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IFCC
Professional Scientific Exchange Programme
Elizabeth A Frank
Biochemical Diagnostic Laboratory, Mysore - 570 021 Karnataka,
India
Fax: +91-821-521948
Dr. Frank, Clinical Chemist, visited as a short time scholar the
Dept. of Pathology and Lab. Medicine at the Louisville School of
Medicine, Kentucky, USA. Her investigations and training was
supervised by Associate Professor James J Miller, Ph.D., and
Stanley S. Levinson, Ph.D.
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Arteriosclerosis is a major cause of morbidity and
mortality not only among North Americans but also amongAsians .
While the initiation and progression of the disease is well
understood, little is known about the cardio protective mechanisms.
The oxidation of LDL is central to current theories concerning the
initiation and progression of atherosclerosis (1) Equally , the
potential protective role of high-density lipoprotein (HDL) has
been emphasized. The inverse correlation that exists between plasma
HDL- cholesterol concentrations and the risk of coronary artery
disease (CAD) has led to the identification of" reverse cholesterol
transport " pathway.( 2). In this pathway the lipoprotein particles
return excess cholesterol to the liver and allow for its excretion
mainly as bile acids. Although it is clear that the reverse
cholesterol transport and the HDL particle are inversely related to
CAD, metabolism of HDL is poorly understood. HDL was shown to
inhibit LDL oxidation in vitro by several laboratories (2). Recent
evidence suggests that several enzymes residing on the HDL could
contribute to this activity. It has been shown that HDL associated
Paraoxonase (PON1) retards the oxidation of LDL by preventing the
generation of lipid peroxides (3).
PON1 is a calcium dependent esterase whose
mechanism of action is incompletely elucidated. It was originally
found to be responsible for the hydrolysis of Paraoxon, a catabolic
product of the insecticide parathion. PON is also able to hydrolyze
other substrates such as phenyl acetate. However the physiological
substrate for PON is still unknown. PON is widely distributed among
tissues such as Liver, kidney, intestine and also serum, were it is
associated with HDL.
This project was designed to study the role of
paraoxonase in the cardio protective mechanism of HDL. Our
objective was to investigate whether the HDL associated paraoxonase
was responsible in retarding oxidation of LDL.This study will
eventually be able to answer many questions like for example
whether possible interactions between Apolipoprotein and
Paraoxonase is responsible in protecting LDL from oxidation. Hence
we first tried to purify paraoxonase from serum and then use it in
studies involving oxidation of LDL in vitro.
We also studied the levels of serum paraoxonase in
relation to lipid profiles in a randomly selected patient
population. Although from this study we are unable to draw definite
conclusions about the role in lipid oxidation, we have made a
number of interesting observations which when pursued will
eventually prove conclusively the in vivo role of paraoxonase in
HDL
All chemicals used were from Sigma - Aldrich USA
.
Patients used in this studies was randomly selected
from among thepatients from Louisville medical school. All patients
selected were between the age group on 21to 80 years. Among the 30
patients studied 17 were female and the rest were males.
Activity of paraoxonase was assayed by following
the formation of p nitro phenyl by its absorbance at 405nm (4, 5).
The assay buffer contains 0.132 M Tris base, 1.32 m M CaCl
2 and 2.63 M NaCl. Addition of 200 m l of 1.2 m M
paraoxon and 10 m l of serum initiated the assay. Absorbance was
continuously monitored at 405 nm. A molar extinction coefficient of
18.05 x 10 3 was used for calculation using paraoxon as
substrate (Biggs 1954) , 3.6 mM of phenyl acetate was also used as
substrate. A molar extinction co-efficient of 1310 was used when
Phenyl acetate was used as a substrate. 1 unit of paraoxonase is
defined as a nmol of 4 nitrophenol formed per minute.
150 ml (or 25 ml) serum is diluted with 150 ml (or
25 ml) of 25 m M tris Hcl buffer pH8.0 containing 2 M Nacl, 0.5 mM
CaCl 2 and 2.5 �M EDTA. This diluted serum was
fractionated on a Cibacron Blue 3 GA - Agarose column (6). The
column was washed with 4 M Nacl. The column was then eluted with
0.2% Sodium deoxycholate in water.
The fractions containing paraoxonase activity were
further fractionated by detergent DEAE - trisacryl M
chromatography. The Trisacryl M columns were equilibrated with
equilibration buffer 15 mM Tris HCl , pH 7.5, 1 m M CaCl
2 and 0.1% nonidetP- 40 . The desalted, concentrated
sample was adjusted to equilibration buffer conditions by the
addition of 25% NP-40. The concentrated sample was loaded on to the
column, which was then washed to baseline OD with equilibration
buffer. The adsorbed protein was eluted with a linear gradient from
0 to 0.125 M Nacl in column equilibration buffer.
Cholesterol Triglycerides, HDL-Cholesterol, APO A,
and APO B were assayed on the Roche Cobas Mira using Sigma
diagnostic kits.
LDL was prepared from pooled human serum using
heparin-agarose affinitycolumns asfollows: . Heparin (1 ml) was
mixedwell by inversion with Agarose and allowed to settle
overnight. The column was allowed to drain . The column was washed
with the elution buffer ( 0.7 % Nacl ) The wash was discarded . Apo
B content of pooled human serum was measured using
immunonephlometry . Serum containing1.25-2 g/L of Apo B (200ul)was
loaded on to the Heparin - agarose column followed by 50 ul of the
alpha elution buffer (o.7% NaCl ) . Alpha proteins were eluted
first with 2 ml of the alpha elution buffer. This allowed complete
elution of albumin and thealpha protein (HDL) . The column was then
eluted with the beta buffer (2.9% NaCl ) to give the LDL fraction.
The heparin agarose column was reusedthree times.
CuSO 4 (20 ul of 250 uM ) was added to
LDL fraction(300ul) and mixed vigorously resulting in oxidized
lipoproteins. particle . Assessment of lipoprotein oxidation was
monitoredby formation of conjugated dienes at 234 nm using Genysis
5 system.
Peroxidation of lipids was monitored using
Xylengthol orange. Peroxides oxidize Fe 2+ to Fe
3+ in acidic solution. Fe 3+ in the presence
of xyelnol orange forms a Fe 3 + - Xylengthol orange
complex which absorbs at 500 nm. In this assay the reagent
contained 900 ml of pure methanol, 100 ml of 250 m M H 2
SO 4 880 mg of BHT ( to inhibit further oxidation within
the assay itself, 76 mg of Xylengthol orange and 98 mg of ammonium
iron (II) sulfate hexahydrate . LDLsamples (0.1 ml , oxidized with
cu 2+) were mixed with 0.9 mlof reagent and incubatedfor 30 minute
at room temperature and the colour developed is measured at 560
nm.
The scheme of purification of human serum
paraoxonaseof a typical run is shown inTable 1
The Lipid components of the fractions are shown
inTable 2
The Fractions eluted from the DEAE column with
arylesterase activity were used as paraoxonase and was used to
study its effect on LDL-Oxidation.
The oxidation of LDL by copper in the absence and
presence of added Paraoxonase and Cibacran blue fraction of a
typical experiment as shown in Fig. 1
Cu- Copper , PON-Paraoxonase , Vit E-Vitamin
E
Formation of conjugated dienes reflects oxidation
of PUFA composing the core of lipoprotein .The process of oxidation
spans three phases.1) An initiationor lag phase . 2) A propagation
phase during which time lipid peroxides are formed and double bonds
produced. 3) Decompositionphase, which is recognizedwhen the
oxidation reaches a plateau during which aldehydesand ketones are
produced from fatty acid degradation.
The Oxidation of LDL by copper ions was followed by
conjugated diene formation and showed a typical hyperbolic curve
with an initial Lag phasefollowed by an propagationphase and
finally a plateau. In the presence of added antioxidants like Vit E
and fibrinogen (Fib) the oxidation of LDLwas abolished.However
paraoxonase (PON) did not abolish the oxidation of LDL.
Peroxidation of LDL in the absence and presence of
PON and HDL are shown in Fig. 2The peroxidation was higher in the presence of PON
alone. However in the presence of PON-HDL the peroxidation
initially increased and then decreased.
A study comparing the antioxidant Vitamins and high
density lipoproteins(HDL) on copper catalysed oxidation of
LDLshowed that antioxidants significantly reduced diene
formationbut did not affect lipid peroxide formation . Conversely
HDL did not effect conjugated diene formation butinhibited the
formation of lipid peroxides by up to 90% (7)
The paroxonase activity of a random population was
studied and cor -related with lipid profiles. A histogram of PON
activity profile among random sample, of US population is shown in
Fig 3A and B. The histograms with PON as substrate showed
three activity peaks while with PA as substrate showed two distinct
peaks of activity. Correlation of PON activity with PON as substrate
and PA as substrate is shown in Fig. 4. PON activity with PON as substrate
negatively correlated (P < 0.01) with PON activity with PA as
substrate. PON activity also correlated positively with HDL as
shown in Fig. 5 (p <.002) but did not correlate with
either cholesterol or Triglycerides. However PON activity with PA
as substrate did not correlate with any of the lipid
parameters.
The object of the study was to assign a role of PON
in cardio protective function. Hence we wanted to test whether PON
activity correlated with antioxidant activity. However in our
experiments, we were unable to separate Apo A or Apo B from the
purified paraoxonase. Our preparation of paraoxonase actually
increased LDL oxidation instead of decreasing oxidation. Addition
of the cibacron blue fraction, which is known to have HDL, also
increased oxidation.This may be due to inefficient purification as
paraoxonase still showed presence of LDL.It is possible that the
concentration of copper in our study may have been too large
resulting in a competition between oxidation by copper and
inhibition of oxidation by paraoxonase. LDL present in our
preparation may also be getting oxidized showing the increase.
Similarly PON did not inhibit peroxide formation, however the
Cibacron blue fraction, which also had HDL, seemed to increase and
then decrease peroxidation.
Our results imply that PON needs to be associated
with HDL to function in its cardio protective role. It is also
possible that the presence of LDL in our preparation may be
confounding the results.
Interestingly there was an inverse relationship
between the PON activity measured with Phenyl acetate as substrate
and paraoxon as substrate. Although aryl esterase activity and
Paraoxonase activities are believed to be in the same enzyme, our
results seems to indicate that aryl esterase activity may not be an
indicator of true paraoxonase activity. This is further supported
by our observation that paraoxonase activity using Paraoxon as
substrate showed a linear correlation with some serum lipid factors
whereas this was not seen with aryl esterase activity. Serum
paraoxonase did not show correlation with any other serum
parameter.
Our results are consistent with literature
reportswhich show polymorphism in the activities of paraoxonase,
one with an allele of high activity and another of low activity.
The heterozygous individuals would show intermediate activity.
However such trimodal activity was seen only with PON using
paraoxon as substrate and not PA as substrate. The high activity in
our study was about 3 times that of the low activity.
Thus because of the presence of both LDL and HDL
contamination in the partially purified PON and possibly the
inadequate concentration of PON in relation to copper, it is not
possible to draw any conclusions about the role of PON as
antioxidant and or antiperoxidant .
Hence as a logical conclusion of the present study
we would like to investigate the following
Role of purified /cloned PON upon lipid oxidation
and peroxidation.
Quantity of PON in the serum by immunoassay method
and not by activity in order to see the correlation if any with
lipid peroxides in relation to PON activity.
Although a natural substrate for PON has not been
identified, we are of the opinion that oxidized or peroxidized
lipids may be the substrates for PON. We would like to investigate
this role to assign a metabolic role for PON.
I am extremely grateful to IFCC for this grant that
enabled me to carry out this work on Paroxonase and its role in
cardiovascular disease.
Besides the work descried in the above report, I
have been successful in many additional activities which are
summarized as follows:
I was able to learn from the clinical laboratory at
the medical school , its day to day functioning, maintenance of
quality control, management of erroneous data, and instrumentation
trouble shooting.
I was able to participate in the clinical meetings
seminars and research meetings and benefited immensely from
them.
I was also successful in setting up the MIRA (fully
automatedChemistry analyzer) and standardizing the instrument. I
also automated the paraoxon assay which is being used presently in
the Laboratory.
I also attended the AACC conference and visited few
other laboratories in the US
I have been able to build up a lot of professional
contacts which will be of mutual benefit in continuing the research
work initiated in the US .
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Mackness M Durrington P N. High density lipoprotein and its
enzymes and its potential to influence lipid peroxidation
.Athereosclerosis 1995; 115: 243 - 245
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Furlong et al. Am J Hum Gen 1988 43: 230 - 238.
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Furlong et al., Analytical Biochemistry 180, 242 - 247
(1989)
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Eckerson ,H W ., wyte C M . And La Du, B N (1983b).The human
serum paraoxonase/arylesterace polymorphism.Identification of
phenotypes by their response to salts. Am J Hum. Genet. 35: 214 -
227
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Michael I Mackness., Caroline Abbott, Sharon Arrol and Paul N
DurringtonRole of high density lipoprotein and lipid soluble
antioxidant vitamins in inhibiting low density lipoprotein
oxidation Bio-Chem j (1993) 294, 829 - 834
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