|Year : 2023 | Volume
| Issue : 3 | Page : 179-184
A prospective study to assess the role of paraoxonase 1 genotype and phenotype on the lipid-lowering and antioxidant activity of statins
Charuta Godbole1, Saket Thaker1, Santosh Salagre2, Vyankatesh Shivane3, Nithya Gogtay1, Urmila Thatte1
1 Department of Clinical Pharmacology, Seth G. S. Medical College and KEM Hospital, Mumbai, Maharashtra, India
2 Department of Medicine, Seth G. S. Medical College and KEM Hospital, Mumbai, Maharashtra, India
3 Department of Endocrinology, Seth G. S. Medical College and KEM Hospital, Mumbai, Maharashtra, India
|Date of Submission||22-Apr-2020|
|Date of Decision||25-Aug-2021|
|Date of Acceptance||06-Aug-2022|
|Date of Web Publication||01-Aug-2023|
Department of Clinical Pharmacology, Seth G.S. Medical College and KEM Hospital, Acharya Donde Marg, Parel, Mumbai - 400 012, Maharashtra
Source of Support: None, Conflict of Interest: None
Human paraoxonase 1 (PON1) enzyme protects against atherosclerosis by preventing low-density lipoprotein from oxidative modification. Upregulation of PON1 enzymatic activity is suggested to contribute to atheroprotective potential of statins. Glutamine (Q) to arginine (R) at site 192 and leucine (L) to methionine (M) substitution at site 55 polymorphisms influence the PON1 activity. The study assessed the role of PON1 polymorphisms on lipid-lowering and PON1-modulating activity of statins in a Western Indian cohort of patients with dyslipidemia. Lipid profile and PON1 activity were determined at baseline and 3 months after initiation of statin treatment. PON1 genotypes (QQ, QR, RR; LL, LM, and MM) were determined by PCR-RFLP. Paraoxon was used as a substrate for assessing PON1 activity by spectrophotometry. A total of 140 statin-naïve patients were enrolled; of them, 116 were available for final analysis. Fifty-seven (50%) had QQ, 39 (35%) had QR, and 17 (15%) had RR genotypes. Seventy-six (67%) patients had LL, 35 (31%) had LM, and 2 (2%) had MM genotypes. We observed no impact of PON1 polymorphisms on lipid parameters posttreatment. A significant increase was observed in the serum PON1 activity from a median (range) of 47.92 U/L (9.03–181.25) to 72.22 U/L (7.64–244.44) (P < 0.05) following statin treatment, which was independent from high-density lipoprotein (HDL) concentration. This increase was significantly greater in QQ compared to QR and RR genotypes (P = 0.01). To conclude, the important antioxidant properties of statins are exerted via the rise in serum PON1 activity, independent of HDL cholesterol concentrations. The increase was greater in individuals with QQ genotype. Future large-scale studies will validate the premise that QQ homozygotes see added benefits from statin treatment compared to R carriers. In the meantime, PON1 enzymatic activity remains an important marker to be measured while assessing pleotropic effects of statins in CAD.
Keywords: Antioxidant, atherosclerosis, high-density lipoprotein, paraoxonase
|How to cite this article:|
Godbole C, Thaker S, Salagre S, Shivane V, Gogtay N, Thatte U. A prospective study to assess the role of paraoxonase 1 genotype and phenotype on the lipid-lowering and antioxidant activity of statins. Indian J Pharmacol 2023;55:179-84
|How to cite this URL:|
Godbole C, Thaker S, Salagre S, Shivane V, Gogtay N, Thatte U. A prospective study to assess the role of paraoxonase 1 genotype and phenotype on the lipid-lowering and antioxidant activity of statins. Indian J Pharmacol [serial online] 2023 [cited 2023 Dec 2];55:179-84. Available from: https://www.ijp-online.com/text.asp?2023/55/3/179/382568
| » Introduction|| |
One of the major risk factors for atherosclerosis is dyslipidemia. The oxidation of low-density lipoprotein cholesterol (LDL-C) represents the fundamental step in initiation and progression of atherosclerosis. Reverse cholesterol transport and prevention of LDL oxidation are the two main mechanisms through which high-density lipoprotein cholesterol (HDL-C) provides protection against atherosclerosis.,,,
Paraoxonase 1 (PON1), associated with HDL particles, is a lipolactonase with a promiscuous esterase activity. It protects LDL from oxidative modification by hydrolyzing lipid peroxides. Substitution of glutamine (Q) to arginine (R) at site 192 (Q192R) and leucine (L) to methionine (M) substitution at site 55 (L55M) attribute to the differences observed in the PON1 activity.,,, It is observed that the greater the PON1 activity, the greater is the progression of atherosclerosis; however, the role of the PON1 polymorphisms on atherosclerosis awaits confirmatory evidence.
Statins provide protection against atherosclerosis by lowering LDL-C, total cholesterol, and triglycerides as well as by elevating the HDL-C level., Treatment with statins upregulates PON1 enzymatic activity, contributing to their atheroprotective potential. Studies assessing the role of PON1 polymorphisms on lipid-lowering and PON1-inducing effect of statins have yielded inconsistent results.
The present study assessed the impact of PON1 polymorphisms on change in lipid levels and PON1 activity following statin treatment in Western Indian patients of dyslipidemia.
| » Methods|| |
The study was initiated after ethics committee approval followed by written informed consent from all trial participants. The trial was also registered at CTRI (CTRI/2017/08/009376).
This was a prospective interventional study.
The study was conducted from March 2016 to May 2018.
Statin-naïve dyslipidemia patients of either gender aged between 18 and 75 years eligible for statin treatment as per the physician's diagnosis were included in the study. The specific statin type and dosages were as per physicians' clinical assessment.
DNA extraction from whole blood was performed using the phenol chloroform method. Amplification of the Q192R (rs662) polymorphism (QQ - wild, QR - heterozygous mutant, RR - homozygous mutant) of the PON1 gene was carried out using the forward and reverse primers; 5'-TAT TGT TGC TGT GGG ACC TGA G-3' and 5'-CAC GCT AAA CCC AAA TAC ATC CTC-3', respectively. The PCR products (99 bp) were electrophoresed on 3% agarose gel. The restriction enzyme AlwI (3 h at 55°C) was used for restriction digestion of the PCR products. The Q allele corresponded to a 99 bp fragment, and the R allele corresponded to 66 bp and 33 bp fragments. Amplification of the L55M (rs854560) polymorphism (LL - wild, LM - heterozygous mutant, MM - homozygous mutant) was carried out using the primers;, 5'-GAA GAG TGA TGT ATA GCC CCA G-3' and 5'-TTT AAT CCA GAG CTA ATG AAA GCC-3'. The PCR products (170 bp) were electrophoresed on 1% agarose gel. The restriction enzyme NlaIII (1 h at 37°C) was used for restriction digestion of the PCR products. The L allele corresponded to a 170 bp fragment, and the M allele corresponded to 126 bp and 44 bp fragments.
Determination of the paraoxonase 1 activity
PON1 paraoxonase activity was measured spectrophotometrically using paraoxon (Sigma, USA) as a substrate after adding 2.22 mmol/1 paraoxon substrate solution in 0.1 mol/l Tris-HCl buffer, pH 8.0, containing 2 mmol/1 CaCl2 and serum sample. The absorbance was regulated spectrophotometrically at 405 nm on a spectrophotometer. The production of p-nitrophenol was monitored every min for 5 min. PON1 activity was stated in international units (IU) where an IU is considered 1 μmol of p-nitrophenol produced per min per liter.,
On the follow-up visit (day 90 + 15), 5 ml of blood was collected and tested for serum lipid levels and PON1 activity.
Change in PON1 enzymatic activity following statin treatment was also standardized for HDL concentration by calculating ratio of PON1 activity and HDL concentration.
A total of 140 patients were required to find a difference of 10 U/l between pre- and post-treatment PON1 activity at 5% alpha error, 80% power, and a dropout rate of 15%. The sample size was calculated using StataCorp. 2009. Stata Statistical Software: Release 11. College Station, TX: StataCorp LP (STATA 11.0).
The study data were analyzed using descriptive and inferential statistics. The Kolmogorov–Smirnov test was used to check the normality of the continuous data. Continuous data were expressed as mean ± standard deviation/median (range) based on the distribution, while categorical data were presented as numbers and proportions. Wilcoxon-matched pairs/paired t-test was used as per the distribution of the data. The categorical variables were evaluated using Chi-square test or Chi-square test for trend. The impact of PON1 genotypes on enzymatic activity following statin treatment was assessed independent of covariates (age, gender, body mass index [BMI], change in serum HDL levels, and history of diabetes and hypertension) in univariate followed by multivariate linear regression analysis. All statistical analyses were done using Statistical Package for the Social Sciences (SPSS) ,Version 20.0. Armonk, NY: IBM Corp. and STATA 11.0 at 5% significance.
| » Results|| |
Baseline clinical and laboratory characteristics
A total of 162 statin-naïve dyslipidemia patients were eligible for the study, of which 22 (13.6%) declined consent, and thus, a total of 140 patients were recruited in the study. Out of 140 patients, 24 (17%) were lost to follow-up, and hence, final analysis was performed on 116 patients. Of them, 59 (51%) were men and 57 (49%) were women. Fifty-four (46.55%) had hypertension, 62 (53.45%) had type 2 diabetes mellitus, and 18 (15.51%) had both as comorbidities.
Out of total 116 patients, genotyping for Q192R and L55M polymorphisms was done for 113 patients as the DNA could not be extracted for three patients. Of these, 57 (50%) had QQ, 41 (36%) had QR, and 15 (14%) had RR genotypes. Seventy-six (67%) had LL, 35 (31%) had LM, and 2 (2%) had MM genotypes. Both the genotype distributions followed Hardy–Weinberg equilibrium (P > 0.05).
Effect of statin treatment on lipid parameters (median)
There was a significant decrease in total cholesterol (mg/dl) (baseline: 220; posttreatment: 157; % change: 28.63%; P < 0.0001), LDL-C (mg/dl) (baseline: 155.35; posttreatment: 89; % change: 42.71%; P < 0.0001), and triglycerides (mg/dl) (baseline: 150.30; posttreatment: 109; % change: 27.48%; P < 0.0001) and increase in HDL-C (mg/dl) (baseline: 40.15; posttreatment: 43; % change: 7.1%; P = 0.0025) levels at 3 months with statin treatment.
Influence of paraoxonase1 polymorphisms on lipid parameters
The change in lipid parameters following statin treatment did not differ between different PON1 genotypes (P > 0.05). Notably, Q192R mutants (QR and RR) had higher serum HDL-C and lower LDL-C concentration at baseline relative to wild (QQ) genotype (P = 0.01 for each) [Table 1].
|Table 1: Baseline and posttreatment lipid parameters according to paraoxonase 1 polymorphisms|
Click here to view
Influence of paraoxonase1 polymorphisms on paraoxonase1 activity
Overall, the PON1 activity increased significantly after statin treatment (U/L) (median [range] – before: 47.92 [9.03–181.25]; after: 72.22 [7.64–244.44]; P < 0.05). The increase however was independent of increase in HDL-C concentration (U/mg) (PON1 activity/HDL ratio: median [range] before: 0.12 [0.02, 0.67]; after: 0.17 [0.02, 0.50]; P < 0.05).
Baseline PON1 activity was significantly higher among the Q192R mutants in comparison to wild genotype though no such difference was observed for L55M polymorphisms. A significant increase in PON1 activity was observed in patients with QQ genotype (wild type) relative to QR and RR genotypes (mutants) in univariate analysis (P < 0.05), while no such difference was observed for L55M polymorphisms (P > 0.05) [Table 2].
|Table 2: Baseline and posttreatment paraoxonase 1 activity according to paraoxonase 1 polymorphisms|
Click here to view
Multiple linear regression analysis
In multivariate analysis, PON1 activity was significantly greater in Q192R wild-type genotype compared to the mutants after adjusting for covariates such as age, gender, BMI, change in HDL-C, and history of diabetes and hypertension (P = 0.01).
| » Discussion|| |
Antioxidant properties of statins contribute to their atheroprotective potential. Augmentation of PON1 activity through pharmacotherapy is a promising approach for protection against cardiovascular diseases. Statin is by far the most extensively studied class of drug for its interaction with PON1 enzyme. We studied the effect of PON1 polymorphisms on lipid-lowering and PON1-modulating (antioxidant) activity of statins in dyslipidemia patients.
We observed a significant rise in PON1 activity in patients with QQ genotype relative to the mutants. Furthermore, there was a significant reduction in LDL-C, cholesterol, and triglyceride and a significant rise in HDL-C levels following statin treatment like Kural et al. In contrast, Mirdamadi et al. and Nagila et al. observed only modest increase in HDL-C levels.
We found similar PON1 genotype distributions to Mirdamadi et al. and Turban et al., who assessed them in the Caucasians.
Several researchers have found no influence of PON1 polymorphisms on change in lipids with statin treatment.,,,, Mirdamadi et al. observed that only simvastatin treatment more effectually reduced the triglyceride levels in patients with R allele compared to QQ genotype. This discordance might be due to the differences in study populations or types of hypercholesterolemia. In addition, the change in lipid parameters following statin treatment can be explained by linkage disequilibrium of PON1 with other genes.,
We observed a significant rise in PON1 activity following statin treatment across all patients regardless of the change in HDL-C levels. This is agreement with Tomás et al. and Harangi et al. Several mechanisms have been put forward to explain statin-induced increase in the PON1 activity. High PON1 activity following statin treatment could be a consequence of reduced oxidative stress since PON1 gets partially deactivated in the presence of oxidized LDL particles. Functional polymorphisms of PON1 gene could also influence PON1-inducing effect of statins., In the present study, we observed a significant increase in the PON1 activity among Q192R wild genotype patients relative to the mutants, while Mirdamadi et al. found the opposite. Christidis et al. and Tomás et al. confirmed that the increase in paraoxonase activity was greater in R allele carriers compared to those with wild-type (QQ) genotype. The discordance in our findings with other studies could be explained by differences in study populations or in the methodology.
The present study has some limitations. First, we did not assess the impact of statin treatment on PON1 arlyesterase activity and its susceptibility to genetic polymorphisms. Second, we did not study the effect of statins on markers of lipid peroxidation (e.g., malonaldehyde) to confirm antioxidant activity. Third, we did not assess the impact of other promoter polymorphisms and haplotypes of the PON1 gene on statin effect.
| » Conclusion|| |
To conclude, the important antioxidant properties of statins are exerted via rise in serum PON1 activity, independent of HDL-C concentrations. The increase was greater in individuals with QQ genotype. Future large-scale studies will validate the premise that QQ homozygotes see added benefits from statin treatment compared to R carriers. In the meantime, PON1 enzymatic activity remains an important marker to be measured while assessing pleotropic effects of statins in CAD.
We thank Ms. Sanchita Ambre, Ms. Lavina Jadhav and Mr. Sukrut Khadke for assistance with recruitment of patients.
Financial support and sponsorship section
This work was supported by the Research Society of the Seth GS Medical College and KEM Hospital, Parel, Mumbai 00 012.
Conflicts of interest
Dr. Urmila Thatte and Dr. Nithya Gogtay are a part of the Editorial board of IJP.
| » References|| |
Kural BV, Orem C, Uydu HA, Alver A, Orem A. The effects of lipid-lowering therapy on paraoxonase activities and their relationships with the oxidant-antioxidant system in patients with dyslipidemia. Coron Artery Dis 2004;15:277-83.
Gupta N, Singh S, Maturu VN, Sharma YP, Gill KD. Paraoxonase 1 (PON1) polymorphisms, haplotypes and activity in predicting cad risk in North-West Indian Punjabis. PLoS One 2011;6:e17805.
Mackness B, Davies GK, Turkie W, Lee E, Roberts DH, Hill E, et al.
Paraoxonase status in coronary heart disease: Are activity and concentration more important than genotype? Arterioscler Thromb Vasc Biol 2001;21:1451-7.
Harangi M, Seres I, Varga Z, Emri G, Szilvássy Z, Paragh G, et al.
Atorvastatin effect on high-density lipoprotein-associated paraoxonase activity and oxidative DNA damage. Eur J Clin Pharmacol 2004;60:685-91.
Turban S, Fuentes F, Ferlic L, Brugada R, Gotto AM, Ballantyne CM, et al.
A prospective study of paraoxonase gene Q/R192 polymorphism and severity, progression and regression of coronary atherosclerosis, plasma lipid levels, clinical events and response to fluvastatin. Atherosclerosis 2001;154:633-40.
Furlong CE, Marsillach J, Jarvik GP, Costa LG. Paraoxonases-1, -2 and -3: What are their functions? Chem Biol Interact 2016;259:51-62.
Tomás M, Sentí M, García-Faria F, Vila J, Torrents A, Covas M, et al.
Effect of simvastatin therapy on paraoxonase activity and related lipoproteins in familial hypercholesterolemic patients. Arterioscler Thromb Vasc Biol 2000;20:2113-9.
Hernández-Díaz Y, Tovilla-Zárate CA, Juárez-Rojop IE, González-Castro TB, Rodríguez-Pérez C, López-Narváez ML, et al.
Effects of paraoxonase 1 gene polymorphisms on heart diseases: Systematic review and meta-analysis of 64 case-control studies. Medicine (Baltimore) 2016;95:e5298.
Mendonça MI, Dos Reis RP, Freitas AI, Sousa AC, Pereira A, Faria P, et al.
Human paraoxonase gene polymorphisms and coronary artery disease risk. Rev Port Cardiol 2008;27:1539-55.
Nagila A, Permpongpaiboon T, Tantrarongroj S, Porapakkham P, Chinwattana K, Deakin S, et al.
Effect of atorvastatin on paraoxonase1 (PON1) and oxidative status. Pharmacol Rep 2009;61:892-8.
Ferretti G, Bacchetti T, Sahebkar A. Effect of statin therapy on paraoxonase-1 status: A systematic review and meta-analysis of 25 clinical trials. Prog Lipid Res 2015;60:50-73.
Christidis DS, Liberopoulos EN, Kakafika AI, Miltiadous GA, Liamis GL, Kakaidi B, et al.
Effect of paraoxonase 1 polymorphisms on the response of lipids and lipoprotein-associated enzymes to treatment with fluvastatin. Arch Med Res 2007;38:403-10.
Sambrook J, Fritsct EF, Maniatis T. Molecular cloning: A laboratory manual, 2nd
ed. New York: Cold Spring Harbour Laboratory; 1989.
Sanghera DK, Saha N, Aston CE, Kamboh MI. Genetic polymorphism of paraoxonase and the risk of coronary heart disease. Arterioscler Thromb Vasc Biol 1997;17:1067-73.
Lakshmy R, Ahmad D, Abraham RA, Sharma M, Vemparala K, Das S, et al.
Paraoxonase gene Q192R & L55M infarction & association with oxidized low density lipoprotein. Indian J Med Res 2010;131:522-9.
] [Full text]
Zama T, Murata M, Matsubara Y, Kawano K, Aoki N, Yoshino H, et al.
A 192Arg variant of the human paraoxonase (HUMPONA) gene polymorphism is associated with an increased risk for coronary artery disease in the Japanese. Arterioscler Thromb Vasc Biol 1997;17:3565-9.
Schiavon R, De Fanti E, Giavarina D, Biasioli S, Cavalcanti G, Guidi G. Serum paraoxonase activity is decreased in uremic patients. Clin Chim Acta 1996;247:71-80.
Kedage V, Muttigi MS, Shetty MS, Suvarna R, Rao SS, Joshi C, et al.
Serum paraoxonase 1 activity status in patients with liver disorders. Saudi J Gastroenterol 2010;16:79-83.
] [Full text]
Mirdamadi HZ, Sztanek F, Derdak Z, Seres I, Harangi M, Paragh G. The human paraoxonase-1 phenotype modifies the effect of statins on paraoxonase activity and lipid parameters. Br J Clin Pharmacol 2008;66:366-74.
de Souza JA, Menin A, Lima LO, Smiderle L, Hutz MH, Van Der Sand CR, et al.
PON1 polymorphisms are predictors of ability to attain HDL-C goals in statin-treated patients. Clin Biochem 2015;48:1039-44.
Malin R, Laaksonen R, Knuuti J, Janatuinen T, Vesalainen R, Nuutila P, et al.
Paraoxonase genotype modifies the effect of pravastatin on high-density lipoprotein cholesterol. Pharmacogenetics 2001;11:625-33.
Deakin S, Guernier S, James RW. Pharmacogenetic interaction between paraoxonase-1 gene promoter polymorphism C-107T and statin. Pharmacogenet Genomics 2007;17:451-7.
[Table 1], [Table 2]