530 Concise Book of Medical Laboratory Technology: Methods and Interpretations Serum alkaline phosphatase Serum acid phosphatase

1. Alkaline optimum

(pH = 10.0)

Acid optimum (pH = 4.9)

2. Three isoenzymes known

— from bone, liver and

intestine

Two isoenzymes known—

prostatic and non-prostatic

3.  Inhibited by EDTA fluoride Inhibited by oxalate and—

Prostatic acid phosphatase is

inhibited by tartrate

4. Tissue sources—Osteoblast

of bone, liver cells, intestine,

kidney and placenta

Bone, liver, spleen, kidney,

prostate and red cells

5. Normal values

 3–13 KA units 1–4 KA units

TRANSAMINASES

Transamination is a process in which an amino group

is transferred from an amino acid to an alpha-ketoacid.

It is an important step in the metabolism of amino

acids. The enzymes responsible for transamination are

called transaminase (now called, amino-transferases).

Two diagnostically useful transaminases are glutamate

oxaloacetate transaminase or GOT or aspartate,

aminotransferase and glutamate pyruvate transaminase or

GPT alanine amino transferase. These enzymes catalyse the

following reactions:

 GOT/AST L-aspartate + Oxoglutarate Oxaloacetate+

(or ketoglutarate) Glutamate

GPT/ALT L-alanine + Oxoglutarate Pyruvate +

(or ketoglutarate) Glutamate

Clinical Significance

Increased serum transaminase activity is seen in liver

dysfunction. Greater activity of GOT (AST) over GPT (ALT)

is typical of myocardial infarction.

Evaluation of Methods

The two methods applied in the analysis of transaminase

activity are colorimetry and ultraviolet spectrophotometry.

The latter procedure requires NADH, coenzyme. The

colorimetric method is discussed below.

Specimen

The serum specimen submitted for the enzyme assay of

SGOT and SGPT should be free from hemolysis. Collect

the serum by the usual procedure described earlier.

Prompt analysis is recommended and if this is not possible,

refrigerate the specimen.

Serum Glutamic oxaloacetic transaminase (SGOT)

(AST) (Reitman and Frankel’s Method)

(Courtesy: Tulip Group of Companies)

For the determination of SGOT (AST) activity in serum

(For in vitro diagnostic use only).

Summary

SGOT is an enzyme found mainly in heart muscle, liver cells,

skeletal muscle and kidneys. Injury to these tissues results

in the release of the enzyme in bloodstream. Elevated levels

are found in myocardial infarction, cardiac operations,

hepatitis, cirrhosis, acute pancreatitis, acute renal diseases,

primary muscle diseases. Decreased levels may be found in

pregnancy, beri beri and diabetic ketoacidosis.

Principle

SGOT converts L-aspartate and α-ketoglutarate to

oxaloacetate and glutamate. The oxaloacetate formed reacts

with 2, 4, Dinitrophenyl hydrazine to produce a hydrazone

derivative, which in an alkaline medium produces a brown

colored complex whose intensity is measured. The reaction

does not obey Beer’s law and hence, a calibration curve

is plotted using a pyruvate standard. The activity of SGOT

(ASAT) is read off this calibration curve.

L-Aspartate SGOT Oxaloacetate

+ +

αKetoglutarate pH 7.4 L-Glutamate

Oxaloacetate Alkaline 2,4,Dinitrophenyl

+ Hydrazone

2,4,DNPH Medium (Brown colored complex)

Normal Reference Values

Serum : 8–40 Units/mL

It is recommended that each laboratory establish its

own normal range representing its patient population.

Contents 40 assays

L1 : Substrate reagent 25 mL

L2 : DNPH reagent 2 × 12.5 mL

L3 : NaOH reagent (4N) 25 mL

S : Pyruvate standard (2 mM) 5 mL

Storage/stability

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

the labels. Sodium hydroxide can be stored at RT till the

expiry mentioned.

Reagent Preparation

All reagents are ready to use except NaOH reagent (4N)

which has to be diluted 1:10 with distilled/deionized water.

Enzymology 531

Working NaOH reagent: Dilute the sodium hydroxide to

250 mL or for every 1.0 mL of NaOH reagent (4N) add 9.0 mL

of distilled water. The working sodium hydroxide reagent is

stable at RT till the expiry mentioned, in a plastic bottle.

Sample Material

Serum. Free from hemolysis SGOT (ASAT) is reported to

be stable in serum for 3 days at 2–8°C.

Procedure

Wavelength/filter : 505 nm (Hg 546 nm)/green

Temperature : 37°C and RT

Light path : 1 cm

Plotting of the calibration curve.

Pipette into five clean dry test tubes labeled as 1, 2, 3, 4,

and 5.

Addition

sequence

Enzyme

activity (U/mL)

1

0

mL

2

24

mL

3

61

mL

4

114

mL

5

190

mL

Substrate reagent (L1) 0.50 0.45 0.40 0.35 0.30

Pyruvate standard (S) - 0.05 0.10 0.15 0.20

Distilled water 0.10 0.10 0.10 0.10 0.10

DNPH reagent (L2) 0.50 0.50 0.50 0.50 0.50

Mix well and allow to stand at RT for 20 minutes

Working NaOH reagent

(L3)

5.00 5.00 5.00 5.00 5.00

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

Measure the absorbances of the tubes 2–5 against tube 1

(blank). Plot a graph of the absorbances of tubes 2–5 on

the ‘Y’ axis versus the corresponding enzyme activity on

the ‘X’ axis.

Assay

Pipette into clean dry test tubes labeled as blank (B) and

Test (T):