ACTIVITY 4

ASPARTATE AMINOTRANSFERASE

(EC 2.6.1.1)

Objective: To properly perform AST testing, and correlate its values with clinical significance of the test.

To enhance laboratory skills in performing lab analysis of AST.

SUMMARY AND EXPLANATION OF TEST#

Aspartate aminotransferase is present both in cytoplasm and mitochondria of the cell. Belonging to the
transamninase family, which catalyzes the conversion of amino acids and a-oxaloglutarate by transfer of amino
groups. AST is commonly found in various human tissues. The heart muscle is found to have the most activity of the
enzyme, secondly in brain, liver, gastric mucosa, skeletal muscle and kidneys.

REACTION PRINCIPLE#
AST
L-aspartate + α-ketoglutarate < > oxaloacetate+ L-glutamate

Oxaloacetate + NADH + H+ < MDH

> L-malate + NAD+

In the assay reaction the AST catalyzes the reversible transamination of L-aspartate and a-oxoglutarate to
oxaloacetate and L-glutamate. The oxaloacetate is then reduced to malate in the presence of malate dehydrogenase
with NADH being oxidized to NAD+. The rateof photometrically determined NADH decrease is directly proportional
to the rate of formation of oxaloacetate and thus the AST activity.

Methodology#: UV assay (IFCC) without pyridoxal phosphate activation

Materials, Reagent, Kits#:

Reactive Ingredients & Concentration :

R1 LDH ≥ 900U/L R2 a-oxoglutarate 60 mmol/L

MDH ≥ 600 U/L NADH 0.9 mmol/L

NADH 0.4 mmol/L

Standard, Reagent Blanks, Calibrators

SPECIMEN COLLECTION AND HANDLING

Serum: Use serum collected by standard venipuncture or capillary collection techniques into glass or plastic tubes
with or without gel barriers. Ensure complete clot formation has taken place prior to centrifugation.

ASSAY PROCEDURE#

Blank Sample
Reagent 1 1000 uL 1000 uL
Dist. Water 100 uL -
Sample - 100 uL
Mix, incubate for 5minutes, then ADD:
Reagent 2 250 uL 250 ul
Mix thoroughly, read the absorbanceafter1min and monitor time. Read the absorbance again for additional 3min.
ΔA/min= (ΔA/min sample) – (ΔA/min blank)
CALCULATIONS *:

Abs: 340 nm

SampleAbsorbance x Concentration of Std = AST ( U/L)

Standard Absorbance

EXPECTED VALUES#

Reference Range

Range (U/L) Range (μkat/L)

Male ≤ 35 ≤0.58

Female ≤ 31 ≤0.52

Conversion Factor:

1 U/L=16.67 x 10-3 μkat/L 1 μkat/L = 60 U/L

#
Shenzhen Mindray Bio-Medical Electrons Co., Ltd. 2020

*Values are for demonstration purposes only.

Illustrate the procedure for AST Assay.

Computations.

QFR :

1. What are the tissue distributions of AST?

2. What are the two forms of AST and how does this affect the assay of AST?

3. What is the clinical significance of AST?

4. How specific is AST in diagnosis of AMI?

5. Discuss the reaction catalyzed by AST? How important is this reaction.

6. Discuss the principle of Karmen method.

7. Illustrate the reaction principle of AST.

8. Discuss the methods for AST analysis.

rrivera Clinical Chemistry 2

group i

std abs : 1.003 std conc’n: 33.5 ΔA/min sample: 1.206 ΔA/min blank: 0.103

group ii

std abs : 2.013 std conc’n: 33.5 ΔA/min sample: 3.001 ΔA/min blank: 0.901

group iii

std abs : 1.102 std conc’n: 33.5 ΔA/min sample: 1.020 ΔA/min blank: 0.001

group iv

std abs : 0.0999 std conc’n: 33.5 ΔA/min sample: 1.018 ΔA/min blank: 0.059

group v

std abs : 1.001 std conc’n: 33.5 ΔA/min sample: 1.000 ΔA/min blank: 0.169