Addis Ababa University

College of Health Sciences

School of Medicine

Department of Biochemistry

Laboratory Report for:

Liver Test -ALT $ AST- (1)

Prepared by: Wondimnew

Walle
ID : UGR/2899/12
August 5,2021
Liver function test
  Alanine aminotransferase/ALT/
Objective:
To investigate whether liver function or damaged by using samples of ALT in
plasma.
Principle:
 ALT is one of the sensitive markers that used as an indicator for liver disease.
When liver cells damaged or destroyed, the enzyme in the liver leaks out into
blood, in the blood raising the enzyme levels in the blood, where they can be
measured by blood test, in this case it increases.
 Alanine aminotransferase (ALT/SGPT) catalyzes the transfer of amino group
from alanine to α-ketoglutarate with the formation of glutamate and pyruvate.
The latter is reduced to malate by lactate dehydrogenase (LDH) in the
presence of reduced nicotinamide adenine dinucleotide (NADH).
 The reaction is monitored kinetically at 340 nm by the rate of decrease in
absorbance resulting from the oxidation of NADH to NAD+ proportional to the
activity of ALT present in the sample.

L-Alanine + α-ketoglutarate ALT/GPT L-Glutamate + Oxaloacetate

Pyruvate + NADH + H+ LDH Lactate + NAD+

Materials and reagents required:

Materials used:

 Spectrophotometer
 Stopwatch
 Micropipettes
 Micropipette tips
 Test tubes
 Test tube racks
 Cuvette

Reagent composition:

RA: Tris’s buffer, L- alanine, LDH

RB: NADH, 2-Oxoglutarate, NaOH, Sodium azide
Reagent preparation:
Working reagent: Mix 4ml of RA+ 1ml of RB
Sample :
Serum and plasma
Procedure:
1. A test tube was labeled as ALT
2. 1000µL of working reagent (based on the ratio of 4mL RA + 1mL RB) was added
in to a test tube
3.We added 50µL of sample in to a test tube
0
4. the result was mixed and incubated for 1 minute at 37 C
5.After 1 minute we read initial absorbance and at 1-minute intervals thereafter
for 3 minutes
6. We calculated the average absorbance difference per minute (∆A/ min)

Results

The results of absorption we obtained when the cuvettes were placed in the
spectrophotometer were:
A0=0.585
A1=0.582

A2=0.572 , A3=0.566

Result calculation
ALT, (U/L) = ∆A/min x 3333(370c)
 = (A0- A1) +(A1- A2) +(A2-A3)/min x 3333

= (0.585-0.582) +(0.582-0.572) +(0.572-0.566)/3 ×3333

=0.019/3×3333=21.109u/l

Discussion:

The normal reference value of ALT in serum at 370c is less than 41u/l and our
result of sample we take is 21.11 which is in normal range.

Error encountered:

During our experiment error may encountered due wrong holding of cuvette,
timing problem

Conclusion:

An ALT test measures the amount of ALT in the blood. High levels of ALT in the
blood can indicate a liver problem, even before you have signs of liver disease,
such as jaundice, a condition that causes your skin and eyes to turn yellow. An
ALT blood test may be helpful in early detection of liver disease .

Since the result obtained was in the normal range, it indicates that the subject
from whom the sample was taken has normal ALT level in his/her blood.

We may say the patient’s liver is normal; however, further investigation is needed
to be certain.

Reference:
  https://www.researchgate.net/publication/
 Awikepidiea.com
 L.Neson David / M.cox Michael : Principle of Biochemistry
6th edition.
 Aspartate aminotransferase (AST)

Objective:
To investigate whether liver function or damaged by using samples of AST
in plasma.

Principle:

L-Aspartate + 2-Oxoglutarate AST L-Glutamate +

Oxaloacetate
+ +
Oxaloacetate + NADH + H MDH L-Malate + NAD
Oxaloacetate is reduced to malate-by-malate dehydrogenase (MDH) in
the presence of reduced nicotinamide adenine dinucleotide (NADH).
+
Decrease in absorbance at 340 nm, due to oxidation of NADH to NAD ,
is directly proportional to the AST activity in the sample.

Material required;
Spectrophotometer
Micropipettes and Micropipettes tips
Test tubes
Test tube racks
Cuvettes
Water bath
Stopwatch

Reagent composition
 RA: Tris’s buffer, L- Aspartae, MDH, Sodium azide, EDTA-Na2
RB: NADH, 2-Oxoglutarate, NaOH
Reagent preparation
Working reagent: Mix 10ml of RA+ 1ml of RB
Sample
Serum and plasma
Procedure
1. A test tube was labled as AST
2. 1000µL of working reagent (based on the ratio of 10mL RA + 1mL
RB) was added in to a test tube
3. 100µL of sample was added in to a test tube
4. It was Mixed and incubated for 1 minute at 370C
5. After 1 minute we recorded initial absorbance and at 1-minute
intervals thereafter for 3 minutes
6. the average absorbance difference per minute (∆A/ min) was
calculated
Result
A0=0.527
A1=0.516
A2=0.544
A3= 0.546
Result calculation
AST, (U/L) = ∆A/min x 1746
= (A0- A1) +(A1- A2) +(A2-A3)/min x 1746
= (0.527-0.516) +(0.516-0.544) +(0.544-0.546) X 1746
=0.011+(-0.028) +(-0.002) X 1746

= -33.17 U/L

Reference value
Discussion

The normal reference value of AST in serum at 370c is given in the above table and
our result of sample we take is - 33.17 u/l which is negative may be due
experimental error that increase absorbance.

Error encountered:

During our experiment error may encountered due wrong holding of cuvette,
timing problem as a result increase absorbance.

Conclusion

Even if the value is in normal range but negative. ALT blood levels are a marker of
liver health, low levels typically indicate a healthy liver, while high levels suggest
liver damage but, to be assure of the liver function further test is needed.

Reference

 https://www.researchgate.net/publication/
 Awikepidiea.com
 L.Neson David / M.cox Michael : Principle of Biochemistry
6th edition