AMINOTRANSFERASES

Transferases catalyze the transfer of an amino group of one amino acid to a hydrocarbon to form a different amino acid. Aspartate aminotransferase (AST) Alanine aminotransferase (ALT)

Aspartate aminotransferase (AST)

Serum Glutamate Oxaloacetate (SGOT) L aspartate ! oxoglutarate aminotransferase ".#. !.$.%.%. o o An enzyme belonging to the class of Transferases. &t is commonly referred to as a transaminase and is in'ol'ed in the transfer of an amino group bet(een Aspartate and )eto acids. The transamination reactions are important in intermediary metabolism because of its function in synthesis and degradation of amino acids. The )etoacids formed by the reaction are ultimately oxidized by the tricarboxylic acid cycle to pro'ide energy
AST

Reaction Catalyzed:

L aspartic * a )etoglutarate Tissue sources+

L glutamic acid * oxaloacetic acid

AST is (idely distributed in human tissues. The highest concentrations are found in cardiac tissue, li'er, and s)eletal muscle, (ith smaller amounts found in the )idney, pancreas, and erythrocytes.
Optimum pH:

-..
Methods of Determination:

o o /eitman and 0ran)el

o o :armen o o <abson et al =ethod Sax and =oore =ethod #abaud et.al.

The oxaloacetate formed under fixed conditions is determined by the reddish bro(n hydrazone it produces (ith !,., 1initrophenylhydrazone in al)aline medium /eagents and /esults+ Substrate+ aspartic alpha )etoglutarate #olor de'eloper+ !,. dinitrophenylhydrazine #olor intensifier 2.. 3 naO4 &ncubation period % hour at 5-# "nd products glutamic acid and oxaloacetic acid 3ormal 6alue+ ..2 %789L The oxaloacetate formed is reacted (ith malic dehydrogenase in the presence of 3A14 producing malic acid (ith the subse;uent oxidation of 3A14 to 3A1. The decreae in absorbance reflects the enzymatic acti'ity of AST (in'erse colorimetry)

3ormal 6alues+ %2 52 &89L >rinciple+ The oxaloacetate formed is treated (ith daizonium salt (Azoene 0ast 6iolet <) to produce a 'iolet colored product o o 3ormal 6alue+ 7 5$ m89ml 8ses Azoene 0ast /ed and True >onceau L

Consideration in AST Assays: Serum is the best specimen 4emolyzed samples must be a'oided Alcohol Lo(ers AST 'alues =uscle trauma li)e intramuscular in?ections, exercise or surgical operation can significantly increase AST le'els

Clinical Significance: &n =yocardial infarction, AST le'els are usually . %2 times the upper limit of normal. These de'elop (ithin . $ hours ($ @ hours for other references) after the onset of pain and pea) on the !. A 5$ the hour. These usually normalize on the .th or Bth day. Although AST increases follo(ing =yocardial infarction, its use is limited by its nonspecificity. The measurement of AST le'els is most helpful for the diagnosis of hepatocellular disease. AST le'els are ele'ated and used in the diagnosis of chronic alcohol abuse and hepatoxicity. &ncreased AST le'els are also seen in pulmonary infarction, pericarditis, acute hepatitis, s)eletal muscle disorders and others 1ecreased AST le'els are seen in pregnant (omen

Alanine Amino Transferases

Serum >yru'ate Transaminase (SG>T) ".#. !.$.%.!
o ALT catalyzes the transfer of the amino group of Alanine to alpha )etoglutaric acid to form pyru'ic acid and glutamic acid. >yridoxal phosphate acts as a coenzyme
Tissue sources+ ALT is found primarily in the li'er but also in the )idneys, heart, s)eletal muscle and

o
o o

the pancreas. &t is considered to be the more li'er specific enzyme of the transferases.
Optimum pH -.. Reaction catalyzed:

ALT

L alanine o

*
o

a )etoglutarate

pyru'ate

* glutamate

Methods of Determination:
>rinciple+ Same (ith AST except for the follo(ing+ o Substrate+ Alanine alpha )etoglutarate o &ncubation period+ 52 mins at 5-# o "nd products + glutamic acid and pyru'ic acid 3ormal 6alue+ !.. %- 89L >rinciple+ This recommended method is based on a coupled enzyme reaction. Thy pyru'ate formed is reacted (ith lactate dehydrogenase producing lactic acid (ith the subse;uent oxidation of 3A14 to 3A1. The decrease in absorbance reflects the enzymatic acti'ity of ALT (in'erse colorimetry) 3ormal 6alue+ %2 5B &89L >rinciple+ Similar (ith Cal)er =ethod+ ALT Alanine * Alpha )etoglutarate L14 pyru'ate * glutamate

/eitman and 0ran)el o o Cal)er et.al =ethod o o

4enry >ollard =ethod

>yru'ate * 3A14 (bro(n)

Lactate * 3A1 (colorless)

Considerations in ALT Assays:

<lood specimen should be dra(n and handled carefully to a'oid hemolysis. 4emolysis falsely increases ALT 'alues Serum ALT is relati'ely unstable and should be )ept at .# for short periods at 5 . days or stored frozen at !2# or less for longer period.

Clinical Significance:
ALT is used in the diagnosis of Acute or chronic 'iral hepatitis since it is increased to a greater degree than AST. <lood 1onor is usually screened for ALT ele'ation prior to transfusion &ncreased le'els are also seen in Acute =yocardial infarction and other hepatocellular disease.

The Transaminases !sing the Reitman and Fran"el method Method#

S$ecific organ S%&strate End 'rod%cts Inc%&ation 'eriod Color De(elo$er Color intensifier AST (S OT! 4eart Aspartic alpha )etoglutaric acid Glutamic acid and oxaloacetic acid % hour at 5-# !,. 1>34 2.. 3 3aO4 ALT (S "T! Li'er Alanine alpha )etoglutaric acid Glutamic acid and pyru'ic acid 52 minutes at 5- # !,. 1>34 2.. 3 3aO4

Lactate Dehydro#enase (LDH!

".# %.%.%.!o L14 is an enzyme that catalyzes the intercon'ersion of Lactic and pyru'ic acids. &t is a hydrogen transfer enzyme that uses the coenzyme 3A1 L14 is (idely distributed in the body. 6ery high acti'ities are found in the heart, li'er, s)eletal muscle, )idney and erythrocytes. &t is present in lesser amounts in the lung, smooth muscle, and brain.
Reaction Catalyzed:

Tissue Sources:

L14 catalyzes the oxidation of lactate to pyru'ate (ith concomitant reduction of 3A14 to 3A1*. The reaction is re'ersible and the bac)(ard reaction is fa'ored at reaction e;uilibrium Lactate
$soenzymes:

3A1

>yru'ate

3A14

4*

The isoenzymes are designated according to their electrophoretic mobility and each of them is a tetramer made up of . subunits (1esignated 4 (heart) and = (muscles)). L14 % L14 ! L14 5 L14 . L14 B 4444 444= 44== 4=== ==== %. !$D !7 57D !2 !$D @ %$D $ %$D

Distri%ution of LD $soenzymes:

LD) and LD* LD+ LD , and LD -

0ast mo'ing fractions and are heat stable, found mostly in the myocardium and erythrocytesE also found in the renal cortex 0ound in a number of tissues predominantly in the (hite blood cells and brain, Lungs, spleen, pancreas Slo( mo'ing and are heat labile found mostly in the li'er and s)eletal musclesE also seen in the ileum of the s)in

The relati'e concentration in normal serum is L1!, L1%, L15, L1. and L1 B in descending order

Techni&ues in Measurin# LD $soenzymes:

'hysical

"lectrophoresis Selecti'e absorption on 1iethyulaminoethy cellulose (1"A") Sol'ent precipitation Techni;ue 4eat denaturation at $B# for 52 mins Substrate end product relationship #oenzyme Affinity 1iffferential chemical inhibition of L1 Acti'ity

Chemical Imm%nologic
Optimum pH:

0or(ard /eaction+ (pyru'ate to lactate)+ @.@ 7.@ <ac)(ard /eaction (lactate to pyhru'ate) -.. -.@
Methods of Determination:

Generally by+ Spectrophotometric method 0lourometric method #olorimetric method Crobleus)i #abaud <erger and <roida Crobleus)i andLa 1ue :ubo(itz and Ott

Laboratory =ethods+

Wrobleuski Cabaud Method

"rinciple:

>yru'ate formed is reacted (ith !,. 13>4 to produce a golden bro(n phenylhydrazone at an al)aline p4.
Rea#ents and Results+

>yru'ic Acid+ substrate

!,. 13>4+ color de'eloper 52 mint at !.# incubation period <ro(n osazone+ end product and color

'ormal (alue+ !22 .B2 Crobleus)i units9ml

Considerations in LD Assays:
o o o /ed cells contain %B2 times more L14 than serum, therefore hemolysis must be a'oided L1 Acti'ity is unstable in serum regardless of the temperature at (hich it is stored. &f the sample cannot be analyzed immediately, it should be stored at !B# and analyzed (ithin .@ hours. L1 B is the most labile isoenzyme. Loss of acti'ity occurs more ;uic)ly at .# that at !B#, L1 isoenzyme analysis should be stored at !B# and analyzed (ithin !. hours of collection.

Clinical Significance:
o o &n myocardial &nfarction, L1 increases @ %! hours after the onset of pain, pea)s at .@ $2 hours and remains ele'ated for %2 %. days. 4ere L1% is higher than L1! calledF flippedF L1 pattern. L1 le'els are moderately to mar)edly ele'ated in the follo(ing =egaloblastic anemia >ulmonary infarction Granulocytic leu)emia 4odg)inGs disease 4emolytic anemia &nfectious mononucleosis >rogressi'e muscular dystrophy

Alpha Hydro)y%utyric Dehydro#enase

A 4<1 A 4<1 is not a separate and distinct enzyme but it is considered to represent the L1 % acti'ity of total L1. 4o(e'er, a 4<1 acti'ity is not entirely specific for the L1 % fraction, because L1 !, L1 5, and L1 . also contain 'arying amounts of 4 subunit. A 4<1 acti'ity is increased in those conditions in (hich the L1 % and L 1! fractions are increased. &n A=&, a 4<1 acti'ity is 'ery similar to the time course of total L1 acti'ity and can be useful in the diagnosis (hen other L1 isoenzyme procedures are not readily a'ailable. =ethod of 1etermination+ /osal)i and Cil)inson A 4<1 o A )etobutyrate * 3A14 * 4

A hydroxybutyrate * 3A1

Creatine *inase (C*!

#reatine >hospho)inase (#>:) AT> #reatine 3 phosphotransferase ".#.!.-.5.!

#reatine )inase (#:) is an enzyme that is generally associated (ith AT> regeneration in contractile or transport systems. &ts predominate physiologic function occurs in muscle calls, (here it is in'ol'ed in the storage of high energy #reatinine phosphate. "'ery contraction cycle of muscle results in #reatinine phosphate use, (ith the production of AT>. This results in relati'ely constant le'els of muscle AT>.

Tiss%e so%rces:
#: is (idely distributed in tissues, (ith highest acti'ities found in s)eletal muscle, hear muscle, and brain tissue. Other tissue sources in (hich #: is present in much smaller ;uantities include the bladder, placenta, gastrointestinal tract, thyroid, uterus, )idney, lung, prostate, spleen, li'er and pancreas.

Isoen.ymes:
Three isoenzymes isolated after electrophoresis << =< == =ost rapidly mo'ing isoenzyme predominantly present in the brain, intestine, thyroid and )idney 0ound in the heart, tongue, esophagus and diaphragm. Slo(est and most common form present in all tissues mostly in s)eletal muscles.

#:% #:! #:5

4eart yields about .2D #:! and $2D #: (hile brain tissues yields 72D of #:% and %2D #:5 =ethods of #: isoenzyme determination+ o o o "lectrophoresis &mmunochemical #hromatographic Separation and "lution Techni;ue

O$tim%m $/:
o o 0or(ard reaction+ 7.2 <ac)(ard reaction+ $.@

Methods of Determination: #olorimetric method >rinciple+ AT> and creatine are incubated (ith a specimen and the reaction is stopped (ith the addition of acid, producing phsophophocreatrine (hich is acid labile. This then dissociates inot creatine and free phosphate ions (hich can be measured for #: acti'ity. Sax and =oore =ethod (0lourometric method)

#reatinine reacts (ith 3inhydrin (tri)etohydrinoene hydrate) solution to produce a flurophor.

4ughes =ethod

#reatine is made to react (ith diacetyl and alpha napthol to produce a pin) end product.
/osal)i and 4ess >rinciple+ This is the most (idely used methos (herein AT> produced in the re'erse reaction pro'ides the high energy phosphate for the follo(ing reaction+ hexo)inase AT> * Glucose G $ >O. * A1> =g G $ > 14 G $ >O. * 3A1>4
lactone

$ phosphogluconolactone * 3A1>

"nzymatic =ethods

A decrease in the absorbance at 5.2 nm reflects the #: acti'ity Tanzer and Gil'arg >rinciple+ The A1> formed in the for(ard reaction is used in the follo(ing reactions+ >yru'ate )inase A1> * >"> AT> * pyru'ate L14 >yru'ate * 3A14 pyru'ate * 3A1 The reduction in absorbance at 5.2 nm reflects the #: acti'ity

Considerations in C* Assays: #: is light sensiti'e and anticoagulants li)e oxalates and fluorides inhibit its action #: in serum is 'ery unstable and rapidly lost during storage. &ts acti'ity is regenerated by the addition of S4 groups li)e cysteine, dithiothreiol or mercaptoethanol (hich stabilize the enzyme by maintaining its AS4 type cross lin)ages. La)ed specimen is not used since it contains cellular products and intermediates li)e adenylate )inase. AT> and Glucose $ phosphate dehyhdrogenase (hich affect the assay. "xercise and intramuscular in?ections cause #: ele'ations.

Clinical Si#nificance:
&n myocardial infarction, #: (ill rise . $ hours after the onset of pain, pea)s at %@ 52 hours and returns to normal on the 5rd day. #) is the most specific indicator for =yocardial infarction. #: le'els are also increased in+ >rogressi'e muscular dystrophy >olymyositis Acute psychosis Alcoholic myopathy 1elirium tremens 4ypothyroidism

=alignant hyperthremia Acute cerebro'ascular disease Trichinosis and dermatomyositis !B 72 &89ml (2..! A%.B% mmol9L) %2 -2 &89ml (2.%- A %.%@ mmol9L)

3ormal 6alue+ =ale 0emale+

+ructose Diphosphate Aldolase (ALD! ,-C- .-/-0-/1

AL1 catalyzes the splitting of 1 fructose diphosphate to 1 glyceraldehydes phosphate and dihydroxy acetone phosphate. Three isoenzymes+ Aldolase A Aldolase < Aldolase # Optimum p4+ $.@ 0ound predominantly in s)eletal muscles 0ound in the li'er, )idney and C<# 0ound in the brain tissues -.!

Methods of Determination:
>into, :aplan and 6an 1real+ Sibley and Lehninger+ 3ormal 6alue+ % $ 89L 3ormal 6alue+ 5 @ 89L

Considerations in ALD Assays: o o La)ing should be a'oided since the red cells contain %2 times as much AL1 as Serum. The AL1 acti'ity of serum and plasma are identical, (ith oxalate, citrate, fluoride, heparin and "1TA as the anticoagulants.

Clinical Si#nificance: o Se'ere ele'ations are seen in+ =uscle degeneration 6iral hepatitis =oderate ele'ations in+ Gangrene =egaloblastic anemia =etastatic li'er #A Granulocytic leu)emia >sychosis Trichinosis

amma
"# !.5.!.!

lutamyl Transferase (

T!

>lays a uni;ue role in the metabolism of amino acids. The basic reaction catalyzed by this enzyme (often referred to as gamma glutamyl transpeptidase is+

GGT Gamma glutamyl cysteinyl glycine * amino acid Gamma glutamyl acid (peptide) * cysteinyl glycine The basic transpeptidation reaction is significant in some aspects of the control of protein synthesis. The synthesis of gluthathione and certain components of the mo'ement of amino acids into cells are in part regulated by the acti'ity of gamma glutamyltranspeptidase. Tissue distribution: Li'er, pancreas and )idneys Determination: 0eneral $rinci$le: The substrate gamma L glutamyl p nitroanilide (ill be acted upon by GGT and (ill transfer the gamma glutamyl portion of the molecule (ould be transferred to the glycylglycine, and the free p nitoraniline could be measured by the spectrophotometer. Cith an absorbance at .2B .!2. The Szasz Assay ma)es use of gamma L glutmayl ! carboxy . nitroanilide, a compound similar to L glutamyl p nitroanilide. One product of the reaction, B amino ! nitrobenzoate is measured at .!2 nm. This pro'ides better absorbance and impro'ed sensiti'ity.

Other methods: Orlo(s)i 1imora and :ulhane) /osal)i and Tarlo( 3ormal 6alue+ =ales+ 0emales+ Clinical Si#nificance: o up to .2 89L up to !B 89L

Li'er 1isease+ 4as been sho(n to be useful mar)er li'er damage. The enzyme is ele'ated in obstructi'e li'er disease, inflammation of the li'er, or obstruction of the biliary tract. "le'ated serum le'els of GGT ha'e been reported in a number of different types of cancer. The increases are more li)ely due to hepatic damage seen as a result of metastasis of the primary tumor =ost useful application is in the detection of alcoholism and monitoring of alcohol consumption by patients during treatment.

o
.

Cholinesterase

cholinesterase is a term (hich refers to one of the t(o enzymes+

o Acetylcholinesterase, also )no(n as RBC cholinesterase, erythrocyte cholinesterase, or

(most formally) acetylcholine acetylhydrolase, found primarily in the blood and neural synapses (".#. 5.%.%.-)

o >seudocholinesterase, also )no(n as plasma cholinesterase, butyrylcholinesterase, or

(most formally) acylcholine acylhydrolase, found primarily in the li'er (".#. 5.%.%@)

<oth of these compounds catalyze the hydrolysis of the neurotransmitter acetylcholine into choline and acetic acid, a reaction necessary to allo( a cholinergic neuron to return to its resting state after acti'ation. The difference bet(een the t(o types of cholinesterase has to do (ith their respecti'e preferences for substrates+ the former hydrolyses acetylcholine more ;uic)lyE the latter hydrolyses butyrylcholine more ;uic)ly. Methods of Determination: Ellman Technique: "rinciple: (#4S) >ropionythiocholine propionic acid * thiocholine colored compound

Thiocholine * B,BGdithiobis A(! nitobenzoic acid) &ncrease in absorbacne is measured at .%2 nm. Sensiti'e, rapid, recommended method

Michel Technique: Principle: based on the hydrolysis of benzoylcholine, change in 86 absorption is measure Temperature sensiti'eE much 'araibility among different laboratories 3eeds special e;uipments

3ormal 6alue+ $,222 %!, 222 89L #linical Significance+ 1ecreased notably in li'er disease A ma?or clinical application of cholinesterase measurments is in screening for possible pesticide exposure. &f acetylcholinesterase acti'ity is bloc)ed by a pesticide, impairement of ner'e transmission occurs.

Other En.ymes: En.ymes

Trypsin 3ucleotidase &socitric 1ehydrogenase Sorbitol 1ehydrogenase

Method
H ray 0ilm Test #ampbell 1ixon and >urdon <elfield and Goldbers <ell and <aron Colfson et.al 3achlas, "llis and Goldberg Gerlach method

Clinical Significance
>ancreatic disorders Li'er disorders &nsecticide poisoning 4epatobillary disease Acute hepatitis

Glucose $ phosphat dehydrogenase

<ishops "llis and :ir)man =otuls)y <entler and =itchell <rom and Grisolia #arriotti /eichard

Ornithine #arbamyl Transferase Leucine Amino >eptidase

Li'er damage &nhalation poisoning >ulmonary and mycoardail infarction >ernicious anemia CerloffGs disease 4yperthyroidism 6iral hepatitis =yolegenous Lu)emia 4epatocellular diseases

4epatobilliary disease