8/21/2019 20.Tools & Tests

1/18

ELECTROPHORESIS

1. Electrophoretic technique and its importance.MGR u! "##$". %escri&e the separation o' Serum Proteins &( paper

electrophoresis. %ra) the pattern o' electrophoresis in i*

Multiple M(eloma ii* +ephrotic S(ndrome. MGR u! "#1#,. Serum protein electrophoresis MGR u! "#11-. Electrophoresis and its applications MGR e& "#1,/. List t)o applications o' electrophoresis in medicine. MGR e&

"#1-0. %ene electrophoresis and mention its applications MGR u!

"#1-2. Electrophoresis MGR e& "#113 "#1"4. M &and MGR u! "#11$. %escri&e the separation o' Serum Proteins &( paper

electrophoresis.1#.%ra) the pattern o' electrophoresis in i* Multiple M(eloma ii*

+ephrotic S(ndrome.11.Ho) are plasma proteins separated &( electroporesis5 6hat is

the si!nicance o' M protein5 Pon +o7 "#111".Gel electrophoresis. Pon Ma( "##$

ELECTROPORESIS1. The term refers to the movement of charged particles through an electrolyte when

subjected to an electric eld. 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 !uids.". #actors a$ecting electrophoresis%

a. &et charge on the particles (ph of proteins)b. 'ass and shape of the particles.c. The p of the medium.d. Strength of electrical eld.e. roperties of the supporting medium.f. Temperature.

*. +lectrophoresis ,pparatus%a. -t consists of the electrophoresis tan to hold the bu$er and tted with the

electrodes as well as a power pac to supply electricity at constant current

and voltage.b. The bu$er is chosen in such a way so as to ensure e$ective separation of the

mixture of proteins. e.g. serum proteins are separated at a p of /.0 using

barbitone bu$er. ,t this p all serum proteins will have a net negative charge

and will migrate towards the anode.

*. Support 'edium%

a. #ilter aper% long time interval and di$usion of particles leading to blurring of

margins are the disadvantagesb. ellulose ,cetate 'embrane% expensive but the process taes less than one

hour and excellent separation2 widely used for separation and identication of 

lipoproteins isoen3ymes and hemoglobins.c. ,gar or ,garose% less expensive2 the gel is prepared in the bu$er and spread

over microscopic slides and allowed to cool. Serum sample or biological !uid

is applied by cutting into the gel. The electrophoretic run taes about 45

minutes. Serum proteins are commonly studied by agar electrophoresis.

d. olyacrylamide gel electrophoresis (,6+)% -t has a high molecular sievinge$ect and so separation is very e7cient2 will show more than "5 di$erent

*/5

8/21/2019 20.Tools & Tests

2/18

bands. The amount of cross lining and thereby the pore si3e can be

controlled. ,nother variant is the S8S9,6+ electrophoresis. ere proteins are

boiled for 19" minutes with a denaturing agent sodium dodecyl sulphate

(S8S). S8S9,6+ is commonly used for molecular weight determination and

for assessing the purity of proteins.

-. 8isuali9ation o' Protein :ands1. ,fter the electrophoretic run is completed the proteins are xed to the solid

support using a xative such as acetone or methanol. Then it is stained by using

dyes e.g ,mido Schwart3 or naphthalene blac and then destained by using

dilute acetic acid.". :esolution after staining of plasma proteins into ve bands designated

albumin α1 α" β and γ  fractions respectively. The stained strip of cellulose

acetate (or other supporting medium) is called an electrophoretogram. The

amounts of these ve bands can be ;uantied by densitometric scanning.

T(pes o' electrophoresis;

1. . The serum proteins are separated into ve distinct bands9albumin a91 a9" b9

and y9globulins

Gel electrophoresis ;

1. This techni;ue involves the separation of molecules based on their si3e in

addition to the electrical charge. The movement of large molecules is slow in gel

electrophoresis (this is in contrast to gel ltration).". The resolution is much higher in this techni;ue. Thus serum proteins can be

separated to about 1? bands instead of ? bands on paper electrophoresis*. The gels commonly used in gel electrophoresis are agarose and polyacrylamide

sodium dodecyl sulfate (S8S). olyacrylamide is employed for the determinationof molecular weights of proteins in a popularly nown electrophoresis

techni;ue nown as S8S9,+

Isoelectric ocusin!

8/21/2019 20.Tools & Tests

3/18

&ormal pattern

Separation o' proteins;1. Several methods are employed to isolate and purify proteins. -nitially proteins

are fractionated by using di$erent concentrations of ammonium sulfate or

sodium sulfate. rotein fractionation may also be carried out by

ultracentrifugation.". rotein separation is achieved by utili3ing%

a. electrophoresisb. isoelectric focussingc. immunoelectrophoresisd. ion9exchange chromatographye. gel9ltrationf. high performance li;uid chromatography (A) etc.

Serum Protein seperation &( paper or !el electrophoresis;1. aper or agar gel electrophoresis with vernol bu$er (p9/.0) is used to separates

plasma proteins by electroporesis.". ,gar gel is prepared in the bu$er and spread over microscopic slides and allowed

to cool. Serum sample or biological !uid is applied by cutting into the gel. The

electrophoretic run taes about 45 minutes.*. -n agar gel electrophoresis serum is separated into ? bands. The concentration of 

each one of these fractions can be estimated by a densitometer as follows%1. Serum albumin  ??B0? C". ,lpha91 globulins "9> C*. ,lpha9" globulins 091" C>. @eta globulins /91" C

?. 6amma globulins  1"9"" CImmuno electrophoresis;

1. ere electrophoretic separation is followed by an antigen antibody reaction. The

electrophoresis is carried out rst by applying the patientDs serum into the wells

cut out in the agar or agarose gel. The proteins are now separated.". To visuali3e them a specic 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.*. Serum is fractionated into more than >5 bands. So it is much more sensitive and

specic than ordinary electrophoresis.

pplications o' electrophoresis in medicine;

1. The relative proportions of plasma proteins can vary in certain diseases andelectrophoretic tracings showing such changes can be a useful diagnostic aid.". haracteristic changes in the amounts of one or more of these ve bands are

*/"

http://en.wikipedia.org/wiki/Serum_albuminhttp://en.wikipedia.org/wiki/Alpha-1_globulinhttp://en.wikipedia.org/w/index.php?title=Alpha-2_globulin&action=edit&redlink=1http://en.wikipedia.org/wiki/Beta_globulinhttp://en.wikipedia.org/wiki/Gamma_globulinhttp://en.wikipedia.org/wiki/Alpha-1_globulinhttp://en.wikipedia.org/w/index.php?title=Alpha-2_globulin&action=edit&redlink=1http://en.wikipedia.org/wiki/Beta_globulinhttp://en.wikipedia.org/wiki/Gamma_globulinhttp://en.wikipedia.org/wiki/Serum_albumin

8/21/2019 20.Tools & Tests

4/18

found in many diseases as follows%a. 'ultiple myeloma % , sharp and distinct ' band appears in the globulin

fraction.b. ,cute infections % a19 and a"9 globulins are increased.c. &ephrotic syndrome% 8ecreaseda lbumin with sharp and prominent a "9

globulin.

d. rimary immune deciency% 8iminished γ  globulin band.

e. a19,ntitrypsin deciency% 8iminished a19 globulin band.

*. -soelectric focussing can be used for the purication of proteins.>. lmmunoelectrophoresis is useful for the analysis of complex mixtures of antigens

and antibodies?. igh voltage electroporesis is now being widely used for separation of proteins

as well as nucleotides from biological !uids.0. S8S9,6+ is used for molecular weight determination as well as for assessing the

purity of proteins.E. ellulose acetate strips are used for separation and identication of lipoproteins

isoen3ymes and hemoglobins.

M=:and;

1. 'ultiple myeloma% -n para9proteinemias a sharp spie is noted and is termed as

'9band. This is due to monoclonal origin of immunoglobulinFs in multiple

myeloma.". 'ultiple 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

(E?C) -g6 and in some cases

("?C) -g, or -g'.*. The plasma of multiple

myeloma patients shows a

characteristic pattern of 

electrophoresis. There is a

sharp and distinct band ('

band for myeloma globulin)between β9and γ   9globulins. #urther this ' band almost replaces the γ 9globulin

band due to the diminished synthesis of normal γ  9 globulins

 

CHROMTOGRPH> 

1. 6hat is the principle o' paper chromato!raph(5 %ene R'

7ale. Pon Ma( "#1#". Thin la(er chromato!raph( principle and applications. Pon

Ma( "#1-,. 6hat is the principle o' a?nit( Chromato!raph( MGR e&

"#1"

i. hromatography is based on the principle of partition of the solute between two

*/*

8/21/2019 20.Tools & Tests

5/18

phasesGsolvents. -t consists of a mobile phase and a stationary phase. The mobile

phase refers to the mixture of substances to be separated dissoved in a li;uid 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. 8epending upon the type of solid support stationary phase and the mobile phase

chromatography can be classied into the following types%1. ,dsorption hromatography". artition hromatography

a. aper hromatographyb. Thin Aayer hromatography

*. -on9exchange>. 'olecular sieving and?. ,7nity.

Partition chromato!raph( ;

1. This includes di$erent types depending on the phases between which the

components are partitioned e.g. solid9li;uid li;uidli;uid gas9li;uid etc.". This is commonly used for the separation of mixtures of amino acids and

peptides.*. There is a stationary phase which may be either solid or li;uid over which a li;uid

or gaseous mobile phase moves.

>. @y this process the components of the mixture to be separated are partitionedbetween the two phases depending on the partition co9e7cient (solubility) of the

particular substances. The redistribution of the substances between the two

phases results in separation of the components of the mixture.?. Types%

a. aper hromatographyb. Thin Aayer hromatography

Paper chromato!raph( %

1. This techni;ue is commonly used for the separation of amino acids sugars sugar

derivatives and peptides.". The stationary phase is water held on a solid support of lter paper (cellulose)*. =hen the migration of the solvent is upwards it is referred to as ascending

chromatography. -n descending chromatography the solvent moves downwards1. , few drops of solution containing a mixture of the compounds to be separated is

*/>

8/21/2019 20.Tools & Tests

6/18

applied as a small compact spot at one corner of the paper about 1 inch from the

edges.". The distance to which each compound moves depends on its partition coe7cient.*. ,fter a su7cient migration of the solvent front the paper (chromatogram) is

removed dried and developed for the identication of the specic spots.

&inhydrin which forms purple complex with cD9amino acidsG is fre;uently used as

a colouring reagent>. Sometimes it is di7cult 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

di$erent solvent system in a direction perpendicular to the rst run. This is

referred to as two dimensional chromatography which enhances the separation

of a mixture into the individual componentsR' 7alue;

1. The migration of a substance is fre;uentlye xpresseda s :f value (ratio of fonts)

:fH 8istancet ravelledb y the substance

  8istance travelled bv solvent front

". The :1 value of each substance characteristic of a given solvents ystemand

paper often helps for the identication of unnown. The :f value is a constantfor a particular solvent system at a given temperature

*. +xamples% :f of ,rginine 12 'ethionine "2 ystine *2

Thin la(er chromato!raph(

8/21/2019 20.Tools & Tests

7/18

". The sample mixture in a solvent is loaded on this column.*. The individual components get di$erentially adsorbed on to the adsorbent. The

elution is carried out by a bu$er system (mobile phase). The individual

compounds come out of the column at di$erent rates which may be separately

collected and identied.>. #or example amino acids can be identied by ninhydrin calorimetric method. ,n

automated column chromatography apparatus9fraction collector9is fre;uently

used nowadays.

lon=e@chan!e chromato!raph( ;

1. -onexchange chromatography involves the separation of molecules on the basis

of their electrical charges. lon9exchange resins are used for this purpose.". ,n anion exchanger exchanges its anion with another anion in solution. Similarly

a cation exchanger exchanges its cation with another cation in solution.*. , mixture of amino acids (protein hydrolysate) or proteins can be separated by

ion9exchange chromatography. The amino acid mixture (at p around *.5) is

passed through a cation exchange and the individual amino acids can be eluted

by using bu$ers of di$erent p.

>. The various fractions eluted containing individual amino acids are allowed toreact with ninhydrin reagent to form coloured complex.

Gel ttration chromato!raph(;

1. ln gel ltration chromatography the separation of molecules is based on their

si3e shape and molecular weight. This techni;ue is also referred to as molecular

sieve or molecular exclusion chromatography.". The apparatus consists of a column paced with spongelie gel beads usually

cross9lined polysaccharides containing pores. The gels serve as molecular

sieves for the separation of smaller and bigger molecules*. The solution mixture containing molecules of di$erent si3es ( eg. proteins) is

applied to column and eluted with a bu$er. 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. @y selecting the

gel beads of di$erent porosity the molecules can be separated.>. The gel9ltration chromatography can be used for an approximate determination

of molecular weights. This is done by using a calibrated column with substances

of nown molecular weight.

?nit( chromato!raph( ;

6hat is the principle o' a?nit( Chromato!raph(5 MGR e& "#1"

1. The techni;ue is based on the high a7nity of specic proteins for specic

chemical groups or ligands.1. +n3ymes bind specically to ligands such as substrates or cofactors. The

techni;ue involves the use of ligands attached to an inert and porous matrix in acolumn. The immobili3ed ligands act as molecular shhoo to selectively pic up

the desired protein while the remaining proteins pass through the column.". The desired protein captured by the ligand can be eluted by using free ligand

molecules. ,lternately some reagents that can brea protein9ligand interaction

can also be employed for the separation.*. ,7nity chromatography is useful for the purication of en3ymes vitamins

nucleic acids drugs hormone receptors antibodies etc.>. #or example &,8 is used to purify dehydrogenases. @y using antibodies

antigens could be easily separated. onversely antibodies can be puried by

passing through a column containing the antigen.

*/0

8/21/2019 20.Tools & Tests

8/18

Hi!h per'ormance liquid chromato!raph(

8/21/2019 20.Tools & Tests

9/18

1. The antibody against the protein (may be antigen or antibody) to be determined

is xed on an inert solid such as polystyrene.". The biological sample containing the protein to be estimated is applied on the

antibody coated surface.*. The protein antibody complex is then reacted with a second protein specic

antibody to which an en3yme is lined. These en3ymes produce colored products.

eroxidase amylase and alaline phosphatase are commonly used.>. ,fter washing the unbound antibody lined en3yme the en3yme bound to the

second antibody complex is assayed.?. The en3yme 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.

PPLICTIO+S;

1. +A-S, is widely used for the determination of small ;uantities of

proteins( hormones antigens antibodies) and other biological substances.". The most commonly used pregnancy test for the detection of human chorionic

gonadotropin (h) in urine is based on +A-S,. @y this test pregnancy can bedetected within few days after conception.

*. +A-S, is also been used for the diagnosis of ,-8S

 HI8 anti&od( Test;

a. ,ntigen from -K is coated in the wells of a plate.b. atientDs serum is added and incubated. -f it contains the antibody it is

xed. The wells are washed. This is to remove excess antibodies in serum.c. &ext a second antibody (antibody against human immunoglobulin)

conjugated with : (en3yme horseradish pero@idase* is added.d. Then color reagent containing hydrogen peroxide and diamino ben3idine

is poured over.e. -f a brown color develops it means that the antibody was originally

present in the patientDs serum. ere the color developed is proportional tothe antibody concentration. Therefore from the color intensity the

concentration of the antibody can be calculated. -K antibody is an

example any antibody could be detected by using the specic antigen.

COLORIMETER

1. %ene spectrophotometr(. Pon ma( "##$". lame photometer MGR e& "##$,. :eer Lam&erts la)-. Colorimeter. MGR u! "##$

Colorimeter;1. olorimeter (or photoelectric colorimeter) is the instrument used for the

measurement of coloured substances. This instrument is operative in the visible

range (>559/55 nm) of the electromagnetic spectrum of light. The woring of

colorimeter is based on the principle of @eer9Aambert law

:eer=Lam&ert la).

1. olored solutions have the property of absorbing light of denite wavelengths.

 The amount of light absorbed or transmitted by a colored solution is in

accordance with the @eer9Aambert law.". ,s per the @eerDs law the intensity of the color is directly proportional to the

concentration of the colored particles in the solution.*. The AambertDs law states that the amount of light absorbed by a colored solution

depends on the length of the column or the depth of the li;uid through which

light passes.

*//

http://en.wikipedia.org/wiki/Enzymehttp://en.wikipedia.org/wiki/Enzyme

8/21/2019 20.Tools & Tests

10/18

>. The @eer9Aambert law combines these two laws. -n the colorimeter the length of

the column through which the light passed is ept constant so that the only

variable is the concentration.?. Lptical density%

a. The ratio of intensity of emergent light to intensity of incident light (+Gi) is

termed as transmittance (T). The absorbance is expressed as Mlog T.b. The Lptical 8ensity is calculated as Mlog T.c. The plot of the concentration versus transmittance is in logarithmic scale

Photoelectric colorimeter;

1. 'ost of the clinical chemistry estimations are done by colorimetric methods. ,

colored derivative of the compound to be measured is prepared and its

absorbance or L8 is measured using a photoelectric colorimeter.". This value is compared with that of a standard of nown concentration.*. The basic components of a photoelectric colorimeter are%

a. Aight source usually a lament lampb. #ilter used for selecting the monochromatic light (mono H single2 chrome

H color). The color of lter should be complementary to the color of the

solution.>. Sample holder called IcuvetteI made up of glass tubes?. 8etector (photocell)0. 8isplay as a digital meter.

Procedure;

1. Serum sample and reagents are mixed and incubated at *Eo for a xed time

say 15 minutes to develop the color optimally.". ,fter the incubation period the L8 is ascertained and the concentration of the

substances is calculated. This is called end point analysis.*. Ln the other hand the serum and reagents are incubated and readings are

taen at " and * minutes exactly2 and from the di$erence in L8 between the two

values the concentration is calculated. This is the inetic analysis. ere the

optimum color is not developed2 but is ;uicer and hence is often used inautoanal(sers.

SPECTROPHOTOMETER

%ene spectrophotometr(. Pon Ma( "##$

a. , spectrophotometer has all the basic components of photoelectric

colorimeter with more sophistication.b. =avelengths in the ultraviolet region are also utili3ed in the

spectrophotometer. Aight 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 1555 times more sensitive. Therefore even minute ;uantities of the

substance (very dilute solution) can be assessed in the spectrophotometer.d. To tae an example protein solutions with high concentration (mgG ml) may

be measured by colorimeter. -f the protein concentration is only

microgramGdl then colorimeter is ine$ective where spectrophotometer can

be used.e. owever spectrophotometer is 155 times more costly than an ordinary

colorimeter.

lame Photometer;

Principle o' Dame photometer

8/21/2019 20.Tools & Tests

11/18

*. Sodium potassium calcium and lithium have the property of emitting a light of

the characteristic wavelength of that particular element when sprayed into a

!ame (incandescence).>. The e;uipment consists of an atomiser which draws sample solution2 and a

compressor which pumps air at high pressure. -t is fed into a !ame. The !ame

will be blue if the sample contains only distilled water. =hen the serum sample

is introduced the !ame ac;uires the color. The emitted light is focussed on to the

photosensor. The electric charge given out by the photosensor is detected

amplied and displayed. -t has to be compared with a standard solution

containing sodiumG potassium

ISOTOPES

6hat are isotopes5 6hat are its applications in &iochemistr(5 MGR

e& "#1#

lsotopes are dened as the elements with same atomic number but di$erent

atomic weights. lsotopes are of two types9sfable and unstable. The latter are

more commonly referred to as radioactive isotopes

Sta&le isotopes

1. They are naturally occurring and do not emit radiations (non9radioactive) e.g.

deuterium (heavy hydrogen) "2 1*2 1s?1D 1/0. ?1I?1I isotopes can be

identied and ;uantitated by mass spectrometry ot nuclear magneti resonance

(&':). They are less fre;uently used in biochemical investigations.

Radioacti7e isotopes

1. The atomic nucleus of radioactive isotopes is unstable and therefore undergoes

decay. The radioactive decay gives rise to one of the following * ioni3ing

radiations

a.   α9:ays9an α particle possessing " protons i.e. helium nuclei.

b.   β9:ays9due to the emission of electrons.

c.   γ 9:ays9due to emission of high energy photons.

". The β and γ  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 en3ymatic

synthesis.*. Jnits of radioactivity % urie (i) is the basic unit of radioactive decay. 'illicurie

(mi) and microcurie (pi) are more commonly used.

Hal'=li7es o' isotopes ;

a. The unstable radio isotopes undergo decay. The radioactivity gets reduced to

half of the original within a xed time. This represents the half9life which is

characteristic for a given isotope.

b. They can be used as tracers in biochemical research since the chemicalproperties of di$erent isotopes of a particular element are identical.

 Therefore the living cells cannot distinguish the radioactive isotope from a

normal atom.c. :adioisotopes are widely used in establishing the precursor9product

relationships in metabolisms and understanding of the complex metabolic

pathways.

pplication o' radioisotopes;

1. @y the use of isotope tracers the metabolic origin of complex molecules such as

heme cholesterol purines and phospholipids can be determined. &itrogen atom

of heme was derived from glycinewas was established by feeding rats with

radioactive glycine and detecting radioactive heme.". The precursor9product relationship in several metabolic pathways has been

investigated by radioisotopes e.g. Nrebs cycle b9oxidation of fatty acids urea

*45

8/21/2019 20.Tools & Tests

12/18

cycle fatty acid synthesis.*. :adioisotopes are used in the study of metabolic pools (e.g. amino acid pool)

and metabolic turnovers (e.g. protein turnover).>. ertain endocrine and immunological studies also depend on the use of

radioisotopes e.g. radioimmunoassay.?. :adioisotopes are employed in elucidating drug metabolism.

Radioisotopes in dia!nosis and treatment

1. ertain radioisotopes are used in the scanning of organs9thyroid gland (1*1-) bone

(45 Sr) and idney (1*1 - hippuran) .". :adioactivity has been employed in the treatment of cancers. This is based on

the principle that radiations produce ioni3ations which damage nucleic acids.

 Thus the uncontrolled proliferation of cells is restricted.

Rotheras test. MGR e& "#1#

%ia!nosis o' etosis

1. ,cetoacetate is the primary etone body while beta9hydroxy butyrate and

acetone are secondary etone bodies1. The presence of etosis can be established by the detection of etone bodies in

urine by :otheraDs test. Supportive evidence may be derived from estimation of

serum electrolytes acid9base parameters glucose and urea estimation.

RotheraFs test;

1. Saturate ? ml of urine with solid ammonium sulfate. ,dd * drops of freshly

prepared sodium nitroprusside followed by " ml of li;uor ammonia along the

sides of the test tube. 8evelopment of a purple ring at the junction of the two

li;uids indicates the presence of acetone or acetoacetic acid in urine. -t is not

answered by beta hydroxy butyrate.". Strip tests based on the same principle are also available.

Li7er unction Tests

%escri&e the li7er 'unction tests and their si!nicance. Pon Ma( "#1-

1. @iochemical tests are of immense value in diagnosis and monitoring of liver

diseases. These tests are usually referred to as Oliver function testsP". 'ajor functions of liver.

a. +xcretion of bile pigments bile salts @S (@romsulphthalein) and -6

(-ndocyanine green).b. 'etabolism of carbohydrates and amino acids.c. Synthesis of serum proteins especially albumin and prothrombin.d. 8etoxication of ammonia and hippuric acid synthesis.

e. Serum en3ymes acting as marers of liver damage.*. linically useful tests are broadly classied as%

a. Tests to detect hepatic injury%i. To detect the disease whether mild or severe2 whether acute or

chronic.ii. To assess the nature of liver injury2 hepatocellular or cholestasis.

a. Tests to assess hepatic function.". roblems in interpretation

a. &ormal A#T values need not indicate absence of liver disease because

liver has very large reserve capacity.b. ,symptomatic people may have abnormal A#T results. So interpretation

should be based on clinical picture.

*. -ndications for Aiver #unction Testsa. Qaundiceb. Suspected liver metastasis

*41

8/21/2019 20.Tools & Tests

13/18

c. ,lcoholic liver diseased. ,ny undiagnosed chronic illnesse. ,nnual chec up of diabetic patientsf. oagulation disordersg. Therapy with statins to chec hepatotoxicity

>. lassication of liver function tests

a. lassication based on laboratory ndingsi. 6roup - (Tests of hepatic excretory function)

1. Serum M @ilirubin2 total conjugated and unconjugated.". Jrine M @ile pigments bile salts and urobilinogen.

ii. 6roup --% Aiver en3yme panel (marers of liver injuryGcholestasis)1. ,lanine amino transferase (,AT)". ,spartate amino transferase (,ST)*. ,laline phosphatase (,A)>. 6amma glutamyl transferase (66T)

iii. 6roup ---% lasma proteins (Tests for synthetic function)1. Total proteins". Serum albumin globulins ,G6 ratio*. rothrombin time

iv. 6roup -K% Special tests1. eruloplasmin". #erritin*. ,lpha919antitrypsin (,,T)>. ,lpha9fetoprotein (,#)

?. lassication based on linical aspectsa. 6roup -% 'arers of liver dysfunction

i. Serum bilirubin total conjugatedii. Jrine% @ile pigments bile salts and J@6iii. Total protein serum albumin and ,G6 ratioiv. rothrombin timev. @lood ammonia when indicated

b. 6roup --% 'arers of hepatocellular injuryi. ,lanine amino transferase (,AT)ii. ,spartate amino transferase (,ST)

c. 6roup ---% 'arers of cholestasisi. ,laline phosphataseii. 6amma glutamyl transferase

'arers of epatic 8ysfunction%

1. Serum @ilirubin%a. &ormal serum bilirubin level varies from 5." to 5./ mgGdl.b. The unconjugated bilirubin varies from 5."M5.E mgGdl andc. conjugated bilirubin (direct bilirubin) 5.1M5.> mgGdl.d. , rise in serum bilirubin above 1 mgGdl is abnormal (latent jaundice)2

". Jrinary Jrobilinogen%

a. -n cases of obstruction bile is not reaching the intestine and sourobilinogen may be decreased or absent in urine.

b. -n hepatocellular jaundice urobilinogen is initially elevated then

decreases or disappears when the obstructive stage sets in and reappears

when obstruction is cleared.d. Jrobilinogen is absent in urine when there is obstruction to bile !ow. The

rst indication of the recovery is the reappearance of urobilinogen in

urine.e. -n hemolytic anemias urobilinogen is increased.f. @ilirubin is detected by #ouchetDs test and urobilinogen by +hrlichDs test.

0. Jrine @ile Saltsa. &ormally bile salts (sodium salts of taurocholic acid and glycocholic acid)

are present in the bile2 but are not seen in urine.b. @ile salts in urine are detected by ayFs test. ositive ayFs test indicates

*4"

8/21/2019 20.Tools & Tests

14/18

the obstruction in the biliary passages causing regurgitation of bile salts

into the systemic circulation leading to its excretion in urine.c. Lbstruction can occur in obstructive jaundice and also in hepatic jaundice

due to obstruction of micro biliary channels caused by in!ammation.

 Tests based on Synthetic #unction of Aiver

1. Serum albumin level%a. plasma proteins except immunoglobulinFs are synthesised by the liver.b. albumin has a fairly long half9life of "5 days in all chronic diseases of the

liver the albumin level is decreased. , reversal in ,G6 ratio is often the

rule in cirrhosis due to hypoalbuminemia and associated

hypergammaglobulinemia.c. &ormal albumin level in blood is *.? to ? gGdl2 and globulin level is ".? to

*.? gGdl.". Serum globulins

a. They constitute immunoglobulins produced by @ lymphocytes as well as

alpha and beta globulins synthesi3ed mainly by hepatocytes.b. 6amma globulins in the serum are increased in chronic liver diseases

(chronic active hepatitis cirrhosis).c. -g6 is increased in autoimmune hepatitis. -g' is increased in primary

biliary cirrhosis. -g, is increased in alcoholic liver disease.*. rothrombin time (T)

a. Since prothrombin is synthesised by the liver it is a useful indicator of

liver function. The half9life of prothrombin is 0 hours only2 therefore T

indicates the present function of the liver. T is prolonged only when liver

loses more than /5C of its reserve capacity.b. Kitamin N deciency is also a cause for prolonged prothrombin time. -n

case of liver disease the T remains prolonged even after parental

administration of vitamin N.>. ,lpha9fetoprotein (,#)

a. -t is a normal component of fetal blood. -t disappears after birth within afew wees. -t is a tumor marer. 'ild elevation is suggestive of chronic

hepatitis or cirrhosis2 drastic increase is seen in hepatocellular carcinoma

germ cell tumors and teratoma of ovary.b. +levated ,# 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. Aow ,# is seen in maternal serum in cases of fetal 8own syndrome.

-mmuno assay is employed to test ,#.d. :eference limits are up to 1 year R *5 ngGml and adults (males and

nonpregnant femalesR1? ngGml.?. eruloplasmin (p)

a. -t is mainly synthesi3ed by the hepatic parenchymal cells anda small part

by lymphocytes. Aevel of p is increased in active hepatitis biliarycirrhosis hemochromatosis and obstructive biliary disease.

b. The level is decreased in =ilsonDs hepatolenticular degeneration0. ,lpha91 antitrypsin (,,T)

a. -t is an acute phase reactant and is synthesi3ed and secreted by the liver.

,,T inactivates serine proteases (elastase and collagenase).b. Aow levels are associated with neonatal cholestasis progressive juvenile

cirrhosis in children and micronodular cirrhosis in adults. Aow levels are

also seen in panlobular emphysema. -t is increased in acute trauma

infections or after estrogen therapy and in many malignancies.E. aptoglobin

a. -t is synthesi3ed in the liver. -t transports free hemoglobin in the plasma to

reticulo endothelial system.b. @eing an acute phase reactant its levels are high in in!ammatory

*4*

8/21/2019 20.Tools & Tests

15/18

processes trauma infections and myocardial infarction. -ts level is low -n

hemolytic jaundice./. +n3ymes indicating hepatocellular damage

a. ,AT%1. &ormal serum ,AT (alanine amino transferase) is 159*? -JGA.". Kery high levels (more than 1555 units) are seen in acute

hepatitis.*. +levation of ,AT is more in cases of hepatic disease compared to

,ST. @ut ,ST may be more than ,AT in alcoholic liver disease. -n

alcoholic liver disease the actual values show only mild elevation2

but a ratio of ,STG,ATmore than " is ;uite suggestive.

&. spartate amino trans'erase

8/21/2019 20.Tools & Tests

16/18

a. To assess glomerular functioni. 6lomerular ltration rateii. learance testsiii. 6lomerular permeabilityiv. roteinuria

b. To assess tubular function

i. :eabsorption studiesii. Secretion testsiii. oncentration and dilution testsi7. :enal acidication

6lomerular function tests%

1. 6#:% The glomerular ltration rate (6#:) is 1"591"? ml per minute in a person with

E5 g body weight. 6#: cannot be measured directly it is estimated from the

clearance tests

Tets o' !lomerular 'unction;

1. learance Tests%a. learance is dened as the volume of blood or plasma completely cleared of

a substance per unit time and is expressed as milliliter per minute.

b. -t is expressed as milliliter of plasma per minute  mg of substance excreted per minute

learance H MMMMMMMMMMMMMMMMMMMMMMMMMMMMM

mg of substance per ml of plasma

". reatinine learance Testa. reatinine is a waste product formed from creatine phosphate.b. Since the production is continuous the blood level will not !uctuate much

maing creatinine an ideal substance for clearance test.c. reatinine excretion is constant in a particular person.d. :eference Kalues of reatinine

i. ,dult males 5.E91.> mgGdlii. ,dult females 5.091.* mgGdl

iii. hildren 5.>91." mgGdl.e. rocedure for reatinine learance Testf. 6ive ?55 ml of water to the patient to promote good urine !ow. ,fter about

*5 minutes as to empty the bladder and discard the urine. +xactly after 05

minutes again void the bladder and collect the urine and note the volume.g. Tae one blood sample. reatinine level in blood and urine are tested and

calculated.h. reatinine clearance could be calculated as%

learance H (JG) x K

(J is the urine creatinine concentration is the plasma creatinine

concentration and K is the urine !ow in mlGmin)

i. -nterpretation of reatinine learance%i. , decreased creatinine clearance is a very sensitive indicator of

reduced glomerular ltration rate.ii. The importance of creatinine clearance is in the early detection of

functional impairment of idneyiii. &ormal value is around E? mlGmin.

*. +stimated 6#: (e6#:) Test%a. , simpler techni;ue of estimating creatinine clearance and thereby 6#: is by

using serum creatinine level.b. , commonly used formula is occroft96ault e;uation.

cr H (1>59age in years) x weight in g GE" x cr in mgGdl

>. ,dvantages%a. ,s the production is continuous the blood level will not !uctuate. @lood may

be collected at anytime.b. -t is not a$ected by diet or exercise.

*4?

8/21/2019 20.Tools & Tests

17/18

?. 8isadvantages%a. reatinine is ltered by glomeruli and actively excreted by the tubules. Lf

the total excretion about 15C is tubular component.b. +arly stages of decrease in 6#: may not be identied by creatinine

clearance.0. rocedure%

a. 6ive ?55 ml of water to the patient to promote good urine !ow. ,fter about*5 minutes as to empty the bladder and discard the urine. +xactly after 05

minutes again void the bladder and collect the urine and note the volume.

 Tae one blood sample. reatinine level in blood and urine are tested and

calculated.b. Jncorrected clearance H ( JG ) x K

where J is the urine creatinine concentration is the plasma creatinine

concentration and K is the urine !ow in mlGmin (The "> hrs urine collection is

not necessary for the creatinine clearance test).

Jrea learance Test%

1. Jrea clearance is dened as the volume (ml) of plasma that would be completely

cleared of urea per minute. -t is calculated by the formulam H JxK

where m. H 'aximum urea clearance

J H Jrea concentration in (mgGml)

K H Jrine excreted oer minute in ml

H Jrea concentration in olasma

(mgGml).

". The above calculation is applicable if the output of urine is more than " ml per

minute. This is referred to as maximum urea clearance and the normal value is

around E? mlGmin.

*. Standard urea clearance % lt is observed that the urea clearance drasticallychanges when the volume of urine is less than " mlGmin. This is nown as

standard urea clearance () and the normal value is around ?> mlGmin. lt is

calculated by a modied formula

>. 8iagnostic importance % , urea clearance value below E? C of the normal is

viewed seriously since it is an indicator of renal damage. @lood urea level as

such is found to increase only when the clearance falls below ?5C normal. ,s

already stated creatinine clearance is a better indicator of renal function.

Other test o' renal 'unctions;

&ormal Serum Jrea Aevel1. &ormal value is "5 to >5 mgGdl.". Serum urea is increased in all forms of idney diseases. -n acute

glomerulonephritis values may be as high as *55 mgGdl.

,3otemia

1. -ncrease in the blood levels of && is referred to as a3otemia and is the hallmar

of idney failure.". &&%urea creatinine and uric acid

Jrine examination

1. The routine urine examination is undoubtedly a guiding factor for renal function.

 The volume of urine excreted its p specic gravity osmolality the

concentration of abnormal constituents (such as proteins etone bodies glucoseand blood) may help to have some preliminary nowledge of idney function

*40

8/21/2019 20.Tools & Tests

18/18

*4E