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ELECTROPHORESIS
1. Electrophoretic technique and its importance.MGR Aug 2009
2. Describe the separation of Serum Proteins by paper
electrophoresis. Draw the pattern of electrophoresis in i)
Multiple Myeloma ii) Nephrotic Syndrome. MGR Aug 2010
3. Serum protein electrophoresis MGR Aug 2011
4. Electrophoresis and its applications MGR Feb 2013
5. List two applications of electrophoresis in medicine. MGR Feb
2014
6. Define electrophoresis and mention its applications MGR Aug
2014
7. Electrophoresis MGR Feb 2011, 2012
8. M band MGR Aug 2011
9. Describe the separation of Serum Proteins by paper
electrophoresis.
10.Draw the pattern of electrophoresis in i) Multiple Myeloma ii)
Nephrotic Syndrome.
11.How are plasma proteins separated by electroporesis? What is
the significance of M protein? Pon Nov 2011
12.Gel electrophoresis. Pon May 2009
ELECTROPORESIS
1. The term refers to the movement of charged particles through an electrolyte when
subjected to an electric field. The positively charged particles (cations) move to
cathode and negatively charged particles (anions) to anode. Since proteins exist as
charged particles, this method is widely used for the separation of proteins in
biological fluids.
2. Factors affecting electrophoresis:
a. Net charge on the particles (ph of proteins)
b. Mass and shape of the particles.
c. The pH of the medium.
d. Strength of electrical field.
e. Properties of the supporting medium.
f. Temperature.
3. Electrophoresis Apparatus:
a. It consists of the electrophoresis tank to hold the buffer and fitted with the
electrodes, as well as a power pack to supply electricity at constant current
and voltage.
b. The buffer is chosen in such a way so as to ensure effective separation of the
mixture of proteins. e.g. serum proteins are separated at a pH of 8.6 using
barbitone buffer. At this pH all serum proteins will have a net negative charge
and will migrate towards the anode.
3. Support Medium:
a. Filter Paper: long time interval and diffusion of particles leading to blurring of
margins are the disadvantages
b. Cellulose Acetate Membrane: expensive, but the process takes less than one
hour and excellent separation; widely used for separation and identification of
lipoproteins, isoenzymes and hemoglobins.
c. Agar or Agarose: less expensive; the gel is prepared in the buffer and spread
over microscopic slides and allowed to cool. Serum sample or biological fluid
is applied by cutting into the gel. The electrophoretic run takes about 90
minutes. Serum proteins are commonly studied by agar electrophoresis.
d. Polyacrylamide gel electrophoresis (PAGE): It has a high molecular sieving
effect and so separation is very efficient; will show more than 20 different

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bands. The amount of cross linking and thereby the pore size can be
controlled. Another variant is the SDS-PAGE electrophoresis. Here proteins are
boiled for 1-2 minutes with a denaturing agent, sodium dodecyl sulphate
(SDS). SDS-PAGE is commonly used for molecular weight determination and
for assessing the purity of proteins.
4. Visualization of Protein Bands
1. After the electrophoretic run is completed, the proteins are fixed to the solid
support using a fixative such as acetone or methanol. Then it is stained by using
dyes e.g Amido Schwartz or naphthalene black, and then destained by using
dilute acetic acid.
2. Resolution, after staining, of plasma proteins into five bands, designated
albumin, 1, 2, and fractions, respectively. The stained strip of cellulose
acetate (or other supporting medium) is called an electrophoretogram. The
amounts of these five bands can be quantified by densitometric scanning.
Types of electrophoresis:
1. Zone electrophoresis
a. Paper
b. Gel
2. Isoelectricf ocussing
3. Immunoelectrophores
Paper electroporesis:
1. The sample is applied on a strip of filter paper wetted with desired buffer
solution. The ends of the strip are dipped into the buffer reservoirs in which the
electrodes are placed. The efectric current is applied allowing the molecules to
migrate for sufficien time.
2. The paper is removed, dried and stained with a dye that specifically colours the
substances to be detected. The coloured spots can be identified by comparing
with a set of standards run simultaneously
3. Eg. For the separation of serum proteins, Whatman No. 1 filter paper, veronal or
tris buffer at pH 8.6 and the stains amido black or bromophenol blue are
employed.
4. The serum proteins are separated into five distinct bands-albumin, a-1, a-2, band y-globulins
Gel electrophoresis :
1. This technique involves the separation of molecules based on their size, in
addition to the electrical charge. The movement of large molecules is slow in gel
electrophoresis (this is in contrast to gel filtration).
2. The resolution is much higher in this technique. Thus, serum proteins can be
separated to about 15 bands, instead of 5 bands on paper electrophoresis
3. The gels commonly used in gel electrophoresis are agarose and polyacrylamide,
sodium dodecyl sulfate (SDS). Polyacrylamide is employed for the determination
of molecular
weights of proteins in a popularly known electrophoresis
technique known as SDS-PACE
Isoelectric Focusing (IEF)
1. In a column, polyacrylamide matrix is filled with ampholytes (substances carrying
both positive and negative charges). Biological fluid containing proteins or
nucleotides are applied. Electricity is passed. The particles migrate, and settle in
the matrix where pH matches the isoelectric pH (net charge is zero) of the
particle.

Electorporesis apparatus

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Normal pattern
Separation of proteins:
1. Several methods are employed to isolate and purify proteins. Initially, proteins
are fractionated by using different concentrations of ammonium sulfate or
sodium sulfate. Protein fractionation may also be carried out by
ultracentrifugation.
2. Protein separation is achieved by utilizing:
a. electrophoresis,
b. isoelectric focussing,
c. immunoelectrophoresis,
d. ion-exchange chromatography,
e. gel-filtration,
f. high performance liquid chromatography (HPLC) etc.
Serum Protein seperation by paper or gel electrophoresis:
1. Paper or agar gel electrophoresis with vernol buffer (pH-8.6) is used to separates
plasma proteins by electroporesis.
2. Agar gel is prepared in the buffer and spread over microscopic slides and allowed
to cool. Serum sample or biological fluid is applied by cutting into the gel. The
electrophoretic run takes about 90 minutes.
3. In agar gel electrophoresis serum is separated into 5 bands. The concentration of
each one of these fractions can be estimated by a densitometer as follows:
1. Serum albumin
5565 %
2. Alpha-1 globulins
2-4 %
3. Alpha-2 globulins
6-12 %
4. Beta globulins
8-12 %
5. Gamma globulins
12-22 %
Immuno electrophoresis:
1. Here electrophoretic separation is followed by an antigen antibody reaction. The
electrophoresis is carried out first by applying the patient's serum into the wells
cut out in the agar or agarose gel. The proteins are now separated.
2. To visualize them, a specific antibody is placed in a trough cut into the gel and
incubated. The precipitation arcs are formed where the antigen and antibody
molecules are at 1:1 ratio.
3. Serum is fractionated into more than 40 bands. So it is much more sensitive and
specific than ordinary electrophoresis.
Applications of electrophoresis in medicine:
1. The relative proportions of plasma proteins can vary in certain diseases and
electrophoretic tracings showing such changes can be a useful diagnostic aid.
2. Characteristic changes in the amounts of one or more of these five bands are

383
found in many diseases as follows:
a. Multiple myeloma : A sharp and distinct M band appears in the globulin
fraction.
b. Acute infections : a1- and a2- globulins are increased.
c. Nephrotic syndrome: Decreaseda lbumin with sharp and prominent a 2globulin.

d. Primary immune deficiency: Diminished globulin band.

e. a1-Antitrypsin deficiency: Diminished a1- globulin band.
3. Isoelectric focussing can be used for the purification of proteins.
4. lmmunoelectrophoresis is useful for the analysis of complex mixtures of antigens
and antibodies
5. High voltage electroporesis is now being widely used for separation of proteins,
as well as nucleotides from biological fluids.
6. SDS-PAGE is used for molecular weight determination as well as for assessing the
purity of proteins.
7. Cellulose acetate strips are used for separation and identification of lipoproteins,
isoenzymes and hemoglobins.
M-Band:
1. Multiple myeloma: In para-proteinemias, a sharp spike is noted and is termed as
M-band. This is due to monoclonal origin of immunoglobulins in multiple
myeloma.
2. Multiple myeloma is due to the malignancy of a single clone of plasma cells in
the bone marrow. This results in the overproduction of abnormal
immunoglobulins,
mostly
(75%) IgG and in some cases
(25%) IgA or IgM.
3. The
plasma
of
multiple
myeloma patients shows a
characteristic
pattern
of
electrophoresis. There is a
sharp and distinct band (M
band, for myeloma globulin)
between -and -globulins. Further, this M band almost replaces the -globulin
band due to the diminished synthesis of normal - globulins
CHROMATOGRAPHY
1. What is the principle of paper chromatography? Define Rf
vale. Pon May 2010
2. Thin layer chromatography principle and applications. Pon
May 2014
3. What is the principle of affinity Chromatography MGR Feb
2012
i. Chromatography is based on the principle of partition of the solute between two

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phases/solvents. It consists of a mobile phase and a stationary phase. The mobile
phase refers to the mixture of substances to be separated, dissoved in a liquid or a
gas. The stationary phase is a porous solid matrix through which the sample
contained in the mobile phase percolates. The interaction between the mobile and
stationary phases results in the separation of the compounds from the mixture.
ii. Depending upon the type of solid support, stationary phase and the mobile phase,
chromatography can be classified into the following types:
1. Adsorption Chromatography
2. Partition Chromatography
a. Paper Chromatography
b. Thin Layer Chromatography
3. Ion-exchange,
4. Molecular sieving and
5. Affinity.

Partition chromatography :
1. This includes different types depending on the phases between which the
components are partitioned, e.g. solid-liquid, liquidliquid, gas-liquid, etc.
2. This is commonly used for the separation of mixtures of amino acids and
peptides.
3. There is a stationary phase which may be either solid or liquid over which a liquid
or gaseous mobile phase moves.
4. By this process, the components of the mixture to be separated are partitioned
between the two phases depending on the partition co-efficient (solubility) of the
particular substances. The redistribution of the substances between the two
phases results in separation of the components of the mixture.
5. Types:
a. Paper Chromatography
b. Thin Layer Chromatography
Paper chromatography :
1. This technique is commonly used for the separation of amino acids, sugars, sugar
derivatives and peptides.
2. The stationary phase is water held on a solid support of filter paper (cellulose)
3. When the migration of the solvent is upwards, it is referred to as ascending
chromatography. In descending chromatography, the solvent moves downwards
1. A few drops of solution containing a mixture of the compounds to be separated is

385
applied as a small compact spot at one corner of the paper about 1 inch from the
edges.
2. The distance to which each compound moves depends on its partition coefficient.
3. After a sufficient migration of the solvent front, the paper (chromatogram) is
removed, dried and developed for the identification of the specific spots.
Ninhydrin, which forms purple complex with c'-amino acids/ is frequently used as
a colouring reagent
4. Sometimes, it is difficult to separate a complex mixture of substances by a single
run with one solvent system. ln such a case, a second run is carried out by a
different solvent system, in a direction perpendicular to the first run. This is
referred to as two dimensional chromatography which enhances the separation
of a mixture into the individual components
Rf value:
1. The migration of a substance is frequentlye xpresseda s Rf value (ratio of fonts)
Rf= Distancet ravelledb y the substance
Distance travelled bv solvent front
2. The R1 value of each substance, characteristic of a given solvents ystemand
paper, often helps for the identification of unknown. The Rf value is a constant
for a particular solvent system at a given temperature
3. Examples: Rf of Arginine 1; Methionine 2; Cystine 3;

Thin layer chromatography (TLC) :

1. The principle of TLC is the same as paper chromatography (partition). ln place of
a paper, cellulose is employed as supporting material.
2. Cellulose is spread as a thin layer on glass or plastic plates. The chromatographic
separation is comparatively rapid in TLC.
3. In case of adsorption thin layer chromatography, adsorbents such as activated
silica gel , alumina, kieselguhr are used.
Gas-liquid chromatography (GLC):
1. This is the method of choice for the separation of volatile substances. ln GLC, the
stationary phase is diatomaceous earth or powdered firebrick, impregnated with
silicon or polyethylene glycol.
2. This is packed in a narrow column and maintained at high temperature around
200"C. A mixture of volatile material is injected into the column along with the
mobile phase, which is an inert gas such as argon, helium or nitrogen.
3. The separated compounds can be identified and quantitated by a detector on the
principles of ionization or thermal conductivity.
4. Gas-liquid chromatography is sensitive, rapid and reliable. lt is frequently used
for the quantitative estimation of biological materials such as lipids, drugs and
vitamins.
Adsorption column chromatography:
1. The adsorbents such as silica gel, alumina, and charcoal powder and calcium
hydroxyapatite are packed into a column in a glass tube. This serves as the
stationary phase.

386
2. The sample mixture in a solvent is loaded on this column.
3. The individual components get differentially adsorbed on to the adsorbent. The
elution is carried out by a buffer system (mobile phase). The individual
compounds come out of the column at different rates which may be separately
collected and identified.
4. For example, amino acids can be identified by ninhydrin calorimetric method. An
automated column chromatography apparatus-fraction collector-is frequently
used nowadays.
lon-exchange chromatography :
1. Ionexchange chromatography involves the separation of molecules on the basis
of their electrical charges. lon-exchange resins are used for this purpose.
2. An anion exchanger exchanges its anion with another anion in solution. Similarly,
a cation exchanger exchanges its cation with another cation in solution.
3. A mixture of amino acids (protein hydrolysate) or proteins can be separated by
ion-exchange chromatography. The amino acid mixture (at pH around 3.0) is
passed through a cation exchange and the individual amino acids can be eluted
by using buffers of different pH.
4. The various fractions eluted, containing individual amino acids, are allowed to
react with ninhydrin reagent to form coloured complex.
Gel fittration chromatography:
1. ln gel filtration chromatography, the separation of molecules is based on their
size, shape and molecular weight. This technique is also referred to as molecular
sieve or molecular exclusion chromatography.
2. The apparatus consists of a column packed with spongelike gel beads, usually
cross-linked polysaccharides containing pores. The gels serve as molecular
sieves, for the separation of smaller and bigger molecules
3. The solution mixture containing molecules of different sizes ( eg. proteins) is
applied to column and eluted with a buffer. The larger molecules cannot pass
through the pores of gel and, therefore, move faster. The smaller molecules
enter the gel beads and are left behind which come out slowly. By selecting the
gel beads of different porosity, the molecules can be separated.
4. The gel-filtration chromatography can be used for an approximate determination
of molecular weights. This is done by using a calibrated column with substances
of known molecular weight.
Affinity chromatography :
What is the principle of affinity Chromatography? MGR Feb 2012
1. The technique is based on the high affinity of specific proteins for specific
chemical groups or ligands.
1. Enzymes bind specifically to ligands such as substrates or cofactors. The
technique involves the use of ligands attached to an inert and porous matrix in a
column. The immobilized ligands act as molecular fishhook to selectively pick up
the desired protein while the remaining proteins pass through the column.
2. The desired protein, captured by the ligand, can be eluted by using free ligand
molecules. Alternately, some reagents that can break protein-ligand interaction
can also be employed for the separation.
3. Affinity chromatography is useful for the purification of enzymes, vitamins,
nucleic acids, drugs, hormone receptors, antibodies etc.
4. For example, NAD is used to purify dehydrogenases. By using antibodies,
antigens could be easily separated. Conversely, antibodies can be purified by
passing through a column containing the antigen.

387

High performance liquid chromatography (HPtC) :

1. The separation can be greatly improved by applying high pressure in the range
of 5,000-10,00 psi (pounds per square inch), hence this technique is also
referred to as high pressure liquid chromatography.
2. HPLC requires the use of of non-compressible resin materials and strong metal
columns. The eluants of the column are detected by methods such as UV
absorption and fluorescence.
ELISA TEST
ELISA MGR Feb 2009; 2013
1. ELISA is the abbreviation for enzyme linked Immuno sorbent assay.
2. Uses:
a. hormone measurements and for
b. detecting growth factors, tumor markers,
c. to detect antigens or antibodies

Principle of ELIZA:

388
1. The antibody against the protein (may be antigen or antibody) to be determined
is fixed on an inert solid such as polystyrene.
2. The biological sample containing the protein to be estimated is applied on the
antibody coated surface.
3. The protein antibody complex is then reacted with a second protein specific
antibody to which an enzyme is linked. These enzymes produce colored products.
Peroxidase, amylase and alkaline phosphatase are commonly used.
4. After washing the unbound antibody linked enzyme, the enzyme bound to the
second antibody complex is assayed.
5. The enzyme activity is determined by its action on a substrate to form a product
(usually coloured). This is related to the concentration of the protein being
estimated.
APPLICATIONS:
1. ELISA is widely used for the determination of small quantities of
proteins( hormones,
antigens, antibodies) and other biological substances.
2. The most commonly used pregnancy test for the detection of human chorionic
gonadotropin (hCC) in urine is based on ELISA. By this test, pregnancy can be
detected within few days after conception.
3. ELISA is also been used for the diagnosis of AIDS
HIV antibody Test:
a. Antigen from HIV is coated in the wells of a plate.
b. Patient's serum is added, and incubated. If it contains the antibody, it is
fixed. The wells are washed. This is to remove excess antibodies in serum.
c. Next a second antibody (antibody against human immunoglobulin)
conjugated with HRP (enzyme horseradish peroxidase) is added.
d. Then color reagent, containing hydrogen peroxide and diamino benzidine
is poured over.
e. If a brown color develops, it means that the antibody was originally
present in the patient's serum. Here the color developed is proportional to
the antibody concentration. Therefore from the color intensity, the
concentration of the antibody can be calculated. HIV antibody is an
example, any antibody could be detected by using the specific antigen.

1.
2.
3.
4.

COLORIMETER
Define spectrophotometry. Pon may 2009
Flame photometer MGR Feb 2009
Beer Lamberts law
Colorimeter. MGR Aug 2009

Colorimeter:
1. Colorimeter (or photoelectric colorimeter) is the instrument used for the
measurement of coloured substances. This instrument is operative in the visible
range (400-800 nm) of the electromagnetic spectrum of light. The working of
colorimeter is based on the principle of Beer-Lambert law
Beer-Lambert law.
1. Colored solutions have the property of absorbing light of definite wavelengths.
The amount of light absorbed or transmitted by a colored solution is in
accordance with the Beer-Lambert law.
2. As per the Beer's law, the intensity of the color is directly proportional to the
concentration of the colored particles in the solution.
3. The Lambert's law states that the amount of light absorbed by a colored solution
depends on the length of the column or the depth of the liquid through which
light passes.

389
4. The Beer-Lambert law combines these two laws. In the colorimeter, the length of
the column through which the light passed is kept constant, so that the only
variable is the concentration.
5. Optical density:
a. The ratio of intensity of emergent light to intensity of incident light (E/i) is
termed as transmittance (T). The absorbance is expressed as log T.
b. The Optical Density is calculated as log T.
c. The plot of the concentration versus transmittance is in logarithmic scale
Photoelectric colorimeter:
1. Most of the clinical chemistry estimations are done by colorimetric methods. A
colored derivative of the compound to be measured is prepared and its
absorbance or OD is measured using a photoelectric colorimeter.
2. This value is compared with that of a standard of known concentration.
3. The basic components of a photoelectric colorimeter are:
a. Light source, usually a filament lamp
b. Filter, used for selecting the monochromatic light (mono = single; chrome
= color). The color of filter should be complementary to the color of the
solution.
4. Sample holder, called "cuvette", made up of glass tubes
5. Detector (photocell)
6. Display as a digital meter.
Procedure:
1. Serum sample and reagents are mixed and incubated at 37oC for a fixed time,
say 10 minutes, to develop the color optimally.
2. After the incubation period, the OD is ascertained and the concentration of the
substances is calculated. This is called end point analysis.
3. On the other hand, the serum and reagents are incubated, and readings are
taken at 2 and 3 minutes exactly; and from the difference in OD between the two
values, the concentration is calculated. This is the kinetic analysis. Here the
optimum color is not developed; but is quicker and hence is often used in
autoanalysers.
SPECTROPHOTOMETER
Define spectrophotometry. Pon May 2009
a. A spectrophotometer has all the basic components of photoelectric
colorimeter with more sophistication.
b. Wavelengths in the ultraviolet region are also utilized in the
spectrophotometer. Light is separated into a continuous spectrum of
wavelengths and passed through the solution.
c. The advantage of the spectrophotometer over the colorimeter, is that the
former is 1000 times more sensitive. Therefore even minute quantities of the
substance (very dilute solution) can be assessed in the spectrophotometer.
d. To take an example, protein solutions with high concentration (mg/ ml) may
be measured by colorimeter. If the protein concentration is only
microgram/dl, then colorimeter is ineffective, where spectrophotometer can
be used.
e. However, spectrophotometer is 100 times more costly than an ordinary
colorimeter.
Flame Photometer:
Principle of flame photometer (S.N)
1. This is an analytical instrument used for quantitative analysis of sodium,
potassium, calcium and lithium in biological fluids like blood, serum and urine.
2. In a colorimeter the optical absorption property is employed, while in a flame
photometer the property of emission spectroscopy is utilized.

390
3. Sodium, potassium, calcium and lithium have the property of emitting a light of
the characteristic wavelength of that particular element, when sprayed into a
flame (incandescence).
4. The equipment consists of an atomiser, which draws sample solution; and a
compressor which pumps air at high pressure. It is fed into a flame. The flame
will be blue, if the sample contains only distilled water. When the serum sample
is introduced, the flame acquires the color. The emitted light is focussed on to the
photosensor. The electric charge given out by the photosensor is detected,
amplified and displayed. It has to be compared with a standard solution
containing sodium/ potassium
ISOTOPES
What are isotopes? What are its applications in biochemistry? MGR
Feb 2010
lsotopes are defined as the elements with same atomic number but different
atomic weights. lsotopes are of two types-sfable and unstable. The latter are
more commonly referred to as radioactive isotopes
Stable isotopes
1. They are naturally occurring and do not emit radiations (non-radioactive) e.g.
deuterium (heavy hydrogen) 2H; 13C; 1s51' 186. 51"51" isotopes can be
identified and quantitated by mass spectrometry ot nuclear magneti resonance
(NMR). They are less frequently used in biochemical investigations.
Radioactive isotopes
1. The atomic nucleus of radioactive isotopes is unstable and, therefore, undergoes
decay. The radioactive decay gives rise to one of the following 3 ionizing
radiations
a. -Rays-an particle possessing 2 protons i.e. helium nuclei.
b. -Rays-due to the emission of electrons.
c. -Rays-due to emission of high energy photons.
2. The and emitting radioisotopes are employed in biochemical research. These
isotopes are produced in nuclear reactors. The simple chemicals so produced are
then converted to radiolabelled biochemicals by chemical or enzymatic
synthesis.
3. Units of radioactivity : Curie (Ci) is the basic unit of radioactive decay. Millicurie
(mCi) and microcurie (pCi) are more commonly used.
Half-lives of isotopes :
a. The unstable radio isotopes undergo decay. The radioactivity gets reduced to
half of the original within a fixed time. This represents the half-life which is
characteristic for a given isotope.
b. They can be used as tracers in biochemical research since the chemical
properties of different isotopes of a particular element are identical.
Therefore, the living cells cannot distinguish the radioactive isotope from a
normal atom.
c. Radioisotopes are widely used in establishing the precursor-product
relationships in metabolisms and understanding of the complex metabolic
pathways.
Application of radioisotopes:
1. By the use of isotope tracers, the metabolic origin of complex molecules such as
heme, cholesterol, purines and phospholipids can be determined. Nitrogen atom
of heme was derived from glycinewas was established by feeding rats with
radioactive glycine and detecting radioactive heme.
2. The precursor-product relationship in several metabolic pathways has been
investigated by radioisotopes e.g. Krebs cycle, b-oxidation of fatty acids, urea

391
cycle, fatty acid synthesis.
3. Radioisotopes are used in the study of metabolic pools (e.g. amino acid pool)
and metabolic turnovers (e.g. protein turnover).
4. Certain endocrine and immunological studies also depend on the use of
radioisotopes e.g. radioimmunoassay.
5. Radioisotopes are employed in elucidating drug metabolism.
Radioisotopes in diagnosis and treatment
1. Certain radioisotopes are used in the scanning of organs-thyroid gland (131I), bone
(90 Sr) and kidney (131 I hippuran) .
2. Radioactivity has been employed in the treatment of cancers. This is based on
the principle that radiations produce ionizations which damage nucleic acids.
Thus, the uncontrolled proliferation of cells is restricted.
Rotheras test. MGR Feb 2010
Diagnosis of Ketosis
1. Acetoacetate is the primary ketone body while beta-hydroxy butyrate and
acetone are secondary ketone bodies
1. The presence of ketosis can be established by the detection of ketone bodies in
urine by Rothera's test. Supportive evidence may be derived from estimation of
serum electrolytes, acid-base parameters, glucose and urea estimation.
Rothera's test:
1. Saturate 5 ml of urine with solid ammonium sulfate. Add 3 drops of freshly
prepared sodium nitroprusside followed by 2 ml of liquor ammonia along the
sides of the test tube. Development of a purple ring at the junction of the two
liquids indicates the presence of acetone or acetoacetic acid in urine. It is not
answered by beta hydroxy butyrate.
2. Strip tests based on the same principle are also available.
Liver Function Tests
Describe the liver function tests and their significance. Pon May 2014
1. Biochemical tests are of immense value in diagnosis and monitoring of liver
diseases. These tests are usually referred to as liver function tests
2. Major functions of liver.
a. Excretion of bile pigments, bile salts, BSP (Bromsulphthalein) and ICG
(Indocyanine green).
b. Metabolism of carbohydrates and amino acids.
c. Synthesis of serum proteins, especially albumin and prothrombin.
d. Detoxification of ammonia and hippuric acid synthesis.
e. Serum enzymes, acting as markers of liver damage.
3. Clinically useful tests are broadly classified as:
a. Tests to detect hepatic injury:
i. To detect the disease, whether mild or severe; whether acute or
chronic.
ii. To assess the nature of liver injury; hepatocellular or cholestasis.
a. Tests to assess hepatic function.
2. Problems in interpretation
a. Normal LFT values need not indicate absence of liver disease, because
liver has very large reserve capacity.
b. Asymptomatic people may have abnormal LFT results. So interpretation
should be based on clinical picture.
3. Indications for Liver Function Tests
a. Jaundice
b. Suspected liver metastasis

392
c. Alcoholic liver disease
d. Any undiagnosed chronic illness
e. Annual check up of diabetic patients
f. Coagulation disorders
g. Therapy with statins to check hepatotoxicity
4. Classification of liver function tests
a. Classification based on laboratory findings
i. Group I (Tests of hepatic excretory function)
1. Serum Bilirubin; total, conjugated, and unconjugated.
2. Urine Bile pigments, bile salts and urobilinogen.
ii. Group II: Liver enzyme panel (markers of liver injury/cholestasis)
1. Alanine amino transferase (ALT)
2. Aspartate amino transferase (AST)
3. Alkaline phosphatase (ALP)
4. Gamma glutamyl transferase (GGT)
iii. Group III: Plasma proteins (Tests for synthetic function)
1. Total proteins
2. Serum albumin, globulins, A/G ratio
3. Prothrombin time
iv. Group IV: Special tests
1. Ceruloplasmin
2. Ferritin
3. Alpha-1-antitrypsin (AAT)
4. Alpha-fetoprotein (AFP)
5. Classification based on Clinical aspects
a. Group I: Markers of liver dysfunction
i. Serum bilirubin, total, conjugated
ii. Urine: Bile pigments, bile salts and UBG
iii. Total protein, serum albumin and A/G ratio
iv. Prothrombin time
v. Blood ammonia, when indicated
b. Group II: Markers of hepatocellular injury
i. Alanine amino transferase (ALT)
ii. Aspartate amino transferase (AST)
c. Group III: Markers of cholestasis
i. Alkaline phosphatase
ii. Gamma glutamyl transferase
Markers of Hepatic Dysfunction:
1. Serum Bilirubin:
a. Normal serum bilirubin level varies from 0.2 to 0.8 mg/dl.
b. The unconjugated bilirubin varies from 0.20.7 mg/dl and
c. conjugated bilirubin (direct bilirubin) 0.10.4 mg/dl.
d. A rise in serum bilirubin above 1 mg/dl is abnormal (latent jaundice);
2. Urinary Urobilinogen:
a. In cases of obstruction, bile is not reaching the intestine and so
urobilinogen may be decreased or absent in urine.
b. In hepatocellular jaundice, urobilinogen is initially elevated, then
decreases or disappears when the obstructive stage sets in and reappears
when obstruction is cleared.
d. Urobilinogen is absent in urine, when there is obstruction to bile flow. The
first indication of the recovery is the reappearance of urobilinogen in
urine.
e. In hemolytic anemias, urobilinogen is increased.
f. Bilirubin is detected by Fouchet's test and urobilinogen by Ehrlich's test.
6. Urine Bile Salts
a. Normally bile salts (sodium salts of taurocholic acid and glycocholic acid)
are present in the bile; but are not seen in urine.
b. Bile salts in urine are detected by Hays test. Positive Hays test indicates

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the obstruction in the biliary passages causing regurgitation of bile salts
into the systemic circulation leading to its excretion in urine.
c. Obstruction can occur in obstructive jaundice and also in hepatic jaundice
due to obstruction of micro biliary channels caused by inflammation.
Tests based on Synthetic Function of Liver
1. Serum albumin level:
a. plasma proteins except immunoglobulins are synthesised by the liver.
b. albumin has a fairly long half-life of 20 days, in all chronic diseases of the
liver, the albumin level is decreased. A reversal in A/G ratio is often the
rule in cirrhosis, due to hypoalbuminemia and associated
hypergammaglobulinemia.
c. Normal albumin level in blood is 3.5 to 5 g/dl; and globulin level is 2.5 to
3.5 g/dl.
2. Serum globulins
a. They constitute immunoglobulins produced by B lymphocytes as well as
alpha and beta globulins synthesized mainly by hepatocytes.
b. Gamma globulins in the serum are increased in chronic liver diseases
(chronic active hepatitis, cirrhosis).
c. IgG is increased in autoimmune hepatitis. IgM is increased in primary
biliary cirrhosis. IgA is increased in alcoholic liver disease.
3. Prothrombin time (PT)
a. Since prothrombin is synthesised by the liver, it is a useful indicator of
liver function. The half-life of prothrombin is 6 hours only; therefore, PT
indicates the present function of the liver. PT is prolonged only when liver
loses more than 80% of its reserve capacity.
b. Vitamin K deficiency is also a cause for prolonged prothrombin time. In
case of liver disease, the PT remains prolonged even after parental
administration of vitamin K.
4. Alpha-fetoprotein (AFP)
a. It is a normal component of fetal blood. It disappears after birth within a
few weeks. It is a tumor marker. Mild elevation is suggestive of chronic
hepatitis or cirrhosis; drastic increase is seen in hepatocellular carcinoma,
germ cell tumors and teratoma of ovary.
b. Elevated AFP in the maternal serum is seen in cases of fetal open neural
tube defects and also in cases with multiple fetuses or fetal death.
c. Low AFP is seen in maternal serum in cases of fetal Down syndrome.
Immuno assay is employed to test AFP.
d. Reference limits are, up to 1 year < 30 ng/ml and adults (males and
nonpregnant females<15 ng/ml.
5. Ceruloplasmin (Cp)
a. It is mainly synthesized by the hepatic parenchymal cells anda small part
by lymphocytes. Level of Cp is increased in active hepatitis, biliary
cirrhosis, hemochromatosis and obstructive biliary disease.
b. The level is decreased in Wilson's hepatolenticular degeneration
6. Alpha-1 antitrypsin (AAT)
a. It is an acute phase reactant and is synthesized and secreted by the liver.
AAT inactivates serine proteases (elastase and collagenase).
b. Low levels are associated with neonatal cholestasis, progressive juvenile
cirrhosis in children and micronodular cirrhosis in adults. Low levels are
also seen in panlobular emphysema. It is increased in acute trauma,
infections or after estrogen therapy and in many malignancies.
7. Haptoglobin
a. It is synthesized in the liver. It transports free hemoglobin in the plasma to
reticulo endothelial system.
b. Being an acute phase reactant, its levels are high in inflammatory

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processes, trauma, infections, and myocardial infarction. Its level is low In
hemolytic jaundice.
8. Enzymes indicating hepatocellular damage
a. ALT:
1. Normal serum ALT (alanine amino transferase) is 10-35 IU/L.
2. Very high levels (more than 1000 units) are seen in acute
hepatitis.
3. Elevation of ALT is more in cases of hepatic disease compared to
AST. But AST may be more than ALT in alcoholic liver disease. In
alcoholic liver disease, the actual values show only mild elevation;
but a ratio of AST/ALTmore than 2 is quite suggestive.
b. Aspartate amino transferase (AST) :
1. It is known as SGOT .
2. Normal serum level is 8-20 U/L
3. It is significantly elevated in myocardial infarction and is
moderately elevated in liver disease; very marked increase in
hepatoma.
9. Markers of Obstructive Liver Disease:
a. Alkaline Phosphatase (ALP)
1. Very high levels of alkaline phosphatase (ALP) are noticed in
patients with cholestasis or hepatic carcinoma. The bile duct
obstruction induces the synthesis of the enzyme by biliary tract
epithelial cells
2. Drastically high levels of ALP (10-25 times of upper limit) are
seen in bone diseases where osteoblastic activity is enhanced.
For example, Paget's disease (osteitis deformans ), rickets,
osteomalacia.
b. Gamma Glutamyl Transferase (GGT)
1. GGT is clinically important because of its sensitivity to detect
alcohol abuse. GGT level in alcoholic liver disease roughly
parallels the alcohol intake.
10. Tests on the Metabolic Capacity of Liver
a. Blood ammonia: It is an index of urea synthesis by liver. It is a useful test
in hepatic encephalopathy
RENAL FUNCTION TEST
1. Renal function test. Pon may 2009
2. Name two tests used to evaluate renal tubular function. Explain one. Pon
may 2014
3. Define clearance. What are the normal values for urea and creatinine
clearance? Pon Nov 2010
Classification of Renal Function Tests
1. To screen for kidney disease
a. Complete urine analysis
b. Plasma urea and creatinine
c. Plasma electrolytes
2. To assess renal function

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a. To assess glomerular function
i. Glomerular filtration rate
ii. Clearance tests
iii. Glomerular permeability
iv. Proteinuria
b. To assess tubular function
i. Reabsorption studies
ii. Secretion tests
iii. Concentration and dilution tests
iv. Renal acidification
Glomerular function tests:
1. GFR: The glomerular filtration rate (GFR) is 120-125 ml per minute in a person with
70 kg body weight. GFR cannot be measured directly, it is estimated from the
clearance tests
Tets of glomerular function:
1. Clearance Tests:
a. Clearance is defined as the volume of blood or plasma completely cleared of
a substance per unit time and is expressed as milliliter per minute.
b. It is expressed as milliliter of plasma per minute
mg of substance excreted per minute
Clearance =
mg of substance per ml of plasma
2. Creatinine Clearance Test
a. Creatinine is a waste product, formed from creatine phosphate.
b. Since the production is continuous, the blood level will not fluctuate much,
making creatinine an ideal substance for clearance test.
c. Creatinine excretion is constant in a particular person.
d. Reference Values of Creatinine
i. Adult males, 0.7-1.4 mg/dl
ii. Adult females, 0.6-1.3 mg/dl
iii. Children, 0.4-1.2 mg/dl.
e. Procedure for Creatinine Clearance Test
f. Give 500 ml of water to the patient, to promote good urine flow. After about
30 minutes, ask to empty the bladder and discard the urine. Exactly after 60
minutes, again void the bladder and collect the urine, and note the volume.
g. Take one blood sample. Creatinine level in blood and urine are tested and
calculated.
h. Creatinine clearance could be calculated as:
Clearance = (U/P) x V
(U is the urine creatinine concentration, P is the plasma creatinine
concentration and V
is the urine flow in ml/min)
i. Interpretation of Creatinine Clearance:
i. A decreased creatinine clearance is a very sensitive indicator of
reduced glomerular filtration rate.
ii. The importance of creatinine clearance is in the early detection of
functional impairment of kidney
iii. Normal value is around 75 ml/min.
3. Estimated GFR (eGFR) Test:
a. A simpler technique of estimating creatinine clearance and thereby GFR is by
using serum creatinine level.
b. A commonly used formula is Cockcroft-Gault equation.
Ccr = (140-age in years) x weight in kg /72 x Pcr in mg/dl
4. Advantages:
a. As the production is continuous, the blood level will not fluctuate. Blood may
be collected at anytime.
b. It is not affected by diet or exercise.

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5. Disadvantages:
a. Creatinine is filtered by glomeruli, and actively excreted by the tubules. Of
the total excretion, about 10% is tubular component.
b. Early stages of decrease in GFR may not be identified by creatinine
clearance.
6. Procedure:
a. Give 500 ml of water to the patient, to promote good urine flow. After about
30 minutes, ask to empty the bladder and discard the urine. Exactly after 60
minutes, again void the bladder and collect the urine, and note the volume.
Take one blood sample. Creatinine level in blood and urine are tested and
calculated.
b. Uncorrected clearance = ( U/P ) x V
where U is the urine creatinine concentration, P is the plasma creatinine
concentration and V is the urine flow in ml/min (The 24 hrs urine collection is
not necessary for the creatinine clearance test).
Urea Clearance Test:
1. Urea clearance is defined as the volume (ml) of plasma that would be completely
cleared of urea per minute. It is calculated by the formula
Cm = UxV
P
where Cm. = Maximum urea clearance
U = Urea concentration in (mg/ml)
V = Urine excreted oer minute in ml
P = Urea concentration in olasma
(mg/ml).
2. The above calculation is applicable if the output of urine is more than 2 ml per
minute. This is referred to as maximum urea clearance and the normal value is
around 75 ml/min.
3. Standard urea clearance : lt is observed that the urea clearance drastically
changes when the volume of urine is less than 2 ml/min. This is known as
standard urea clearance (C) and the normal value is around 54 ml/min. lt is
calculated by a modified formula

4. Diagnostic importance : A urea clearance value below 75 % of the normal is

viewed seriously, since it is an indicator of renal damage. Blood urea level as
such is found to increase only when the clearance falls below 50% normal. As
already stated, creatinine clearance is a better indicator of renal function.
Other test of renal functions:
Normal Serum Urea Level
1. Normal value is 20 to 40 mg/dl.
2. Serum urea is increased in all forms of kidney diseases. In acute
glomerulonephritis values may be as high as 300 mg/dl.
Azotemia
1. Increase in the blood levels of NPN is referred to as azotemia and is the hallmark
of kidney failure.
2. NPN:urea, creatinine and uric acid
Urine examination
1. The routine urine examination is undoubtedly a guiding factor for renal function.
The volume of urine excreted, its pH, specific gravity, osmolality, the
concentration of abnormal constituents (such as proteins, ketone bodies, glucose
and blood) may help to have some preliminary knowledge of kidney function

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