sulphate until pale permanent pink colour is
produced.

Contents of conical Burette reading Titre value Indicator

flask
Initial Final

Calculation:

20 ml of AgNO3 were used to precipitate chloride.

This was diluted to 100ml.

50 ml of filtrate used in titration=10 ml of AgNO3

10 ml of thiocyanate used 0.1 m eq chloride in 5 ml urine.

Chloride present in 1000ml urine= (10-x)*0.1*1000/5

Report: The amount of chloride present in 1000ml of urine =

21
 CHLORIDE IN URINE

Adult: 140-250 mEq per 24 hours or 140-250 mmol per day

Child (10-14
64-176 mEq/24 hours or 64-176 mmol/day
years):

Child
(younger than 15-40 mEq/24 hours or 15-40 mmol/day
6 years):

Abnormal
High chloride levels may be caused by:

• Dehydration, such as from diarrhea or vomiting.

• Eating a lot of salt.

• Kidney disease.

• An overactive parathyroid gland (hyperparathyroidism).

Low chloride levels may be caused by:

• Conditions that cause too much water to build up in the body, such as
with syndrome of inappropriate antidiuretic hormone secretion
(SIADH).

▪ Addison's disease.
▪ A condition that raises the pH of the blood above the normal range
(metabolic alkalosis).
▪ Heart failure.
▪ Ongoing vomiting.

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 EXP.NO 8 Quantitative Estimation of urine creatinine by Jaffe’s method

Aim:

To estimate the amount of creatinine present in urine byJaffe’s method.

Principle:

Creatinine reacts with alkaline picrate reagent to form a reddish orange coloure complex
called creatinine picrate. Alkaline is measured at green filter at 540 nm. The intensity of
colour is proportional to amount of creatinine present.

Reagents:

0.75N sodium hydroxide

0.04M picric acid.

Procedure:

Take three test tubes. Label them as test, standard and blank. To the tube labeled as test add
1ml of urine. To the tube labeled as standard add 1ml of standard solution
(concentration:2mg/100ml). to the tube labeled as blank add 1 ml of water. To all the three
tubes add 1ml picric acid and 1ml sodium hydroxide. Wait for 10 minutes at room
temperature. Measure the optical density at 540 nm.

Abnormal results of urine creatinine are nonspecific, but may be due to any of the following
conditions:

• Glomerulonephritis
• High meat diet
• Kidney infection (pyelonephritis)
• Kidney failure
• Muscular dystrophy (late stage)
• Myasthenia gravis
• Prerenal azotemia
• Reduced kidney blood flow (as in shock or congestive heart failure)
• Urinary tract obstruction
23
Calculation:

Concentration of creatinine in solution

= [OD of test-OD of blank] / OD of Std-OD of blank* concentration of std

Report: The amount of creatinine present in 100ml of urine=

Normal value

Urine creatinine (24-hour sample) values can range from 500 to 2000 mg/day. Results depend
greatly on your age and amount of lean body mass.

Another way of expressing the normal range for these test results are:

14 to 26 mg per kg of body mass per day for men

11 to 20 mg per kg of body mass per day for women

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EXP.NO 9 Quantitative Estimation of urine calcium by precipitation method

Aim:

To estimate the amount of calcium present in urine by precipitation method.

Reagents:

Ammonia 0.88

Con HCl

Oxalic acid (2.5%)

Potassium permanganate (0.1N)

Procedure:

If necessary make the urine to slightly acidic to litmus paper. The measure 100ml into a
suitable flask, make neutral to litmus wuth ammonia and add 5 drops of concentrated Hcl.
Add 5 ml 2.5% Oxalic acid followed by 4ml sodium acetate allow to stand for 30 minutes.
Filter wash the precipitate with distill water, dissolve the precipitate in warm 2N sulphuric
acid, titrate with 0.1N potassium permanganate.

High levels of urine calcium (above 300 mg/day) may be due to:

• Chronic kidney disease

• Leaking of urine from the kidneys, which causes calcium kidney stones
• Sarcoidosis
• Takiong too much calcium
• Too much production of parathyroid hormone by the parathyroid glands in the neck
• Use of water pills called loop diuretics
• Very highvitamin D levels

Low levels of urine calcium may be due to:

• Disorders in which the body does not absorb nutrients from food well
• Parathyroid glands in the neck do not produce enough parathyroid hormone (PTH)
• Very low levels ofvitamin D
25
Calculation:

Mg calcium excreted per day=ml of KMNO4 require*2* Vol of 24 hr urine/100

Report: The amount of calcium excreted per day =

Normal value: If you are eating a normal diet, the expected amount of calcium in the urine is
100 to 300 mg/day. If you are eating a diet low in calcium, the amount of calcium in the urine
will be 50 to 150 mg/day.

26
 EXP.NO 10 Quantitative Estimation of serum cholesterol by Libermann Burchard’s
method

Aim:

To estimate the amount of cholesterol present in saerum by Libermann Burchard’s method.

Principle:

Cholesterol reacts with ferric chloride in acetic acid and con. H2SO4 to give reddish pink
colour which is measured colorimetrically at 540 nm.

Reagents:

Stock ferric chloride (2.5 mg/dl)

Working ferric chloride (0.05 mg/dl)
Cholesterol standard (100mg/dl)
Concentrated sulphuric acid
Serum
2/3 N sulphuric acid
10% sodium tungstate
Procedure:

Take 3 test tubes. Label them as test, standard and blank. Pipette out the contents according
to table 1. Mix well by stirring with glass rod vigorously. Stand for five minutes. Centrifuge.
Take 3 test tubes. Label them as test, standard and blank. Pipette out the contents according
to table 2. Mix well by stirring with glass rod vigorously. Stand for five minutes. Measure the
optical density at 540 nm.

Table of Cholesterol Values

mg/dL: mmol/L:
Total Cholesterol
Desirable < 200 < 5.1
Borderline high 200 – 239 5.1 - 6.1
High > 239 > 6.1

27
Clincal conditions:
Hypercholesterimia
▪ Arthreoscelerosis
▪ myocardial infarction
▪ stroke
▪ peripheral vascular disease
Hypocholesterimia
▪ cancer
▪ depression
▪ cerebral haemerrohage
Table 1
S.NO Reagents STD Test Blank

1 Plasma/ serum 0.1 ml

2 Std cholesterol 0.1 ml

3 Working FeCl3 10 ml 10 ml 10 ml

4 10% sodium tungstate 0.5 ml 0.5ml 0.5 ml

5 2/3 N H2SO4 0.5 ml 0.5ml 0.5 ml

Table 2

S.NO Reagents STD Test Blank

1 Supernatant 5 ml 5 ml 5ml

2 Con H2SO4 3 ml 3 ml 3 ml

Report:

Calculation:

Concentration of standard= 1 mg/ml

Concentration of test= OD of test-OD of Blank/OD of std-OD of Blank* Conc of standard.

Normal value=140-250 mg/dl

28
 EXP.NO 11 Preparation of Folin wu filtrate from blood

Aim:

To prepare folin wu filtrate from blood.

Principle

I n 1919 Folin and Wu (1) proposed the preparation of protein-free blood filtrates by
precipitation of the proteins with the tungstic acid formed by the addition of 1 part 10 per
cent sodium tungstate and 1 part 8 N sulfuric acid to 1 part blood and 7 parts water. They
stated that the filtrates are nearly “neutral,” and that 10 ml. of filtrate should require only
about 0.2 ml. of 0.1 N NaOH for neutralization; Tungstic acid precipitation is usually thought
to result from combination of the acid anion with the cationic form of protein (protein+),
requiring the protein to be on the acid side of its isoelectric point. The isoelectric points of
serum proteins fall in the range of approximately pH 5 to 7 (3). It would be expected,
therefore, that Folin-Wu filtrates would have to have a pH of about 5 or less for clarity and
minimal protein content.

Reagents:

2/3 N Sulphuric acid

Sodium hydroxide

10% Sodium tungstate

20% Potassium oxalate

Procedure

Collect 5 ml of venous blood in a test tube. Add 1 drop of 20% Potassium oxalate. Add 5ml
of 2/3N sulfuric acid and 5 ml of 10 per cent sodium tungstate . Mix well. Allow to stand for
10 minutes. Filter through whattman filter paper.

29
 EXP.NO 12 Quantitative Estimation of blood creatinine by Jaffe’s method

Aim:

To estimate the amount of creatinine present in urine byJaffe’s method.

Principle:

Creatinine reacts with alkaline picrate reagent to form a reddish orange coloure complex
called creatinine picrate. Alkaline is measured at green filter at 540 nm. The intensity of
colour is proportional to amount of creatinine present.

Reagents:
10% sodium tunstate
2/3 N H2SO4
0.75N sodium hydroxide
0.04M picric acid.
Procedure:

S.NO Reagent Blank Standard Test

1 Water 4 ml 3.5 ml 3.5 ml
2 Serum 0.5 ml
3 Working std 0.5 ml
4 10% sodium tunstate 0.5 ml 0.5 ml 0.5 ml
5 2/3 N H2SO4 0.5 ml 0.5 ml 0.5 ml

Mix it and wait for 10 minutes and centrifuge.

S.NO Reagent Blank Standard Test

1 Water 4.0 ml 2.0 ml 2.0 ml
2 Working std 2.0 ml
3 Filtrate test 2.0 ml
4 Picric acid 1 ml 1ml 1ml
5 NaOH 1 ml 1ml 1ml

30
Mix it and wait for 10 minutes and take the reading at 540 nm.

Normal value

A normal result is 0.7 to 1.3 mg/dL for men and 0.6 to 1.1 mg/dL for women.

Clinical significance:

Higher than normal levels may be due to

• Acute tubular necrosis

• Dehydration
• Diabetic nephropathy
• Eclampsia (a condition of pregnancy that includes seizures)
• Glomerulonephritis
• Kidney failure
• Muscular dystrophy
• Preeclampsia (pregnancy-induced hypertension)
• Pyelonephritis
• Reduced kidney blood flow (shock, congestive heart failure)
• Rhabdomyolysis
• Urinary tract obstruction

Lower than normal levels may be due to:

Muscular dystrophy (late stage)

• Myasthenia gravis
• Alport syndrome
• Amyloidosis
• Atheroembolic kidney disease
• Chronic kidney disease
• Cushing syndrome
• Dementia due to metabolic causes
• Diabetes
• Digitalis toxicity
• Ectopic Cushing syndrome
• Generalized tonic-clonic seizure
• Goodpasture syndrome

31
Calculation:

Concentration of standard= 1 mg/ml

Concentration of test= OD of test-OD of Blank/OD of std-OD of Blank* Conc of standard.

Report:

32
 EXP.NO 13 Quantitative Estimation of blood sugar by Folin Wu tube method

Aim: To estimate the amount of blood sugar by Folin Wu method

Principle: Glucose reduces the cupric ions present in the alkaline copper reagent to cuprous
ions or the cupric sulfate is converted into cuprous oxide, which reduces the
phosphomolybdic acid to phosphomolybdous acid, which is blue when optical density is
measured at 420 nm.

Reagents Required:

Alkaline copper-reagent: Dissolve 40 gms of anhydrous sodium carbonate (Na2CO3) in about

400 mL of water and transfer it into a 1-liter flask. Dissolve 7.5 gms of tartaric acid in this
solution and 4.5 gms of CuSO4, which is dissolved in 100 mL water. Mix the solution and
make it up to 1 liter.

1. Phosphomolybdic acid reagent: Add 5 gms of sodium tungstate to 35 gm of

phosphomolybdic acid. Add 20 mL 10% NaOH and 50 mL distilled water boiled
vigorously for 20 to 40 minutes so as to remove the NH3 present in molybdic acid.
Cool it and dilute to 350 mL. Add 125 mL of o-phosphoric acid and make the volume
up to 500 mL.
2. Standard glucose sodium: Dissolve 100 mg of glucose in 100 mL distilled water and
take 10 mL from this sodium and make it up to 100 mL.

Procedure:
Pipette out standard glucose sodium 0 to 1-mL range and make up the sodium to 2 mL with
distilled water. Add 2 mL of alkaline Cu-reagent to all the test tubes. Mix the contents and
keep them in a boiling water bath for 8 minutes. Cool and then add 2 mL of
phosphomolybdic acid reagent to all the test tubes. Wait for 10 minutes and mix the content.
Make up the volume to 25 mL with distilled water. Take the optical density at 420 nm.

S.No Reagent Blank Standard Test

1 Water 4 ml 3.5 ml 3.5 ml
2 Serum 0.5 ml
3 Working std 0.5 ml
4 10% sodium tunstate 0.5 ml 0.5 ml 0.5 ml

33
 5 2/3 N H2SO4 0.5 ml 0.5 ml 0.5 ml
S.No Reagent Blank Standard Test
1 Test solution 1 ml
2 Standard solution 1ml
3 Water 1ml
4 Alkaline Cu-reagent 2 ml 2 ml 2 ml
5 Phosphomolybdic acid 2 ml 2 ml 2 ml
reagent

Calculation:

Concentration of standard= 1 mg/ml

Concentration of test= OD of test-OD of Blank/OD of std-OD of Blank* Conc of standard.

Normal value

Blood sugar calculator provides a description of value of blood sugar in terms of mg/dl
depending on the test type – Fasting sugar, post-meal or post prandial and Glucose tolerance
test (GTT) for a normal person, in early diabetes and in established diabetes.

Category of a Fasting Value Post Prandial

person
Minimum Value Maximum Value Value 2 hours after
consuming glucose
Normal 70 100 Less than 140
Early Diabetes 101 126 140 to 200
Established Diabetes More than 126 - More than 200

* All values are in Milligrams

Uncontrolled or high blood sugar levels can lead to health complications such as blindness,
heart disease, kidney disease.vIncrease in concentration of glucose in the blood leads to a
condition called "diabetic coma" or hyperglycemia.Note: Folin Wu tube is a specially
designed tube for preventing reoxidation of Cu2O by atomospheric oxygen
Report:
34
 EXP.NO 14 Estimation of SGOT in serum

Aim:

To estimate the amount of SGOT in the given serum

Principle:

SGOT catalyses the transfer of amine group between L-Aspartate and ∞ keto glutarate to
form oxaloacetate and glutamate. The oxaloacetate formed reacts with NADH in the presence
of malate dehydrogenase to form NAD. The rate of oxidation of NADH to NAD is measured
as a decrease in the absorbance which is proportional to the SGOT activity in the sample.

L-Aspartate + α keto glutarate Oxaloacetate+ L-glumate

Oxaloacetate+ NADH + H Malate+ NAD

Reagents:
Starter reagent
Enzyme reagent
Procedure:

Pipette into a clean dry test tube labeled as test (T). Add 0.8 ml of enzyme reagent to 0.1 ml
of sample. Incubate at the assy temperature for 1 minute and add 0.2 ml 0f starter reagent.
Mix well and calculate the mean absorbance change/minute.

Addition sequence T 25◦ C T 37◦ C

Enzyme reagent 0.8 ml 0.8 ml
Sample 2 ml 2 ml
Incubate at the assay temperature for 1 min and add
Starter reagent L2 0.2 ml 0.2 ml
Addition sequence T 25◦ C T 37◦ C
Enzyme reagent
Incubate at the assay temperature for 1 min and add
Sample 0.2 ml 0.2 ml

35
Normal Value

Serum (male): upto 37 µ/L at 37◦ C

Serum (female): upto 31 µ/L at 37◦ C

SGOT increased in

▪ Myocardial infarction,

▪ Acute pancreatitis,

▪ Acute hemolytic anemia,

▪ Severe burns,

▪ acute renal disease,

▪ Musculoskeletal diseases, and trauma

Report:

The amount of SGOT was found to be

36
 EXP.NO 15 Estimation of SGPT in serum

Aim:
To estimate the amount of SGPT in the given serum
Principle:
SGPT catalyses the transfer of amine group between L-Alanine and ∞ keto glutarate to form
pyruvate and glutamate. The pyruvate formed reacts with NADH in the presence of lactate
dehydrogenase to form NAD. The rate of oxidation of NADH to NAD is measured as a
decrease in the absorbance which is proportional to the SGPT activity in the sample. SGPT is
found in a variety of tissues but is mainly found in the liver. Increased levels are found in
hepatitis, cirrhosis, obstructive jaundice and other hepatic disease. Slight elevation on the also
seen in myocardial infarction.
L-Alanine + ∞ keto glutarate Pyruvate+ L-glumate
Pyruvate+ NADH + H Lactate+ NAD
Reagent:
Working reagent: for sample , start assys a single reagent is required. Pour the contents of
the bottle starter into bottle of Li(enzyme reagent). Working reagent is stable for atleast three
weeks, when stored at 2-8 ◦ C. Alternatively flexibility as much of working may be made as
and when desired mixing together 4 parts of Li and 1 part of starter reagent(L2).
Procedure
Pipette the contents mentioned in table. Add 0.8 ml of enzyme reagent and 0.1 ml of sample.
Incubate at assay temperature for 1 min and add 0.2 ml of starter reagent. Mix well. Read the
absorbance at 340 nm.
Addition sequence T 25◦ C T 37◦ C
Enzyme reagent 0.8 ml 0.8 ml
Sample 2 ml 2 ml
Incubate at the assay temperature for 1 min and add
Starter reagent L2 0.2 ml 0.2 ml

Addition sequence T 25◦ C T 37◦ C

Enzyme reagent
Incubate at the assay temperature for 1 min and add
Sample 0.2 ml 0.2 ml

37
Normal values:

3-15 Iu/L

Clinical significance

Increased in

▪ Tissue damage

▪ Viral hepatitis

▪ Malignant cholestasis

Report:

38
 EXP.NO 16 Estimation of urea in serum

Aim:

To estimate urea in serum.

Principle:

Urea reacts with diacetyl monoxime under acidic condition in the presence of ferric ions and
thiosemicarbazide to give pink colour complex. The intensity of pink colour is a measure of
amount of urea under present in blood.

Reagents:

10% Sodium tungstate

2/3 N H2SO4

DAM/TSC

Acid mixture

Std urea solution –Stock 1 G%, Std-50 mg%(5ml of stock diluted to 100ml with
water.

Procedure:

Take 3 centrifuge tubes. Label them as B(Blank),S(Standard),T (test).

S.No Reagent Blank Standard Test

1 Water 4 ml 3.5 ml 3.5 ml
2 Serum 0.5 ml
3 Working std 0.5 ml
4 10% sodium 0.5 ml 0.5 ml 0.5 ml
tunstate
5 2/3 N H2SO4 0.5 ml 0.5 ml 0.5 ml

Mix and keep them for 5 minutes. Centrifuge to get a clear filtrate. Take 3 test tubes. Label
them as B(Blank),S(Standard),T (test).

39
S.No Reagent Blank Standard Test
1 DAM TSC reagent 1.5ml 1.5 ml 1.5 ml
2 Urea acid reagent 1.5ml 1.5 ml 1.5 ml
3 Distilled water 20 µl
4 Standard 20 µl

5 Un known sample 20 µl
Normal range

Blood urea-15 -40 mg/dl

Urinary excretion/day-20-30gm/day

Clinical significance

Increase in blood urea level is called uremia.It occurs mainly in kidney disease. It may be
renal, pre renal, post renal.

Pre renal causes Renal causes Post renal causes

Dehydration Acute glomerulo nephritis Prostatic enlargement

Haemetamesis Chronic nephritis Stones in kidney

Haemorrhage Renal failure Tumours of bladder

Shock due to burns Malignant hyper tension

Cardiac failure Renal tuberculosis

Report:

40
 EXP.NO 17. Estimation of protein in serum

Aim:

To estimate protein in serum.

Principle:

The Biuret reaction involves a reagent containing copper (cupric) ions in alkaline solution.
Molecules containing 2 or more peptide bonds associate with the cupric ions to form a
coordination complex that imparts a purple color to the solution at 540 nm. The purple color
of the complex can be measured independently of the blue color of the reagent itself with a
spectrophotometer or colorimeter.

Reagents

Biurett reagent

Procedure:

Take 3 centrifuge tubes. Label them as B(Blank),S(Standard),T (test).

S.No Reagent Blank Standard Test

1 water 4 ml 3.5 ml 3.5 ml
2 Serum 0.5 ml
3 Working std 0.5 ml
4 10% sodium tunstate 0.5 ml 0.5 ml 0.5 ml
5 2/3 N H2SO4 0.5 ml 0.5 ml 0.5 ml
Mix and keep them for 5 minutes. Centrifuge to get a clear filtrate. Take 3 test tubes. Label
them as B(Blank), S(Standard), T (test).

S.No Reagent Blank Standard Test

1 Biurett reagent 1.0ml 1.0 ml 1.0 ml
2 Distilled water 20 µl
3 Standard 20 µl
4 Un known sample 20 µl
The normal range for total serum protein is 6-8 g/dl.

41
Report:

42
 EXP.NO 18. Determination of serum bilirubin

Aim:

To estimate bilirubin in serum.

Principle:

Bilirubin in serum reacts with diazotized sulfanilinic acid(Vanden Burg reagent) to form
purple coloured complex , azobilirubin. The intensity of purple colour is measured at 540 nm.
To solubize un conjugated bilirubin , methanol is used as an accelerator.

Procedure: Take 3 centrifuge tubes. Label them as B(Blank),S(Standard),T (test).

S.No Reagent Blank Standard Test

1 Water 4 ml 3.5 ml 3.5 ml
2 Serum 0.5 ml
3 Working std 0.5 ml
4 10% sodium tungstate 0.5 ml 0.5 ml 0.5 ml

5 2/3 N H2SO4 0.5 ml 0.5 ml 0.5 ml

Mix and keep them for 5 minutes. Centrifuge to get a clear filtrate. Take 3 test tubes. Label
them as B(Blank),S(Standard),T (test).

S.No Reagent Blank Standard Test

1 Vandenburgreagent 0.5ml 0.5 ml 0.5 ml
2 Distilled water 0.2ml
3 Standard 0.2ml
4 Un known sample 0.2ml

Clinical significance:

Present in jaundice.

43
Report:

44
 EXP.NO 19. Determination of Glucose by glucose oxidase

Aim:

To estimate glucosein serum.

Principle:

Glucose oxidase oxidises glucose to glucoronic acid andhydrogen peroxide. In presence of

enzyme peroxidise hydrogen peroxide is coupled with ortho dianisidine oxidises to red
chromophore. Absorbance of dye is measured at 505nm

Reagents:

Glucose oxidase peroxidase reagent: Dissolve 25 mg ortho dianisidine in 1 ml ethanol. Add

49 ml of 0.1 M phosphate buffer (pH6.5). Add 5 mg of peroxidise and 5 mg of glucose
peroxidise.

Procedure:

Take 3 centrifuge tubes. Label them as B (Blank),S(Standard),T (test).

S.No Reagent Blank Standard Test

1 Water 4 ml 3.5 ml 3.5 ml
2 Serum 0.5 ml
3 Working std 0.5 ml
4 10% sodium tunstate 0.5 ml 0.5 ml 0.5 ml
5 2/3 N H2SO4 0.5 ml 0.5 ml 0.5 ml
Mix and keep them for 5 minutes. Centrifuge to get a clear filtrate. Take 3 test tubes. Label
them as B(Blank),S(Standard),T (test).

S.No Reagent Blank Standard Test

1 Glucose oxidase peroxidise reagent 1.0ml 1.0 ml 1.0 ml
2 Distilled water 0.2ml
3 Standard 0.2ml
4 Un known sample 0.2ml

45
 EXP.NO 20. Hydrolysis of starch by amylase

Aim: To determine the hydrolysis of starch by salivary amylase .

Procedure:

Collect approximately 5 mL of saliva in a medium test tube. Place 2 droppers full of 1 %

starch solution in each of 5 medium test tubes. Number the test tubes 1-5. Place the tubes in
a 37-40 °C water bath. After 5 minutes, add the following amounts of saliva to the test tubes
as quickly as possible, mixing each solution thoroughly, and then returning the tubes to the
water bath. Do not overheat the bath or you will inactivate the enzyme.

Test tube Number of drops of

number saliva
1 1

2 2

3 5

4 10

5 20

Prepare a spot plate for testing for the presence of starch in the samples by placing one drop
of iodine reagent in each of five depressions on the spot plate. Two minutes after the addition
of saliva, transfer one drop from each test tube (using a different pipet for each tube) to a
separate drop of iodine in the spot plate. Note the color produced for each. Remember that the
complex formed by starch and iodine is an indigo blue. If the color of the iodine solution
remains red or gold after adding the starch solution, the starch has been completely
hydrolyzed. As soon as one of your starch solutions has hydrolyzed, use it to begin preparing
the solutions for Parts B and C below. Continue to test the remaining starch solutions that
have not yet hydrolyzed. Clean the spot plate and then prepare it for the next testing by
placing one drop of iodine reagent in each of five depressions. Repeat the testing at 5 minutes
after the addition of saliva, and at 5 minute intervals thereafter. Continue testing for 20
minutes, or until the blue-black color no longer appears for each sample. Record the time
required for the hydrolysis of starch in each sample.

Report:

46
 EXP NO 21. Determination of Na+ and K+ in solution by flame photometry

Aim: To determine Na+ and K+ in solution by flame photometry

Reagents:

Oral rehydration sachet

NaCl standards: 0.25, 0.5, 1.0, 2.0, 4.0 and 5.0 mM

KCl standards: 0.1, 0.2, 0.5, 1.0, 1.5, 2.0 mM

Procedure:

1. Carefully open an oral rehydration sachet and empty the contents into a clean 250 ml
beaker. Add about 150 ml distilled water and gently swirl the contents until dissolved.

2. Pour the solution into a 200 ml volumetric flask and rinse out the beaker with small
amounts of distilled water, adding the washings to the flask. Finally, make up the flask to
exactly 200 ml and mix thoroughly.

3. Make a 1/50 dilution of the redissolved sachet solution by accurately pipetting 2 ml of the
solution into a 100 ml volumetric flask and making up to 100 ml with distilled water.
Retain the undiluted solution for Experiment B.

Instructions for use of the flame photometer:

4. Ensure that the photometer drain is leading into a sink and that the instrument is
connected to gas, air and electricity supplies. Ensure the mains supply gas tap is off.

5. Turn the "Sensitivity" and instrument "Gas" controls control fully counterclockwise
(towards you).

6. Insert the sodium optical filter.

7. Switch on the instrument and unclamp the galvanometer by turning counterclockwise.

8. Open the mica window, turn on the mains gas supply, light the gas and close the window.

(CAUTION: DO NOT LEAN OVER THE INTRUMENT OR YOU WILL SET YOUR
HAIR ALIGHT)

9. Turn on the air supply control and adjust the air pressure to 10 lb/in2.

Leave for 1-2 minutes to stabilise.

10. Place a beaker of distilled water into position at the left hand side of the instrument and
insert the narrow draw tube into it to allow water to pass through the photometer.
47
 (NOTE: once set up, the photometer must have water running through it at all times
when a salt solution is not being measured. The rate of uptake is fast, so make sure there
is always enough water in the beaker).

11. Adjust the gas control to give a flame with a large central blue cone then, with water
passing through the instrument, slowly close the gas control until ten separate blue cones
just form.

12. Set the galvanometer to zero using the "Set zero" control.13. Replace the distilled water
with the 5 mM NaCl standard and adjust the "Sensitivity" control till the galvanometer
reads 100.

14. Quickly but carefully, replace the 5 mM NaCl standard with standards of decreasing
concentration from 4 mM to 0.25 mM and note the readings in the Table below.

15. Run water through the instrument again for 1-2 min then place the draw tube into a
beaker containing the 1 in 50 diluted rehydration sachet solution and note the
galvanometer reading.

16. Run water through the instrument again and replace the sodium with the potassium filter.

17. Repeat the above procedure with the KCl standards, setting to 100 with 2.0 mM KCl, then
reading the others in reverse order. Then read the 1 in 50 diluted rehydration sachet
solution.

18. Finally, run water through the instrument until the flame appears free of colour again.

19. When the instrument is no longer required, switch off in the following

▪ Turn off the gas control and the mains gas supply

▪ Wait for the flame to die out.

▪ Turn off the air supply.

▪ Switch off the electricity

▪ Clamp the galvanometer

[Na +]mM 5.0 4.0 2.0 1.0 0.5 0.25 0

Galvoreading 100 0
[K +]mM 5.0 4.0 2.0 1.0 0.5 0.25 0
Galvoreading 100 0

48
Plot the galvanometer readings against Na+ and K+ concentrations on the graph paper
provided (separate graph for each ion) and from these calibration curves determine the Na+
and K+ concentrations in the diluted sachet solution. Finally, calculate the Na+ and K+
concentrations in the undiluted sachet solution.

Galvonometer Diluted Undiluted

reading concentration concentration
Sodium ion
Potassium ion

Report:

The normal range for blood sodium levels is 135 to 145 milliequivalents per liter (mEq/L)

Abnormal sodium levels can be due to many different conditions.

A higher than normal sodium level is called hypernatremia. It may be due to:

• Cushing syndrome
• Diabetes insipidus
• Hyperaldosteronism
• Increased fluid loss due to excessive sweating, diarrhea, use of diuretics, or burns
• Too much salt or sodium bicarbonate in your diet
• Use of certain medicines, including birth control pills, corticosteroids, laxatives,
lithium, and NSAIDs such as ibuprofen or naproxen

A lower than normal sodium level is called hyponatremia. This may be due to:

• Addison's disease
• Dehydration, vomiting, diarrhea
• An increase in total body water seen in those with heart failure, certain kidney
diseases, or cirrhosis of the liver
• Ketonuria
• SIADH

49
50
 EXPNO 22. Determination of serum calcium

Aim:

To determine serum calcium

Principle:

Serum calcium combines with calcein-thymolphthalein indicator to form a bluish green

coloured complex in alkaline medium. On titration with EDTA calium is releasedfrom the
complex. The released calium gets chelated with EDTA. The titer value is compared with that
of standard solution treated in the same way.

Reagents:

1. Alkaline buffer: pH12.66 Mix 0.1M glycine (1.5 gm per 200ml) and 80ml of
0.1M sodium hydroxide.

2. EDTA disodium salt solution: dissolve 0.93 gms of disodium EDTAin water
and make upto 1 litre.

3. Stock Standard solution: weigh 2.5 gms of calcium carbonate. add 200ml
water and 50 ml HCl. Allow it stand overnight. Make upto 1 litre with water.

4. Workig standard: 10ml stock to 100ml with distill water.

5. calcein-thymolphthalein indicator: grind 0.2 gm calcein, 0.12g of

thymolphthalein 20 gm KCl together in mortor.

Procedure

Pipette 1ml of serum into 5ml of buffer solution in a china dish and add a
small amount of indicator (1mg). Titrate with EDTA. End point is colour change from orange
green to pink. Repeat this with standard solution. Here colour change is from yellowish green
to mauve. Carry out a blank determination using water.

Calculation

Mg Calcium/100ml serum = tv of test- tv of blank / tv of std-tv of blank

51
Clinical significance

Normal range: Normal values range from 8.5 to 10.2 mg/dL.

Higher than normal levels may be due to a number of health conditions. Common causes
include:

• Being on bed rest for a long time

• Taking too much calcium or vitamin D
• HIV/AIDS
• Hyperparathyroidism
• Infections that cause granulomas such as tuberculosis and certain fungal and
mycobacterial infections
• Metastatic bone tumor
• Multiple myeloma
• Overactive thyroid gland (hyperthyroidism) or too much thyroid hormone
replacement medication
• Paget's disease
• Sarcoidosis
• Tumors producing a parathyroid hormone-like substance
• Use of certain medications such as lithium, tamoxifen, and thiazides

Lower than normal levels may be due to:

• Hypoparathyroidism
• Kidney failure
• Liver disease
• Magnesium deficiency
• Disorders that affect absorption of nutrients from your intestines
• Osteomalacia
• Pancreatitis
• Vitamin D deficiency

Report:

52
 EXP NO 23 Preparation of buffer and measurement of pH

Aim: to prepare buffer solution and measure their pH

Procedure:

Phosphate salts are known by several names and the correct phosphate must be used to
prepare buffer solutions.One phosphate cannot be substituted for another phosphate.

pH=7.00 :
Add 29.1 ml of 0.1 molar NaOH to 50 ml 0.1 molar potassium dihydrogen phosphate.

Alternatively :
Dissolve 1.20g of sodium dihydrogen phosphate and 0.885g of disidium hydrogen phosphate in
1 liter volume distilled water.

For pH= 4.00 :

Add 0.1 ml of 0.1 molar NaOH to 50 ml of 0.1 molar potassium hydrogen phthalate .

Alternatively :
Dissolve 8.954g of disodium hydrogen phosphste.12 H2O and 3.4023g of potassium
dihydrogen phosphate in 1 liter volume distilled water.

Measuring the pH of an buffer Solution

1. Calibrate the electrode.

2. Rinse the electrode with distilled water.
3. Rinse the electrode with a small portion of the analyte solution.
4. Place the electrode in the analyte solution.
5. Wait a moment for the meter to stabilize, then record the pH from the meter's display.
6. Remove the electrode and rinse it with distilled water.

Report:
53
54
 EXP NO 24 Determination of Total Cholesterol

AIM: To determine the total cholesterol concentration in plsma.

REAGENTS:

1. 4-Amino antipyrine
2. Phenol
3. 3,5-dichlorophenol
4. Cholesterol esterase
5. Cholesterol oxidase
6. Peroxidase
7. Phosphate buffer.

PROCEDURE:

(i) To 1000ml of reagents in groups meant for sample,standard and blank, 10ml of
sample, standard and distilled water is added respectively.
(ii) The contents were mixed and incubated for 5 mins at 37oC.
(iii) The absorbance are measured at 546nm.

CALCULATION:

Conc.of cholesterol = absorbance of test

in plasma (mg/dl) absorbance of std

Report:

55
56
 EXP NO 25 Estimation of HDL Cholesterol

AIM: To estimate the HDL cholesterol concentration in plasma.

PRINCIPLE:

LDL and chylomicrons fractions are precipitated quantitatively by the addition of

phospholugstic acid in the presence of magnesium ions. After centrifugation of the cholesterol,
concentration in the HDL fraction which remains in the supernatant is determined.

PROCEDURE:

(i) Into centrifuged tubes, 500ml of sample and 100ml of HDL cholesterol
precipitant was added.
(ii) Mixed and allowed to stand for 10mins at room temperature.
(iii) Then the tubes were centrifuged at 4000 rpm.
(iv) 100 ml of supernatant were assayed for total cholesterol estimation with the
procedure given above for total cholesterol estimation.

CALCULATION:

Conc. of HDL = absorbance of test

in (mg/dl) X conc.of std
absorbance of std

57
58
 EXP NO 26 ESTIMATION OF LDL

AIM: To estimate the amount of LDL(Low Density Lipoprotein) present in the given serum

PRINCIPLE:

When the serum is reacted with polyethylene glycol obtained in the precipitating
reagent,all the VLDL and LDL are precipitated.The HDL remins in the supernatant and is
then assayed as a sample for cholesterol using the cholesterol reagent. Lipoproyeins are the
proteins which mainly transport fats in the blood stream. They can be grouped into
chylomicrons, VDL, low density lipoproteins(LDL) and HDL. Chylomicrons and VLDL also
transport mainly triglycerides, though VLDL also transport some amount of cholesterol. LDL
carries cholesterol to the peripheral tissues where it can be deposited and increase the risk of
arterisclerotic heart and peripheral vascular diseases . Hence high levels of LDL are atheroge

REAGENTS:

Precipitating reagent 10ml

LDL cholesterol standard(25mg/dl) 5ml

PROCEDURE:

Pipette into the test tube according to the table. Add 1ml of working reagent in the
blank , standard and test; distilled water(.05) in blank and .05ml of LDL standard in standard
and also the supernatant about .05ml.

REPORT:

The amount of LDL present in the given serum was found to be

59