Fully Automated Paraoxonase
Activity Measurement Kit
Paraoxonase-1 (PON1) is an high density
lipoprotein (HDL)-associated enzyme with
antioxidant and antiatherogenic functions,
protecting lipoproteins against oxidative
modification. It also catalyzes the
hydrolysis of organophosphates such as
paraoxon and aromatic carboxylic acid
esters of fatty acids. It has been shown
that serum paraoxonase activity decrease
in diabetes mellitus, coronary artery
disease, hypercholesterolaemia, iron
deficiency anemia, hepatitis, cirrhosis,
prostate cancer, tuberculosis and
inflammations.

Principle of Assay
Fully automated paraoxonase activity
measurement method consists of two
different sequential reagents. The first
reagent is an appropriate Tris buffer and it
also contains calcium ion, which is a
cofactor of PON1 enzyme. The second
reagent is a new developed stabile
substrate solution. The sample is mixed
with the Reagent 1 and the substrate
solution is added. Linear increase of the
absorbance of p-nitrophenol, produced
from paraoxon, is followed at kinetic
measurement mode. Nonenzymatic
hydrolysis of paraoxon was substracted
from the total rate of hydrolysis. The molar
absorptivity of p-nitrophenol is 18,290 M-1
cm-1 and one unit of paraoxonase activity
is equal to 1 mol of paraoxon hydrolyzed
per liter per minute at 37oC

Components
All reagents are ready to use.

• Reagent 1 (buffer solution) = 50 ml
• Reagent 2 (substrate solution) = 5 ml

Storage Conditions
This kit should be stored at 2-8oC.

Samples
Blood serum, heparinized plasma, semen
plasma, cell lysates and tissue
homogenates can be used as sample.

Procedure
The assay format of the test is given below.

Reagent 1 volume 500 L.
Sample volume 25 L.
Reagent 2 volume 25 L.
Wavelength 412 nm.
Reading point Kinetic (rate-up) measurement.
Calibration type Factor ( 1294 ) 

Manual measurement
In manual working, the volumes of the
sample and the reagents are increased at
same ratio according to the above values.

Interference and stability
Calcium chelators such as EDTA and
citrate inhibited paraoxonase activity.
Heparin, hemolysis and bilirubin did not
interfere the the assay. Uremic plasma
samples did not interfere with the assay.
No significant difference was observed
between fresh and non fresh serum
arylesterase activities.