Biochemistry

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, or" Ie de terrnin a11on of SGPT ( ALA r) (For lnvilro Diagnostic Use O ,

Summar y SGPT i:, found in a variety of tissues but is main fou tJ in lhe' hver. Increased levels are found in hepatitis , cirrhosis, obstructive j ndice and other hepahc diseases . Slight elevation of th e enzymes is also seen in rd ia l infarcti on.

Principle SG PT ( ALAT ) catalyze s the transfer of amino group between L-Alanin e and a.Ketoglutarate to form Pyruvate and _Gluta_rnate. The Pyruvate fo rmed reacts with NADH in the presence of Lactate Dehydiogenase to form NAO.The rate of oxidation of NADH to NAD is measured as a decrease in absorbance which is proportional to the SGPT ( ALAT ) activity in the sar:nple .

L- Alanine + aKetoglutarate

Pyruvete + NADH + W

Normal reference values

SGPT

LOH

Serum ( males ) upto 40 U/l at 37°C

Pyruvate + L-Glutamate

Lactate + NAO•

. ( le.ma!es ) upto 31 U/1.. at 31°C ft is recommended that each laboratory establish its own normal range representing its patient population.

Contents L 1 : Enzyme Reagent L2 : Starter Reagent

25 ml 20 ml 5 ml

75 ml 60 ml 15 ml

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Reagents are read y to use. Working re agent : For sample start ass"ys ·1 single rea gent rs required . Pou r ti,e contents of 1 bottle oi L2 (Starter Reagent) into t bottle of L 1 (Enzyme Reagent ) Tl115 wo rking reagent is stable for at least 3 weeks when stored at 2-8~ C. Alt emahvely for fl exibr lity as much of working reagent may be made as and when desired by mixing together 4 parts of L 1 (Enzyme Reagent ) and 1 part of L2 (Starter Reagent ). Alterna tively 0.8ml of L1 and 0.2 ml of L2 may also be used instead ot 1 ml of the working reagent directly during the assay

Sample ma ter ia l Serum . Free from hemolysis. SG PT (ALAT ) Is reported to be stable in serum for 3 days at 2-8° C.

Procedure Wavelength I filter Temperature Light path

Substrate Start Assay :

340 nm 3 ?"C / 30"C I 25"C 1 cm

Pipette into a clean dry lest lube labeled as Test ( T ) .

Addition ( T) Sequence 2soe , 30•c Enzyme Reagent ( L 1 ) 0.8 ml Sample 0.2 ml ~)4 .c, .·

Incubate at the ossay temperature for 1 minute and add

Starter Reagent ( L2 ) 0.2 ml

(T) 37°C

0.8 ml 0.1ml

0.2 ml

Mix well and read the ilitlal absolbance A, & repeat the absorbance reading alter every 1, 2, & 3 minutes. Calculate the mean absorbance change per minute ( t,. A I min. )

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' Mix well and keep the test tubes at R.T. lo exactly 20 minutes . Measure the absorbance of the Stand.mi /Abs~\ ~no TAst Sampl, IAbs.n aaainst the Blank. ·- ~-·· --.. ·--~- - -. . - -· ' L · - --- --- · - ·-· - ·-··· ·· • -• Addi tion ( 1 ) ( l j Seq uenc e 25"C .' 30°C 37°c

Working Reagen I I 10 ml 1 o mi

Incuba te at the assay temperat ure for 1 r111nu te and add

Sample 0 2 ml 0.1 ml

Mix well and read th e initial abso rban ce A 0

af ler !minute & repeat tile absorbance reading after eve1y 1, 2, & 3 minutes. CalOJ late lhe mean abs01bance change per minule ( M / mo. )

Calculations Substrate I Sample start SG PT ( ALAT ) Activity in U/L 25°C / 30°C

31°c

!'iA I min. x 952

L\A I min x 174 6

TEMPERATURE CONVERSION FACTORS

Assay Desired Reporting Temperature Temperature 2s•c 30°c 37°c

2s•c 1.00 1.32 1.82 Jo•c 0.76 1. 00 1.38 31°C 0.55 0.72 1.00

Linearity The procedure is linear upto 500 U/L al 37°C. If the absorbance change ( ti.A I min.} exceeds 0.250, use only the valife of the first lwo minutes to calcufai~-ihe result, oi dilute the sample 1 + 9 with normal saline ( NaCl 0.9%) and repeal the assay (Re sults x 10).

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Thr- working ,ea genl or lh e combined r0<1g~nl shoul d ~i;;ve ~n al):;o rl1 a .. ce aho1 I 000 againsl dis lill erJ wal er at 1'10 mn Discard lhic, 1eagr1n t 11 lh e abc.01oance below I IJQO

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System Parameters

Reaction U. V l(ine lic Int er val 60 Wavelength 340 nm Sample Vol. 0. 10 ml Zero Setting Distilled water Reagent Vol. 1.00 ml lncub . Temp . 37°C Standard lncub. Time Factor 1746 Delay Time 60 sec . React. Slope Decreasing Read Time 180 sec . Linearity 500 U/L No . of read. 4 Units U/L

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