Semmelweis University

Department of Medical Biochemistry Practical manual: Transaminase enzyme activities

TRANSAMINASE ENZYME ACTIVITIES

The α-amino group of an amino acid is transferred to an α-ketoacid by transaminase enzymes. The
prostetic group of transaminase enzymes is pyridoxal-5'-phosphate, a derivative of B6 vitamin. The most important
transaminase enzymes in diagnostics are the aspartate-aminotransferase and alanine-aminotransferase.

The aspartate-aminotransferase, ASAT (EC 2.6.1.1)

The aspartate-aminotransferase (former glutamate-oxaloacetate-transaminase GOT, GOT activity of

serum is called sGOT) localising to all organs, - its concentration is extremely high in the liver and heart - catalyses
the following reaction (Fig. 1.):

- - - -
COO COO COO COO
CH + CH ASAT CH + CH
2 2 2 2
+
CH NH CH C O CH
3 2 - 2 +
-
COO C O COO CH NH
- - 3
COO COO

L-Aspartate α-ketoglutarate Oxaloacetate L-Glutamate

Figure 1: The ASAT (GOT) enzyme activity

The alanine-aminotransferase, ALAT (EC 2.6.1.2)

The alanine-aminotransferase (former glutamate-pyruvate-transaminase GPT, GPT activity of serum is

called sGPT) enzyme activity is localized to the liver, kidney, heart, and skeletal muscle cells. Its activity is high in
the liver cells in particular. The ALAT enzyme catalyses the following reaction (Fig. 2.):

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 - - - -
COO COO COO COO
CH NH + CH ALAT C O + CH
2 2 2
CH CH CH CH
3 2 3 2 +
C O CH NH
- - 3
COO COO

L-Alanin α-ketoglutarate Pyruvate L-Glutamate

Figure 2: The ALAT (GPT) enzyme activity

The ALAT activity is detectable in both mitochondria and about 50-85 % of total enzyme activity of cells in
the cytosol.

Determination of the aspartate-aminotransferase, ASAT [GOT] activity

The principe of the determination

ASAT
L - Aspartate + α - ketoglutar ate  → Oxaloacetate + L - Glutamate
MDH
Oxaloacetate + NADH + H +  
→ Malate + NAD +

MDH = malate-dehydrogenase

Reagents

Reagent 'ASAT': 80.00 mmole/l TRIS buffer pH=7.8

200.00 mmole/l L-aspartate
12.00 mmole/l α-ketoglutarate
0.60 kU/l Malate dehydrogenase (MDH)
0.18 mmole/l NADH
Test samples: serum of a 'normal' and an 'ill' patient

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 Semmelweis University
Department of Medical Biochemistry Practical manual: Transaminase enzyme activities

Procedure

Please, determine the ASAT activity of the 'ill' patient only after the complete determination - incubation,
photometry - of the ‘normal’ patient.

Set up the reaction in photometer cuvettes

Reagents 1 2
Reagens 'ASAT' ml 1,0 1,0
Serum of 'normal' patient ml *0,1 0,0
Serum of 'ill' patient ml 0,0 *0,1
*Start of reaction

Read the optical densities of the samples each minute for a 5 min. period at 340 nm. Distilled water serves as
blank. Calculate the ∆A/min for each tube.

Calculation of the ASAT (SGOT) enzyme activity

An enzyme unit is defined as the amount of enzyme catalyzing the transamination of 1 µMol aspartate in 1 min.

1000 ∗ V
IU/l = ∆A/min ∗
ε ∗ d∗ v
Where
V volume of reaction mixture [ml]
ε NADH340 nm: 6.22 [cm2 × µmol-1]

d the length of the optical way in [cm]

v volume of serum added to the test [ml]

Linearity: If the ∆A/min values of samples are higher than 0.150 (340 nm), dilute samples fivefold or tenfold with a
0.9 % solution of NaCl. Determine the enzyme activity with the diluted sample and multiply the results by 5 or 10.

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The alanine amino transferase, ALAT [GPT]

The principle of determination

ALAT
L - Alanine + α - ketoglutar ate  → Pyruvate + L - Glutamate
LDH
Pyruvate + NADH + H +  → L - Lactate + NAD +

LDH = lactate-dehydrogenase

Reagents

Reagent 'ALAT' 100.00 mmole/l TRIS buffer pH=7.5

500.00 mmole/l L-alanine
15.00 mmole/l α-ketoglutarate
1.20 kU/l Lactate dehydrogenase (LDH)
0.18 mmole/l NADH
Test samples serum of a 'normal' and an 'ill' patient

Procedure

Determine the ASAT activity of the 'ill' patient only after the complete determination - incubation, photometry - of
'normal' patient.
Set up the reaction in photometer cuvettes

Reagent 1 2
Reagent 'AL' ml 1,0 1,0

Serum of 'normal' patient ml *0,1 0,0

Serum of 'ill' patient ml 0,0 *0,1

*Start of reaction

Read optical activities for 5 minutes at 34o nm. Distilled water serves as blank. Calculate the ∆A/min for each
tubes.

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 Semmelweis University
Department of Medical Biochemistry Practical manual: Transaminase enzyme activities

Calculation of the ALAT (SGPT) enzym activity

An enzyme unit is defined as the amount of enzyme catalyzing the transamination of 1 µMole alanine in 1 min.

1000 ∗ V
IU/l = ∆A/min ∗
ε ∗ d∗ v
Where
V volume of reaction mixture [ml]
ε NADH340 nm: 6.22 [cm2 × µmol-1]
d the length of the optical way in [cm]
v volume of serum added to the test [ml]

Linearity: If the ∆A/min values of samples are higher than 0.150 (340 nm), dilute samples fivefold or tenfold with a
0.9 % solution of NaCl. Determine the enzyme activity with the diluted sample and multiply the results by 5 or 10.

Biomedical aspects of transaminase activity

The transaminase enzymes show a low level in the plasma under normal circumstancens. With the
accelerated death of cells - virus infection, necrosis - soluble enzymes enter the bloodstream. Parallel
determination of both transaminase activities is of great importance in the diagnosis and evalution of liver and heart
diseases.
Myocardial infarction significantly enhances the activity of ASAT (SGOT) while the activity of ALAT (SGPT)
is elavated moderately. On the other hand, hepatocellular tissue destruction enhances both activity, but the level of
ALAT (SGPT) is higher, than that of ASAT (SGOT). Acute hepatitis is likely when the quotient of activities of
ASAT/ALAT is under the value of 1.3; on the other hand, acute myocardial infarction is followed by a higher value
of ASAT/ALAT quotient.

The ASAT activity is enhanced

1. In myocardial infarction.
2. In acute rheumatoid carditis.

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 3. In the first 10 days of heart surgical interventions.
4. After catheter treatment of the heart.
5. After heart massage.
6. During the first 48 hours of acute pancreatitis.
7. Acute destruction of liver cells

U/l 25 oC U/l 37 oC
Adult: men ≤ 19 ≤ 35
women ≤ 15 ≤ 40

Table 1. The normal values of ASAT [GOT] activities.

The SGPT activity is enhanced

1. In acute hepatitis.
2. In liver chirrosis
3. Mononucleosis

Normal ALAT (SGPT) activities are summarized in the table 2.

U/l 25 oC U/l 37 oC
Adult: men ≤ 23 ≤ 35
women ≤ 19 ≤ 40

Table 2: The normal values of ALAT [GPT] activities