534 Concise Book of Medical Laboratory Technology: Methods and Interpretations Addition

Sequence

(B)

(mL)

(T)

(mL)

Sample – 0.10

Mix well and incubate at 37°C for 30 minutes

DNPH reagent (L2) 0.50 0.50

Mix well and allow to stand at RT for 20 minutes

Distilled water 0.10

Working NaOH reagent (L3) 5.00 5.00

Mix well and allow to stand at RT for 10 minutes.

Measure the absorbances of the Test (T) against Blank

(Blank) and read the activity of the test from the calibration

curve plotted earlier.

Note

One sample blank is sufficient for each assay series.

If enzymes activity exceeds 150 U/mL dilute the sample

with distilled water and repeat the assay. Multiply the

value with proper dilution factor.

High concentration of aldehydes and ketones in the

sample or icteric or lipemic, samples may cause slightly

elevated results. It is recommended to run a sample blank

for these samples using serum instead of distilled water in

the blank. High levels of serum pyruvate may interfere with

the results.

System Parameters

Reaction : End point Interval : —

Wavelength : 505 nm Sample volume : 0.10 mL

Zero setting : Reagent blank Reagent volume : 6.00 mL

Incubation

temperature

: 37°C Standard : Calib curve

Incubation

time

: 50 min Factor : —

Delay time : — Reac. slope : Increasing

Read time : — Linearity : 150 U/mL

No. of read : — Units : U/mL

SGPT (ALT) (Mod. IFCC Method)

(Courtesy: Tulip Group of Companies)

For the determination of SGPT (ALT) activity in serum (For

in vitro diagnostic use only).

Summary

SGPT is found in a variety of tissues but is mainly found in

the liver. Increased levels are found in hepatitis, cirrhosis,

obstructive jaundice and other hepatic diseases. Slight

elevation of the enzymes is also seen in myocardial infarction.

Principle

SGPT (ALT) catalyzes the transfer of amino group between

L-alanine and α ketoglutarate to form pyruvate and

glutamate. The pyruvate formed reacts with NADH in the

presence of Lactate Dehydrogenase to form NAD. The rate

of oxidation of NADH to NAD is measured as a decrease

in absorbance which is proportional to the SGPT (ALT)

activity in the sample.

SGPT L- Alanine + α Ketoglutarate Pyruvate + L-Glutamate

 LDH Pyruvate + NADH + H Lactate + NAD+

Normal Reference Values

Serum (males) : Up to 40 U/L at 37°C

(females) : Up to 31 U/L at 37°C.

It is recommended that each laboratory establish its

own normal range representing its patient population.

Contents 25 mL 75 mL

L1 : Enzyme reagent 20 mL 60 mL

L2 : Starter reagent 5 mL 15 mL

Storage/stability

Contents are stable at 2–8°C till the expiry mentioned on

the labels.

Reagent Preparation

Reagents are ready to use.

Working reagent : For sample start assays a single reagent

is required. Pour the contents of 1 bottle of L2 (Starter

Reagent) into 1 bottle of L1 (Enzyme Reagent). This working

reagent is stable for at least 3 weeks when stored at 2–8°C.

Alternatively for flexibility as much of working reagent may

be made as and when desired by mixing together 4 parts of

L1 (Enzyme Reagent ) and 1 part of L2 (Starter Reagent).

Alternatively, 0.8 mL of L1 and 0.2 mL of L2 may also be

used instead of 1 mL of the working reagent directly during

the assay.

Sample Material

Serum. Free from hemolysis. SGPT (ALT) is reported to be

stable in serum for 3 days at 2–8°C.

Procedure

Wavelength/filter : 340 nm

Temperature : 37°C/30°C/25°C

Light path : 1 cm

Contd...

Enzymology 535

Substrate Start Assay

Pipette into a clean dry test tube labeled as Test (T):

Addition

Sequence

(T)

25°C / 30°C

(T)

37°C

Enzyme reagent (L1) 0.8 mL 0.8 mL

Sample 0.2 mL 0.1 mL

Incubate at the assay temperature for 1 minute and add

Starter reagent (L2) 0.2 mL 0.2 mL

Mix well and read the initial absorbance A0 and repeat

the absorbance reading after every 1, 2, and 3 minutes.

Calculate the mean absorbance change per minute

(∆A/min).

Sample Start Assay

Pipette into a clean dry test tube labeled as Test (T):

Addition (T) (T)

Sequence 25°C / 30°C 37°C

Working reagent 1.0 mL 1.0 mL

Incubate at the assay temperature for 1 minute and add

Sample 0.2 mL 0.1 mL

Mix well and read the initial absorbance A0 after 1

minute and repeat the absorbance reading after every 1, 2,

and 3 minutes. Calculate the mean absorbance change per

minute (∆A/min).

Calculations

Substrate/sample start

SGPT (ALT) Activity in = ∆A/min × 952

U/L 25°C/30°C

SGPT (ALT) Activity in = ∆A/min × 1746

U/L 37°C.

Temperature Conversion Factors

Assay

Temperature

Desired

25°C

Reporting

30°C

Temperature

37°C

25°C 1.00 1.32 1.82

30°C 0.76 1.00 1.38

37°C 0.55 0.72 1.00

Linearity

The procedure is linear up to 500 U/L at 37°C. If the

absorbance change (∆A/min) exceeds 0.250, use only the

value of the first 2 minutes to calculate the result, or dilute

the sample 1 + 9 with normal saline (NaCI 0.9%) and repeat

the assay (Results ×10).

Note

Samples having a very high activity show a very low initial

absorbance as most of the NADH is consumed prior to the

start of measurement. If this is suspected then dilute the

sample and repeat the assay.

The working reagent or the combined reagent should

have an absorbance above 1.000 against distilled water

at 340 mn. Discard the reagent if the absorbance is below

1.000.

System Parameters

Reaction : UV kinetic Interval : 60

Wavelength : 340 nm Sample volume : 0.10 mL

Zero setting : Distilled water Reagent volume : 1.00 mL

Incubation

temperature

: 37°C Standard :

Incubation

time

: — Factor : 1746

Delay time : 60 sec. Reac slope : Decreasing

Read time : 180 sec. Linearity : 500 U/L

No. of read : 4 Units : U/L

Clinical Relevance of SGPT/ALT/ALAT

A. Increased levels are found in:

1. Hepatocellular disease (moderate to high increase)

2. Active cirrhosis (mild increase)

3. Metastatic liver tumor (mild increase)

4. Obstructive jaundice/biliary obstruction (mild to

moderate increase)

5. Infection or toxic hepatitis (markedly increased)

6. Liver congestion (mild to moderate increase)

7. Pancreatitis (mild increase)

8. Hepatic injury in myocardial infarction complicated

by shock

9. Infectious mononucleosis (moderate increase)

10. Chronic active hepatitis (moderate increase)

11. Reye’s syndrome (moderate increase)

12. Laennec’s cirrhosis (mild increase)

13. Alcoholic fatty liver (mild increase).

B. SGOT/SGPT comparison:

1. Although SGOT level is always increased in acute

myocardial infarction, the SGPT level does not

always increase proportionately.

2. SGPT is usually increased more than SGOT in acute

extrahepatic biliary obstruction.

536 Concise Book of Medical Laboratory Technology: Methods and Interpretations Clinical Relevance of SGOT/AST/ASAT

A. Increased levels occur in:

1. Myocardial infarction (MI):

 a. In MI, the SGOT level may be increased 4 to 10

times the normal values.

 b. The SGOT level reaches a peak in 24 hours and returns to normal by the 3rd or 4th day. Secondary

rises in SGOT levels suggest extension or recurrence of MI.

 c. The SGOT curve in MI parallels that of CPK.

 d. Elevated levels do not always indicate MI in suspected patients. Severe arrhythmias and severe

angina can also cause elevation.

2. Liver disease:

 a. Level is always enhanced in cirrhosis of the liver.

 b. In liver disease, the level may be 10 to 100 times

the normal.

 c. Liver disorders associated with elevated SGOT

levels

 1. Acute hepatitis

 2. Active cirrhosis

 3. Infectious mononucleosis with hepatitis

 4. Hepatic necrosis

 5. Metastatic or primary tumor of liver.

3. Other diseases associated with elevated SGOT levels:

 a. Acute pancreatitis

 b. Trauma and irradiation of skeletal muscle

 c. Acute hemolytic anemia

 d. Acute renal disease

 e. Severe tumors

 f. Cardiac catheterization and angiography

 g. Recent brain trauma with brain necrosis

 h. Crushing injuries

 i. Progressive muscular dystrophy

 j. Delirium tremens

 k. Pulmonary infarction

 l. Pericarditis

 m. Cerebrovascular accident.

B. Decreased levels occur in:

1. Beriberi.

2. Uncontrolled diabetes mellitus with acidosis.

3. Occasional liver disease may cause a decrease

instead of the expected increase.

C. Interfering factors

1. Slight decreases occur during pregnancy when there

is abnormal metabolism of pyridoxine.

2. Drugs that can cause elevated levels:

a. Aspirin

b. Codeine

c. Cortisone

d. Cholinergics

e. Theophylline

f. Vitamin A

g. Large doses of nicotinic acid

h. Hydralazine

i. Meperidine

j. Erythromycin

k. Morphine

l. Tolbutamide

m. Guanethidine analogs

n. Griseofulvin.

3. Salicylates may cause falsely decreased or increased

SGOT levels.

For diagnosis of myocardial infarction, the SGOT levels

should be done on three consecutive days because the

peak is reached in 24 hours and levels are back to normal

in 3 to 4 days.

GAMMA-GLUTAMYL TRANSPEPTIDASE (GGTP)