INTRODUCTION:

Atherosclerosis is one of the most common cause of mortality¹. It is associated with increased concentrations of low-density lipoprotein (LDL) and eventually results in sudden plaque ruptures². The development and progression of athersclerosis is due to the oxidative modification of LDL in the artery wall^3,4. Monocyte adhesion, endothelial injury, activity of endothelial nitric oxide synthase (eNOS) expression, platelet aggregation and inhibition of apoptosis, all of which induced by Ox-LDL contribute to atherosclerotic process and hypertension^5,6. Some protective properties of high density lipoprotein (HDL) have been shown to inhibit the propagation of LDL oxidation.

HDL-associated PON seems to play a major role in the protection of LDL against peroxidation6. It is accountable for most of the antioxidant role of HDL and can prevent atherogenesis by reverse cholesterol transport, and inhibition of lipid peroxidation. HDL-associated PON1, an esterase, is a ~43 kDa polypeptide made up of 355 amino acids^7,8. It is synthesized in the liver and is secreted into the bloodstream where it is associated with HDL particles^9-12. PON1 plays a protective role in CAD, other atherosclerosis-related disease and ischemic stroke have been studied¹³.

Myeloperoxidase (MPO), enzyme derived from the leukocyte. It acts as a catalyst in the formation of various reactive oxidant species and is associated with multitude of diseases, including atherosclerosis^14-18. Oxidants generated by the myeloperoxidase plays a key role in oxidative modification of LDL and the progression to atherosclerosis¹⁹. Epidemiological studies clearly indicate the importance of MPO as CVD risk marker in addition to traditional markers²⁰. MPO measurement in plasma may contribute to CVD risk stratification. The status of oxidant and the antioxidant enzymes in subjects with elevated LDL is not well documented. The present study aimed to assess the antioxidant activity of enzyme paraoxonase1 and the oxidant activity of myeloperoxidase in subjects with elevated LDL.

MATERIALS AND METHODS:

Subjects visiting the Clinical Biochemistry Laboratory, KMC Hospitals, Mangalore were included in the study and were aged between 35 -75 years. They were divided into two groups. Participants with elevated LDL (>100mg/dl) constituted the test group and participants with normal LDL (< 100mg/dl) constituted the control group. Approved from the Institutional Ethics Committee (IEC KMC MLR-04-17/59) was obtained before the conduct of the study. Subjects with hypertension, diabetes mellitus, hepatic disease, smokers, and those on statin treatment were not included. Consent was obtained from all the study participants.

Sample collection and storage: Five ml of blood (venous) was collected from the subjects after 12 hours of fasting using disposable syringes and needles. Blood samples were centrifuged for 10 min at 3000rpm. The division of serum samples into two aliquots were done. One of the aliquots was used for estimation of HDL & LDL. The other aliquot was stored at -20°C for a period two weeks and used for the assay of Ox-LDL, Paraoxonase and Myeloperoxidase.

Methods for the measurement of HDL, LDL and Ox-LDL: Analysis of LDL and HDL was done in the COBAS autoanalyzer by using the ROCHE Diagnostic kits. HDL cholesterol was estimated by CHOD – PAP method after precipitation of LDL and chylomicrons^{21, 22}. LDL cholesterol was done by direct determination using immune separation reagent and enzymatic cholesterol assay²³. Ox-LDLwas measured using ELISA kit provided by CLOUD-CLONE CORP²⁴.

Myeloperoxidase activity: MPO activity was measured spectrophotometrically by using 4-AAP (4-Amino Antipyrine) as the Hydrogen donor. MPO in the presence of Hydrogen Peroxide oxidizes phenol and forms a red coloured product with 4 Amino Antipyrene (quinoneimine). Rate of formation of quinoneimine was determined by measuring the increase in absorbance observed /min at 510nm²⁵.

Paroxonase activity: Was measured spectrophotometrically using 4-nitrophenyl phosphate as the substrate. The increase in absorbance due to the formation of the yellow 4-nitrophenol was monitored for 3 minutes at 412nm²⁶. The activity of both, basal and salt stimulated PON1 were estimated.

Statistical analysis: It was carried out using SPSS version 20. The results are expressed as Mean±standard deviation. The values of the two groups compared by the Independent student t test. Karl Pearson’s correlation was used for correlation analysis. p<0.05 was considered statistically significant.

RESULTS:

There was no significant difference in age between the cases and controls. The number of males and females in both the groups were almost similar. There was no significant difference in the levels of HDL. The levels of LDL and Ox-LDL are significantly high (p<0.001) in the cases as compared to the controls (Table 1).

Table 1: Clinical-demographic details of the subjects

Parameters	Controls (n=50)	Cases (n=50)	p value
Age	55.24 ± 11.41	52.86 ± 13.11	-
Gender (male/female)	28/22	27/23	-
HDL (mg/dL)	44.61 ± 15.09	41.64 ±14.35	0.316
LDL (mg/dL)	88.16 ± 18.08	148.87 ± 26.64	< 0.001
Ox-LDL (ng/ml)	555.56 ± 24.901	763.083 ± 75.59	< 0.001

Values are shown as Mean ± Standard deviation. n=no of subjects.p< 0.05 was considered significant.

A significant decrease in the levels of SPON and BPON are seen in cases (p<0.001) as compared to the controls. A significant increase in levels of MPO is seen in cases (p<0.001) as compared to controls (Table 2).

Table 2: Serum levels of PON1 and MPO

Parameters	Controls (n=50)	Cases (n=50)	p value
SPON (U/L)	6.535 ± 2.154	3.266 ± 1.794	< 0.001
BPON (U/L)	98.34 ± 25.549	41.311 ± 31.058	< 0.001
MPO (U/L)	6.839 ± 3.932	49.396 ± 22.661	< 0.001

All values are expressed as Mean ± SD. p< 0.05 was considered significant. n = no of subjects.

A significant positive correlation of LDL with MPO and a negative correlation with BPON are seen in both cases and controls. However, the negative correlation of LDL with SPON is not statistically significant (Table 3 and figure 1).

Table 3: Correlation Coefficient of LDL with Oxidant and Antioxidant enzymes

	p and (r) values
Parameters	Controls (n=50)	Cases (n=50)
MPO (U/L)	0.001 (r = 0.671)	0.001 ( r = 0.926)
BPON (U/L)	0.001 ( r = -0.631)	0.001 ( r = -0.861)
SPON (U/L)	0.737 ( r = -0.049)	0.291 ( r = -0.156)

All values are expressed as Mean ± SD.

p < 0.05 was considered significant. n = no of subjects.

Fig 1: Correlation of LDL with Oxidant and Antioxidant Enzymes in all study participants

The association of Ox-LDL with MPO, SPON and BPON was not significant in controls. However in cases a significant positive correlation with MPO and negative correlation with BPON is seen (Table 4 and figure 2).

Table 4: Correlation of Ox-LDL with Oxidant and Antioxidant enzymes

Parameters	Controls (n=50)	Cases (n=50)
MPO (U/ L)	0.889 (r = 0.023)	<0.001* (r = 0.948)
BPON (U/ L)	0.810 (r = -0.173)	<0.001* (r = -0.863)
SPON (U/ L)	0.292 (r = -0.040)	0.791 (r = 0.042)

All values are expressed as Mean ± SD. * p < 0.05 – significant.

Fig 2: Correlation of Oxidised LDL with Oxidant and Antioxidant Enzymes in all study participants

DISCUSSION:

Hypercholesterolemia as an important cause of coronary heart disease is well established. Various studies have proved that elevation in the plasma LDL concentration and decreased HDL levels are associated with atherogenesis. HDL levels were not significantly different in the two groups. However there was 1.6 fold upsurge in the LDL levels and 1.37 folds in the levels of Ox-LDL in the subjects with elevated cholesterol as compared to control. The oxidant enzyme, MPO showed a 7 fold increase in the test group. The levels of BPON and SPON were significantly decreased (2.4 and 2 folds). A positive association was found between Ox-LDL and MPO whereas a negative association of Ox-LDL was found with BPON while there was no significant association of the same in the control subjects. Sigurdardottir et al 27 have reported the association of Ox-LDL with factors of metabolic syndrome such as BMI, triacylglycerol and HDL cholesterol. Similar results were found by Holvoet et al and Kondo et al^28,29.

LDL showed a positive association with MPO and a negative association with BPON in both cases and controls. No association of HDL with MPO, BPON and SPON was seen in either of the groups. It has been shown that in vitro purified paraoxonase decreased LDL peroxidation³⁰. Decreased Paraoxonase activity was noticed in cardiovascular risk patients, in diabetes mellitus, myocardial infarction and heterogenous familial hypercholesterolemia^31,32. Miljkovic et al have shown that PON activity decreases with age³³. PON activity was also found to be lowered in patients suffering from chronic renal failure. A meta-analysis inclusive of 47 studies with CAD subjects confirmed the association of lower plasma PON1 activity with increased CAD risk. They have also shown the importance of assessing the PON1 activity in the identification of CHD over paraoxonase genetic polymorphisms³⁴.Studies in humans are consistent and hypothesize that PON1 decreases the risk of cardiovascular disease by lowering the levels of Ox-LDL. The decrease in PON1 activity in subjects with elevated LDL found in the present studies is in agreement with the studies mentioned above.

A positive association between elevated MPO levels and Ox-LDL with a seven fold increase in MPO levels observed in cases is an important finding. Basati et al have reported a positive association between elevated MPO levels and the severity of coronary artery disease³⁵. Heslop et al reported the doubling of CVD risk with elevated MPO over a period of 13 years³⁶. This allows us to hypothesise that activity of PON1is associated with the prevention of LDL oxidation. The decrease in PON1 activity and increase in Ox-LDL levels in subjects with elevated LDL could be an important factor in vascular ageing and cardiovascular diseases. Further studies on the correlation of Ox-LDL with other potential risk factors such as Lp(a) and homocysteine may improve the CVD risk prediction.

CONCLUSION:

Positive correlation of both Ox-LDL and LDL with MPO and a negative association of Ox-LDL with PON1 was observed in only subjects with high LDL while the correlation of LDL with PON1 was negative in both cases and controls. The results of the study prove that the Ox-LDL is a more sensitive indicator of CVD risk compared to LDL or HDL cholesterol. HDL associated PON1 exerts a protective effect in control subjects. Measurement of, Myeloperoxidase, Paraoxonase and Ox-LDL and may provide further details in the context of cardiovascular risk prediction and aid in the treatment.

ACKNOWLEDGEMENT:

The authors are grateful to the Manipal Academy of Higher Education, Manipal and Kasturba Medical College, Mangalore for the encouragement and support.

CONFLICTS OF INTERESTS:

The authors declare that they have no conflicts of interests

REFERENCES:

1. Prabha JL, Sankari M. Role of Il-1 in Atherosclerosis. Research J. Pharm. and Tech 2018; 11(7): 3163-3166.

2. Kattoor AJ, Kanuri SH, Mehta JL. Role of Ox-LDL and LOX-1 in Atherogenesis. Curr Med Chem 2019;26(9):1693-1700.

3. Khosravi M, Poursaleh A, Ghasempour G, Farhad S, Najafi M. The effects of oxidative stress on the development of atherosclerosis. Biol Chem 2019;400(6):711-732.

4. Ndanusa AH, Rohini K, Uma SA, Khin MA. Effect of Zingiber officinale on Lipid profile of Sprague-Dawley rats induced with Streptozotocin. Research J. Pharm. and Tech. 2020; 13(8):3577-3579.

5. Dayuan Li, Jawahar Mehta L. 3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors protect against oxidized low density lipoprotein induced endothelial dysfunction. Endothelium Journal of endothelial Cell Research 2003; 10: 17– 21.

6. Deakin S, Bioletto S, Bochaton-Piallat M, James R. HDL-associated paraoxonase-1 can redistribute to cell membranes and influence sensitivity to oxidative stress. Free Radical Biology and Medicine 2011; 50: 102- 109.

7. Suematsu Y, Goto M, Park C, Nunes ACF, Jing W, Streja E,et al. Association of Serum Paraoxonase/Arylesterase Activity With All-Cause Mortality in Maintenance Hemodialysis Patients. J Clin Endocrinol Metab 2019 Oct 1;104(10):4848-4856.

8. Ferretti G, Bacchetti T, Campanati A, Simonetti O, Liberati G, Offidani A. Correlation between lipoprotein(a) and lipid peroxidation in psoriasis: role of the enzyme paraoxonase-1. Br J Dermatol 2012;166(1):204-207.

9. Rosenblat M, L Gaidukov, O Khersonsky et al. The catalytic histidine dyad of high density Lipoprotein-associated serum paraoxonase-1 (PON1) is essential for PON1-mediated inhibition of low density lipoprotein oxidation and stimulation of macrophage cholesterol efflux. J Biol Chem 2006; 281:7657– 7665.

10. Boshtam M, Razavi A. E, Pourfarzam M , Ani M, Naderi GA, Basati G et al .Serum paraoxonase 1 activity is associated with fatty acid composition of high density lipoprotein. Disease Markers .2013; 35: 273–280.

11. A Gugliucci, M Numaguchi, R Caccavello, S Kimura. Paraoxonase1 lactonase activity and distribution in the HDL subclasses in the cord blood. Redox Report 2014; 19: 124–132.

12. Aviram M, Rosenblat M, Bisgaier C, Newton R, Primo-Parmo S, La Du BN et al. Paraoxonase inhibits high- density lipoprotein oxidation and preserves its functions. A possible peroxidative role for paraoxonase. Journal of Clinical Investigation 1998; 101:1581-90.

13. Basati G, Emami Razavi A, Abdi S, Sarrafzadegan N. Association between adipokine and myeloperoxidase levels in patients with coronary artery disease. Acta Med Iran 2015;53(1):25-29.

14. Shih DM, Gu L, Hama S, Xia YR, Navab M, Fogelman AM etal . Genetic dietary regulation of serum paraoxonase expression and its role in atherogenesis in a mouse model. J Clin Invest 1996; 97:1630-1639.

15. Nahrendorf M, Sosnovik D, Chen J, Panizzi P, Figueiredo J, Aikawa E et al. Activatable Magnetic Resonance Imaging Agent Reports Myeloperoxidase Activity in Healing Infarcts and Noninvasively Detects the Anti- inflammatory Effects of Atorvastatin on Ischemia- Reperfusion Injury. Circulation 2008; 117: 1153-1160.

16. Kacprzak M, Zielinska M. Prognostic value of myeloperoxidase concentration in patients with ST-segment elevation myocardial infarction treated with primary percutaneous coronary intervention. Int J Cardiol 2016;223: 452-457.

17. Rudolph V, Andrie RP, Rudolph TK, Friedrichs K, Klinke A et al. Myeloperoxidase acts as a profibrotic mediator of atrial fibrillation. Nat Med 2010; 16: 470–474.

18. Reynolds WF, Rhees J, Maciejewski D, Paladino T, Sieburg H, et al. Myeloperoxidase polymorphism is associated with gender specific risk for Alzheimer’s disease. Exp Neurol 1999; 155: 31–41.

19. Malle E, Marsche G, Arnhold J, Davies M. Modification of low-density lipoprotein by Myeloperoxidase-derived oxidants and reagent hypochlorous acid. Biochimica Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids 2006; 1761: 392-415.

20. Schindhelm R, van der Zwan L, Teerlink T, Scheffer P. Myeloperoxidase:A Useful Biomarker for Cardiovascular Disease Risk Stratification?Clinical Chemistry 2009; 55: 1462-1470.

21. Suguichi H, Uji Y, Okabe H, Irie T, Uekama K, Kayahara N etal. Direct measurement of HDL cholesterol in serum with polyethylene glycol- modified enzymes and sulfated α-Cyclodextrin. Clin Chem 1995; 41: 717-723.

22. Matsuzaki Y, Kawaguchi E, Morita Y, Mashige F, Ohisa S, Nakahara K, et al. Evaluation of two kinds of reagents for direct determination of HDL–cholesterol.J Anal Bio-Sc 1996; 19: 419-427.

23. Pisani T, Gebski CP, Leary ET, Warnock GR, Ollington JF. Accurate direct determination of low- density lipoprotein cholesterol using an immune separation reagent and enzymatic cholesterol assay. Arch Pathol Lab Med 1995; 119: 1127- 1135.

24. Alamdari DH, Kostidou E, Paletas K, Sarigianni M, Konstas AG, Karapiperidou A, Koliakos G. High sensitivity enzyme-linked immunosorbent assay (ELISA) method for measuring protein carbonyl in samples with low amounts of protein. Free Radic Biol Med 2005; 39:1362-7.

25. Souparnika S. Emerging Role of Myeloperoxidase in the Prognosis of Nephrotic Syndrome in Patients Before and After Steroid Therapy.Journal of clinical and diagnostic research 2015; 9: 1-4.

26. Bełtowski J e. Species- and substrate-specific stimulation of human plasma paraoxonase 1 (PON1) activity by high chloride concentration. Acta Biochim Pol 2002; 49: 927-936.

27. Sigurdardottir V, Fagerberg B, Hulthe J. Circulating oxidized low-density lipoprotein (LDL) is associated with risk factors of the metabolic syndrome and LDL size in clinically healthy 58-years-old men (AIR study). J Intern Med 2002; 252: 440–447

28. Holvoet P, Mertens A, Verhamme P, Bogaerts K, Beyens G, Verhaeghe R etal . Circulating oxidized LDL is a useful marker for identifying patients with coronary artery disease. Arterioscler Thromb Vasc Biol 2001; 21: 844–848.

29. Kondo A, Muranaka Y, Ohta I, Notsu K, Manabe M, Kotani K et al .Relationship between triglyceride concentrations and LDL size evaluated by Malondialdehyde- modified LDL . Clin Chem 2001; 47: 893–900.

30. Mackness MI, Arrol S, Durrington PN. Paraoxonase prevents accumulation of lipoperoxides in low density lipoprotein .FEBS Lett 1991; 286: 152-154.

31. McElveen J, Mackness MI, Colley CM, Peard T, Warner S, Walker CH: Distribution of paraoxon hydrolytic activity of patients after myocardial infarction. Clin Chem 1986; 32:671-673.

32. Idrees M, Siddiq AR, Ajmal M, Akram M, Khalid RR, Hussain A, et al. Decreased serum PON1 arylesterase activity in familial hypercholesterolemia patients with a mutated LDLR gene. Genet Mol Biol 2018;41(3):570-577.

33. Miljkovic M, Stefanovic A, Vekic J, Zeljkovic A, Gojkovic T, Simic-Ogrizovic S, Bogavac-Stanojevic N, Cerne D, Ilic J, Stefanovic I, Jelic-Ivanovic Z, Spasojevic-Kalimanovska V, Kotur-Stevuljevic J. Activity of paraoxonase 1 (PON1) on HDL₂ and HDL₃ subclasses in renal disease. Clin Biochem 2018 Sep;60:52-58.

34. Mackness B, Davies GK, Turkie W, Lee E, Roberts DH, Durrington PN et al .Paraoxonase status in coronary heart disease.: are activity and concentration more important than genotype? Arterioscler Thromb Vasc.Biol 2001; 21: 1451 -1457.

35. Basati G, Emami Razavi A, Abdi S, Sarrafzadegan N. Association between adipokine and myeloperoxidase levels in patients with coronary artery disease. Acta Med Iran 2015;53(1):25-29.

36. Heslop CL, Frohlich JJ, Hill JS. Myeloperoxidase and C-reactive protein have combined utility for long-term prediction of cardiovascular mortality after coronary angiography.Journal of the American College of Cardiology 2010; 55: 1102–1109.

Received on 25.09.2020 Modified on 17.11.2021

Research J. Pharm. and Tech 2022; 15(9):3836-3840.

DOI: 10.52711/0974-360X.2022.00643