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Paroxonase and its role in cardiovascular disease

Paroxonase and its Role in Cardiovascular Disease

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
Materials and Methods
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.
Paraoxonase assay:
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.
Purification of serum Paraoxonase:
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.
Preparation of LDL:
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.
Lipoprotein Oxidation
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 Assay
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.

Purification of Serum Paraoxonase

The scheme of purification of human serum paraoxonaseof a typical run is shown inTable 1
Table 1
Volume ml
25 ml
Cibacran Blue Wash
25 ml
DEAE Column
Flow through
Re-eluted on DEAE Flow through
20 ml
Eluted with 0.35M Nacl
The Lipid components of the fractions are shown inTable 2
Table 2
Cholesterol mg/dl
APO A1 Presence of HDL
APO B Presence of LDL
Cibacran Blue sepharose fraction
1 st DEAE Fraction
2 nd DEAE Fraction
The Fractions eluted from the DEAE column with arylesterase activity were used as paraoxonase and was used to study its effect on LDL-Oxidation.
Oxidation of LDL
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
Peroxidation of LDL in the absence and presence of PON and HDL are shown in Fig. 2
The 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)
Serum Paroxonase Activity
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 .
  1. Steinberg D Parthasarathy S, Carew TE, Khoo JC, Witztum JL. Beyond cholesterol modifications of low-density lipoprotein that increase its atherogenicity. New Engl J Med 1989 320 : 915 - 924
  2. Mackness M Durrington P N. High density lipoprotein and its enzymes and its potential to influence lipid peroxidation .Athereosclerosis 1995; 115: 243 - 245
  3. Furlong et al. Am J Hum Gen 1988 43: 230 - 238.
  4. Furlong et al., Analytical Biochemistry 180, 242 - 247 (1989)
  5. 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
  6. 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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