Department of Biochemistry

Government medical college Surat

Student Journal for Practical Biochemistry
 Certificate

This is to certify that _________________________________

student of 1st MBBS,Roll No ____________,Year of
Admission _________ ,has completed training in
practical biochemistry at Department of Biochemistry
Government Medical College, Surat.

Tutor Professor and Head

Department of Biochemisty Department of Biochemistry
Government Medical College Government Medical College
Surat Surat

2
INDEX
Page
No Exercise Date Sign
No
1 Introduction to Practical Biochemistry 04
2 Chemistry of Carbohydrates 06

3 Chemistry of Proteins and Amino acids 17

4 Chemistry of lipids 27

5 Physiological urine 30

6 Pathological urine 37

7 Estimation of acid output by stomach 44

8 Secretion and buffering of acids by kidney 51

9 Colorimetry 56

10 Estimation of Serum Creatinine 61

11 Estimation of Plasma Glucose 64

12 Estimation of Serum Cholesterol 66

13 Estimation of Serum Bilirubin 68

14 Estimation of Serum Total Protein 71

15 Estimation of Serum Albumin 73

16 Estimation of Cerebro Spinal fluid protein 76

17 Estimation of serum uric acid 78

18 Electrophoresis 80

19 Chromatography 83

20 Clinical Cases 85
21 Medical Biochemistry – Syllabus 94
22 Subject distribution and Paper Style 96

3
4
 1. Introduction to Practical Biochemistry
Practical Biochemistry serves several purposes to medical students.
Practical Biochemistry augments concepts learnt in classroom. e.g.
Study of properties of basic biomolecules. e.g Carbohydrate, Proteins
Study of biochemical investigative tools e.g colorimetry, chromatography,
electrophoresis
Study of patient case histories in light of its laboratory investigations is
fundamental to understanding medical aspects of biochemistry.
It prepares the student for possible use of the practical techniques in
clinical practice. e.g.
Many bedside biochemistry diagnostic technologies are used by
physicians themselves. Such technologies are called point-of-care-
technologies (POCT). They are based on many simple concepts studied in
practical biochemistry.
Many biochemistry diagnostic technologies are used by patients
themselves. Such home monitoring by patients require support from their
physicians. Practical biochemistry help medical students for supporting
their patient’s for such support.
Hazards in Clinical Biochemistry laboratory
Hazards arises from three main basic sources
1. From dangerous chemicals
2. From infected specimen sent for analysis
3. From faulty apparatus & instruments
These are further increased by carelessness, untidiness, faulty hygiene,
conduct of staff, unsatisfactory working condition.
Wide variety of articles are used like conical flasks, ,volumetric flasks,
tube, measuring cylinders, pipettes, reagent bottles.

Disposal of laboratory Waste

There are guidelines to dispose waste.It is recommended that waste
should be segregated at the point of generation & disposed in bags with
correct colour coding.

5
Questions:
Describe any laboratory accident you or your schoolmate has suffered in
your school days. How will/was it be first-aid? How will you prevent it?

Give list of Biochemistry POCT and home-monitoring technologies.

Explain each of them.

Mention five more points to be noted for laboratory safety.

6
7
2.Chemistry of Carbohydrates
Test solution
Glucose solution(400mg/dl) : Dissolve 4 gm of glucose powder in 1000
ml water
Starch solution(1%): Add 10 gm of starch powder in 100 ml of water
.Boil till solution become clear. Make up to 1 litre.
Sucrose solution( (400mg/dl) : Dissolve 4 gm of Sucrose powder in 1000
ml water
Fructose solution( (400mg/dl) : Dissolve 4 gm of Fructose in 1000 ml
water
Maltose solution( (400mg/dl) : Dissolve 4 gm of Maltose powder in
1000 ml water

Molisch’s test:
Reagent
1 % -Naphthol: Dissove 1 gm -Naphthol powder in 100 ml methanol
Conc.H2SO4
Principle
All carbohydrates when treated with conc. sulphuric acid undergo
dehydration to give fufural compounds. These compounds condense with
Alpha-napthol to form colored compounds.
Molish test is given by sugars with at least five carbons because it
involves furfurl derivatives,which are five carbon compounds.

8
9
Benedict’s Test:
All Reducing sugars give positive benedict's test.Reducing sugars have a
free aldehyde or keto group.
Reagent
Benedict’s Reagent:One liter of Benedict's solution contains ,
173 grams --------------------> sodium citrate,
100 grams ---------------------> sodium carbonate
17.3 grams -------------------->cupric sulphate pentahydrate.

With the help of heat,dissolve 173 gm of sodium citrate & 100 gm of

sodium carbonate in 800 ml of water.Dissolve 17.3 gm cupric sulphate
pentahydrate in 100 ml of water in different container.
Pour cupric sulfate solution in carbonate- citrate solution with constant
stirring& make upto 1000ml.

Role of ingradient of benedict's solution:

1.Sodium citrate:Holding of cupric oxide in alkaline solution
2.Sodium carbonate:provide alkaline pH
3.cupric sulphate pentahydrate:Reducing Agent

Principle
Glucose (R-CHO) + 2Cu2+ + 2H2O (Boil)
Gluconic acid ( R-COOH) +
Cu2O + 4H+

The principle of Benedict's test is that when reducing sugars are heated in

the presence of an alkali(pH 10.6), they get converted to powerful reducing

2+
compounds known as enediols. Enediols reduce the cupric ions (Cu )

+
present in the Benedict's reagent to cuprous ions (Cu ) which get

precipitated as insoluble red copper(I) oxide.

The color of the obtained precipitate gives an idea about the quantity of

sugar present in the solution, hence the test is semi-quantitative.

10
Carbohydrates giving positive Benedict’s test:
Glucose, Fructose, Galactose,
Ribose, Glucuronic acid,
Lactose, Maltose
Note: Sucrose with no free reducing group give negative test.

Non-Carbohydrates giving positive Benedict’s test:

High concentration of Uric acid , Creatinine and Ketones
Homogentisic acid (solution turns black due to black colored oxidized
homogentisic acid)
Vitamin C (even without Boiling)
Certain drugs like aspirin, cephalosporins
Starches
Starches do not react or react very poorly with Benedict's reagent, due to
the relatively small number of reducing sugar moieties, which occur only
at the ends of carbohydrate chains.
Different concentration of glucose gives different color of solution with
Benedict’s test, depending on amount of precipitate and residual cupric
sulphate.

Grade Color of Reaction Mixture Approximate Glucose

concentration
+ Green 0.5-1 gm%
++ Yellow 1-1.5 gm%
+++ Orange 1.5-2 gm%
++++ Red >2 gm%

Benedict’s test is frequently used to detect glucose in urine. Although

glucose is most frequent reducing substance present in urine, in some
patient positive Benedict’s test may be due to non-glucose reducing
substances listed above. This phenomenon may be called false positive
result.
Following test based on glucose oxidase is positive only with glucose in
urine.
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12
 Glucose oxidase test:
Reagent:
Glucose strip or liquid reagent based on GOD-POD method
Principle
Glucose Oxidase
Glucose + O2 Gluconolactone + H2O2

Spontaneous
Gluconolactone + H2O Gluconate

Peroxidase
H2O2 + (reduced colorless dye) Oxidized colored dye

Some of the dyes used are O-tolidine, tetramethylbenzidine, and

potassium iodide, 4-aminophenazome + phenol .

Reagents for this test are present on a strip of paper in solid form. When
the paper is wet with urine, the reagents dissolve in urine on paper and
react with glucose in urine. The darkness of color can be correlated with
amount of glucose present in urine.

Because Glucose oxidase enzyme can act only on beta-D-Glucose,

other reducing substances do not give this test positive. (Exception:
Galactose can react with glucose oxidase, but very slowly)

Following reaction occur when urine contain compounds reacting with

H2O2.

Glucose Oxidase
Glucose + O2 Gluconolactone + H2O2

Peroxidase
H2O2 + Vitamin C Oxidized Vitamin C + H2O

Thus, compounds like Vitamin C, Aspirin utilize H2O2 produced in the

reaction. Due to lack of H2O2, peroxidase can not oxidize dye. Thus,
glucose may not be detected even if present, if urine contain Vitamin C or
Aspirin in large amount. This phenomenon is called false negative
result.
In neonate, positive Benedict’s test in urine, in presence of negative
Glucose oxidase test, indicate possible presence of Fructose or
Galactose in urine. (But note the exception mentioned above). Fructose
and galactose are found in some inborn deficiency of enzymes of their
metabolic pathways.
Performed Benedict’s test and Glucose oxidase strip test with following
compounds and fill up the table given.
Compound Benedict test Glucose Oxidase Inference
13
 Strip Test
Fructose
Vitamin C
Glucose with Vitamin C
Cephalosporin Drugs

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Barfoed’s Test:
This test is based on the same principle as Benedict’s test. But, the test
medium is acidic. In acidic medium(pH 4.6) monosaccharides react
faster than disaccharide. Monosaccharides react fast within 1-2 minutes
but disaccharides take longer i.e. 7-12 minutes.
Reagent:
Barfoed's reagent: Dissolve 70 gm of cupric acetate monohydrate in 800
ml of water. Add 9 ml glacial acetic acid & make to 1000 ml with water.
Principle
Acidic pH(4.6),Heat
RCHO + 2Cu2+ + 2H2O RCOOH + Cu2O↓ + 4H+

Seliwanof’s Test
Seliwanoff’s test is a chemical test which distinguishes between aldose
and ketose sugars. This test is based on the fact that, when heated,
ketoses are more rapidly dehydrated than aldoses.
Reagent
Seliwanoff's reagent: Add 50mg of Resorcinol in 66 ml of water. Add 33 ml
concentrated HCL. Wear goggles. The reagent is colorless if red color develop,
discard it.
Principle
Ketohexoses like fructose on treatment with HCl form 5-
hydroxymethylfurfural, which on condensation with resorcinol gives a
cherry red complex.
Sucrose is hydrolyzed into glucose and fructose when boiled in acidic
medium of Seliwanoff’s reagent. Fructose, present in hydrolysate gives
positive Seliwanoff’s test.

15
Inversion Test:
Reagent
10 % HCL
40% NaOH : dissolve 40 gm of NaOH pellet in 100ml Water
Benedict's reagent
Seliwanoff's reagent
Principle
When sucrose is boiled with conc. HCl, It is hydrolyzed into its
constituent monosaccharides i.e. fructose and glucose. The hydrolyzed
glucose and fructose give Benedict’s test. Fructose gives seliwanoff’s test.

Sucrose is dextrorotatory. The optical rotation changes from

dextrorotatory to leavorotatory on hydrolysis, since fructose causes a
much greater leavorotation than the dextrorotation caused by glucose.
This is known as inversion. The resultant hydrolysate is called invert
sugar, which is sweeter than sucrose because fructose is sweeter than
sucrose.

Iodine test for starch

Reagent:
Iodine solution : Dissolve 1.27 gm Iodine and 3 gm potassium iodide
crystals in 100 ml water.Dilute 1:10 in water before use.
Iodine by itself is very poorly soluble in water. One way to dissolve iodine
in water is to add potassium or sodium iodine. Those salts dissolve into
potassium or sodium ions and iodine ions. The iodine ion (I-) reacts with
the free iodine (I2) to form a triiodide ion (I3-) which is soluble in water
and can react with glucose chains.

Principle
Iodine binds starch to give blue colored complex.

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When glucose chains are sufficiently long they coil up like springs. This
coil is supported by weak links between the glucose molecules. These
links break down at high temperatures and the glucose chains uncoil.
When the chains are longer than about 9 glucose molecules a triiodide
ion (I3-) fits inside the coil (Figure ).The longer the glucose chains are the
more iodine molecules fit into the coils and the more intense the color
reaction will be.

The resulting color depends on the length of the glucose chains. Shorter
chains (starting at about 9 glucose molecules in unbranched chains and
up to 60 glucose molecules in branches chains) give a red color .

Amylose, which consists of very long glucose chains between occasional

branch points and very large dextrines give a dark blue color .

while amylopectin, which has much more branch points and shorter
glucose chains between these branch points, gives a more reddish color in
the presence of iodine.

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 Hydrolysis Test for starch
When starch/dextrin is boiled with HCl, It is hydrolyzed into its
constituent monosaccharides i.e. glucose. Glucose, thus formed, gives
Benedict’s test.
TEST METHOD OBSERVATION INFERENCE
Molisch’s 1ml OS + 2 drops of α- Purple ring is Carbohydrate
Test napthol solution mix. formed at the present.
add 2 ml. of conc. junction of acid
Sulphuric acid carefully and solution.
through the side of the test
tube without shaking.
Benedict’s 5ml of Benedict’s reagent + Green / Yellow / Reducing Group
Test 8 drops of OS, mix Orange / Red / present.
Boil and cool. Brick Red
precipitates seen
Barfoed’s 1 ml OS + 1 ml Barfoed’s Red colored Disaccharides
Test reagent Boil for 30 sec , precipitates. At absent.
Cool the bottom of the Monosaccharide
Excess boiling or may give tube. present
false positive results.
Seliwanoff’s 1 ml O.S. + 1 ml Red colored Keto sugars
Test Seliwanoff’s reagent. formed. present e.g.
Boil and cool for 5 min Fructose
Iodine Test 1 ml OS + 2 drops of iodine Blue color Starch present.
solution, Mix develops.
Dextrine
Violet colour present.
develops.
Inversion 1 ml OS + 1ml of 10% HCl. Benedict’s and Sucrose is
Test Boil for 2 mins. Cool. Saliwanoff’s test present if OS
Make it alkaline with 5 are positive give negative
drops of 40% NaOH. Benedict’s test.
From this solution perform
Benedict’s test and
Saliwanoff’s test.
Hydrolysis Step-1: Perform Benedict’s Benedict’s test is Starch present
test for Test with OS. negative/ weakly (weak
starch/dextr Step-2: 5 ml OS + 2 drops positive Benedict’s test
in of conc. HCl . Boil for 2 with OS is due
mins. Cool. Benedict’s test is to free reducing
Make alkaline with 5 drops positive groups at end of
of 40% NaOH. starch
From this solution perform molecules.)

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 Benedict’s test
Glucose Method for the test will be Observation will Glucose present
oxidase test provided in the laboratory be explained in in the solution
( on strip or the laboratory
with liquid
reagents)
What you will do:

Perform tests mentioned in above table with various carbohydrates given

to you. Note down your observation and inference in tables as shown
below.

TEST OBSERVATION INFERENCE

Molisch’s Test

Benedict’s Test

Barfoed’s Test

Seliwanoff’s Test

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Iodine Test

Inversion Test

Hydrolysis test for

starch/dextrin

Glucose oxidase test (

on strip or with
liquid reagents)

Questions:

Explain biochemical reason why Sucrose gives negative Benedict’s test.

Why the hydrolysis of sucrose is called ‘Inversion test’?

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Does alpha-D-Glucose in the solution react with Glucose Oxidase?
Explain.

3. Chemistry of Proteins and Amino acid

Proteins are made up of amino acids. Amino acids differ from
each other in their side chain (-R group). The differing –R groups in
different amino acids are responsible for many reactions mentioned
below.

Preparation of Protein solutions:

Egg albumin solution (1:21): Mix 50 ml of egg(both white and yellow) in
1 liter of tap water. Use only for 24 hours
Gelatine solution(0.5%): Dissolve 5 gm of Gelatin powder in 50 ml of
water by slight Heating & make upto 1 liter
Peptone solution(0.5%) :Dissolve 5 gm of Peptone powder in 50 ml of
water by slight Heating & make upto 1 liter
Casein solution(0.5%) : Dissolve 5 gm of Casein powder in 20 ml of 40%
NaOH & make upto 1 Liter with water
Biuret Test
This test is given by all peptides having at least two peptide bonds. So, it
is given by all proteins.
Reagents:
10% NaOH: Take 10gm NaOH pellets and make it up to 100ml with DI
water.
1% CuSO4: 1 gm of
CuSO4 in 100 ml DI water.

Principle:

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Cupric ions of copper sulphate solutions in alkaline medium form
coordinate complex with at least two nitrogens of the peptide bonds to
form purple colored complex. Thus color intensity is proportionate to the
presence of number of peptide linkages.

Minimum of 2 peptide bonds (3 amino acids) are required for binding of

Cu2 + with peptide. single amino acids and dipeptides do not give positive
test.

The name of reaction is derived from organic compound biuret which is

formed by condensation of 2 urea molecules at high temperature.

Figure of Biuret
Biurat is formed when solid urea powder is heated in a tube. The
resultant Biurat is solid at room temperature and soluble in water.
The test produces color proportionate to number of peptide bonds which
can be correlated with amount of protein. Similar reagent is used for
estimation of serum proteins quantitatively.

Ninhydrin Test

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This test is given by all compounds having free α-Amino groups. ex:
peptides, proteins, free α- Amino acid. Different
Proline and hydroxyproline give yellow color in this test.
Prepare reagent:
1 % Ninhydrine solution : 1 gm of Ninhydrine powder disolved in 100 ml
DI water.
Principle:
Ninhydrine +α- Amino acid  hydrindantin + aldehyde + CO2 + NH3
Hydrindantin + NH3 + Ninhydrine  blue colored complex

Ninhydrin oxidises an α-amino acid to an aldehyde liberating NH3 and

CO2 and is itself reduced to hydrindantin. Hydrindantin then react with
NH3 and another molecule of ninhydrine to form a purple colored
complex.

All amino acids that have a free amino group will give positive result
(purple color) .While not free amino group-proline and hydroxy-proline
(amino acids) will give a (yellow color).
Note: Many substances other than amino acids, such as amines will yield
a blue color with ninhydrin, particularly if reaction is carried out on filter
paper.

Xanthoproteic Test:
This test is answered by aromatic amino acids. ( Tyrosine, Tryptophane)
Reagent:
Concentrated HNO3
40 % NAOH : 40 gm NAOH in 100 ml DI water.
Principle
Concentrated nitric acid causes nitration of activated benzene ring of
tyrosine and tryptophan. (Benzene ring is considered activated when
additonal groups are attached to it) The nitrated activated benzene is
yellow in color. It turns to orange in alkaline medium.. Phenylalanine also
contains benzene ring, but ring is not activated, so it does not undergo
nitration. The reaction can be hastened by heating. The heat may be
produced by dilution of concentrated HNO3 with OS or may require
heating.

23
Aldehyde Test
Reagents
1:500 Formaldehyde Reagent:
Take 1 ml of Formaldehyde solution (37-41 % W/V) and make upto 500
ml with DI water.Use only for 1 week. Old Formaldehyde may not give
test.
1 % Sodium Nitrite solution :
Take 1 gm sodium nitrite powder and make upto 100 ml with DI water.
Use only for 1 week. Old Sodium nitrite may not give test.
Sulphuric acid AR :
Use sulphuric acid Bottle directly for use as reagent. Use for 1 week.Old
Sulphuric acid may not give test

Principle
Indole ring is present in tryptophan. Formaldehyde react with
indole ring to give violet colored complexes in presence of H2SO4.
Addition of Sodium nitrite intensify and stabilize colour.

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Millon’s reagent
Reagent:
Millon's reagent:
Dissolve 10 gm of mercuric sulphate(HgSO4) +100ml DI water + 7 ml
Conc.H2SO4
1% sodium nitrite:1 gm in 100 ml DI water

Principle

Tyrosine has hydroxyphenyl(Phenol) group. The hydrophobic group is in

the core of protein. The protein is denatured by mercuric sulphate in
boiling water exposing hydroxyphenyl group. Sodium nitrite reacts with
sulfuric acid to form nitrous acid. The exposed hydroxyphenyl groups
react with nitrous acid. The compound formed chelates Hg2+ & give red
colour precipitates.

Sakaguchi’s Test
This test is for Guanido group Which is the R-group of arginine.
Reagent:
1%w/v α-Napthol: Dissolve 1 gm α-Napthol in 100 ml of methanol
10%w/v NaOH: Dissolve 10gm of NaOH & make it upto 100ml with DI
water.
Alkaline hypochloride : Make 100 ml 10 % NaOH & add 8 ml 5-6 %
Analytical grade Sodium hypochloride.
Principle
In an alkaline medium, alpha-Napthol combines with guanidino group of
arginine to form a complex, which is oxidized by bromine/chlorine.

Sulphur Test (Lead acetate test):

Reagent:

25
2% Lead acetate in 10% NaOH: add 20 gm lead acetate, 100 gm NaOH in
1 liter of water. There is no need to make exactly up to 1 liter. Above
solution will be more than 1 liter in volume.
Principle:
When protein containing cysteine & cystine is boiled with
strong alkali, organic sulphur(R-SH) is converted to sulphide (Na2S].
Addition of lead acetate to this solution causes precipitation of insoluble
lead sulphide (PbS), which is black-gray in colour. Methionine does not
give this test due to the presence of thioether linkage (H3C-S-CH2-R)
which does not allow the release of sulphur in this reaction.

Heat coagulation test:

Reagent:
1% acetic acid: 1 ml acetic acid up to 100 ml with DI water.
Principle
Proteins have net zero charge at their iso-electric pH (pI). So, at pI, protein
molecules have minimum repelling force. Thus proteins are easily
precipitated at pI. When proteins are heated, weak bonds like hydrogen-
bonds, salt bonds and van-der-wal forces are broken. Proteins are said to
be denatured.
Core hydrophobic regions of denatured Albumin can form
intermolecular associations and cause precipitation.Thus, in order to
precipitate proteins like albumin, two conditions are required. 1) Bring
albumin to its pI(5.4) by adding few drops of 1% acetic acid. 2)Heat the
solution

26
Half & Full Saturation Test:

Reagent:
Saturated ammonium sulphate [(NH4)2SO4]: Add ammonium sulphate in
500 ml DI water till it stops dissolving.
Ammonium sulphate [(NH4)2SO4] power

Principle
When ammonium sulphate is added to protein solution, water
concentration decreases. This removes shell of water from outer surface of
protein molecules, favoring formation of hydrogen bonds among protein
molecules and causing their precipitation. While proteins like globulin,
gelatin and casein are precipitated in half-saturated ammonium sulphate
solutions, albumin is precipitated in full-saturated ammonium sulphate
solution.

Protein molecules contain both hydrophilic & hydrophobic aminoacids.

In aqueous medium, hydrophobic amino acids form protected areas
while hydrophilic amino acids form hydrogen bonds with surrounding
water molecules (solvation layer).When proteins are present in salt
solutions (e.g.ammonium sulfate), some of the water molecules in the
solvation layer are attracted by salt ions. When salt concentration
gradually increases, the number of water molecules in the solvation layer
gradually decreases until protein molecules coagulate forming a
precipitate; this is known as “salting out”.
For example,albumin requires higher salt concentration for
precipitation than casein or gelatin.Albumin particals are smaller in
size & so have larger surface area,so they hold more water molecules
around them.so a higher concentration of Ammonium sulphate is
required.The salt concentration used is described as 'half saturation'(for
27
casein,gelatin,globulin) or ' full saturation' (for albumin).

PROCEDURES
TEST METHOD OBSERVATION INFERENCE

 10% NaOH (2 ml ) +
Pink or Violet
1% CuSO4 (2 ml) Two or more
BIURET Colour develops
 divide above mixture in peptide linkages
in part 1. No
TEST two parts of 2 ml present. Protein
such color
 part 1: add 2 ml OS present
develop in part 2
part 2: add 2 ml H2O

 OS (0.5 ml) + HNO3con (1

ml) Mix it. (Solution
turns yellow) +
40%NaOH (1 ml) Aromatic Amino
XANTHO-
in above mixture. Yellow-Orange Acids Tyrosine and
PROTEIC
 Solution turns orange colour develops. Tryptophan present
TEST Note: Use Fresh(tightly in protein.
packed) conc.HNO
otherwise test come
negative.
Alpha Amino
 OS (1 ml) +
groups of proteins
NINHYDRI
1% Ninhydrine
Blue or Purple at N-terminal are
(2 drops)
N TEST colour develops. responsible for
 Mix, Boil (1 min).
positive test with
 Cool.
proteins.

28
  1 ml Protein Solution +
1 drop of 1:500 formalin.
Mix.
 Slant the test tube and
slowly add 1 ml of conc.
H2SO4 . Mix. Indole group
 Add 1 drop of 1% sodium Violet color is present in protein.
Aldehyde
nitrite solution in Test formed. Tryptophan present
Test tube. Mix. in the protein.
 Use Fresh(tightly
packed) conc.H2SO4
&1:500formaline
otherwise test come
negative.
 0.5 ml protein sol. +50 ul Hydroxyphenyl
MILLION’S
Red coloured
sodium nitrate sol.n+100 group present in
precipitate
TEST ul Millon’s reagent. mix protein. Tyrosine
0bserved.
well & Heat present in protein.
1 ml Protein sol.n + 2 drops Guanidino group
SAKAGUCHI of alpha Napthol + 1 ml Carmine Red present in protein.
TEST Alkaline sodium colour observed. Arginine present in
Hypoochloride protein.
SULPHER
Sulfhydryl group
TEST  0.5 ml OS + 0.5 ml Lead (-SH) present in
Black- Grey
(Lead acetate reagent protein.
colour seen.
acetate  Boil for 1 minute Cysteine & Cystine
present in protein
test)
5 ml Protein solution + 1- 2
drops of chlorophenol red.
 If faint pink colour
appears, add 1% acetic
White
acid drop wise till you get
precipitates seen
HEAT
faint pink colour with Albumin is
in upper part of
yellowish tinge (pH 5.4 is precipitated when
COAGULAT solution, as
obtained) denatured at its
ION TEST compared to
 If yellow colour appears, pI~5.4
clear lower part
add 2% Na2CO3 solution
of solution
drop wise till you get
faint pink colour with
yellowish tinge (pH 5.4 is
obtained)

29
 Casein, Gelatin and
HALF 2 ml of the protein sol.n + 2
Globulin are
ml of saturated sol.n of White precipitate
SATURATI precipitated at half
(NH4)2 SO4 (Thus, saturated formed.
ON TEST saturation with
(NH4)2 SO4 is half diluted)
(NH4)2 SO4
5 ml. Of protein sol.n + a
pinch of Ammonium
FULL Albumin
Sulphate powder, Shake
White precipitate precipitates at full
SATURATI Repeat above steps till some
formed saturation with
ON TEST undissolved (NH4)2 SO4
(NH4)2 SO4
remains at the bottom of
the test tube.
 1ml OS + 2 drops of α-
napthol solution, mix
 Add 2 ml. of conc. Purple ring is
MOLISCH’S Sulphuric acid carefully formed at the Proteins contain
TEST through the side of the junction of acid Carbohydrates
test tube without and solution.
shaking.

What you will do:

 Perform tests mentioned in above table with various Protein Solutions
given to you. Note down your observation and inference in tables as
shown below.

TEST OBSERVATION INFERENCE

Biuret Test

XANTHO-PROTEIC TEST

NINHYDRIN TEST

Aldehyde Test

30
MILLION’S TEST

SAKAGUCHI’S TEST

SULPHER TEST

HEAT COAGULATION TEST

HALF SATURATION TEST

FULL SATURATION TEST

MOLISCH’S TEST

 Fill up the table given below.

Use: ‘P’ for positive test ‘N’ for negative test ‘W’ for weakly positive test

Test Amino acids Albumin Casein Gelatin Peptone

responsible for
the test
Xanthoproteic test

Ninhydrin test

Hopkin’s and Cole test

Million’s test

Sakaguchi’s test
Lead acetate test

 Mention food sources of Albumin, Casein and Gelatin.

31
 Which of the Albumin, Casein and Gelatin is nutritionally best?
Explain.

 If by mistake Ninhydrin touches your skin while doing the ninhydrin

test, skin gets bluish stain. Explain.

32
 4. Chemistry of lipids
Lipids are heterogeneous group of compounds soluble in non-polar
solvents like chloroform but not soluble in polar solvents like water.
While body is water medium, lipids of body require specialized
methods for digestion, absorption and transport.
Bile salts cause emulsification of oil due to their amphipathic
nature and ability to reduce surface tension. Thus making bile salts
essential for digestion and absorption of lipids of food.
Lipids of blood are transported as lipoproteins. Without
lipoproteins, lipids would be insoluble is plasma (93% water).

Reagent
Any oil : Ground nut oil, coconut oil
Non polar Solvent : Acetone/ Methanol
Bile salt solution : Dissolve 0.6 gm sodium deoxycholate in 100 ml DI
water. Donot take tape water for making bile salt solution,Precipitation
occur due to interference by calcium.

TEST METHOD OBSERVATION

Solubility of oil in  0.1 ml of oil + 1 ml
water, mix, for 15 Big oil drops are observed
water sec.
Solubility of oil in  0.1 ml of oil + 1 ml
oil droplets are not
Acetone/Methanol,
non-polar solvent observed
mix, for 15 sec.
 Take 2 test tubes T1
and T2
 Take 1 ml H2O in
Compare size of oil drops
T1 test tube.
and turbidity immediately
 Take 1 ml Bile salt
.
solution(Sodium
T1: Big oil drops, Clear
Emulsification of oil deoxycholate
water(Compared to T2)
solution) in T2 test
in Bile salts. tube.
T2: Small oil drops,
 Add 0.1 ml of oil in
Turbid solution
T1and T2.
(Compared to T1)
 Mix T1 & T2,all
together for 15 sec.
against palm of
your hand.

33
34
What will you do:
 Perform the test shown above with the oil provided. Draw table showing
the tests and your observations.

TEST OBSERVATION INTERFERENCE

Solubility of oil in

water
Solubility of oil in

non-polar solvent
Emulsification of oil

in Bile salts.

Draw structure of bile salt

Draw structure of an oil droplet in a bile salt solution.

35
Draw structure of a micelle. Write its function in body.

Draw structure of a lipoprotein particle. Write its function in body.

36
 5. Physiological Urine
Artificial Urine sample:
Ammonium sulfate 2 gm
Sodium phosphate dibasic(monobasic 2 gm
Pottasium dihydrogen phosphate 2 gm
Urea powder 2 gm
creatinine powder 2 gm
Uric acid powder 1 gm
Calcium carbonate/Calcium chloride : 1 gm.
NaCl 4 gm,
And make upto 2 liters

Physical characteristics of urine

Volume:
Normal adult excretes 800-2000 ml of urine daily

Factors afecting urine volume:

 According to quantity of fluid ingested
 Environment temperature
 Physical activity
 Loss of water in feces, via skin, in vomitus etc.

Collection of urine to measure volume:

Discard the first morning urine. Then collect urine during each
micturition in a vessel up to, including the next morning urine.

Some conditions with increased urine volume:

o Diabetes mellitus
o Diabetes insipidus (low specific gravity of urine)
o Diuretics drug therapy
Some conditions with decreased urine volume:
o Dehydration
o Renal failure
Appearance:
Normal urine is clear and transparent when freshly voided. On standing
bacterial urease converts urea into CO2 and Ammonia. Ammonia makes
urine alkaline. Phosphates precipitate in alkaline urine making it turbid.
Color:
Fresh urine is amber yellow. This colour is due to urobillin.
Odour:
Fresh urine has an aromatic odor due to presence of volatile organic acids
produced by body and intestinal bacteria.

37
Reaction:
Fresh urine is normally acidic (pH<7.0). Post-prandial urine is alkaline
due to secretion of HCl in stomach, the condition known as “Alkaline
Tide”.

Specific gravity:
Normal range-1.003 to 1.035 gm/ml of urine. The greater the amount of
solutes per unit volume of urine, the greater the specific gravity. It is high
in diabetes mellitus, while low in diabetes insipidus.

Determination of specific gravity: Wipe the urinometer by a filter paper

and allow it to float in the urine contained in the cylinder. See carefully
that the apparatus do not touch the sides or bottom of the cylinder, when
it is at rest take the reading from lower meniscus (true surface) of urine.
Note the temperature of urine. If it differs from the standard temperature
written on the urinometer, add one unit (0.001) for every 3 degree rise
from the standard temperature.

Chemical analysis of urine

A. Inorganic Chemical constituents

Ammonia:
Reagent:
1% phenolphthalein : Dissolve 0.5 gm of phenolphthalein in 50 ml of
methanol. Phenolphthalein is insoluble in water
2% sodium carbonate : Dissolve 10 gm of sodium carbonate in 500 ml of
water
Principle:
Urinary ammonia is derived from glutamine in kidney. It is secreted as a
buffer against H+ secreted by tubules.

NH4+ + OH-  NH3 + H2O

On heating NH3 evaporate, dissolve in water around a glass road and

make it alkaline. At alkaline pH phenolphthalein ions are formed which is
pink coloured.
Phenolphthalein is a weak acid, which can lose H+ ions in solution. The
phenolphthalein molecule(HIn) is colorless, and the phenolphthalein

38
ion( In-) is pink. When a base is added to the phenolphthalein, the
molecule ⇌ ions equilibrium shifts to the right, leading to more ionization
as H+ ions are removed.

HIn --------------------------------------> H+ + In-

colourless PH >8.2 Red colour

For phenolphthalein: pH 8.2 = colorless; pH 10 = red

Procedure:
 Take 5ml urine in a test tube and add a drop of phenolphthalein. Add
drop wise 2% sodium carbonate solution till the solution turns faint
pink. Boil and hold a glass rod dipped in phenolphthalein at the mouth
of the test tube. Phenolphthalein turns pink due to gaseous ammonia.

Chloride:
Reagent:
Concentrated HNO3
3% AgNO3 : Dissolve 15 gm of AgNO3 in 500 ml of water.
Principle:

AgNO3(aq) + NaCl(aq) → AgCl(s) + NaNO3(aq)

White precipitation
When acidified urine reacts with silver nitrate,a white precipitate of silver
chloride is formed.

Procedure:
 [3 ml of urine] + [1.0ml concentrated HNO3] + [1.0 ml 3% AgNO3]
Curdy white precipitate of AgCl is formed.

(Concentrated HNO3 is added to prevent precipitation of urate and acid

phosphates by AgNO3)

Calcium:
Reagent:
Saturated ammonium oxlate solution: Dissolve ammonium oxalate
powder in 500 ml of water till it become undissolved.
Principle:
Calcium precipitated as insoluble calcium oxalate with ammonium
oxalate
39
 CaCl2(aq) + (NH4)2C2O4(aq) ------------------- CaC2O4(s) + 2
NH4Cl(aq)

Procedure:
 Sulkowitch Test: To 5 ml urine and add 3 ml saturated ammonium
oxalate solution.
Calcium precipitated as insoluble calcium oxalate is observed as
turbidity.

Phosphorus:
Reagent:
Concentrated HNO3
5% Ammonium Molybdate : Dissolve 5 gm of Ammonium Molybdate in
100 ml of water

Principle:Inorganic phosphorus reacts with ammonium molybdate in an

acidic medium to form a phosphomolybdate complex.

Procedure:
 [2-ml of urine] + [0.5 ml concentrated HNO3] + [3 ml of 5% Ammonium
Molybdate], Heat
Canary yellow precipitate of Ammonium phosphomolybdate are formed

Sulphate:
Reagent:
1 % HCL : Take 1 ml of concentrated HCL & make upto 100 ml

40
10% Barium chloride :Dissolve 50 gm of Barium chloride in 500 ml of
water

Principle:
HCL
-2
SO 4 + BaCl2 ------------------BaSO4 + KCL

Procedure:
[5 ml urine] + [1 ml 1 % diluted HCL] + [2 ml of 10% Barium chloride].
White precipitate of BaSO4 are formed

Organic Chemical constituents

Urea:
(Specific Urease Test)
Reagent:
1% phenolphthalein :Dissolve 1 gm of phenolphthalein in 100 ml
methanol
Principle:
Urea NH3 + CO2
Urease
CO2 evaporates.

NH3 + H2O NH4+ + OH-

In this reaction the liberation of NH3 changes the pH to alkaline side,

turning phenolphthalein to pink colour.

Procedure:
[2 ml Urine] + [2 drops phenolphthalein]
Add 2% Na2CO3 till faint pink color is seen.
Add acetic acid, one drop at a time, with mixing, till faint pink color just
disappears.
41
Add a spatula of Urease powder( Jack Bean Meal Powder), mix.
Pink color develops after few minutes.

Uric acid:
Phosphotungstic acid reduction test:
Reagent:
10% Sodium carbonate: Dissolve 10gm of sodium carbonate in 100ml of
water
Phosphotungstic acid Reagent :
Stock : Dissolve 50 gm of sodium tungstate in 400 ml of water & add
40ml of 85% phosphoric acid .Make final volume to 500ml.
Working : Dilute 50ml of stock to 500ml with water.
Principle:
Uric acid is reducing agent in alkaline medium.It reduced
phosphotungstic acid into tungsten blue.

Procedure
To 2.5 ml of urine add 0.5 ml of sodium carbonate and 0.5 ml of
Phosphotungstic acid reagent working reagent

Creatinine
Reagent:
Refer SOP in dokuwiki document
Creatinine R1 (NaOH)
1. Weigh 24 gm NaOH.
2. Dissolve in approximately 500 ml DI water.
3. Add 20 ml of 30% brij in above mixture.
4. Weigh 2 gm SDS and pour it into approximately 200 ml water in
beaker. Heat the solution until SDS dissolve.
5. Add SDS containing solution in main mixture.
6. Make upto 2 liter with DI water.

Creatinine R2 (Picric acid)

1. Dry picric acid between filter paper pieces

2. Weight 9.16 gm dry picric acid
3. Dissolve in approx. 600 ml water
4. Add 20 ml of 30% Brij in above mixture.

42
 5. Remove froth with a clean object of glass or plastic dipped in capryl
alcohol
6. Make 2 liter with water

Alkaline picrate reagent:

Mix R1 and R2 1:1 to make working alkaline picrate reagent.
Principle:
Creatinine forms creatinine picrate in alkaline medium which is orange in
colour
Procedure
2 ml alkaline picrate solution + 1 drop of urine & mix

Physical Characteristics of Urine.

Physical characteristics of Observation Interference
urine
Volume

Appearance

Colour

Odour

Reaction

Specific gravity

Inorganic constituents of urine.

Inorganic constituents of Observation Interference

Urine
Ammonia

Chloride

Calcium

Phosphorus

43
Sulphate

Organic constituents of urine

Organic constituents of Observation Interference

Urine
Urea

Uric acid

Creatinine

44
 6. Pathological Urine
Appearance:
Turbid: infection (cells make urine turbid)
Color:
Yellow: Hepatic jaundice & obstructive jaundice (Conjugated bilirubin)
Red: Hematuria, rifampicin therapy
Red on exposure to air: porphyria
Black on exposure to air: alkaptonuria
Odour:
Fruity: diabetic ketoacidosis (acetone)
Mousy smell: Phenylketonuria. (Phenylacetyl glutamine)
Foul smell: Urinary tract infections. (H2S etc.)
Specific Gravity :
High Specific Gravity
 Diabetes mellitus
 Diarrohea
 Dehydration

Low Specific Gravity

 Diabetes insipidus.
 Renal failure
 Excessive fluid intake
 Acute tubular necrosis
Abnormal Constituents of Urine
I.Protein:
Reagent :
A. Sample preparation :
10 mg% albumin : Dissolve 100 mg bovin albumin in 1000 ml of water
50 mg% albumin: Dissolve 500 mg bovin albumin in 1000 ml of water
100 mg% albumin: Dissolve 1000 mg bovin albumin in 1000 ml of water
B. 1% Acetic acid:
 5 ml of acetic acid in 500 ml of water
C. 30% Sulphosalisylic acid:
 Dissolve 150 gm of Sulphosalisylic acid in 500 ml of water
Proteinuria and albuminuria
Proteinuria
Normal Adult <150 mg /day
Proteinuria >=150 mg /day
Proteinuria >3500 mg / day

Albuminuria

45
Normal Adult <30 mg /day
Microalbuminuria 30-300 mg /day
Macroalbuminuria >300 mg /day

Albumin (Filtered but not reabsorbed) and Tamm-Horsfall protein

(secreted by renal tubules) are normally present.
Causes of Proteinuria:
 Pre-renal: (overload proteinuria)
o (Many non-Albumin proteins)
o Multiple myeloma (light chains of immunoglobulins)
o Severe hemolysis (Hemoglobin)
o Severe muscle injury (Myoglobinuria)
 Renal:Glomerular diseases (Mainly albumin, being small)
o After streptococcal infection
o Diabetes mellitus
o Hypertension
o Lipoid Nephrosis (Nephrotic range proteinuria)
o Tubular diseases (decreased reabsorption of proteins)
o (Small, normally reabsorbed, proteins like Beta2
microglobulin, Retinol Binding protein)
o Tubular necrosis due to Drugs and toxins
 Post Renal: (various blood and cellular proteins)
o Bleeding in urinary tract
o Infection in urinary tract
o Tumor in urinary tract
 Other causes:
o Postural: on standing posture.
o Exposure to cold, physical activity, fever.
o Last weeks of pregnancy

Heat coagulation Test:

Principle :
Proteins have net zero charge at their iso-electric pH (pI). So, at pI, protein
molecules have minimum repelling force. Thus proteins are easily
precipitated at pI.
When proteins are heated, weak bonds like hydrogen-bonds, salt bonds
and van-der-wal forces are broken. Proteins are said to be denatured.Core
hydrophobic regions of denatured Albumin can form intermolecular
associations and cause precipitation.Thus, in order to precipitate proteins
like albumin, two conditions are required.
1. Bring albumin to its pI(5.4) by adding few drops of 1% acetic acid
2. Heat the solution
Procedure
46
Fill 3/4 th of the test tube with urine sample,Heat the upper part on the
flame till either turbidity appears or urine starts boiling.Then add few
drops of 1% acetic acid if turbidity develops & note change.

In case of multiple myeloma, light chains of immunoglobulin precipitate

between 40-60 degrees centigrade. With further heating turbidity
disappears. Turbidity appears again on cooling to 40-60 degree
centigrade.

Sulphosalisylic Test:
Principle
Test is based on the precipitation of urine protein by a strong acid,
sulfosalicylic acid. Precipitation of protein in the sample seen as
increasing turbidity)
Unlike the routine urine protein chemistry dipstick pad, the SSA reaction
will
detect globulin and Bence-Jones proteins, in addition to albumin

Method: 3 ml of urine + 0.3 ml of 30% Sulphosalisylic acid, mix.

Turbidity indicates presence of urinary proteins.
Iodinated contrast agents used for evaluation of renal disorders can give
the test positive.
False positives:
X-ray contrast media
High concentration of antibiotics, such as penicillin and cephalosporin
derivatives.
False negatives:
Highly buffered alkaline urine. (The urine may require acidification to a
pH of 7.0 before performing the SSA test.)
Dilute urine
Turbid urine - may mask a positive reaction. Again, best practice is to
always used supernatant from a properly spun urine sample.

Dip-Stick Test:
Principle
Testing for protein is based on the phenomenon called the "Protein Error
of Indicators" (ability of protein to alter the color of some acid-base
indicators without altering the pH).
This principle is based on the fact that proteins alter the colour of some
pH indicators even though the pH of the media remains constant. This
occurs because proteins (and particularly albumin) acquire hydrogen
47
 ions at the expense of the indicator as the protein’s amino groups are
highly efficient acceptors of H+ ions.
Indicator-H+(Yellow) + Protein → Indicator(Blue-green) + Protein-H+
At pH 3 and in the absence of proteins both indicators are yellow, as pro-
tein concentration increases the colour changes through various shades
of green until it becomes a dark blue.

According to the manufacturer, the strip’s protein pad contains tetrabro-

mophenol blue or 3',3,5',5-tetrachlorophenol-3,4,5,5-tetrabromosulphon-
phthalein, as well as an acid buffer substance to maintain pH at a con-
stant level.

The main problem with the protein tests found on urine test strips is
that very alkali urine can neutralise the acid buffer and produce a false
positive reading that is unrelated to the presence of proteins. Another
similar error occurs if the strip is left submerged in the urine sample for
too long.

This method is more sensitive to albumin than to globulin, Bence Jones

protein and mucoprotein are examples of globulin components that are
sometimes present in urine, but are not distinguishable by the dipstick
method for protein

Method: Dip the strip for Albumin in urine. Drain excess urine from
strip. Read the color chart. (Read instruction manual provided with the
strips for time of reading after dip.).

Because the dipstick test detect albumin, it can not identify many
pre-renal proteinuria caused by Hb, Mb and light chains of Igs.

All the three tests mentioned above are qualitative and used for
screening proteinuria and albuminuria. Once proteinuria is found
quantitative estimation of proteinuria and albuminuria is required for
clinical decision making.

What Will You Do:

Perform all three tests with urine. Draw table of your observations.

Sr. Concentration Heat Dipstick Sulphosali- Interference

no coagulation test sylic acid
test
1 10 mg %
48
2 50 mg %
3 100 mg %
4 Urine sample

Which of the three tests is most sensitive?

Write biochemical explanation of proteinuria in diabetes mellitus and

hypertension.

II.Acetone & acetoacetic acid (Ketone Bodies):

Reagent
1. Ammonium sulphate powder
2. Small crystals of sodium nitroprusside
3. liquor Ammonia
Rothera’s powdered reagent :
 Sodium Nitroprusside 1 gm
 Sodium carbonate 20 gm
 Ammonium sulphate 20 gm
Mix & grind all in fine particales & stored in air-tight container.
Sample Preparation
0.1 ml/L Acetone : Take 0.1 ml Acetone in 1000 ml DI water
1 ml/L Acetone : Take 1 ml of Acetone in 1000 ml DI water
10 ml/L Acetone :Take 10 ml of Acetone in 1000 ml DI water

Principle
Acetoacetic acid and acetone form a violet coloured complex with sodium
nitroprusside in alkaline medium. Acetoacetic acid reacts more
sensitively than acetone. Values of 10 mg/dl of acetoacetic acid or 50
mg/dl acetone are indicated. Phenylketones in higher concentrations
interfere with the test, and will produce deviating colours. ß-
hydroxybutyric acid (not a ketone) is not detected.

Sodium Nitroprusside : acetone form a violet coloured complex with

sodium nitroprusside in alkaline medium
49
Sodium carbonate: Provide Alkaline medium
Ammonium sulphate : Precipitate other protein which give purple colour
with sodium nitroprusside & make solution Heavier than liquire
Ammonia, so Ammonia may be remain on top of solution ,so purple ring
is formed.

Rothera’s test, liquid reagent

Saturate 2ml urine with ammonium sulphate powder.
Add a small crystal of sodium nitroprusside. Mix.
Add 0.5 ml liquor ammonia by side of the tube to form a ring.
Permanganate/Purple color ring is formed

Rothera’s test, powdered reagent

Take a pinch of Rothera’s powdered reagent
Add 1-2 drops of urine on powder.
Permanganate/purple color is formed

What Will You Do:

Perform both tests with given sample of urine.
Perform both tests with 0.1ml/L , 1/ml/L , 10 ml/L acetone

Sr. Concentration Rothera’s Rothera’s Interference

no test,powder test, Liquid
reagent reagent
1 0.1 ml/L
2 1 ml/L
3 10 ml/L
4 Urine sample

Perform both tests with given sample of urine.

Perform both tests with 0.1ml/L , 1/ml/L , 10 ml/L Acetoacetate

Sr. Concentration Rothera’s Rothera’s Interference

no test,powder test, Liquid
reagent reagent
1 0.1 ml/L
2 1 ml/L
3 10 ml/L

50
4 Urine sample

Which other tests in blood and urine are usually done when tests for
ketone bodies are positive?

III.Bile Salts:
REAGENT
Bile salt sample : Dissolve 2 gm of Bile salt powder into 1000 ml of water.
Sulfur powder
Principle
Sulphur powder is non-polar. It floats on water surface due to surface
tension of water. Bile salt reduces surface tension of water and thereby
sulphur powder sinks.
Procedure
Hay’s sulfur flower Test:
Sprinkle a pinch of sulphur powder over 2 ml urine in a test tube &
Sprinkle a pinch of sulphur powder over 2 ml Water in a test tube.
Observed & compare immediately without shaking of test tubes.
Sulphur powder sink to the bottom of the test tube if bile salts are
present.

What Will You Do:

51
Perform the Hay’s sulfur flower test with given sample

Sample Observation Interference

Bile salt solution
Water
Urine sample

IV : Glucose
Perform both the tests with urine. Draw table of your observation.
Perform both tests with 100 mg%, 500 mg%, 1 gm% glucose. Note
color of the test. Draw table showing the results as follows.

Glucose % Benedict’s Test color GOD Strip test color Interference

100 mg%
500 mg%
1000 mg%
Urine sample

52
7. Estimation of acid output by stomach.
Parietal cells of gastric mucosa secrete H+ using H+-K+-ATPase.
Gastrin, acetylcholine (from vagus) and histamine stimulate H+ secretion.
Thus, abnormality of parietal cells, G cells and Vegas are important in
disturbances of gastric acid secretion.
Collection of Gastric juice for Analysis
Preparation of the Patient:
1. Patient should be fasting 10 to 12 hours (preferably since bed time
night
before).
2. Patient should have not received any medications specially
anticholinergic
agent, H2 blocker, Antacids since night before as they are liable to alter
the
results.
3. Procedure should be explained to the patient in simple words.
Procedure:
1. Remove dentures if there are any from patient’s mouth.Take the
Nasogastric tube and lubricate it.
2. Check patient’s nostrils and choose the one (while patient still
sitting up right with neck flexed) through which breathing is easier
and nostril is wider.
3. Begin intubation by gently pushing the tube, some times tube gets-
curled up in the pharynx and there is excessive coughing or gaging
which may prevent further passage, at this time tube is drawn back
a few inches, patient, is reassured and intubation is resumed.
4. During this insertion patient is instructed to swallow and continue
to swallow throughout the intubation period.
5. After the tube has progressed to approximately 40 cm (the first
mark on the tube), the head may be allowed to resume its
comfortable position.
6. Continue intubation by gently pushing the tube with the patient
still swallowing until the fourth mark or 65 cm is reached.
7. Tape the tube to the patient’s nose with adhesive tape.At this point
patient is sent to the X-ray for fluoroscopy to check position.
8. The tube should lie along the lesser curvature with the tip in the
antrum of the stomach.
9. In patient with partial gastrectomy tip of tube should be in the most
dependent portion of the stomach.
Collection of Gastric Juice For Analysis
1. Empty the stomach of its contents with a 50 cc. Syringe.
2. After recording the pH volume and colour, this residual volume may
be discarded.
3. After emptying stomach of the residual volume, collection of gastric
juice is begun under Basal conditions.
4. At least four samples are collected each 15 minutes apart in
separate containers.
53
 5. Collection may be carried out either manually with the syringe or
by using a suction pump.
6. During this procedure patency of nasogastric tube is maintained by
injecting about 50 cc of air down the tube.
7. Gastric fluid specimen should be spot checked as a guide to
whether the patient is making-acid or is achiorohydric.
8. After having collected gastric juice under basal conditions
augmented or stimulated gastric analysis may be carried out as
follows:
9. Pentagastrin administered by sub-cutaneous injection in the dose
6 mg per kg body weight. It is a synthetic peptide having the
biologically active sequence of gastrin.
10. The gastric secreation is collected every 15 minutes for next 1
hour.

1. Basal acid output (B.A.0):

It is the acid output in milimol per hour in the basal secretion.
2. Maximal acid output (M.A.0)
It is the acid output in milimol per hour, given by the sum of acid output
of the four 15 minute sample after the stimulation

Hypochlorhydria: (decreased acid output, pH>4)

 Pernicious anemia
 Autoimmunity to parietal cells destroys them.
 Antibodies to Na+-K+-ATPase are found
 Chronic Helicobacter Pylori infection of gastric mucosa.
 Treatment with Proton pump inhibitors, H2-Blocker
 Vagotomy
Hyperchlorhydria: (increased acid output)
 Zollinger-Ellision Syndrome
 G cells tumors in GIT
Reagent
0.1 mol/L NaOH : Dissolve 20 gm of NaOH in 5000 ml of water
1 % phenolphthalein : Dissolve 1 gm of phenolphthalein in 100 ml
methanol
Sample preparation
Gastric juice Sample : 0.1mol/L HCL solution
How 0.1 mol/L HcL will be prepared?
1000ml of HCL solution contain=11.5 mol H+
??????? =0.08 mol H+
=1000x0.1/11.5
=8.6 ml
So add 17 ml of concentrated HCL & make upto 2 liter with water.
54
Examples
Example-1:
If you want your result will be Gastric Acid Output (mmol/hr) = 5
mmol/hr and You give Fasting Gastric juice output in 1 hour =100 ml/hr
then prepare gastric juice sample as follow,

Fasting Gastric juice output =100 ml/hr

BAO = 5 mmol/L

100 ml of fasting gastric juice contain = 5 mmol/L HCL

1000 ml of fasting gastric juice contain = ???
= 1000 x 5
-----------
100
= 50 mmol/L HCL
= 0.05 mol/L HCL
Now We use fixed 10 ml of Gastric juice sample & titrate with fixed 0.1
mol/L NaOH
10 ml of 0.05 mol/L HCL =-------ml of 0.1 mol/L NaOH
V1=10 ml of Gastric juice V2=???? ml of NaOH
NI=0.05 mol/L HCL N2=0.1 mol/L NaOH

V2=10 x 0.05/0.1
=5 ml of 0.1 mol/L NaoH
Thus 5 ml of 0.1 mol/L NaOH is required to titrate 10 ml of 0.05 mol/L
HCL.
Now ,Check your sample of gastric juice is made proper or not by
following formula,

Gastric Acid Output =

[Average Reading R] *[Gastric Juice Output in one hour]
---------------------------------------------------------------------
100
We require 5 ml of NaOH & give 100 ml/hr Gastric output,so our result is
Gastric Acid Output = 5 x 100/100
=5 mmol/hr ,that is our BAO.

Example-2:

55
If you want your result will be Gastric Acid Output (mmol/hr) = 8
mmol/hr and You give Fasting Gastric juice output in 1 hour =80 ml/hr
then prepare gastric juice sample as follow,

Fasting Gastric juice output =80 ml/hr

BAO = 8 mmol/L

80 ml of fasting gastric juice contain = 8 mmol/L HCL

1000 ml of fasting gastric juice contain = ???
= 1000 x 8/80
= 100mmol/L HCL
= 0.1 mol/L HCL
Thus Take 8.6 ml of Concentrated HCL solution and make upto 1000ml
with water is made to 8.6 mol/L HCL solution.
Now We use fixed 10 ml of Gastric juice sample & titrate with fixed 0.1
mol/L NaOH

10 ml of 0.1 mol/L HCL = __________ ml of 0.1 mol/L NaOH

V1=10 ml of Gastric juice V2=???? ml of NaOH
NI=0.08 mol/L HCL N2=0.1 mol/L NaOH

V2 =10 x 0.1/0.1
=10 ml of 0.1 mol/L NaoH
Thus 10 ml of 0.1 mol/L NaOH is required to titrate 10 ml of 0.1 mol/L
HCL.

Now , Check your sample of gastric juice is made proper or not by

following formula,

Gastric Acid Output =

[Average Reading R]* [Gastric Juice Output in one hour]
---------------------------------------------------------------------
100
We require 10 ml of NaOH & give 80 ml/hr Gastric output, so our result
is Gastric Acid Output = 10 x 80/100
= 8 mmol/hr ,that is our BAO.
Principle:

56
 Acid output in stomach is measured as mmol/hour. For its
measurement, amount of gastric juice output as well as amount of acid
in gastric juice needs to be measured.
Amount of Gastric juice output is measured by suction of gastric juice
using Ryle’s tube inserted in to stomach.
Amount of acid in gastric juice is measured as follows.
Free Acidity:
Due to H+ (H3O+) ions.
Combined Acidity:
Some of the H+ in gastric juice are bound to other anions like proteins
and lactic acids at low pH of Gastric Juice. These represent combined
acidity.
(Proteins-).(H+) , (Lactate-).(H+)
Free Acidity + Combined Acidity = Total acidity
On addition of alkali, initially free H+ and later on combined H+ are
neutralized.
When not much H+ remain in solution (at pH 8.6), Phenolphthalein
indicator becomes pink. The requirement of alkali is used to calculate
acid output.

Procedure:
First Reading:
 Step 1
o Take10 ml gastric juice in a flask/beaker.
o Add 1 drop of phenolphthalein.
o (Do not mouth pipette anything)
 Step 2
o Fill burette with 0.1 mol/L NaOH up to zero mark.
o Perform as follows.
 Step 3
o Add 1 ml of NaOH from burette, mix, and watch for pink
color.
o Repeat above step till pink color develops.
o Suppose reading is X1 ml of NaOH
Second Reading:

57
  Repeat-step 1 and step-2 of first reading.
 Add [X1 - 1] ml of NaOH from burette mix.
 Add NaOH drop wise till pink color develops.
 Take Reading will be X2.
Third Reading:
 Repeat-step 1 and step-2 of first reading.
 Add [X2 - 1] ml of NaOH from burette mix.
 Add NaOH drop wise till pink color develops.
 Take Reading will be X3.
Find Average(R) of X2 & X3 .

Calculation:
Explanation of calculation:
1 mol NaOH  1 mol HCl
R ml of 0.1 mol/L NaOH  R ml of 0.1 mol/L HCl
 R /10 ml of 1 mol/L HCl
 R /(10*1000) mol/ ml of HCl
 (R /10) mmol/ml of HCl

Thus, 10 ml of Gastric Juice will have (R/10) mmol HCl equivalents.

Thus, 1 ml of Gastric Juice will have (R/100) mmol HCl equivalents.

Gastric acid output = (R/100) * G mmol/hr

G = Gastric Juice Output (ml/hr)
Normal Gastric Juice Output (ml/hr) = 80 ml/hr

Result:
Gastric Acid Output (mmol/hr) =

Reference Ranges:
 Fasting Gastric Juice Output: 20-100 ml /hr
 Basal Acid Output (BAO): Measured in fasting state
o Normal 1-6 mmol/hr
o ZE Syndrome >15 mmol/hr (M)
>10 mmol/hr (F)
 Maximum Acid Output (MAO): Measured after pentagastrin
stimulation
o Normal 5-40 mmol/hr
In pernicious anemia, both MAO and BAO are almost zero.

58
 Above reference ranges are not universally accepted. Serum gastrin level,
pH of gastric juice and other clinical finding e.g megaloblastic anemia are
important to establish diagnosis.
What will you do:
Estimate gastric acid output in given sample or gastric juice.
Consider Gastric juice output 80 ml/hr.
Initial reading (X) Next reading (Y) Volume of NaOH used (X-Y) ml

Average of (X-Y)

Gastric Acid Output =

[Average Reading R] * [Gastric Juice Output in onehour]
---------------------------------------------------------------------
100
Result : Your Gastric acid output is --------------------------

Comment on your result

Q-1 What is Zollinger-Ellision syndrome?

Q-2 What happens to Gastric acid output in the ZE syndrome? Why?

Q-3 Write complications of the ZE syndrome.

59
Q-4 Write cause of destruction of parietal cells in pernicious anemia.

Q-5 What happens to Gastric acid output in the pernicious anemia?

Why?

Q-6 Which other important products are formed and secreted by parietal
cells?

Q-7 Why should destruction of parietal cell lead to anemia?

Q-8 What is difference between gastrin and pentagastrin.

Q-9 Both pernicious anemia and ZE syndrome result in high serum

gastrin level. Explain.

Q-10 Explain mechanism of action and use of ranitidine and omeprazole

as drugs.

60
8.Secretion and bufering of acids by kidney.

Reagent
1. 1 % phenolphthalein :Dissolve 0.5 gm of phenolphthalein in 50 ml
of Methanol.
2. Neutral formalin (formaldehyde):Take 500ml of formaldehyde & add
0.1ml of phenolphthalein in solution. Then add 0.1 mol/L NaOH
till colorless formaldehyde solution become slight pink coloured.
3. 0.1mol/L NaOH : Dissolve 20 gm of NaOH & make upto 5000 ml
with Water.

Urine Sample Preparation:

Urine output ml/day = U

Titrable acidity mmol/day = A

A
Take ---- x 68 gm of KH2PO4 MW of KH2PO4 = 68 gm/L
U

Ammonia bound acidity mmol/day = B

B
Take ---- x 66 gm of (NH4)2SO4 MW of (NH4)2SO4 = 132 gm/L
U

Here two NH4+ is released when 1 molecule of (NH4)2SO4 will be

dissociated.

Example
You want to give Titrable acidity = 30 mmol HCL /day &
Ammonia bound acidity = 40 mmol HCl /day ,then prepare Urine sample
as follow,
Urine output U = 1500 ml/day
Titrable acidity mmol/day A = 30 mmol HCL/day
= A/U x 68
=30/1500 x 68
=1.36 gm of KH2PO4

Ammonia Bound acidity mmol/day B = 40 mmol HCL/day

= B/U x 66
=40/1500 x 66

61
 =1.76 gm of (NH4)2SO4
Finally dissolve 1.36 gm of KH2PO4 and 1.76 gm of (NH4)2SO4 &
make upto 1000 ml with water.

Principle:

Catabolism of food substances produces H+ and OH-. In the process,

there is excess of H+ over OH-. Excess H+ is excreted by kidney. NH3 and
Phosphate buffer the H+ secreted by renal tubules.
NH3 + H+ ------------- NH4+ ----(1)
HPO42- + H+ ------------- H2PO4- ----(2)

You will estimate total Acids in urine and proportions buffered by

ammonia and phosphate.
Correlate the experiment with theoretical concepts of renal regulation of
pH learnt in the classroom.

pK of reaction (1) is 9.25.

pK of reaction (2) is 6.8.
For phenolphthalein: pH 8.2 = colorless; pH 10 = red
HIn --------------------------------------> H+ + In-
colourless PH >8.2 Red colour
Phenolphthalein is a weak acid, which can lose H+ ions in solution. The
phenolphthalein molecule(HIn) is colorless, and the phenolphthalein
ion( In-) is pink. When a base is added to the phenolphthalein, the
molecule ⇌ ions equilibrium shifts to the right, leading to more ionization
as H+ ions are removed

When urine, acidic in nature, is titrated with NaOH, initially reaction (2)
goes towards left. When all H2PO4- is converted into HPO42-, pH rises to
8.6, causing ionization of phenolphthalein .Phenolphathalein ion
proceduced pink colour,sosolution turn into pink coloured. NaOH
required to reach this stage represent H+ bound to phosphate, called
“Titrable Acidity”.

Neutral formalin is added to urine. We will convert formalin (Acid) to

Neutral formalin, otherwise formalin(acid) itself react with NaOH when
we measure H+ of NH4+

Now, Formaldehyde is added to urine. Following reaction occur.

4NH4Cl + 6HCHO  N4(CH2)6 + 6H2O + HCl ----(3)

62
Released H+ decrease pH of urine, making phenolphthalein colorless
again.
Further titration with NaOH, till phenolphthalein become pink, will
actually represent H+ bound with ammonia released during reaction (3).
It is called “Ammonia bound acidity”.
H+ bound to NH3 can not be titrated without adding formaldehyde.
Hence, H+ bound to phosphate is called titrable acidity.

63
Procedure:
First Reading:
 Step -1
o Take 25 ml urine in a flask/beaker.
o Add 1 drop of phenolphthalein. (Do not mouth pipette
anything)
 Step - 2
o Fill burette with 0.1 mol/L NaOH up to zero mark.
 Step - 3
o Perform as follows.
o Add 1 ml of NaOH from burette, mix, and watch for pink
color.
o Repeat above step (adding 1 ml NaOH) till pink color
develops.
o Suppose reading is X1 ml of NaOH
 Step - 4
o Add 10 ml of neutral formalin & Mix.
o The pink color disappears.
o Repeat step-3.
o Suppose the reading is Y1
Second Reading:
 Repeat-step 1 and step-2 of above.
 Add [X1 - 1] ml of NaOH from burette & mix.
 Add NaOH drop wise till pink color develops.
 Take reading X2.
 Add 10 ml of neutral formalin & Mix.
 Add [Y1-1] ml of NaOH from burette & Mix.
 Add NaOH one drop wise till pink color develops. Take reading Y2.
Third Reading:
 Repeat-step 1 and step-2 of above.
 Add [X2 - 1] ml of NaOH from burette & mix.
 Add NaOH drop wise till pink color develops.
 Take reading X3.
 Add 10 ml of neutral formalin & Mix.
 Add [Y2-1] ml of NaOH from burette & Mix.
 Add NaOH one drop wise till pink color develops. Take reading Y3.

Find X (Average of X2 & X3 ) and Y (Average of Y2 & Y3 ) .

Explanation of calculation:
Titrable acidity: reading X ml
1 mol NaOH  1 mol HCl

64
X ml of 0.1 mol/L NaOH X ml of 0.1 mol/L HCl
 X /10 ml of 1 mol/L HCl
 X /(10*1000) mol/ml of HCl
 (X /10) mmol/ml of HCl
As titration is done with 25 ml of urine,
Titrable acidity in 25 ml of urine = (X /10) mmol HCl
Titrable acidity in 1 ml of urine = X /(10*25) mmol HCl

If urine output per day is U ml

Excreted Titrable acidity /day = (U * X) / 250 mmol HCl

Ammonia bound acidity: reading Y ml

It is expressed either as mmol of HCl or mg of ammonia

Ammonia bound acidity / day = (U * Y) / 250 mmol HCl

H+ + NH3  NH4+

1 mmol of NH3 binds 1 mmol of H+ to form 1 mmol of NH4+ ---(b)

MW of Ammonia (NH3) = 17 gm
1 mmol NH3 = 17 mg of NH3 ---(a)

From (a) and (b)

Excreted Ammonia / day = (U * Y) /250 mmol NH3

= ((U * Y) /250)*(17) mg NH3

Excreted Ammonia / day = U * Y * (0.068) mg NH3

Reference Range:
Titrable acidity = 20-50 mmol HCl / day
Ammonia bound acidity =[30-50 mmol HCl/day] or [510-850 mg
NH3/day]
Total acid excretion =70-100 mmol/day

What will you do:

Estimate Titrable and ammonia bound acidity in given sample of urine.

Titrable acidity

No. Initial reading(ml) Final reading(ml) Difference(ml)

X2

65
X3
Average X

Ammonia bound acidity

No. Initial reading(ml) Final reading(ml) Difference(ml)

Y2
Y3
Average Y
Result & conclusion

Titrable acidity =

Ammonia bound acidity =

What is the source of phosphate in urine?

What is the source of ammonia in urine?

Diabetic ketoacidosis elevate urinary ammonia. Explain.

66
9. Colorimetry
Colored molecule absorbs various wavelength of light passing through
their solution.

Colore
Imparted light  immerging light

soluti
on
d


For a given wavelength of light, ratio of (immerging light intensity) to

(imparted light intensity) is called Transmittance T.

-kct
T=e c = concentration of colored molecule
t = length of light path
k = constant
-kct = log e T
log 10 T
-kct = log 10 e

-k’ct = log T (common logarithm) k’ =constant

-k’ct = log (T*100/100)

-k’ct = log (T*100) – 2

k’ct = 2 - log (T%) (T% is called percentage Transmittance)

k’ct = A (2 – logT%) is called Absorbance, denoted as A

Following graph describe relationship between T% and A.

67
A = k’ct. (Beer’s and Lambert’s law)
Hence,
A ∞ t Absorbance is proportional to length of light path

A ∞ c Absorbance is proportional to concentration of substance

Therefore, If light path is constant, for concentration (C1 and C2) and
respective absorbance (A1 and A2)

C1/C2 = A1/A2

If A1 and A2 is measured and C2 is known

C1= (A1/A2)*C2 can be calculated. --------(1)

This principle is utilized by biochemistry laboratory to measure various

substances in biological materials.

Various instruments based on the principle are colorimeter and

spectrophotometer.

Instrument:

Light source emit light of all wavelengths

Monochromator allow only certain wavelength of light to pass. (Mono +
color)
Cuvette is a transparent vessel holding colored solution
I0 = Imparted light
It = Immerging light

68
Photocell converts light in to current. Current is proportional to light
intensity.
Galvanometer measures current.
General procedure to use colorimeter:
Suppose concentration of Glucose in plasma is to be estimated.

Glucose is colorless, hence can not be measured directly.

Add fixed amount of Y in fixed amount of plasma. P and Q are produced
Glucose + Y  P + Q
Suppose Q is colored compound and absorbs light of a particular
wavelength. Its concentration will be proportional to concentration of
glucose.
Take a solution of glucose with known concentration C (it is called
calibrator) and process as above in 2.
Take a water (it is called blank) and process as above in 2.
Measure absorbance of color produced by Sample and Standard and
blank.
Blank Absorbance, amount of color produced with no glucose, needs to
be deducted from absorbance of Sample and Standard.
Using equation (1)
(Asample - Ablank)
Glucose concentration in plasma (mg%) = -------------------- *C
(Astandard - Ablank)

What will you do:

Reagent:
Buffer:
pH Chemical drug Mol/L MW Gm/L
6.853 Na2HPO4 0.025 141.96 3.549
KH2PO4 0.025 136.09 3.402
9.139 Na2 tetraborate 0.01 381.37 3.814
Stock 1
Red coloured solution: Dissolve 20 mg of Phenol red in 50 ml of 9.139 pH
buffer
Stock 2
Blue coloured solution: Dissolve 20 mg of BCG(Bromocresol green) in 50
ml of 6.8 pH buffer.
Note: Dilution of stock coloured solution will be always done with
respective Buffer.
Diluted Red Dye from Stock-1:
Dilute 1:30 times of red coloured stock-1 solution with buffer of pH=9.1
by Adding 20 ml of stock-1 red coloured solution into 580 ml of Buffer of
pH=9.1

69
Diluted Blue Dye from Stock-2:
Dilute 1:20 times of Blue coloured stock solution with buffer of pH=6.8
by Adding 10 ml of stock Blue coloured solution into 290 ml of Buffer of
pH=6.8

Exercise:1
You will be given a concentrated colored solution.
Dilute it in a series of test tubes as follows. Measure absorbance.
Test Diluted Red pH=9.139 Absorbance (A) on
tube Dye from Buffer(pH=9.139) 505 nm Filter
Stock-1 (micro-liter)
(micro-liter)
0 0 1000
1 200 800
2 400 600
3 600 400
4 800 200
5 1000 0
Draw Graph of various Dilution of dye versus its absorbance

Result & Conclusion:

70
Exerscise-2
[1]: Diluted Red Dye from Stock-1: (Phenol red dye)
Measure Absorbance of this red coloured solution on different filters.

Filters(nm) Absorbance
340
405
450
505
546
578
630
670

Absorbance spectrum of phenol red dye solution (Red coloured)

[2]: Diluted Blue Dye from Stock-2: [Bromocresol green dye]

Measure Absorbance of these diluted Blue coloured solution on different

filters.
Filters(nm) Absorbance
71
340
405
450
505
546
578
630
670

Absorbance spectrum of BCG(Bromocresol green) dye solution (Blue

coloured)

Draw Graph of various filter versus absorption on that filter for red

colored solution & Blue coloured solution .

Note : Diferent colored solutions absorb light at diferent
wavelengths in diferent proportions.

72
10. Estimation of serum creatinine

Creatinine is produced from creatine present mainly in muscles. It is

filtered by glomerulus of kidney.

Principle:
Picrate + OH- ---------- activated [ Picrate-OH- ]* complex
[ Picrate-OH- ]* + creatinine ------ Creatinine-Picrate complex + OH-

Red colored Creatinine-Picrate complex, also called Janovaski complex, is

measured at 505 nm.
The rate of reaction is proportional to concentration of creatinine.
The rate of reaction is also indicated by rate of rise in Absorbance (ΔA)
Thus, [creatinine] ∞ ΔA
ΔA is difference of absorbances in 60 seconds of reaction
A for standard is A standard and A for sample is Asample

 Asample
Conc. of Creatinine in sample = ------------- X Standard
Astandard
Reagents
The timed measurements of Absorbance require sophisticated
colorimeters with flow-through cuvette. The reaction mixture is aspirated
in the cuvette and Absorbance is measured at different time.
The Laboratory technologist will help to carry out following steps:
 NaOH solution :Refer to SOP for creatinine reagent
 Picric acid solution : Refer to SOP for creatinine reagent

Creatinine Standard
2 mg/dl Creratinine :
Dissolve 0.010 gm of creatinine powder in 500 ml of 0.1 mol/L HCl
solution
Creatinine Test sample.
4 mg/dl Creratinine :
Dissolve 0.020 gm of creatinine powder in 500 ml of 0.1 mol/L HCl
solution
6 mg/dl creatinine :
Dissolve 0.030 gm of creatinine powder in 500 ml of 0.1 mol/L HCl
solution
0.1mol/l HCL solution
Add 17.4 ml of Conc. HCL solution (11.5 molar Conc.HCL solu.) & make

73
upto 2000 ml with DI water.

Creatinine R 1(NAOH)
1. Weigh 12gmNaOH.
2. Dissolve in approximately 500 ml DI water.
3. Add10ml of 30% brij in above mixture.
4. Weigh 1gm SDS and pour it into approximately 100 ml water in
beaker.Heat the solution until SDS dissolve.
5. Add SDS containing solution in main mixture.
6. Make upto 1liter with DI water.

Creatinine R2 (picric acid)

Dry picric acid between filter paper pieces
1. Weight 4.58gm dry picric acid.
2. Dissolve in approx. 300 ml water
3. Add 10 ml of 30% Brij in above mixture.
4. Remove froth with a clean object of glass or plastic dipped in capryl
alcohol
5. Make 1 liter with water.

Working alkaline -picrate reagent:

Mix 50 ml R1 & 50 ml R2 on the day of practical for 50 student.
Procedure
For sample and standard perform following.
1 ml of Alkaline Picrate Reagent
+
0.1 ml sample.
Mix it and Analyzed sample immediately at 505 nm wave length.
Note the Absorption (Optical Density=O.D.) at starting of reaction and at
end of reaction (after 60 second)
Calculate : Change in O.D. in 6o second = A = A0 - A60

Calculation and Result:

 Asample
Conc. of Creatinine in sample = ------------- X Standard
Astandard

Your result will be ---------------------------------

74
Comment on your result :

Reference Range:
Male 0.7 - 1.3 mg%
Female 0.6 - 1.2 mg%
1 mmol = 1000 micromole
Creatinine Molecular weight = 113.12

What will you do:

Express adult plasma creatinine reference range in micromole/L.

Write clinical conditions affecting plasma creatinine concentration.

75
 11. Estimation of plasma glucose
Reagent:
Glucose reagent : Dissolve 100mg of 4- Aminoantipyrine dye in 1000ml of
DI water and add 1 ml of phenol saturated water .

Water saturated Phenol

Phenol saturated water

Note :
Wear goggles & Glove while taking phenol.
Senior person must be present.
Glucose test sample :
Add 3 ml of analytical grade Sodium Hypochlorite solution
& make upto 10 ml with DI water
Glucose standard sample :
Add 1 ml of analytical grade Sodium Hypochlorite solution
& make upto 10 ml with DI water.

Principle:
Glucose + O2 Glucose Oxidase Gluconolactone + H2O2

Gluconolactone + H2O Spontaneous Gluconate

H2O2 + 4-aminophenazone + phenol Peroxidase Quinonamine (red

color

Procedure
Reagents Blank standard Plasma
H2O 0.1 ml
Glucose Standard 0.1 ml
Plasma 0.1 ml
Glucose oxidase + 1 ml 1 ml 1 ml
Peroxidase Reagent
(GOD POD reagent)
Mix & Incubate at room temperature for 30 min.
Read absorbance at 505 nm
Absorbance Ablank Astandard Aplasma
Calculation:

76
 (Aplasma
Ablank) -

Glucose concentration in plasma = -------------------- * Standard

(Astd. - Ablank)
Ablank : ---------------------------- A standard : -------------------------

Aplasma : ------------------------------ Stardard conc.:--------------

Glucose concentration in plasma =

Your result will be ----------------------------------

Comment on your result:

Reference Ranges:
Fasting Plasma Interpretation Oral Glucose Interpretation
Glucose Tolerance
<=110 mg% Normal <139 mg% Normal
111-125 mg% Impaired Fasting 140-199 mg% Impaired Glucose
Glucose Tolerance
>=126 mg% Diabetes >=200 mg% Diabetes mellitus
mellitus

Fasting = No food intake for at least 8 hours

Oral Glucose Tolerance = 75 gm glucose orally after 8 hrs of fasting.
Above results are valid if found on two or more occasions.

What will you do:

Measure Glucose concentration in given sample of plasma.

Draw above table again with mmol/L format. (Glucose MW=180 gm).

77
78
 12. Estimation of serum cholesterol
Reagent
Cholesterol reagent :
Dissolve 100mg of 4- Aminophenabenzene & 1 ml of phenol saturated
water and make upto 1000ml with DI water.
Cholesterol test sample :
Add 3 ml of HOCL(analytical grade) & make upto 10 ml with DI water
Cholesterol standard sample :
Add 1 ml of HOCL(analytical grade) & make upto 10 ml with DI water
Principle:
Cholesterol ester Cholesterol esterase Cholesterol + Fatty acid

Cholesterol + O2 Cholesterol Oxidase Cholest-4-en-3-one + H2O2

H2O2 + 4-aminophenazone + phenol Peroxidase Quinonamine

Reagents and procedure:

Reagents Blank Standard Plasma
H2O 0.1 ml
Cholesterol Standard 0.1 ml
Plasma 0.1 ml
Cholesterol oxidase + 1 ml 1 ml 1 ml
Peroxidase Reagent
(COD POD reagent)
Mix, incubate at room temperature for 30 min.
Read absorbance at 505 nm
Absorbance Ablank Astandard Aplasma

Calculation :
(A plasma - Ablank)
Cholesterol concentration in plasma = -------------------- * Standard
(Astandard - Ablank)

Result:
Ablank : ____________ Astd : _____________

Aplasma : _____________ Standard Conc. _______

Glucose concentration in plasma =

79
Your result will be ----------------------------------
Comment on your result:

Reference Ranges:
Desirable: <200 mg/dL
Borderline: 200-239 mg/dL
High: >=240 mg/dL

What will you do:

Measure Cholesterol concentration in given sample of plasma.

Rewrite reference ranges in mmol/L format. Cholesterol MW = 386.64 gm

80
13. Estimation of Serum Total bilirubin
Reagent:
Refer to SOP for billirubine reagent:

R1 (Cafeine)

1. Dissolved 75 gm caffeine in 900 ml deionised water with constant

mixing
2. Add 112 gm Na Benzoate in above mixer with constant mixing
3. Add 112 gm anhydrous Na Acetate in above mixer with constant
mixing
4. Add 2 gm disodium EDTA in above mixer with constant mixing
5. Make upto 2 liter with deionised water
6. Filter if turbid
7. Store in glass container in freeze
8. If crystalline precipitation are seen at 2-8'C, bring solution to room
temperature to redisolve it before use

Diazo A

1. Dissolve 10 gm sulfanilic acid in 900 ml deionised water

2. Add 30 ml concentrated HCL in above mixer
3. Make upto 2 liter with deionised water
4. Store in glass container

Diazo B

1. Dissolve 1.25 gm Na nitrite(NaNo2) and make upto 250 ml with

deionised water.
2. Store in brown glass container

R2 (Diazo Mix)

Mix Diazo A 10 ml & Diazo B 0.3 ml

Billirubin test solution

Dissolve 2 mg of billirubin powder & make upto 100 ml with DI water

Billirubin test solution :

Dissolve 4 mg of billirubin powder & make upto 100 ml with DI water

81
 Principle
One molecule of bilirubin reacts with two molecules of diazotized
sulfanilic acid (diazo mix) in an acid solution to form two purple
azobilirubin molecules (560 nm). Direct bilirubin reacts in water as well
as with acceletor (e.g. caffeine, methanol) , while indirect bilirubin react
only in presence of acceletor.

Reagents

Reagents Test Test Standard Standard

Blank Blank
Sample 0.1 ml 0.1 ml

Standard 0.1 ml 0.1 ml

R1 (Cafeine Reagent) 1 ml 1 ml 1 ml 1 ml

Incubate for 10 minute

R2 (Diazo Mix) 0.2 ml 0.2 ml

Diazo Blank Reagent (Diazo A) 0.2 ml 0.2 ml

82
Absorbance A Test Blank A Test A Standard Blank A Standard

Blanks are taken to subtract absorbance caused by hemolysis (resulting

in presence of red color of hemoglobin in serum).
Diazo blank reagent does not have sodium nitrite, hence do not produce
azobilirubin.

Calculation:

(ATest – ATest Blank)

Total Bilirubin (mg/dL) = ---------------------------- X Std Conc.
(AStandard – AStandard Blank)

Result
Your result will be--------------

Comment

Reference ranges: (For Adults)

Total Bilirubin 0.2-1.2 mg/dl
Direct Bilirubin 0.1-0.4 mg/dl
Indirect Bilirubin 0.2-0.7 mg/dl
Bilirubin = MW 584.67 gm
1 mmol=1000 micromole

What will you do:

Enumerate causes of unconjugated hyperbilirubinemia and mixed
hyperbilirubinemia.

Express Reference ranges in micromole/Liter.

83
Sample for bilirubin should not be exposed to light. Phototherapy is used
in treatment of neonatal jaundice. Explain and correlate.

84
14. Estimation of serum total protein
Except immunoglobulins, majorities of plasma proteins are synthesized
by liver. Various tissues catabolize plasma proteins. Plasma protein
concentration reflects balance between their synthesis and catabolism.
Under certain conditions intact proteins from plasma are also lost
through GIT, urine and skin. Proteins from intravascular compartment
may reach other body compartments. Protein concentration may also be
affected by change in plasma water.
Reagent:
Refer to SOP for total protein.
1. Weight 3 gm Cuso4.5H2O.
2. Dissolve in approx. 500 ml water.
3. Weight 9 gm (Na K Tartrate).(4H2O) and 5 gm KI.
4. Add sequentially 9 gm (Na K Tartrate).(4H2O) and 5 gm KI in copper
sulphate solution.
5. Weight 24 gm NaOH.
6. Add slowly with mixing 24 gm NaOH in 100ml of water.
7. Add slowly with mixing NaOH solution in copper sulphate solution.
8. Make upto 1 liter with water

Total Protein Standard:

Dilute Serum pool with DI water(1:20 ratio)

Prepare 10 ml of standard (0.5 ml pool serum+ 9.5 ml DI water)

Total Protein Test:

Dilute Serum pool with Nine part of DI water(1:10 ratio)

Prepare 10 ml of Test (1 ml pool serum+ 9 ml DI water)

Principle :
Two or more peptide bonds of proteins form coordination complex
with one cu2+ in alkaline solutions to form a colored product. The
absorbance of the product is determined spectrophotometrically at 540
nm.

85
 Procedure:
Reagents Blank Standard Sample
H2O 0.1 ml - -
Protein standard - 0.1 ml -
Sample - - 0.1 ml
Biuret reagent 1 ml 1 ml 1 ml
0
Mix and incubate at 37 C temperature for 30 min.
Read Absorbance at 540 nm
Absorbance Ablank Astandard. Asample

Calculation and result:

(Aserum - Ablank)
Total Protein concentration in plasma = -------------------- * Standard
(Astandard - Ablank)

Your result will be ---------------------------

Comment :
86
Reference ranges: Serum proteins 6.0-8.0 g/dL
Albumin 3.5-5.5 g/dL
Globulins 2.0-3.6 g/dL
Fibrinogen 0.2-0.6 g/dL

What will you do:

Q-1 Serum protein reference ranges are lower than that of plasma.
Explain.

Q-2 Why reference ranges for plasma proteins can not be expressed in
mmol?

QA-3 Enumerate conditions affecting plasma protein level.

87
15. Estimation of serum albumin
Different disorders affect different plasma proteins differently.
Thus, it is useful to know albumin and globulin concentration in serum,
in addition to total protein. Once total protein and albumin (as shown
below) are estimated, serum globulin can be calculated.

Reagent :
Albumin Standard:Dilute Serum pool with DI water(1:20 ratio).Prepare
10 ml of standard (0.5 ml pool serum+ 9.5 ml DI water.

Total Protein Test:Dilute Serum pool with Nine part of DI water(1:10

ratio).Prepare 10 ml of Test (1 ml pool serum+ 9 ml DI water).

BCG reagent : Refer SOP for Albumin reagent preparation.

Add 42mg BCG(MW=698) in approx. 250 ml DI water.
Add 5.9 gm succinic acid (MW=118.09 ,pKA1=4.2 ,pKA2 = 5.6) in above
mixer while constantly mixing.
Add 1.8 ml of 30% Brij-35 In above mixer while constantly miximg.
Add 1 gm NAOH in above mixter while constatly mixing
Add 200 mg Na azide in above mixer while constatly mixing.
If required adjust pH to 4.2
Make upto 1000 ml with volumetric flask with deionised water.

88
 Principle:
BCG = BromoCresol Green

At pH 4.2: [Albumin+] + [BCG-]  [Albumin+ BCG- complex]

At pH 4.2 BCG is yellowish, while Albumin+ BCG- complex is greenish.

The green color is measured at 630 nm.

Procedure:

Reagents Blank Standard Sample

H2O 0.1 ml - -
Standard - 0.1 ml -
Sample - - 0.1 ml
BCG reagent 1 ml 1 ml 1 ml
Mix, and read immediately at 630 nm.
Absorbance Ablank Astandard Asample

Calculation and result:

(Asample - Ablank)
Albumin concentration in serum = -------------------- * Standard
(Astandard - Ablank)

89
Your Result will be --------------------------
Comment on your result

Reference ranges:
Serum Total proteins 6.0-8.0 g/dL
Serum Albumin 3.5-5.5 g/dL
Serum Globulins 2.0-3.6 g/dL
Plasma Fibrinogen 0.2-0.6 g/dL

What will you do:

Calculate S.Globulin from Sample’s Albumin conc. & If Total Protein = 7
gm/dl.

Enumerate conditions of alter A:G Ratio.

90
16.Estimation of Cerebrospinal fluid protein.
Cerebrospinal fluid is not freely permeable to plasma proteins. Hence, its
concentration is almost 1/100 times the plasma. Some proteins are
synthesized by the pia matter itself. Under various CNS inflammatory
conditions, CSF protein is increased due to increased permeability of pia
matter as well as due to increased synthesis by it.
Reagent:
Pyrogallol Red reagent:Refer to SOP for Pyrogallol red reagent

PR(Pyrogallol red)
Making Reagent

1. Dissolve pyrogallol red 60 mg in 100 ml of methanol.

2. Store in plastic container.

MB(molybabdate)

Making Reagent

91
 1. Dissolve disodium molybdate 0.24 gm in 100 ml of deionized water.
2. Store in plastic container.

Final microprotein Reagent

Making Reagent

1. Dissolve succinic acid 5.9 gm in 900 ml of deionized water.

2. Add sodium oxolate 0.14 gm in above mixture with constantly
mixing.
3. Add sodium banzoate 0.5 gm in above mixture with constantly
mixing.
4. Add PR(Pyrogallol red) 40 ml in above mixture with constantly
mixing.Discard other 60 ml PR(Pyrogallol red).
5. Add (molybabdate) 4 ml in above mixture with constantly
mixing.Discard other 96 ml (molybabdate).
6. Calibrate PH meter and if required adjust PH to 2.5.
7. Make up to above mixture 1 L with deionized water.

CSF Protein Calibrator: Take 0.02 ml of serum protein & make upto 10
ml with DI water
CSF protein Sample :Take 0.04 ml of serum Protein & make upto 10 ml
with DI Water

Principle:
pyrogallol red-molybldate complex combine with protein and give
colour which is mesure at 630 nm.

Procedure:

Reagents Blank Standard Sample

H2O 0.1 ml - -
CSF Protein Standard - 0.1 ml -
CSF Sample - - 0.1 ml
Pyrogallol Red reagent 1 ml 1 ml 1 ml
Mix, wait for10 min, mix before reading at 630 nm.
Absorbance Ablank Astandard Asample

(Asample - Ablank)
92
Protein concentration in CSF = -------------------- * Standard
(Astandard - Ablank)
Your result & comment

Reference ranges: 15-45 mg%

What will you do:

Enumerate conditions affecting CSF protein level.

93
 17. Estimation of plasma uric acid
Uric acid is formed by catabolism of purines. Uric acid is excreted by
kidney.
Reagent:
Uric acid test sample :
Add 1.5 ml of analytical grade Sodium Hypochlorite solution& make upto
10 ml with DI water.
Uric acid standard sample :
Add 0.5 ml of analytical grade Sodium Hypochlorite solution& make upto
10 ml with DI water.
Principle:
Uric acid yields allantoin and H2O2 on action by uricase. Peroxidase use
hydrogen peroxide to oxidize various colorless dyes to red colored
quinonimine like dyes measured at 505 nm by absorption photometry .
Uricase
Uric acid + 2H2O + O2 ------------ Allantoine + CO2 + 2H2O2

H2O2 + 4-aminophenazone + phenol Peroxidase Quinonamine (red

color)

Procedure:

Test Standard Blank

Serum 0.1 ml
Standard 0.1 ml
Water 0.1 ml
Reagent 1 ml 1 ml 1 ml
Measure absorbance at 505 nm
Absorbance ASerum AStd. ABlank

Calculation
(ASerum - Ablank)
Uric acid concentration in Sample = -------------------- * Standard
(Astandar. - Ablank)

Result & Comment:

94
Reference ranges:
Male : 3.6 - 7.7 mg/dL (214 to 458 micromole/L)
Female : 2.5 – 6.8 mg/dL (149 to 405 micromole/L)

What will you do:

Enumerate conditions affecting plasma uric acid level.

Calculate molecular weight of uric acid from reference ranges given.

95
18.Electrophoresis
Reagent: Refer to Sop for Serum & Hb electrophoresis
Principle:
Electrophoresis is a refers to the migration of charged molecules under
electrical field.
Procedure:
Prepare thin 1 % Agarose gel in appropriate buffer.
Apply appropriate sample in thin line over agarose gel.
Keep gel with sample applied in electrophoretic chamber & connect the
gel with buffer through strips of filter paper and apply appropriate
voltage.
After sample run is completed, switch off the power supply and remove
slide from chamber.
Denature proteins in methanol and dry the gel with heating.
Stain slide with appropriate stain.

Bufer :
Barbiturate Buffer (for protein electrophoresis)
Tris Buffer (for protein electrophoresis)
TEB Buffer (for Haemoglobin electrophoresis)

Supporting Media:
Whatman Filter Paper
Cellulose Acetate Paper
Agarose Gel
Polyacrylamide Gel

96
Clinical Applications:
Diagnosis of sickle trait and sickle disease.
Diagnosis of multiple myeloma

Questions:

What is agarose? Why it is used to prepare gel.

Name other electrophoresis support media.

How much agarose is required to prepare 15 ml of 1% agarose?

Which sample is used for hemoglobin electrophoreis?

97
What was voltage, current and duration of electrophoresis demonstrated
to you?

What are major hazard of electrophoresis procedure?

What precautions must be taken to avoid them?

What is difference between electrophorogram of serum protein and

plasma protein?

Name stain used during demonstration.

Draw hemoglobin electrophoretic patten in normal, sickle trait and sickle

cell disease patients. Explain its biochemical basis.

98
Draw hemoglobin electrophoretic patten in HbC and HbD carrier patients.
Explain its biochemical basis.

Draw serum protein electrophoretic patten in multiple myeloma. Explain

its biochemical basis.

What is monoclonal antibody?

99
19. Chromatography

Principle : Chromatography is a process in which components of a

mixture are separated by differential distribution between a mobile phase
and a stationary phase. Components with greater distribution into the
stationary phase are retained and move through the system more slowly.

Requirements :

Amino acid standard : 1% amino acid standard

Mobile phase : 12(Butanol):3(Glacial acetic acid):5 (Di water)
Sample Type : Serum,Urine .
Equipments & consumables :Chromatography chamber(air tight),
Glass road, clips, Whatman fiter paper, Gloves, pencil, scale, centrifuge,
pipettes
Stain : Ninhydrin solution (0.25 %)(250 mg of Ninhydrin powder in 100
ml of methanol/acetone)

Procedure :
Clean hands throughly with soap.
Wear gloves before handling filter paper.
Take a Whatman filter paper ,make a horizontal line at one end of filter
paper,around 1.5-2 cm above from the edge of the paper.
Put marking at 3.5 cm apart for each sample for sample application.
Repeat sample & standard application for twice once previous sample
gets dried .
Take 500 ml of mobile phase reagent in reagent chamber.
Clip the dried filter paper on glass rod,Make sure that distance between
each sample & road is equal.
Put glass rod in chromatography chamber, make sure that sample
application sites do not get dipped in the solvent.
Close the chamber air tight .Note the time & allow the separation for 4
hours.
Remove the paper from chamber after 4 hours, allow the paper to dry at
room temperature.
Take 0.25% Ninhydrin solution in shallow plastic container big enough to
accommodate the entire filter paper. Dip paper in it for few seconds.
Put the paper in incubator at 100oc for 20-25 minute/ till purple bands
are seen
Preserve the paper in dark room for latter use.

Calculate Rf value

100
 Distance from application point to solute center
Rf= ---------------------------------------------------------
Distance from application point to solvent front

QUESTIONS :

Name stationary phase in the experiment. Is it mainly hydrophobic or

mainly hydrophilic? Explain.

Name mobile phase in the experiment. Is it mainly hydrophobic or mainly

hydrophilic? Explain.

List hydrophobic amino acids used in the experiment.

List hydrophobic amino acids used in the experiment.

Comment why some amino acids move faster and other slower during the
chromatography.

Why wearing gloves is essential in the experiment?

Why wearing protective eye glass is essential in the experiment?

Name few conditions where abnormal amount of some amino acids are
lost in urine. Explain their biochemical basis.

101
102
 Clinical Case - 1
Early in the morning, 40 years old male patient came in emergency with
complain of chest pain, perspiration and altered consciousness for 4
hours. Patient also had diabetes mellitus for 10 years. He was taking
medicine for diabetes mellitus irregularly. In history, it was found that he
was chronic alcoholic and a day before chest pain , he also had heavy
alcohol ingestion., with no feed intake.Doctor asked for few blood
investigations. From ECG finding and abnormal cardiac function test.
Diagnosis of myocardial infarction was confirmed.

Following treatment was given

 loading dose of anti-platelet drug (Aspirin)
 loading dose of hypocholesterolemic (Statin group) drug
 Fibrinolytic drug (streptokinase)
 50% dextrose saline with Thiamine (Vitamin B1)
After complete management and recovery after 7 days of admission in
hospital, at time discharge from hospital, physician advised to take
medicines regularly and to take more amount of fruit and fiber food.
 Random Blood Sugar = 30 mg %
 HbA1C =9%
 S. Cholesterol = 350 mg %
 S. Triglyceride = 250 mg %
 S. HDL Cholesterol = 25 mg %
Question
1. What are chronic complication of DM?
2. Why uncontroled diabetic mellitus increase chances of
atherosclerosis?
3. What is cardiac function test?
4. Which test will you prefer to do for diagnosis of myocardial
infarction, if patient come after 4 day of onset of chest pain?
5. How statin reduce cholesterol level?
6. What is biochemical explanation of hypoglycemia?
7. Why physician asked to give injectable 50% Dextrose saline with
Thiamine (Vitamin B1)?
8. What is role of fruits and fiber in chronic diabetes mellitus and
atherosclerosis?
9. Why blood sample for blood sugar estimation is collected in fluoride
containing vial?
10. What is re-perfusion injury ? And what is role of allopurinol
to prevent it?
11. How will you calculate patient’s LDL cholecterol?
12. What is role of fibrinolytic drugs (streptokinase) in myocardial
infarction?

103
 Clinical Case – 2
56 year male patient came in emergency with alter-conciuosness &
haemetemesis . He was suffering from chronic cirrhotic liver disease due
to chronic alcoholism. On examination , it was found that he has edema
on both lower limb, fluid collection in peritoneal cavity (Ascites),
yellowish discolouration of skin & sclera (icterus), with hypotension
(decrease Blood Pressure).
On blood investigation following was found.
 Blood Glucose : 50 mg%
 Serum Protein : 5.5 gm %
 Serm Albumin : 2.0 gm%
 Serum Ammonia : Very High
 Serum Total Billirubin : 20 mg%
 APTT – Test : 60 second
 APTT – Control : 30 second
 APTT – INR : 2
 Haemogloin : 6 gm%
Ultra Sono-Graphy detected
 Cirrhosis of Liver
 Fatty Liver
Physician advise to give Following treatment
 Injection 10% Dextrose
 Injection Thiamine (B1)
 Injection Vitamin K
 Injection 10% Albumin
 Oral Neomycin (Anti-microbial, Antibiotic)
 Liq Lactulose (Laxative)
 Oral Phenylbutarate

1. Biochemical explaination about following symptoms in chronic

alcoholic
a. Alter conciousness
b. Haemetemesis
2. Biochemical explaination about following signs in chronic alcoholic
a. Edeme
b. Ascites
c. Hypotension
3. What is hepato-renal syndrome?
4. Biochemical reason for giving following in patient of chronic
alcoholic
a. Dextrose plus thiamine
b. Vitamin K
c. 10% Albumin
d. Oral Neomycin (Anti-microbial, Antibiotic)
e. Liq Lactulose (Laxative)
f. Oral Phenylbutarate

104
 Clinical Case – 3

A 54 year old obese person come in emergency with altered consciousness

level and increase respiratory rate (tachypnia) for last 4 hours.
He is having history of uncontrolled diabetes mellitus since 15 years, as
he was not following any medical advice from physician. He was on
insulin therapy for 3 years, but he was not taking regular dose of insulin.
Patient's relative is telling that he is also having complain of weakness
and decrease urine output for last 2 days.
On General examination, physician noted
 Dryness of mouth
 Pale & dry conjunctive
 Shrunken eye ball.
 Feeble (low volume) pulse
 Tachypnea (increase respiratory rate)
 Tachycardia (increase heart rate)
 Very low blood pressure (70/40 mm Hg).
Doctor makes admission in ICU and asked immediately for blood
investigation.
Parameter Value Reference range
RBS 500 mg/dl 140 mg/dl
Serum Acetone 10 mg/dl <1 mg/dl
Serum Creatinine 2.5 mg/dl 0.4 - 1.4 mg/dl
Blood Urea 150 mg/dl 15 - 45 mg/dl
Serum Na+ 120 mmol/l 135 - 145 mmol/l
Serum K+ 6.0 mmol/l 3.5 - 5.0 mmol/l
pH 7.1 7.35 - 7.45
pO2 95 mmHg 90 - 100 mmHg
pCO2 24 mmHg 32 - 40 mmHg
HCO3- (Bicarbonate) 12 mmol/l 24 - 32 mmol/l
Diagnosed = “Diabetic ketoacidosis with acute renal failure”
Advised to following treatment.
 Inj normal saline fast I.V. (4-5 litre in 1st 24 hrs) Until systolic
blood pressure reaches to normal
 Inj Human Insulin injection slow infusion I.V.As per blood sugar
level
 Inj Bicarbonate 200 ml I.V.
 K+ Binding resin Sachets Orally.
Urinary catheterization done.
But urine output is nil
105
To follow below protocol for treatment of this patient.
 If RBS > 200 mg/dl ---> Give Normal Saline + Human Insulin
 If RBS < 200 mg/dl ---> Give Dextrose Saline + Human Insulin
Doctor asked to repeat following investigation during management
 RBS every 2 hourly.
 Serum K+ level after 4 hours.
 Arterial Blood Gas analysis after 6 hours (if require)

24 hours after admission and intensive care

 He get consciousness, normal respiration ,
 normal blood pressure & 1200 ml of urine output.
 RBS = 150 mg% with Human insulin infusion
 Serum acetone = 2 mg/dl
 Electrolyte and ABG = Normal.
He shifted to ward & remained admitted for 5 days in hospital.

On discharge, physician advises to take prescribe insulin dose regularly

as well as regular follow up with FBS & PP2BS.

1. Give explanation for altered consciousness and increase respiratory

rate in this case.
2. What metabolic and functional abnormality can occur due to
increase acetone level?
3. Why after 24 hours serum acetone came down nearer to normal
level?
4. What is patho-physiology behind decrease urine output in this
patient?
5. Give comment on patient ABG report.
6. Give biochemical reason for increase K+ level in this case.
7. What is biochemical reason for giving dextrose saline plus human
insulin infusion if RBS is below 200 mg%?
8. How bicarbonate, insulin and K+ binding resin reduce serum
potassium level?

106
Medical Biochemistry – Syllabus
Medical Biochemistry encompasses any topic of biochemistry relevant to
human health and diseases. As medicine is an ever expanding body of
knowledge, Medical Biochemistry syllabus is continuously expanding.

At bare minimum, you are expected to get integrated knowledge of

theoretical and practical aspects of following in context of the field of
Medicine. In addition, newer advances in the field of medical biochemistry
needs to be studied.

Carbohydrates:
Chemistry, Nutrition, Digestion, Absorption, Transport, metabolism and
biochemical basis of related diseases, their treatment and prevention.
Alcohol metabolism

Amino acids and Proteins:

Chemistry, Nutrition, Digestion, Absorption, Transport, metabolism and
biochemical basis of various diseases, their treatment and prevention.
Enzymes
Hemoglobin and Heme metabolism
Plasma proteins
Collagen, elastin and extracellular matrix proteins

Lipids:
Chemistry, Nutrition, Digestion, Absorption, Transport, metabolism and
biochemical basis of various diseases, their treatment and prevention.
Prostaglandins

Nucleic Acids:
Chemistry, Nutrition, Digestion, Absorption, Transport, metabolism and
biochemical basis of various diseases, their treatment and prevention.
Genetics
DNA and RNA structure and functions
Genome and Chromatin
Replication, Transcription, Genetic code and Translation
DNA Damage and repair
Mutations
Recombinant DNA Technology
Cell cycle and its regulation
Biochemistry of cancer
Biochemical basis of genetic diseases, their treatment and prevention.

107
Integration of metabolism:
Bioenergetics
Cellular Respiration
Interrelationship among metabolic pathways.
Biochemical basis of related diseases, their treatment and prevention.

Vitamins:
Chemistry, Nutrition, Digestion, Absorption, Transport, metabolism and
biochemical basis of various diseases, their treatment and prevention.

Minerals:
Chemistry, Nutrition, Digestion, Absorption, Transport, metabolism and
biochemical basis of various diseases, their treatment and prevention.

Water and pH:

Water biochemistry and biochemical basis of related disorders.
Blood buffers, regulation of blood pH and biochemical basis of related
disorders.

Xenobiotics:
Chemistry, Metabolism and excretion of xenobiotics.
biochemical basis of related disorders

Tools for study of Biochemistry:

Colorimetry
Chromatography
Electrophoresis
ELISA
RIA
PCR and blotting techniques

Biochemistry of supramolecular structures (overlapping above topics):

Biochemical characteristics of various organelles, cells, tissues and
organs e.g. Mitochondria, perioxisomes, general cell structure, RBC,
Liver, Brain, Heart, Skeletal muscles etc.

108
 Subject distribution and paper style
Paper distribution:
Paper 1:
Chemistry, digestion, absorption and metabolism of Carbohydrate, Lipid,
Water, pH, Minerals
Paper 2:
Chemistry, digestion, absorption and metabolism of Protein( including
hemoglobin, plasma proteins and enzymes), Nucleic acids including
genetics, Vitamins, Xenobiotics
Note: Overlapping common topics are acceptable in any paper e.g
integration of metabolism, nutrition, tissue and organ biochemistry,
biochemistry laboratory techniques, biochemistry of microorganisms (e.g
HIV), environmental biochemistry and Cancer.

Paper style( paper 1 and 2)

Section 1
Q-1 Short notes (2 out of 3) 08 marks
Q-2 Describe in brief (4 out of 6) 12 marks
Q-3 Write answer in few line(5 out of 6) 05 mqrks

Section 2
Q-4 Case with 5 questions 10 marks
Q-5 Answer in few lines(5 out of 7) 10 marks
Q-6 Write answer in few line(5 out of 6) 05 marks

109
 Important Short Notes
General
1.Blood buffer mechanism
2.Renal Buffer mechanism for acid base balance.
3.Arterial Blood Gas Analysis & interpretation.
4.H2O2 – Myeloperoxidase (MPO) – Halide system of ROS (reactive Oxygen species)
5.Fluidic Model of Cell membrane
6.Type and Example of Transport mechanism.
7.Primary & Secondary cause of Hyperuricemia (Gout)
8.Chemi-osmotic hypothesis.
9.Energy production in TCA cycle.
10.Uncouplers of Oxidative phosphorylation
11. Principle, Type and utility of Electrophoresis.
12. Principle, Type and utility of ELISA.
13.Principle and utility of Colorimeter
14.Biochemical changes in Liver,Adipose tissue and muscle in well fed state
15.Biochemical changes in Liver,Adipose tissue and muscle in well fasting.

Carbohydrate
16.Mucopolysaccharide
17.Proteoglycans
18.Digestion & absorption of carbohydrate
19.Lactose intolerance
20.Energy production of Glycolysis
21.Regulation of Glycolysis
22.Amphibolic role of TCA cycle
23.Significant of NADPH
24.Significant of HMP Shunt pathway
25.Substrate & Regulation of Gluconeogenesis
26.Sorbitol Pathway
27.Polyol pathway and it’s significant
28.Effect of alcoholism on gluconeogenesis, oxidation of fatty acid & TCA cycle.
29.Diagnostic criteria for Diabetes Mellitus
30.Define and significant of Glycate haemoglobin
31.Metabolic alteration in Diabetes Mellitus
32.Acute and Chronic complication of Diabetes Mellitus
33.Biochemical explaination of Diabetic Ketoacidosis
34.Define C-peptide & it's significant.
35.Define and significant of Glycated (HbA1c) haemoglobin
36.Advance glycated end product.
37.Type of Diabetes Mellitus . Explain LADA, MODY,Gestational diabetes & Secondary
diabetes
38.Von Gierke’s Disease
39.G6PD deficiency
40.Fate of Acetyl CoA.
41.Ketone body synthesis & utilization
42.Alcohol metabolism
43.Epimer & Isomer

110
Lipid
44.Name and Significant of Essential Fatty acid
45.Type ,Difference & clinical significant of saturated & unsaturated fatty acid.
46.Fate of cholesterol
47.Ketogenesis and ketolysis
48.Metabolism of LDL
49.Regulation of LDL receptor
50.Formation of Eicosanoid & explain its inhibitor with significance.
51.Pathogenesis of atheroscerosis in contex of 'oxidised LDL'.
52.Type and differenciation of oxidation of fatty acid
53.Rancidity of Fatty acid
54.Liposome & Micelle
55.Function of Phospholipids
56.Lung surfactant
57.Role of Phospholipase A2 of Snake venum in RBC lysis.
58.Role of phospholipid in signal transmission
59.Lipid digestion –absorption.
60.Significance and Regulation of Cholesterol.
61.Risk factor for Atherosclerosis
62.Prevention of Atherosclerosis
63.Type and Function Lipoproteins
64.Type and function of Apo-lipoproteins
65.Metabolism of HDL
66.Reverse cholesterol transport.
67.Role of' Lipoprotein-a in Atherosclerosis
68.Energy production of long (16 carbon ) saturated fatty acid through Beta oxidation
69.Carnitine shuttle
70.Assessment,Metabolic changes and influencing factors of obesity.
71.Cause of Fatty liver
72.Name the Lipotrophic Factor. Explain it’s effect

Protein and Amino acid

73.Zwitter ion
74.Functional classification of protein
75.Protein structural –functional relationship
76.Primary , Secondary , Tertiary and Quarternary structure of Protein.
77.Define Chaperon & Prion protein
78.Protein folding & unfolding.
79.Define Protein Denaturation. Give It’s significant & causative factor
80.Digestion & Absorption of Protein
81.Urea cycle & Ammonia detoxification.
82.Define & give significant of transamination, transdeamination & deamination.
83.Overview of tyrosine & phenylalanine
84.Fates of Tyrosine & Phenylalanine & it’s related disorder
85.Biochemical explanation of Phenylketonuria
86.Biochemical explanation of Albinism & Alkaptonuria
87.Fates of Tryptophan & it’s related disorder.
88.Maple Syrup Urine Disease

111
89.Folate trap
90.Collagen-Homocystineuria-Ectopia lentis
91.Nitrogen disposal-GDH and Alpha ketoglutarate
92.Role of Glutathione & NADPH for maintain RBC membrane
93.Fates of Glycin
94.Fates of Glutamic acid
95.Transport and Detoxification of Ammonia
96.Role of 2-3 BPG on oxygen diffusion-dissociation and effect during hypoxia
97.Mechanism of the Halden & Bohr effect
98.Developmental changes in Hemoglobin gene expression from intrauterine life to
adult.
99.Regulation of Hemoglobin synthesis.
100.Haemoglobin degradation pathway & it's related disorder.
101.Types , Causes and differentiation of Jaundice by serum and urine examination.
102.Haemoglobin synthesis pathway & it's related disorder.
103.Define Porphyria. Explain Causes, Clinical Feature and diagnosis of Acute
intermittent porphyria and Congenital erythropoietic porphyria.
104.Molecular and Biochemical explanation for pathogenesis of Sickle cell disease
105.Molecular and Biochemical bases of Thalassemia.
106.Define and explain cause & effect of Met-haemoglobinemia
107.Transport Plasma proteins
108.Storage proteins

Enzyme
109.Write and Explain Factor affecting enzyme activity with example.
110.Explain First order & zero order enzyme kinetics.
111.Explain Difference in Function of Glucokinase and Hexokinase on bases of it’s
Vmax and Km.
112.Difference between Competitive inhibition and Noncompetitive inhibition.
113.Diagnostic importance of isoenzyme
114.Type of Enzyme Inhibition. Explain with example.
115.Regulation of Enzyme activity
116.Define Co-Enzyme, Cofactor , Apo-Enzyme , Prosthetic group & Holoenzyme
117.Enumerate Liver Function Test & Write it’s significant.
118.Enumerate Cardiac Function Test & Write it’s significant.

Nutrition & Vitamin

119.Protein Energy Malnutrition (PEM)
120.Difference between Kwashiorkor & Murasmus
121.Factor affecting Basal metabolic rate
122.Assessment of obesity.
123.People having Mediterranean diet show low incidence of CHD
124.Clinical significance of Dietary fibre
125.Vitamin B12 & folic acid deficiency can cause hyperhomocysteinemia
126.Folate trap
127.Effect of Warfarin & Dicoumarol on Vitamin K metabolism
128.Function of Vitamin K
129.Function of vitamin C

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130.Role of Active form of Vitamin B1 (Thiamine) in metabolism and give it's
significance.
131.Visual cycle of Vitamin A
132. Name and write clinical manifestation occur in Vitamin A deficiency.
133.Metabolism, Function and Clinical significance of Vitamin D
134.Homoestasis changes in calcium , vitamin D & parathyroid hormone in case of
renal failure.
135.Regulation of calcium.
136.Hypocalcaemia
137.Mucosal block theory of iron absorption.
138.Iron deficiency Anemia
139.Type and clinical features of Beriberi.
140.Pernicious anaemia.
141.Metabolic changes during starvation
142.Metabolic role of Vitamin B12.
143.Name Riboflavin (FAD) & Niacin (NAD+ & NADP+) dependant enzymatic reaction.

Molecular
144.Type and Watson & Crick Model Of DNA
145.Organisation of eukaryotic DNA.
146.t-RNA.
147.Degeneracy & wobbling phenomena
148.Genetic codon
149.Molecular basis of Sickle cell anaemia.
150.Type of DNA polymerase & specify it's fuction.
151.Name & role of the component of the DNA replication fork
152.DNA repair mechanism.
153.Define Telomer & Telomerase. It’s significant
154.Effect and Type of Mutation with examples.
155.Initiation of Transcription
156.Post-transcription modification.
157.Post translation modification.
158.Protein synthesis inhibition by drugs.
159.Salvage pathway of Purine synthesis and related disease.
160.Lysch Nyhan Syndrome
161.Adenosine deaminase deficiency.
162.Lac operon
163.Procedure & Significant of PCR
164.Significant of RFLP in diagnosis of Sickle cell disease
165.Microarray
166.Recombinant DNA Technology
167.DNA Library
168.Uric acid synthesis and its inhibitors.
169.Causes and management ( biochemical aspect ) of Gout
170.Type and function of Topoisomerase
171.DNA dependent RNA polymerase.
172.Ribozymes
173.Define Autosomal dominant & Autosomal recessive & Draw pedgree chart.

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