Introduction
Human serum paraoxonase (PON1, EC 3.1.8.1)
is a Ca2+ dependent, 45 kDa glycoprotein that is
associated with high density lipoprotein (HDL). PON1 hydrolyses
organophosphates, insecticides and nerve gases. Although PON1
can offer protection against the toxicity of some
organophosphates, its physiological role is still not known.
However, evidence exists for a protective effect of PON1
against oxidative damage. It retards the oxidation of low
density lipoprotein (LDL), both in vivo and in vitro, by
hydrolyzing the lipid peroxides formed in plasma (1-4). It has
been suggested that PON1 is related to coronary heart disease
risk. PON1 activity was reported to be lower in subjects with
familial hypercholesterolemia, the disease that lead to the
development of atherosclerosis. PON1 activity is under genetic
and environmental regulation and appears to vary widely among
individuals and populations (5).
Beta-blockers are widely used to treat
cardiovascular diseases. It has been shown that non-selective
beta-blockers affect the concentration and oxidizability of
plasma lipids. They tend to increase triglycerides and LDL,
while decreasing the atheroprotective HDL (10). There is no
previous report on the effect of beta-blockers on PON1
activity. Propranolol is a well known non-selective
beta-blocker widely used in the treatment of arrhythmia, angina
and hypertension.
The aim of the present study was to
investigate whether propranolol, a non-selective beta-blocker
could affect PON1 activity.
Experimental
PON1 activity was measured
spectrophotometrically. PON1 hydrolyses paraoxon (substrate) in
the presence of Ca2+ and Na+ and as a result p-nitrophenol is liberated (11, 12). An Increase
in absorbance at 412 nm is related to PON1 activity. No
absorbance was observed in visible range for plasma itself.
Materials
Paraoxon was purchased from Sigma-Aldrich
chemie GmbH (Germany).
Propranolol was obtained from Tolid Daru
(Iran). Other chemical compounds were from Merck Co. (Germany).
UV absorbance was measured with a SHIMADZU 160-A
spectrophotometer. Serum was obtained from a fast, healthy,
non-smoker, male volunteer.
Solutions
Solution A: glycine/NaOH buffer (50 mM, pH=
10.0) containing 1.0 M NaCl and 1.0 mM CaCl2 was prepared.
Solutions B1-B6: Paraoxon was added to
solution A to reach final concentrations of2.5, 5.0, 7.5, 10,
12.5 and 15.0 mM respectively.
Solutions C1-C6: Propranolol (as the base)
was dissolved in methanol to prepare concentrations of 0.25,
0.5, 0.75, 1.0, 1.25 and 1.5 mM respectively.
Methods
PON1 activity measurement
Four hundred ml of a 1/5 prediluted serum sample with
distilled water, was added to 4.1 ml solution A and then 500 ml of one of the
solutions B1-B6 was added. The rate of paraoxon hydrolysis was
assessed by measuring liberation of p-nitrophenol at 412 nm at
25 C (e=
17000, pH=10.0). For subtraction of non-enzymatic hydrolysis,
blanks (samples without serum) were used. Enzyme activities
were expressed in international units (U) per milliliter of
serum. One U corresponds to the quantity of enzyme that
hydrolyses 1 mmol of substrate per minute at the given pH and
temperature.
Inhibition assay
1) Four hundred ml of a 1/5 prediluted
serum sample with distilled water, was added to 4.0 ml solution
A and then 100 ml one of the solutions C1-C6 was added. After 10 min incubation, 500 ml of solution B6
was added and the absorbance measured at 412 nm. 2) Four
hundred ml of a 1/5 prediluted serum sample was added to 4.0 ml
solution A followed by the addition of 100 ml of solution C2. After
10 min incubation, 500 ml of one of the solutions B1-B6 was added and
absorbance measured at 412 nm.
Results And Discussion
The enzyme kinetic (with and without
propranolol) has been shown as the Lineweaver-Burk plot in
figure 1. Figure 1 shows that propranolol is a mixed
non-competitive inhibitor of PON1. Hence the equation
describing the kinetics is a modified form of the
Lineweaver-Burk equation:
Secondary plots were drawn and shown in
figures 2-a and 2-b. PON1 kinetic parameters were calculated
(Km=0.4 mM, Vm=0.255 mol.min-1.ml-1 serum) and inhibition parameters were obtained
through Figures 2-a and 2-b (Ki=37 mm, aKi=KI=79 mM, a=2.13).
Based on the previous reports, PON1
activity is under genetic and environmental regulation.
Regarding the environmental parameters, it has been reported
that mice which had consumed red wine had less oxidized LDL,
presumably related to an enhanced serum PON1 activity in these
polyphenol-treated mice (6).
The inhibition of LDL oxidation by HDL is
due to the hydrolysis of lipid peroxidases and the resulting
inhibition of lipid peroxides appears to be, at least in part,
a function of the enzyme paraoxonase, which is a component of
HDL (2, 13). It has been shown that PON1 destroys the
multioxygenated molecules found in oxidized phosphatidycholine.
Furthermore, it has been demonstrated that inactivation of PON1
reduces the ability of HDL to inhibit LDL modification. It also
reduces the ability of HDL to inhibit monocytic-endothelial
interactions. They both appear to be important in the
inflammatory response of arterial wall cells, which promotes
atherogenesis (3). PON1 reduces mildly oxidized phospholipids
by eliminating oxidized derivatives of unsaturated fatty acids
(2, 3). It has also been shown that smoking is associated with
a reduced serum PON1 activity and concentration (7). However,
vitamins C and E intake are associated with an increased PON1
activity (8).
Serum PON1 activity is significantly
increased during treatment with simvastatin (9). These
evidences imply the importance of jointly considering
environmental factors that modify PON1 activity.
Overall, in this study we found that
propranolol, a well known non-selective beta-blocker, is a
mixed non-competitive human serum paraoxonase inhibitor. Thus,
it is possible that the use of propranolol could be influencing
PON1 activity and its effect on the concentration and
oxidizability of plasma lipids may be related to this property.
Acknowledgement
This work was partially supported by a
grant from Tehran University of Medical Sciences Research
council.
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