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Clinical StudyParaoxonase-1 and Simvastatin Treatment in Patients withStable Coronary Artery Disease

RafaB Januszek

Department of Cardiology University Hospital Kopernika Street 17 31-501 Cracow Poland

Correspondence should be addressed to Rafał Januszek jaanrafinteriapl

Received 24 September 2015 Revised 14 January 2016 Accepted 20 March 2016

Academic Editor Karlheinz Peter

Copyright copy 2016 Rafał JanuszekThis is an open access article distributed under theCreativeCommonsAttribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Background Paraoxonase-1 (PON1) is the crucial antioxidant marker of high-density lipoproteins The present study is aimed atassessing the effect of simvastatin treatment on PON1 activity and its relationship to Q192R and M55L polymorphisms in subjectswith stable coronary artery disease (CAD)MethodsThe patient group was composed of 53 individuals with stable CAD and thecontrol group included 53 sex-matched police officers without CAD CAD patients were treated with simvastatin 40mgday for12 months Respectively flow mediated dilatation (FMD) serum hs-CRP and TNF-120572 levels urinary 8-iso-PGF

2120572concentrations

and PON1 activity were evaluated in definitive intervals Results There was no effect of simvastatin treatment on urinary 8-iso-PGF2120572 Simvastatin treatment significantly increased FMD value decreased CRP and TNF-120572 concentration After adjusting for

PON1 genotypes significantly higher PON1 activity was noted in the 192R allele carriers in both groups Regardless of genotypePON1 activity remained stable after simvastatin treatment ConclusionsThe present study confirms a positive effect of simvastatintherapy on endothelial function and inflammatory markers in secondary prevention Simvastatin treatment shows no effects onPON1 activity and 8-isoprostanes level The effect of simvastatin therapy on PON1 activity is not modulated by Q192R and M55Lpolymorphisms

1 Introduction

Statins reversibly inhibit HMG-CoA reductase also attenu-ating production of important isoprenoid intermediates usedfor posttranslational modification of different proteins [1]The main pleiotropic effects of statin therapy are improve-ment of endothelial function stabilisation of atheroscle-rotic plaques anti-inflammatory properties antithromboticactivity and antioxidative effects [2] All paraoxonases arelactonases characterised by the widest known substratespecificity and due to the involvement in various signal-ing pathways they play an important role in the anti-inflammatory and antioxidant response [3] Paraoxonase-1(PON1) is the first described esterase that hydrolyzes pesti-cides as toxic metabolites of parathion-paraoxon [4] PON1is almost exclusively present in HDL particles [5] Peroxidaseand esterase PON1 activity are crucial in detoxifying oxidativestress mediators whereas lactonase activity is responsible forthe hydrolysis of various compounds Furthermore PON1presenting esterase activity hydrolyzes the ester bond present

in oxidized cholesterol esters thereby scavenging oxidizedfats from atherosclerotic lesions [6] In PON1 knockout miceit has been shown that HDL fails to prevent oxidation ofLDL andmonocyte chemotaxis On the other hand the over-expression of PON1 in transgenic mice increased the effectof HDL on oxidation and attenuated the rate of formationof atherosclerotic lesions [7 8] Five polymorphisms wererecognized in the PON1 gene promoter region Some ofthe polymorphisms significantly affect gene expression andimpact serum PON1 concentration Currently there are twoknown polymorphisms within the coding region of PON1which leads to the conversion of glutaminerarrarginine atposition 192 (Q192R) and leucinerarrmethionine at position 55(L55M)The genetic polymorphisms of PON1 gene influencePON1 enzyme concentration and specific activityThe Q192Rpolymorphismdoes not affect the serumPON1 concentrationbut significantly modifies its activity 192R carriers show thehighest activity of PON1 against paraxone whereas 192QQhomozygotes show the highest PON1 activity against pheny-lacetate [9] The L55M polymorphism mostly influences

Hindawi Publishing CorporationInternational Journal of Vascular MedicineVolume 2016 Article ID 6312478 10 pageshttpdxdoiorg10115520166312478

2 International Journal of Vascular Medicine

binding affinity of HDL and PON1 [10] Homozygotes 192QQand 55MM show the highest protection from LDL oxidationwhile 192RR and 55LL homozygotes show LDL with theleast resistance to oxidation [11] Genetic factors includingpolymorphisms are responsible for more than 60 of thephenotypic variability of PON1 Demographic factors suchas age and sex account for 1ndash6 of PON1 variability whilemetabolic variables account for about 4ndash19 [12] Eatinghabits physical activity and smoking also modify serumPON1 activity and oxidative stress [13] Thus far publishedstudies highlighted that statins can have a beneficial influencenot only by their lipid-lowering effect but also throughdiminishing oxidative stress and enhancing PON1 activityThe improvement in PON1 activity after statin therapycould be generated by oxidative stress reduction a directeffect on promoter activity and PON1 gene expression [14]Additionally patients with coronary artery disease (CAD)present lower serum PON1 activity This was found to be apredictor of CAD although not all studies have confirmedthis relationship [15] A weak association of CADwith Q192Rpolymorphism was demonstrated in prior research [16] Theleading inflammation marker is high sensitivity C-reactiveprotein (hs-CRP) which is substantially regulated by tumornecrosis factor 120572 (TNF-120572) The increased expression of TNF-120572 intensifies production of reactive oxygen species (ROS) anddecreases bioavailability of NO leading to endothelial dys-function [17] Statins present anti-inflammatory propertiesHowever the effect of statins on fibrinogen is inconsistent[18 19] Risk factors of atherosclerosis enhance expressionof NADPH oxidase leading to significant generation ofO2

minus and consequently depletion of NO bioavailabilityStatins improve endothelial function in patients with hyper-cholesterolemia and atherosclerosis [20] The ldquofree radicalchain reactionrdquo initiated by ROS results in the formation ofadditional reactive compounds including F

2120572-isoprostanes

which is largely represented by 8-iso-prostaglandin-F2120572

(8-iso-PGF

2120572) Higher concentrations of 8-iso-PGF

2120572and its

metabolites were demonstrated in patients with CAD and8-iso-PGF

2120572is a marker of CAD [21] Statins decrease

oxidative stress by inhibition of oxidative stress in theendothelium their effects on cardiac muscle cells and theirinfluence on PON1 activity At this time the dominatingtheory is that statin therapy reduces oxidative stress markers[22] The mechanism of PON1rsquos protective effects seems tobe tightly associated with the antioxidant potential of theenzyme PON1 scavenges oxidative stress products leadingto improved endothelial function especially in patients withCAD The effect of statin therapy on PON1 activity in CADpatients treated with simvastatin for 12 months has yet to beevaluated while shorter studies did not provide conclusiveresults Also research assessing the relationship of Q192Rand L55M polymorphisms with CAD produced inconclusiveresults

The main goal of the present study was to assess theeffect of 12 months of simvastatin treatment on serum PON1activity and urinary 8-isoprostanes in patients with CADThepresent study also aimed to evaluate the association of Q192Rand L55M PON1 gene polymorphisms on PON1 activity withand without simvastatin treatment The effect of simvastatin

treatment on endothelial function intima-media thickness(IMT) proinflammatory marker concentrations and lipidprofile was also assessed to confirm parallel simvastatinefficacy

2 Subjects and Methods

21 Subjects Thepresent studywas performed on 53 patientsaged 35 to 65 years with stable CAD (based on imagingstudy angiocomputed tomography of coronary arteries orcoronarography echocardiography and exercise treadmilltest with electrocardiogram) The control group consisted of53 active professional police officers without CAD Twenty-two obese individuals were included in the patients groupand 18 in the control group All participants of the presentstudy qualified for the simvastatin therapy were permitted tocontinue treatment with aspirin 75ndash100mgday 120573-blockersangiotensin-converting enzyme inhibitors calcium channelblockers diuretics and nitrates at a stable dose throughoutthe study period Some participants in the control groupremained on treatment due to concomitant diseases Patientswho required a change in chronic therapy during the studyperiod were excluded from the study Patients were qualifiedfor simvastatin treatment according to the current ESCguidelines Individuals who had an acute coronary episodein the last 3 months heart failure LDL lt 18mmolLand triglycerides level gt 45mmolL and were previouslytreated with lipid-lowering agents or oral anticoagulantswere excluded from the study Also patients with recentlyperacted infections diabetes impaired renal and liver func-tion myopathy and neoplastic and thyroid disease were notincluded in the study Participants with CAD were treatedwith simvastatin 40mg daily for 12 months Throughout thepresent study participants were restricted from nonsteroidalanti-inflammatory drugs and maintained a stable level ofphysical activity and diet The percentage of smokers inthe two groups also did not change significantly duringthe present study The lipid profile flow-mediated dilation(FMD) intima-media thickness (IMT) serum fibrinogenCRP and TNF-120572 as well as urine 8-iso-PGF

2120572and serum

PON1 activity were evaluated at baseline after 6 and 12months of simvastatin treatment in the CAD group and atbaseline and after 12months in the control group PON1 poly-morphisms (Q192R and M55L) were assessed at baseline inall participants Blood samples were collected after a 14-hourovernight fast and FMD measurements were assessed thefollowing day Patients were asked to refrain from smoking 12hours prior to the blood test Blood samples for the analysis offibrinogen hs-CRP lipid profile and creatinine were drawnfrom the forearm vein (between 600 and 800 am) after30min rest and immediately sent to the laboratory Blood andurine samples for further analysis (PON1 activity PON1 genepolymorphisms TNF-120572 urine 8-iso-PGF

2120572 and creatinine)

were frozen and stored at minus 80 degrees centigradeNo signs of myopathy or liver function impairment wereobserved during the studyThepresent studywas approved bythe local Ethics Committee and conducted according to theethical standards outlined in the Declaration of Helsinki and

International Journal of Vascular Medicine 3

its amendments All participants of the present study signeda written informed letter of consent

22 Imaging Studies The FMDmeasurement was performedaccording to the previously published protocol [23] Themean IMT value was established on the basis of three ultra-sound measurements in the distal section of the commoncarotid artery approximately 2 cm from the bifurcation atcross-sectional and longitudinal sections and in the antero-lateral and posterolateral presentations

23 PON1 Activity and Polymorphisms Serum PON1 activitywas determined spectrophotometrically using a spectropho-tometer 8730UV PUVIS Paraoxon hydrolysis rate to p-nitrophenol was monitored (120576 = 13 000minus1 cmminus1) in K-HEPESbuffer at 28∘C at length 412 nm One unit of PON1 catalyzesthe formation of 335 pmol of p-nitrophenol in 10 minutesat 25∘C with 05120583L of serum in 05mL of the reactionmixture (equivalent to 0001 A

412) The L55M and Q192R

polymorphisms in the PON1 coding gene were tested byanalysis of restricted fragment length polymorphism (RFLP)of amplified gene segment by polymerase chain reaction(PCR) PON1 genetic variants that cause amino acid substitu-tion Q192R and M55L were genotyped by PCR amplificationand further restriction digestion (PCR-RFLP) with sectiondigestion on an agarose gel Replacement of glutamine (Q)to arginine (R) at position 192 of the polypeptide chain isdue to the transition of adenine to guanine detected byrestriction digestion using the MboI enzyme Genomic DNAfragment of 291 bp in length was amplified under standardconditions of 34 cycles using primers with the followingsequence 51015840-ATA GAA GAG GAC AGT CAG TGC TTand 51015840-CAT CAT CGG TGC ACT GTG AA TT In thecase of the AA genotype (homozygous 192QQ) the size ofdigestion products was 154 and 137 base pairs For the GGgenotype (homozygous 192RR) the length of the digestionproducts was 137 126 and 28 base pairs Heterozygote (AG192QR) had a combination of these passages 154 137 126and 28 base pairs Replacement of methionine (M) to leucine(L) at position 55 in the amino acid chain results fromthe substitution of adenine to thymine detected with therestriction enzyme NlaIII Genomic DNA with a lengthof 293 bp was amplified under standard conditions usingprimers of appropriate sequences 51015840-TTT CCA CGC TATAAT CAT ATT C and 51015840-AAT TTA AAC GAC TGC TCCCAG TA In the case of the AA genotype (homozygous55MM) the size of digestion products was 192 and 101base pairs In the case of TT genotype (homozygous 55LL)amplified fragment was not subject to restrictase cutting andits lengthwas 293 bpHeterozygotes 55MLhad a combinationof all of these fragments although largely 293 192 and 101base pairs

24 Markers of Inflammation and Oxidative Stress Serumultrasensitive CRP protein was determined by nephelome-try (Dade-Behring Germany) TNF-120572 levels were measuredby ELISA (RampD Systems United Kingdom) Plasma fib-rinogen concentration was determined by the use of BCT

coagulometer (Behring Coagulation Timer Dade-BehringGermany) and Multifibren U analyzer Urine 8-iso-PGF

2120572

concentrations were determined by ELISA (EIA Kit UrinaryIsoprostane NucliLab BV Netherlands)

25 Lipids Total cholesterol and triglycerides were deter-mined by an enzymatic method (Technicon RA-1000 ana-lyzer)

26 Statistical Analysis STATISTICA for Windows Release10 (Statsoft Inc 2011) was used for data analysis The testchoice depended on the distribution of particular data Tocompare measurable variables (or to assess the statisticalsignificance of the observed differences) parametric testswere used the two-sided Studentrsquos 119905-test and Spearmanlinear correlation In case ofmissingmentioned assumptionsnonparametric tests were used (Friedman ANOVA Mann-Whitney 119880 test Kruskal-Wallis and Chi2 test) Multivariateanalysis of variance was also performed 119901 values lt 005 wereconsidered significant

3 Results

31 Baseline Characteristics of the Study Groups Table 1shows clinical characteristics of the investigated groups

32 Effects of Simvastatin Treatment on Lipid Profile Atbaseline significantly higher total (119901 = 000004) and LDLcholesterol levels (119901 lt 0000001) were found in the patientgroup No significant differences were demonstrated betweenpatient and control groups in HDL cholesterol and triglyc-erides concentrations The effect of simvastatin treatment onlipid profile is presented in Table 2

33 Proinflammatory Markers At baseline serum hs-CRPand TNF-120572 concentration in the CAD group were signifi-cantly higher compared to the control group (119901 = 00005and 119901 = 000002) In the control group the mean fibrinogenconcentration was similar in both time points and wassignificantly lower than in the patient group (119901 = 002 119901 =0005) The effect of simvastatin treatment on hs-CRP TNF-120572 and fibrinogen concentration is presented in Table 2

34 The Urine 8-Iso-PGF2120572 Initially the urine 8-iso-PGF

2120572

concentration was significantly higher in the treated group(119901 lt 00001) Furthermore after 12 months of simvastatintreatment the urine 8-iso-PGF

2120572concentration in the patient

group remained significantly higher compared to that inthe control group (119901 lt 00001) The effect of simvastatintreatment on urine 8-iso-PGF

2120572is presented in Table 2

35 Endothelial Function and Intima-Media Thickness Atbaseline themean FMDwas significantly lower in the patientgroup in comparison to the control group (119901 = 0015)Compared to the baseline there was a significant increaseof the FMD after 6 (119901 = 00001) and 12 months (119901 =0006) of treatment The FMD values observed after 6 and 12


Table 1 Characteristics of the study groups

CAD group Controls 119901

Number of patients females 53 (9) 53 (6) 04lowastlowast

Age years 513 plusmn 78 (38 divide 65) 4203 plusmn 482 (35 divide 55) lt0000001lowast

Framingham Risk Score 121 plusmn 64 (18 divide 28) 49 plusmn 36 (02 divide 198) lt0000001lowast

BMI kgm2 294 plusmn 49 (195 divide 447) 292 plusmn 38 (217 divide 407) 081lowast

Waist-hip ratio 096 plusmn 006 (077 divide 109) 094 plusmn 006 (074 divide 11) 019lowast

Overweight 119899 () 20 (3773) 28 (5283) 011lowastlowast

Obesity 119899 () 22 (4145) 18 (3396) 072lowastlowast

Hypertension 119899 () 37 (6981) 19 (3584) 00005lowastlowast

CAD 119899 () 53 (100) mdash mdashHistory of

Myocardial infarction 119899 () 7 (132) mdash mdashPCI 119899 () 9 (1698) mdash mdashCABG 119899 () 1 (188) mdash mdash

Smoking historyWhenever 119899 () 35 (6604) 27 (5094) 052lowastlowast

Number of pack-years 2575 plusmn 904 (1 divide 40) 1525 plusmn 704 (1 divide 26) 0007lowast

Currently 119899 () 17 (3207) 16 (3018) 083lowastlowast

COPD 119899 () 6 (1132) 0 001lowastlowast

Familial history of cardiovascular diseases 119899 () 40 (7547) 29 (5471) 002lowastlowast

MedicationsAspirin 119899 () 53 (100) 7 (132) lt000001lowastlowast

ACEIARB 119899 () 34 (6415) 10 (1886) lt000001lowastlowast

120573-blocker 119899 () 29 (5471) 12 (2264) 00007lowastlowast

Calcium channel blocker 119899 () 11 (2075) 3 (566) 002lowastlowast

Diuretics 119899 () 5 (943) 7 (132) 053lowastlowast

Nitrates 119899 () 11 (2075) mdashData is presented as arithmetic means plusmn SD (min divide max) lowastStudents 119905-test lowastlowastChi2 test Explanation of abbreviations used ACEI angiotensin-convertingenzyme inhibitor ARB angiotensin receptor blocker BMI body-mass index PCI percutaneous coronary intervention CAD coronary artery disease CABGcoronary artery bypass graft and COPD chronic obstructive pulmonary disease

Table 2 Lipid profile concentrations of selected markers of inflammation oxidative stress FMD and IMT in patients and controls beforeand after treatment with simvastatin

CAD group ControlsAt baseline After 6 months After 12 months 119901 At baseline After 12 months 119901

Number of subjects females 53 (9) 53 (9) 53 (9) 53 (6) 53 (6)Total cholesterol mmolL 602 plusmn 081m 454 plusmn 073yen 497 plusmn 097sectpara 0000001lowast 518 plusmn 095 526 plusmn 094 069lowastlowast

LDL cholesterol mmolL 379 plusmn 076m 236 plusmn 064yen 285 plusmn 082sect 0000001lowast 293 plusmn 073 308 plusmn 075 046lowastlowast

HDL cholesterol mmolL 129 plusmn 034 136 plusmn 046 135 plusmn 044 032lowast 132 plusmn 041 127 plusmn 034 044lowastlowast

Triglycerides mmolL 211 plusmn 105 183 plusmn 11 177 plusmn 095 006lowast 201 plusmn 142 22 plusmn 247 051lowastlowast

Fibrinogen gL 373 plusmn 102m 393 plusmn 119 385 plusmn 119para 065lowast 334 plusmn 074 326 plusmn 087 098lowastlowast

hs-CRP mgL 293 plusmn 253m 157 plusmn 146yen 167 plusmn 119sectpara 0021lowast 131 plusmn 114 134 plusmn 154 081lowastlowast

TNF-120572 pgmL 182 plusmn 141m 166 plusmn 132 131 plusmn 095sectpara 0001lowast 085 plusmn 045 088 plusmn 043 067lowastlowast

8-Iso-PGF2120572 pgmg creat 13052 plusmn 8171m 13330 plusmn 8295 12855 plusmn 7903para 086lowast 5726 plusmn 3148 54749 plusmn 3068 079lowastlowast

FMD 676 plusmn 253m 825 plusmn 33yen 812 plusmn 228sect 00004lowast 808 plusmn 28 826 plusmn 312 047lowastlowast

IMT mm 07 plusmn 014m 064 plusmn 014yen 063 plusmn 016sect 0008lowast 057 plusmn 008 055 plusmn 008 027lowastlowast

Data is presented as arithmetic means plusmn SD Studentrsquos 119905-test Mann-Whitney 119880 test lowastANOVA Friedman test lowastlowastWilcoxon signed-rank test yen119901 lt 005 after 6months of treatment compared to baseline sect119901 lt 005 after 12 months of treatment compared to baseline para119901 lt 005 after 12 months of treatment comparedto controls m119901 lt 005 at baseline compared to controls


41732

CAD group

41831

Controls

RRQR

QQ RRQR

QQ

(a)

CAD groupControls

p lt 00001

0

50

100

150

200

250

300

350

400

450

PON

1 ac

tivity

(UL

)

QQ QR RR 192R alleleAll subjectscarriers

(b)

Figure 1 The L55M polymorphism of the PON1 gene (a) the percentage distribution of PON1 genotypes in patient and control groups (b)the association of Q192R polymorphism with serum PON1 activity

Table 3 The effect of Q192R and L55M polymorphisms on serum PON1 activity

CAD group Controls119901

PON1 activity (UL)119901

119873 () 119873 () CAD group ControlsNumber of subjects () 53 (100) 53 (100) 10lowastlowast 12658 plusmn 8595 12594 plusmn 7956 091lowast

Q192R polymorphismGenotype

QQ 32 (604) 31 (585) 084lowastlowast 6604 plusmn 964 7228 plusmn 1419 014lowast

QR 17 (321) 18 (34) 083lowastlowast 19654 plusmn 4824 18276 plusmn 405 043lowast

RR 4 (75) 4 (75) 10lowastlowast 30275 plusmn 9165 31375 plusmn 7228 085lowast

192R allele carriers 21 (396) 22 (415) 084lowastlowast 21505 plusmn 7035 20771 plusmn 6723 065lowast

L55M polymorphismGenotype

LL 14 (246) 18 (34) 039lowastlowast 12613 plusmn 7726 13606 plusmn 8335 044lowast

LM 31 (585) 18 (34) 001lowastlowast 11385 plusmn 686 1128 plusmn 6943 054lowast

MM 8 (151) 17 (32) 0039lowastlowast 16390 plusmn 13464 12282 plusmn 9562 074lowast

Data is presented as arithmetic means plusmn SD lowastlowastChi2 test lowastMann-Whitney 119880 test

months of treatment with simvastatin in the patient groupwere similar to those found in the control group In thepatient group the FMD negatively correlated with age (119901 =003) and IMT (119901 = 0002) and correlated weakly with hs-CRP (119901 = 002) At baseline IMT values were significantlyhigher in the patient group (119901 lt 0000001) In the patientgroup significant reduction of IMT after 6 and 12 monthsof simvastatin treatment was observed (119901 = 0008 and 119901 =0004) although IMT still remained higher after treatmentcompared to controls (119901 = 0003) It was also found that IMTcorrelated positively with age (119901 = 00002) and negativelywith FMD (119901 = 0002 Table 2) Multivariate ANOVAanalysis confirmed the significance of simvastatin treatmentto decrease lipid profiles (total and LDL cholesterol) hs-CRP concentration and IMT ANOVA also showed a slight

reduction in TNF-120572 concentration but no reduction inFMD

36 Genetic Polymorphisms and PON1 Activity No devia-tion from genetic equilibrium in Q192R polymorphism wasobserved (according to the Hardy-Weinberg principle) bothin the patient and in control groups (119901 = 081 119901 = 013)Similar percentage distribution of the particular genotypeswas noticed in the patient and control groups (Figure 1Table 3) Also frequency of the 192R allele was similar in bothgroups (0235 in patients and 0245 in controls) althoughgenetic deviation from equilibrium of L55M polymorphismwas observed in the control group (119901 = 001) The 55LLgenotype was found in 264 and 34 of individuals 55LMin 585 and 34 and 55MM in 151 and 32 (in patients


14

31

8

CAD group

LLLM

MM LLLM

MM

18

18

17

Controls

(a)

CAD groupControls

NS

LL LM MMAll subjects0

50

100

150

200

250

PON

1 ac

tivity

(UL

)

(b)

Figure 2The Q192R polymorphism of the PON1 gene (a) the percentage distribution of PON1 genotypes in the patient and control groups(b) the association of Q192R polymorphism with serum PON1 activity

Table 4 The effect of Q192R and L55M polymorphisms on serum PON1 activity before and after treatment with simvastatin

GenotypeCAD group Controls

PON1 activity (UL) PON1 activity (UL)119873 At baseline After 6 months After 12 months 119901 119873 At baseline After 12 months 119901

All subjects 53 12658 plusmn 8595 12546 plusmn 9313 12285 plusmn 8771 065lowast 53 12594 plusmn 7956 12834 plusmn 868 032lowastlowast

192QQ 32 6604 plusmn 964 6267 plusmn 969 646 plusmn 937 011lowast 31 7228 plusmn 1419 709 plusmn 1654 023lowastlowast

192QR 17 19654 plusmn 4824 20433 plusmn 1374 19550 plusmn 1853 07lowast 18 18276 plusmn 405 19356 plusmn 1012 038lowastlowast

192RR 4 30275 plusmn 9165 30425 plusmn 6323 287 plusmn 7597 036lowast 4 31375 plusmn 7228 327 plusmn 4052 027lowastlowast

192R allele carriers 21 21505 plusmn 7035 2205 plusmn 149 21583 plusmn 186 038lowast 22 20771 plusmn 6723 22025 plusmn 1632 022lowastlowast

55LL 14 12613 plusmn 7726 12613 plusmn 7726 12966 plusmn 2557 076lowast 18 13606 plusmn 8335 15171 plusmn 9207 087lowastlowast

55LM 31 11385 plusmn 686 11187 plusmn 1356 10443 plusmn 124 022lowast 18 1128 plusmn 6943 12358 plusmn 6943 081lowastlowast

55MM 8 1639 plusmn 13464 1559 plusmn 4035 15900 plusmn 4592 079lowast 17 12282 plusmn 9562 13356 plusmn 10529 045lowastlowast

Data is presented as arithmetic means plusmn SD lowastANOVA Friedman test lowastlowastWilcoxon signed-rank test

and controls resp Figure 2 Table 3) At baseline the averageserum PON1 activity in the patient group was comparable tothat in the control group (Table 4) No significant differenceswere found between the patient and control groups regardingPON1 activity in different genotypes (119901 = 014 119901 = 043119901 = 085 for genotypes 192QQ 192QR and 192RR respTable 3) Higher PON1 activity was found in patients with192RR genotype as compared to heterozygotes 192QR andhomozygotes 192QQ (119901 lt 00001 Figure 1 Table 3) Due tothe small number of individuals with genotype 192RR theywere included in the group with genotype 192QR creatinga ldquo192R allele carriersrdquo group The average PON1 activityin 192R allele carriers was significantly higher compared tothose with genotype 192QQ (119901 lt 00001 and 119901 lt 00001in patients and controls resp Table 3 Figure 1) Table 4

presents the effect of simvastatin treatment on serum PON1activity according to genotype After 6 and 12 months oftreatment with simvastatin the mean PON1 activity did notchange significantly compared to baseline values in eachgenotype In the control group the average PON1 activitywas similar at baseline and after 12 months There were nosignificant differences in PON1 activity between individualswith genotype 55LL 55LM or 55MM in both patient andcontrol groups (Table 3) PON1 activity in each genotypegroup for the L55Mpolymorphismdid not differ significantlybetween individuals in patient and control groups (Figure 2Table 3) Patients in each genotypic group (55LL 55LM and55MM) showed no significant changes in PON1 activity after6 and 12 months of simvastatin treatment when comparedto baseline (Table 4) In controls the average PON1 activity


Table 5 Combined genotypes and PON1 activity

GenotypeCAD patients Controls


192QQ55LL 9 6777 plusmn 754 6666 plusmn 479 66 plusmn 821 087lowast 8 7662 plusmn 1025 7575 plusmn 1251 078lowastlowast

192QQ55LM 20 6842 plusmn 951 6347 plusmn 92yen 6942 plusmn 1494 0041lowast 14 7628 plusmn 1517 7478 plusmn 172 044lowastlowast

192QQ55MM 3 5550 plusmn 869 49 plusmn 1112 55 plusmn 624 053lowast 10 6320 plusmn 1209 6160 plusmn 1591 031lowastlowast

192QR55LL 4 20980 plusmn 3892 22114 plusmn 6569 239 plusmn 9244 062lowast 6 19033 plusmn 4126 1942 plusmn 5644 05lowastlowast

192QR55LM 10 19172 plusmn 5412 1979 plusmn 6148 18162 plusmn 5618 088lowast 5 1846 plusmn 1301 2002 plusmn 2873 05lowastlowast

192QR55MM 3 19233 plusmn 5173 19733 plusmn 4801 164 plusmn 6081 013lowast 6 17366 plusmn 4274 1875 plusmn 4025 013lowastlowast

192RR55LL 1 233 238 214 2 288 plusmn 4384 3064 plusmn 4384 018lowastlowast

192RR55LM 1 211 266 230 1 265 254192RR55MM 2 2835 plusmn 5868 3565 plusmn 2616 352 plusmn 1697 06lowast 1 414 441Data is presented as arithmetic means plusmn SD lowastANOVA Friedman test lowastlowastthe Wilcoxon signed-rank testyen119901 = 005 after 6 months of treatment compared to the baseline value

was similar at baseline and after 12 months in each genotypicgroup (Table 4) PON1 activity after simvastatin treatmentwas analyzed to examine the impact of lifestyle choicesand genetic polymorphisms among participants Analysisrevealed no significant differences in PON1 activity aftersimvastatin treatment among smokers and nonsmokers andobese and nonobese participants and after adjusting for bothL55M and Q192R polymorphisms

Analysis of all study participants revealed all 9 possiblePON1 gene combination genotypes (Table 5) Consideringboth of the analysed PON1 gene polymorphisms signifi-cantly higher (119901 lt 00001) initial PON1 activity was foundin patients with combined genotype containing 192R allele(192QR55LL 192QR55LM 192QR55MM 192RR55LL192RR55LM and 192RR55MM) compared to Q allelehomozygotes (192QQ55LL 192QQ55LM and 192QQ55MM)There were no significant differences between PON1activities at baseline and after 6 and 12 months of treat-ment with simvastatin in patients with combined genotypescontaining 192R allele The lowest PON1 activity in bothpatients and control group was found in individuals with thecombined genotype 192QQ55MM compared to genotypes192QQ55LL and 192QQ55LM After 6 months of treatmentwith simvastatin significant decreases in PON1 activity wereonly observed in individuals from the patient group withcombined genotype 192QQ55LM (119901 = 0007) (Table 5)

4 Discussion

41 Simvastatin Treatment and Lipid Profile In the presentstudy simvastatin significantly reduced total cholesterol (by245 and 174) and LDL (by 377 and 248) levels after6 and 12 months of treatment Similar results were publishedin large randomized trials although a greater lipid-loweringeffect was observed in trials compared with the present study[24] A relatively low initial mean cardiovascular risk and lowbaseline total cholesterol and LDL undoubtedly influencedthe lipid-lowering effect of simvastatin in the present study

42 Extra-Lipid Effects of Simvastatin Therapy Simvastatintreatment significantly reduced hs-CRP and TNF-120572 levels

which confirmed pleiotropic properties of statins The effectof statin therapy on fibrinogen is still not established [25]Currently there are no specific cut-off points for urineF2120572-isoprostane levels Analysing the results of published

studies we observed a large variability in the range of F2120572-

isoprostane concentrations which may be affected by mea-surement methods the specific substrate used the substanceinvestigated and coexisting medications and diseases The 8-iso-PGF

2120572values in the present study were higher compared

to one publication and comparable to other papers [26 27]Most of the published studies found that statin therapyreduced concentration of 8-iso-PGF

2120572 However many of

these studies were conducted on small populations withhypercholesterolemia and excluded atherosclerosis [28] Thepresent results remain in line with results reported by otherresearchers [29] Perhaps simvastatin therapy has not beenefficient enough to prevent lipid peroxidation and indirectlythe burden of atherosclerosis It has been previously proventhat statins improve FMD in patients with stable angina [30]A recently published large meta-analysis showed that statinshave a significant effect on IMT in secondary prevention butnot in primary prevention which we found as well in thepresent study [31]

43 PON1 Activity and CAD The most convincing evi-dence for the PON1rsquos association with atherosclerosis wasdiscovered with transgenic mice [8] Human studies alsoindicated that low serum PON1 activity is a predictor ofadverse cardiovascular events However conflicting resultswere published shortly thereafter [32 33] Currently theprevailing view is that PON1 activity is lower in patientswith CAD [32]TheQ192R polymorphism does not influenceserum concentrations of PON1 but affects its activity fora specific substrate The highest activity against paraoxonoccurs in 192R allele carriers while the highest activity againstphenylacetate occurs in individuals with genotype 192QQ[12] In the present study paraoxonwas used as a substrate forPON1 activity assessment and the highest activity was foundin the group of 192R allele carriers Analysis of combinedgenotypes confirmed the effect of 192R allele influence onPON1 serum activity The average PON1 activity in CAD


patients and in different genotype groups was comparable tothat in controls which is consistent with recently publishedresearch [33] In most case-control studies participants inthe control group were younger than patients in treatmentgroups However few studies investigated the effect of ageon serum PON1 activity [34] The relatively small size ofthe study groups affected the results Our results show noassociation of the L55M polymorphism with serum PON1activity and CAD risk This conclusion is supported by theresults of a large meta-analysis which found only a weakassociation of Q192R polymorphism with CAD [35]

44 The Genetic Polymorphisms of PON1 and SimvastatinEffects At present little is known about the pharmacologicalregulation of PON1 in humans Antioxidant properties ofstatins demonstrated on animals were also verified in humanstudies where individuals with hypercholesterolemia pre-sented significant reduction in oxidative stress markers andincreased PON1 activity after statin therapy [36]The increasein PON1 activity is the consequence of oxidative stressreduction enhanced expression and attenuated inactivationof PON1These results were based on research demonstratinga direct effect of statin therapy on the promoter activityand expression of the PON1 gene [37] It has been shownthat sterol regulatory element-binding proteins (SREBP-2)increase promoter region activity proportionally to the statindose suggesting a relationship between PON1 intracellularcholesterol homeostasis and lipid metabolism Statins addi-tionally inhibit isoprenoid mediators which influence PON1activity Statins increase the activity of an enzyme associatedwithHDL and PON1HDL ratio [38] However in the presentstudy there was no effect of simvastatin on PON1 activity inthe patient group neither before nor after adjusting PON1activity for the Q192R polymorphism Maintained elevationof the urine 8-iso-PGF

2120572may explain why our results show

that PON1 activity did not increase after simvastatin treat-ment Regarding published data it should be noted that it isdifficult to compare particular results due to differences in theassay methods investigated populations selection bias andethnic differences Inclusion criteria of participants often didnot address smoking obesity or age and the number of theparticipants was usually small Only a few studies focused onthe effect of statins on PON1 activity in patients with CADand they revealed increases in HDL concentration and PON1activity [39] Aspirin treatment could influence our results Incontrast to published works all individuals from the patientgroup were treated with aspirin at the dose of 75mgdayAspirinrsquos dose-dependent inhibitory effect on the formationof p-nitrophenol indicates that aspirin is also hydrolyzedwithparaoxon and may compete for the active site of PON1 [40]Furthermore aspirin hydrolysis to salicylic acid occurredunder the influence of isolated HDL showing that in addi-tion to hepatocytic hydrolysis aspirin is also hydrolyzed incirculation Based on this data it could be concluded thatresults are underestimated in patients treated with aspirinespecially when paraoxon is used for the assessment of PON1activity Although this hypothesis requires further studiesdeactivation changes of the enzyme induced by acetylation oftyrosine in the active center of PON1 could not be excluded

In another study which focused on aspirinrsquos relation to PON1activity in patients with CAD higher PON1 activity wasdemonstrated in individuals treated with aspirin althoughthe results and PON1 activity were not adjusted to evaluatetheQ192R polymorphism [41] On the other handWatermanet al found prooxidative properties of aspirin at dose of75mgday in healthy volunteers increased ex vivo sensitivityof LDL to oxidative modification triggering the growth ofproatherogenic properties of LDL cholesterol The oxidativestress hypothesis is supported by evidence linking oxidativestress with aspirin and PON1 [42] An alternative thesis isan anti-inflammatory activity of aspirin though the PON1concentration is reduced during an inflammatory responsein animal models [43] Daily aspirin therapy was introducedin most participants 2-3 weeks before initiating simvastatintherapy Perhaps increased PON1 activity after aspirin ther-apymasked the expected difference in PON1 activity betweenpatients and controls The potential improvement of PON1enzyme activity under the influence of pharmacologicalagents is presumably low around 7ndash11 [36] Hypotheticallyassuming that PON1 activity was to improve after aspirintreatment further improvement after simvastatin may havebeen imperceptible We cannot exclude that the effect ofstatins was attenuated by the prooxidative effect of aspirin orother factors associated with CAD which decreased PON1activity Plasma PON1 concentration was not measured inthe present study This is a methodological imperfection andmay have significance in determining the effect of statinson PON1 activity Additionally the atheroprotective effectsof PON1 activity may vary considerably if PON1 reactswith a compound other than paraoxon substrates such aslipid hydroperoxides Hydrolysis of paraoxon in vitro whichis an expression of PON1 activity may be a suboptimalmethod of measuring its current antioxidant capacity It ispossible that the use of different assays to determine PON1activity could give dissimilar results Thus far there is noknown satisfactory method for accurate measurement of theantioxidant potential of PON1 in vivo The quantificationof isoprostanes by gas chromatographymass spectrometrycould also amend the results

In conclusion the present study supports the theory thatthe effect of 12 months of simvastatin treatment on PON1activity and urine 8-isoprostanes is not significant in patientswith stable coronary artery disease The positive effect ofsimvastatin treatment on PON1 activity and reduction inoxidative stress markers could be disguised by other factorswhich reduce the effectiveness of simvastatin such as coexist-ing medical treatment atherosclerosis burden or coexistingrisk factors for atherosclerosis

The decrease of inflammatory markers intima-mediathickness and improvement of endothelial function aftersimvastatin treatment confirms the pleiotropic effects ofsimvastatin in patients with stable CAD

Q192R and M55L PON1 gene polymorphisms are notassociated with an altered effect of simvastatin treatment onPON1 activity and urine 8-isoprostanes in patients with stableCAD treated for 12 months with simvastatin The R allele ofQ192R polymorphism is associatedwith higher PON1 activityin patients with CAD Neither Q192R nor M55L PON1 gene


polymorphismwas associatedwith increased coronary arterydisease incidence

Competing Interests

The author declares that he has no competing interests

Acknowledgments

The present study was sponsored by the Ministry of Scienceand Higher Education Poland for the purposes of researchand education as part of a PhD thesis

References

[1] J L Goldstein and M S Brown ldquoRegulation of the mevalonatepathwayrdquo Nature vol 343 no 6257 pp 425ndash430 1990

[2] A Undas K E Brummel J Musial K G Mann and ASzczeklik ldquoSimvastatin depresses blood clotting by inhibitingactivation of prothrombin factor V and factor XIII and byenhancing factor Va inactivationrdquo Circulation vol 103 no 18pp 2248ndash2253 2001

[3] D I Draganov J F Teiber A Speelman Y Osawa R Sunaharaand B N La Du ldquoHuman paraoxonases (PON1 PON2 andPON3) are lactonases with overlapping and distinct substratespecificitiesrdquo Journal of Lipid Research vol 46 no 6 pp 1239ndash1247 2005

[4] A Mazur ldquoAn enzyme in animal tissues capable of hydrolysingthe phosphorus-fluorine bond of alkyl fluorophosphatesrdquo TheJournal of Biological Chemistry vol 164 pp 271ndash289 1946

[5] J Marsillach B Mackness M Mackness et al ldquoImmunohis-tochemical analysis of paraoxonases-1 2 and 3 expression innormal mouse tissuesrdquo Free Radical Biology and Medicine vol45 no 2 pp 146ndash157 2008

[6] M Aviram E Hardak J Vaya et al ldquoHuman serum paraox-onases (PON1) Q and R selectively decrease lipid peroxidesin human coronary and carotid atherosclerotic lesions PON1esterase and peroxidase-like activitiesrdquo Circulation vol 101 no21 pp 2510ndash2517 2000

[7] D M Shih L Gu Y-R Xia et al ldquoMice lacking serumparaoxonase are susceptible to organophosphate toxicity andatherosclerosisrdquo Nature vol 394 no 6690 pp 284ndash287 1998

[8] A Tward Y-R Xia X-P Wang et al ldquoDecreased atheroscle-rotic lesion formation in human serum paraoxonase transgenicmicerdquo Circulation vol 106 no 4 pp 484ndash490 2002

[9] I Leviev and RW James ldquoPromoter polymorphisms of humanparaoxonase PON1 gene and serum paraoxonase activitiesand concentrationsrdquo Arteriosclerosis Thrombosis and VascularBiology vol 20 no 2 pp 516ndash521 2000

[10] I Leviev S Deakin and R W James ldquoDecreased stabilityof the M54 isoform of paraoxonase as a contributory factorto variations in human serum paraoxonase concentrationsrdquoJournal of Lipid Research vol 42 no 4 pp 528ndash535 2001

[11] B MacKness M I MacKness S Arrol W Turkie and PN Durrington ldquoEffect of the human serum paraoxonase 55and 192 genetic polymorphisms on the protection by highdensity lipoprotein against low density lipoprotein oxidativemodificationrdquo FEBS Letters vol 423 no 1 pp 57ndash60 1998

[12] D L Rainwater S Rutherford T D Dyer et al ldquoDeterminantsof variation in human serum paraoxonase activityrdquo Heredityvol 102 no 2 pp 147ndash154 2009

[13] P Kleemola R Freese M Jauhiainen R Pahlman G Alfthanand M Mutanen ldquoDietary determinants of serum paraoxonaseactivity in healthy humansrdquo Atherosclerosis vol 160 no 2 pp425ndash432 2002

[14] S Deakin I Leviev S Guernier and R W James ldquoSimvas-tatin modulates expression of the PON1 gene and increasesserumparaoxonase a role for sterol regulatory element-bindingprotein-2rdquo Arteriosclerosis Thrombosis and Vascular Biologyvol 23 no 11 pp 2083ndash2089 2003

[15] MKerkeni F AddadMChauffert et al ldquoHyperhomocysteine-mia paraoxonase activity and risk of coronary artery diseaserdquoClinical Biochemistry vol 39 no 8 pp 821ndash825 2006

[16] J G Wheeler B D Keavney H Watkins R Collins and JDanesh ldquoFour paraoxonase gene polymorphisms in 11212 casesof coronary heart disease and 12786 controls meta-analysis of43 studiesrdquoThe Lancet vol 363 no 9410 pp 689ndash695 2004

[17] B L Goodwin L C Pendleton M M Levy L P Solomonsonand D C Eichler ldquoTumor necrosis factor-120572 reduces argini-nosuccinate synthase expression and nitric oxide production inaortic endothelial cellsrdquoAmerican Journal of PhysiologymdashHeartand Circulatory Physiology vol 293 no 2 pp H1115ndashH11212007

[18] MR Schroeter THumboldt K Schafer and S KonstantinidesldquoRosuvastatin reduces atherosclerotic lesions and promotesprogenitor cell mobilisation and recruitment in apolipoproteinE knockout micerdquo Atherosclerosis vol 205 no 1 pp 63ndash732009

[19] AUndas K E Brummel-Ziedins andKGMann ldquoStatins andblood coagulationrdquo Arteriosclerosis Thrombosis and VascularBiology vol 25 no 2 pp 287ndash294 2005

[20] J G Robinson B Smith N Maheshwari and H SchrottldquoPleiotropic effects of statins benefit beyond cholesterol reduc-tion A meta-regression analysisrdquo Journal of the AmericanCollege of Cardiology vol 46 no 10 pp 1855ndash1862 2005

[21] E Schwedhelm A Bartling H Lenzen et al ldquoUrinary 8-iso-prostaglandin f2120572 as a risk marker in patients with coronaryheart disease a matched case-control studyrdquo Circulation vol109 no 7 pp 843ndash848 2004

[22] P Pignatelli R Carnevale D Pastori et al ldquoImmediate antiox-idant and antiplatelet effect of atorvastatin via inhibition ofNox2rdquo Circulation vol 126 no 1 pp 92ndash103 2012

[23] R Januszek P Mika A Konik T Petriczek R Nowobilski andR Nizankowski ldquoThe effect of treadmill training on endothelialfunction and walking abilities in patients with peripheralarterial diseaserdquo Journal of Cardiology vol 64 no 2 pp 145ndash151 2014

[24] Scandinavian Simvastatin Survival Study Group ldquoRandomisedtrial of cholesterol lowering in 4444 patients with coronaryheart disease the Scandinavian Simvastatin Survival Study(4S)rdquoThe Lancet vol 344 no 8934 pp 1383ndash1389 1994

[25] R S Rosenson C C Tangney and E J Schaefer ldquoCompar-ative study of HMG-CoA reductase inhibitors on fibrinogenrdquoAtherosclerosis vol 155 no 2 pp 463ndash466 2001

[26] R Cangemi L Loffredo R Carnevale et al ldquoEarly decreaseof oxidative stress by atorvastatin in hypercholesterolaemicpatients effect on circulating vitamin Erdquo European HeartJournal vol 29 no 1 pp 54ndash62 2008

[27] X Wu H Cai Y-B Xiang et al ldquoIntra-person variationof urinary biomarkers of oxidative stress and inflammationrdquoCancer Epidemiology Biomarkers and Prevention vol 19 no 4pp 947ndash952 2010


[28] L Puccetti F Santilli A L Pasqui et al ldquoEffects of atorvastatinand rosuvastatin on thromboxane-dependent platelet activa-tion and oxidative stress in hypercholesterolemiardquo Atheroscle-rosis vol 214 no 1 pp 122ndash128 2011

[29] G D Kom E Schwedhelm RMaas L Schneider R Benndorfand R H Boger ldquoImpact of atorvastatin treatment on platelet-activating factor acetylhydrolase and 15-F

2119905119903119886119899119904-isoprostane in

hypercholesterolaemic patientsrdquo British Journal of ClinicalPharmacology vol 63 no 6 pp 672ndash679 2007

[30] M A Ostad S Eggeling P Tschentscher et al ldquoFlow-mediateddilation in patients with coronary artery disease is enhancedby high dose atorvastatin compared to combined low doseatorvastatin and ezetimibe results of the CEZAR studyrdquoAtherosclerosis vol 205 no 1 pp 227ndash232 2009

[31] Y HuangW Li L Dong R Li and YWu ldquoEffect of statin ther-apy on the progression of common carotid artery intima-mediathickness an updated systematic review and meta-analysis ofrandomized controlled trialsrdquo Journal of Atherosclerosis andThrombosis vol 20 no 1 pp 108ndash121 2013

[32] B Mackness P Durrington P McElduff et al ldquoLow paraox-onase activity predicts coronary events in the CaerphillyProspective studyrdquo Circulation vol 107 no 22 pp 2775ndash27792003

[33] R S Birjmohun M Vergeer E S G Stroes et al ldquoBothparaoxonase-1 genotype and activity do not predict the risk offuture coronary artery disease the EPIC-Norfolk prospectivepopulation studyrdquo PLoS ONE vol 4 no 8 Article ID e68092009

[34] M Sentı M Tomas J Vila et al ldquoRelationship of age-relatedmyocardial infarction risk and GlnArg 192 variants of thehuman paraoxonase1 gene the REGICOR studyrdquo Atherosclero-sis vol 156 no 2 pp 443ndash449 2001

[35] M Wang X Lang L Zou S Huang and Z Xu ldquoFour geneticpolymorphisms of paraoxonase gene and risk of coronary heartdisease a meta-analysis based on 88 case-control studiesrdquoAtherosclerosis vol 214 no 2 pp 377ndash385 2011

[36] A Kassai L Illyes H ZMirdamadi et al ldquoThe effect of atorvas-tatin therapy on lecithin cholesterol acyltransferase cholesterylester transfer protein and the antioxidant paraoxonaserdquoClinicalBiochemistry vol 40 no 1-2 pp 1ndash5 2007

[37] S Deakin S Guernier and R W James ldquoPharmacogeneticinteraction between paraoxonase-1 gene promoter polymor-phism C-107T and statinrdquo Pharmacogenetics and Genomics vol17 no 6 pp 451ndash457 2007

[38] M Tomas M Sentı F Garcıa-Faria et al ldquoEffect of simvastatintherapy on paraoxonase activity and related lipoproteins infamilial hypercholesterolemic patientsrdquoArteriosclerosisThrom-bosis and Vascular Biology vol 20 no 9 pp 2113ndash2119 2000

[39] S C Bergheanu A Van Tol G M Dallinga-Thie et al ldquoEffectof rosuvastatin versus atorvastatin treatment on paraoxonase-1 activity in men with established cardiovascular disease and alow HDL-cholesterolrdquo Current Medical Research and Opinionvol 23 no 9 pp 2235ndash2240 2007

[40] N Santanam and S Parthasarathy ldquoAspirin is a substratefor paraoxonase-like activity implications in atherosclerosisrdquoAtherosclerosis vol 191 no 2 pp 272ndash275 2007

[41] M C Blatter-Garin B Kalix S De Pree and R W JamesldquoAspirin use is associated with higher serum concentrations ofthe anti-oxidant enzyme paraoxonase-1rdquo Diabetologia vol 46no 4 pp 593ndash594 2003

[42] M Waterman B Fuhrman S Keidar and T Hayek ldquoAspirinpromotes low density lipoprotein susceptibility to oxidative

modification in healthy volunteersrdquo Israel Medical AssociationJournal vol 11 no 12 pp 730ndash734 2009

[43] J Marsillach J Camps N Ferre et al ldquoParaoxonase-1 is relatedto inflammation fibrosis and PPAR delta in experimental liverdiseaserdquo BMC Gastroenterology vol 9 article 3 2009

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


binding affinity of HDL and PON1 [10] Homozygotes 192QQand 55MM show the highest protection from LDL oxidationwhile 192RR and 55LL homozygotes show LDL with theleast resistance to oxidation [11] Genetic factors includingpolymorphisms are responsible for more than 60 of thephenotypic variability of PON1 Demographic factors suchas age and sex account for 1ndash6 of PON1 variability whilemetabolic variables account for about 4ndash19 [12] Eatinghabits physical activity and smoking also modify serumPON1 activity and oxidative stress [13] Thus far publishedstudies highlighted that statins can have a beneficial influencenot only by their lipid-lowering effect but also throughdiminishing oxidative stress and enhancing PON1 activityThe improvement in PON1 activity after statin therapycould be generated by oxidative stress reduction a directeffect on promoter activity and PON1 gene expression [14]Additionally patients with coronary artery disease (CAD)present lower serum PON1 activity This was found to be apredictor of CAD although not all studies have confirmedthis relationship [15] A weak association of CADwith Q192Rpolymorphism was demonstrated in prior research [16] Theleading inflammation marker is high sensitivity C-reactiveprotein (hs-CRP) which is substantially regulated by tumornecrosis factor 120572 (TNF-120572) The increased expression of TNF-120572 intensifies production of reactive oxygen species (ROS) anddecreases bioavailability of NO leading to endothelial dys-function [17] Statins present anti-inflammatory propertiesHowever the effect of statins on fibrinogen is inconsistent[18 19] Risk factors of atherosclerosis enhance expressionof NADPH oxidase leading to significant generation ofO2

minus and consequently depletion of NO bioavailabilityStatins improve endothelial function in patients with hyper-cholesterolemia and atherosclerosis [20] The ldquofree radicalchain reactionrdquo initiated by ROS results in the formation ofadditional reactive compounds including F

2120572-isoprostanes

which is largely represented by 8-iso-prostaglandin-F2120572

(8-iso-PGF

2120572) Higher concentrations of 8-iso-PGF

2120572and its

metabolites were demonstrated in patients with CAD and8-iso-PGF

2120572is a marker of CAD [21] Statins decrease

oxidative stress by inhibition of oxidative stress in theendothelium their effects on cardiac muscle cells and theirinfluence on PON1 activity At this time the dominatingtheory is that statin therapy reduces oxidative stress markers[22] The mechanism of PON1rsquos protective effects seems tobe tightly associated with the antioxidant potential of theenzyme PON1 scavenges oxidative stress products leadingto improved endothelial function especially in patients withCAD The effect of statin therapy on PON1 activity in CADpatients treated with simvastatin for 12 months has yet to beevaluated while shorter studies did not provide conclusiveresults Also research assessing the relationship of Q192Rand L55M polymorphisms with CAD produced inconclusiveresults

The main goal of the present study was to assess theeffect of 12 months of simvastatin treatment on serum PON1activity and urinary 8-isoprostanes in patients with CADThepresent study also aimed to evaluate the association of Q192Rand L55M PON1 gene polymorphisms on PON1 activity withand without simvastatin treatment The effect of simvastatin

treatment on endothelial function intima-media thickness(IMT) proinflammatory marker concentrations and lipidprofile was also assessed to confirm parallel simvastatinefficacy

2 Subjects and Methods

21 Subjects Thepresent studywas performed on 53 patientsaged 35 to 65 years with stable CAD (based on imagingstudy angiocomputed tomography of coronary arteries orcoronarography echocardiography and exercise treadmilltest with electrocardiogram) The control group consisted of53 active professional police officers without CAD Twenty-two obese individuals were included in the patients groupand 18 in the control group All participants of the presentstudy qualified for the simvastatin therapy were permitted tocontinue treatment with aspirin 75ndash100mgday 120573-blockersangiotensin-converting enzyme inhibitors calcium channelblockers diuretics and nitrates at a stable dose throughoutthe study period Some participants in the control groupremained on treatment due to concomitant diseases Patientswho required a change in chronic therapy during the studyperiod were excluded from the study Patients were qualifiedfor simvastatin treatment according to the current ESCguidelines Individuals who had an acute coronary episodein the last 3 months heart failure LDL lt 18mmolLand triglycerides level gt 45mmolL and were previouslytreated with lipid-lowering agents or oral anticoagulantswere excluded from the study Also patients with recentlyperacted infections diabetes impaired renal and liver func-tion myopathy and neoplastic and thyroid disease were notincluded in the study Participants with CAD were treatedwith simvastatin 40mg daily for 12 months Throughout thepresent study participants were restricted from nonsteroidalanti-inflammatory drugs and maintained a stable level ofphysical activity and diet The percentage of smokers inthe two groups also did not change significantly duringthe present study The lipid profile flow-mediated dilation(FMD) intima-media thickness (IMT) serum fibrinogenCRP and TNF-120572 as well as urine 8-iso-PGF

2120572and serum

PON1 activity were evaluated at baseline after 6 and 12months of simvastatin treatment in the CAD group and atbaseline and after 12months in the control group PON1 poly-morphisms (Q192R and M55L) were assessed at baseline inall participants Blood samples were collected after a 14-hourovernight fast and FMD measurements were assessed thefollowing day Patients were asked to refrain from smoking 12hours prior to the blood test Blood samples for the analysis offibrinogen hs-CRP lipid profile and creatinine were drawnfrom the forearm vein (between 600 and 800 am) after30min rest and immediately sent to the laboratory Blood andurine samples for further analysis (PON1 activity PON1 genepolymorphisms TNF-120572 urine 8-iso-PGF

2120572 and creatinine)

were frozen and stored at minus 80 degrees centigradeNo signs of myopathy or liver function impairment wereobserved during the studyThepresent studywas approved bythe local Ethics Committee and conducted according to theethical standards outlined in the Declaration of Helsinki and









2120572




3 Results





2120572












































41732

CAD group

41831

Controls

RRQR

QQ RRQR

QQ

(a)

CAD groupControls

p lt 00001

0

50

100

150

200

250

300

350

400

450

PON

1 ac

tivity

(UL

)


(b)




















14

31

8

CAD group

LLLM

MM LLLM

MM

18

18

17

Controls

(a)

CAD groupControls

NS


50

100

150

200

250

PON

1 ac

tivity

(UL

)

(b)






























4 Discussion






















Competing Interests


Acknowledgments


References































2119905119903119886119899119904-isoprostane in






















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























2120572




3 Results





2120572












































41732

CAD group

41831

Controls

RRQR

QQ RRQR

QQ

(a)

CAD groupControls

p lt 00001

0

50

100

150

200

250

300

350

400

450

PON

1 ac

tivity

(UL

)


(b)




















14

31

8

CAD group

LLLM

MM LLLM

MM

18

18

17

Controls

(a)

CAD groupControls

NS


50

100

150

200

250

PON

1 ac

tivity

(UL

)

(b)






























4 Discussion






















Competing Interests


Acknowledgments


References































2119905119903119886119899119904-isoprostane in






















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















































41732

CAD group

41831

Controls

RRQR

QQ RRQR

QQ

(a)

CAD groupControls

p lt 00001

0

50

100

150

200

250

300

350

400

450

PON

1 ac

tivity

(UL

)


(b)




















14

31

8

CAD group

LLLM

MM LLLM

MM

18

18

17

Controls

(a)

CAD groupControls

NS


50

100

150

200

250

PON

1 ac

tivity

(UL

)

(b)






























4 Discussion






















Competing Interests


Acknowledgments


References































2119905119903119886119899119904-isoprostane in






















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















41732

CAD group

41831

Controls

RRQR

QQ RRQR

QQ

(a)

CAD groupControls

p lt 00001

0

50

100

150

200

250

300

350

400

450

PON

1 ac

tivity

(UL

)


(b)




















14

31

8

CAD group

LLLM

MM LLLM

MM

18

18

17

Controls

(a)

CAD groupControls

NS


50

100

150

200

250

PON

1 ac

tivity

(UL

)

(b)






























4 Discussion






















Competing Interests


Acknowledgments


References































2119905119903119886119899119904-isoprostane in






















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















14

31

8

CAD group

LLLM

MM LLLM

MM

18

18

17

Controls

(a)

CAD groupControls

NS


50

100

150

200

250

PON

1 ac

tivity

(UL

)

(b)






























4 Discussion






















Competing Interests


Acknowledgments


References































2119905119903119886119899119904-isoprostane in






















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






























4 Discussion






















Competing Interests


Acknowledgments


References































2119905119903119886119899119904-isoprostane in






















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























Competing Interests


Acknowledgments


References































2119905119903119886119899119904-isoprostane in






















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Competing Interests


Acknowledgments


References































2119905119903119886119899119904-isoprostane in






















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















2119905119903119886119899119904-isoprostane in






















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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Clinical Study Paraoxonase-1 and Simvastatin Treatment in ...downloads.hindawi.com/journals/ijvm/2016/6312478.pdf · Clinical Study Paraoxonase-1 and Simvastatin Treatment in Patients