Gender-specific correlation between plasma myeloperoxidase levels and serum high-density lipoprotein-associated paraoxonase-1 levels in patients with stable and unstable coronary artery disease

Atherosclerosis 231 (2013) 308e314

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Gender-specific correlation between plasma myeloperoxidase levels and serum high-density lipoprotein-associated paraoxonase-1 levels in patients with stable and unstable coronary artery disease Kei Yunoki a, Takahiko Naruko a, Mayumi Inaba b, Takeshi Inoue c, Masashi Nakagawa d, Kenichi Sugioka d, Masahiko Ohsawa b, Yoko Iwasa b, Ryushi Komatsu a, Akira Itoh a, Kazuo Haze a, Minoru Yoshiyama d, Anton E. Becker e, Makiko Ueda b, * a

Department of Cardiology, Osaka City General Hospital, Osaka, Japan Department of Pathology, Osaka City University Graduate School of Medicine, Osaka, Japan c Department of Pathology, Osaka City General Hospital, Osaka, Japan d Department of Cardiology and Internal Medicine, Osaka City University Graduate School of Medicine, Osaka, Japan e Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 20 February 2013 Received in revised form 3 July 2013 Accepted 27 August 2013 Available online 8 October 2013

Objective: Low high-density lipoprotein (HDL) cholesterol is well-established as a negative risk factor for coronary artery disease (CAD) and its anti-oxidant property has been attributed mainly to the HDLbound enzyme paraoxonase-1 (PON-1). Recently, myeloperoxidase (MPO), a pro-oxidant enzyme released from activated neutrophils, has been shown to alter the atheroprotective function of HDL to a dysfunctional form. This study investigated the relationship between plasma MPO and serum PON-1 levels in patients with stable (SAP) and unstable angina pectoris (UAP). Methods: Plasma MPO levels and serum PON-1 concentration/activity were measured in patients with SAP (n ¼ 226), UAP (n ¼ 151) and in control subjects (n ¼ 99). Results: Plasma MPO levels in UAP patients were significantly higher than those in SAP patients or in control subjects (UAP, 21.6[16.7e44.6]; SAP, 19.3[15.7e29.1]; control, 15.9[14.7e18.7] ng/mL; P < 0.0001). Serum PON-1 concentrations in UAP and SAP patients were significantly lower than those in control subjects (UAP, 55.6[45.9e69.7]; SAP, 55.0[46.9e64.9]; control, 62.5[51.1e78.8] mg/mL; P ¼ 0.0002). Plasma MPO levels showed a weak inverse correlation with serum PON-1 concentrations in all subjects (R ¼ 0.163, P < 0.0005). Moreover, in women, plasma MPO levels showed a significant inverse correlation with serum PON-1 concentrations and PON-arylesterase activity in SAP (concentration: R ¼ 0.537, P < 0.0001; arylesterase-activity: R ¼ 0.469, P < 0.001) and UAP (concentration: R ¼ 0.340, P < 0.05; arylesterase-activity: R ¼ 0.350, P < 0.05) patients, but not in men. Conclusion: This study demonstrates that plasma MPO levels have a significant inverse correlation with PON-1 levels, especially in women, in SAP and UAP patients, and suggests that an imbalance between pro-oxidants and anti-oxidants may contribute to the progression of coronary plaque instability. Ó 2013 Elsevier Ireland Ltd. All rights reserved.

Keywords: Angina Myeloperoxidase Oxidative stress Paraoxonase

1. Introduction Inflammation and oxidative stress play a key role in the progression of atherosclerosis and plaque instability. Recent studies have focused on an identification of biomarkers for risk

* Corresponding author. Department of Pathology, Osaka City University Graduate School of Medicine, 1-4-3 Asahi-machi, Abeno-ku, Osaka 545-8585, Japan. Tel.: þ81 6 66453740; fax: þ81 6 66453742. E-mail address: [email protected] (M. Ueda). 0021-9150/$ e see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.atherosclerosis.2013.08.037

stratification in patients with acute coronary syndrome (ACS) and for improved understanding of the pathophysiology of ACS [1]. Myeloperoxidase (MPO) is a hemeprotein stored in azurophilic granules of neutrophils and monocytes. Accumulating evidence suggests that MPO, released mainly from activated neutrophils, may play a key role in mediating destabilization of atherosclerotic plaques [2]. This enzyme has been implicated in the oxidation of lipids contained within low-density lipoprotein (LDL) and thereby promoted lipid-rich plaque formation [3]. We previously demonstrated that abundant MPO-positive neutrophils are infiltrated at sites of plaque rupture or erosion in coronary culprit lesions of ACS

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patients [4]. Recently, mass assays based on an enzyme-linked immunoassay have been developed (Oxis Research and Assay Design). Using this method, several clinical studies showed the usefulness of plasma MPO levels for risk stratification in subjects presenting with chest pain [5] or with ACS [6]. Our recent studies also demonstrated that circulating MPO concentrations were higher in patients with ACS than those in patients with stable angina pectoris (SAP) [7,8] and that plasma MPO levels showed a positive correlation with plasma oxidized LDL levels in patients with acute ST-elevation myocardial infarction [9]. High-density lipoproteins (HDLs) have a well-established inverse relationship with the risk for coronary artery disease (CAD). The atheroprotective effects of HDL are mediated by its role in reverse cholesterol transport and its anti-inflammatory properties. HDLs have been shown to retard the oxidation of LDL, and this antioxidant property of HDL has been attributed largely to the HDLbound enzyme paraoxonase-1 (PON-1) which catalyses the breakdown of oxidized phospholipids in LDL [10]. Recent studies have shown that PON-1 activity and concentration are significantly lower in subjects with CAD compared with controls and are associated with severity and extent of CAD [11]. Moreover, low serum PON-1 activity has been shown to be an independent risk factor for coronary events [12]. Recently, there is growing evidence that MPO converts the normally atheroprotective HDL molecules into a dysfunctional form [13]. MPO has been shown to oxidize apolipoprotein A-I (apoA-I), the major HDL protein, and this oxidized apoA-_ inhibits cholesterol efflux by the ATP-binding cassette transporter A1 (ABCA1) pathway and impairs lecithin:cholesterol acyltransferase (LCAT), which rapidly converts free cholesterol to cholesterol ester, a critical step in HDL maturation [13]. Meanwhile, it has been shown that PON-1 enhances cholesterol efflux from macrophages by HDL binding mediated by ABCA1 [14]. Thus, MPO and HDL-associated PON-1 are potentially associated with each other and considered to be biomarkers which could reflect the pathophisiology of ACS, i.e. acute inflammatory responses, and oxidative and anti-oxidative stress. However, no headto-head comparisons between those markers have been carried out in patients with CAD. In this study, we measured blood levels of MPO and PON-1 activity and mass concentration in patients with SAP and unstable angina pectoris (UAP), and evaluated those relationships.

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antioxidant drugs. A total of 99 age- and gender-matched healthy volunteers served as controls (71 men, aged 64  10 years). Among the control subjects, none had diabetes mellitus, 25 had a history of hypertension, 40 met the diagnostic criteria for hypercholesterolemia and 34 were smokers. All 25 hypertensives were in stage I according to the criteria established by the Joint National Committee VII [17]; none used anti-hypertensive medication. Antioxidants were not administered to any controls. Serum levels of total cholesterol, HDL cholesterol, LDL cholesterol, triglyceride (TG), and creatinine, plasma levels of MPO, serum PON-1 concentration, PON activity, and PON-arylesterase activity were measured in the 2 groups of patients and in the control subjects. Serum high-sensitivity C-reactive protein (hs-CRP) levels, a leukocyte count, and a neutrophil count were also measured in the 2 groups of patients. The following data were also obtained: age, gender, body mass index, medications on admission and the presence of risk factors (cigarette smoking, hypertension as defined by the Joint National Committee VII [17], diabetes mellitus (DM) as defined by the WHO Study Group [18], and hypercholesterolemia defined by Japan Atherosclerotic Society Guideline 2002) [19], multivessel disease (2 the number of disease vessels narrowed >70% detected angiographically in the major coronary artery), arteriosclerosis obliterans, and old cerebral infarction. All patients provided written informed consent and the study was approved by the hospital ethics committee. 2.2. Biochemical analysis Venous blood samples from all patients were obtained on admission to the hospital, prior to heparin administration. For measuring total cholesterol, HDL cholesterol, LDL cholesterol and TG levels, blood samples were obtained after an overnight fast. The concentration of hs-CRP was measured by the latex agglutination photometric immunoassay with an automated immunochemistry analyzer (LXz-6000; Eiken Chemical Co., Tokyo, Japan) with normal values <0.3 mg/dL. The serum troponin T (TnT) level was determined by an enzyme-linked immunosorbent assay (ELISA) using an ES-300 immunoassay analyzer (Boehringer-Mannheim, Mannheim, Germany) with normal values <0.1 mg/L. 2.3. Measurements of MPO, PON-1 concentration, and PON-1 enzyme activities

2. Methods 2.1. Study populations Study population contained 377 patients with either SAP or UAP, who admitted to our hospital (Osaka City General Hospital). SAP was diagnosed in 226 patients and defined as chest pain typical of cardiac ischemia on exertion [15]. UAP was diagnosed in 151 patients, who were defined as new-onset angina within 2 months after a previous bout; angina with a progressive crescendo pattern, with the anginal episodes increasing in frequency and/or duration; angina that occurred at rest [16]. The UAP patients were further divided into class I (n ¼ 58), class II (n ¼ 9), and class III (n ¼ 84), according to Braunwald’s criteria [16]. All patients had primary unstable angina, corresponding to subclass “B”. All patients had undergone coronary angiography and had angiographically documented narrowing of at least 70% of the luminal diameter of a major coronary artery. We excluded patients with variant angina, concomitant inflammatory diseases, or malignant tumors, and patients undergoing dialysis. Of all 377 patients, only 3 patients with SAP were treated with antioxidant drugs (3 patients with probucol); the remaining 374 patients did not receive any

Plasma MPO levels were measured with an ELISA method (Oxis) according to procedures previously reported [5]. PON-1 activity and mass concentration were measured at the clinical reference laboratory of BML Inc. (Saitama, Japan). PON-1 concentration was determined by sandwich ELISA using two different monoclonal antibodies, as previously described by Kujiraoka et al. [20]. The inter- and intra-assay coefficients of variation were <3.8% and <8.7%, respectively. PON-1 enzyme activities were analyzed according to Eckerson et al. [21], using both paraoxon (PON activity) and phenylacetate (PON-arylesterase activity) as substrates with a modification by adapting the procedure to a Prestage autoanalyzer (TOKYO BOEKI LTD., Tokyo, Japan). PON activity was defined as 1 nmol of p-nitrophenol liberated by the hydrolysis of paraoxon per minute at 412 nm at 37  C at pH 10.5 [22]. The ε412 for p-nitrophenol was 18,290 M1 cm1. PON-arylesterase activity was defined as 1 mmol of phenol liberated by the hydrolysis of phenylacetate per minute at 37  C at pH 8.0 [22]. The liberated phenol by hydrolysis was converted to the colored compound of 4– (p-benzoqunone-monoimino)-phenazone2,6-dichloro-4acetylphenol with 4-aminoantipyrine and measured the absorbance at l ¼ 510 nm [22]. The inter- and intra-assay coefficients of

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variation were <3.8% and <5.2% for PON activity, and <3.9% and <4.6% for PON-arylesterase activity, respectively [20]. Furthermore, the polymorphism (rs662) of glutamine (Q) or arginine (R) at codon 192 in the PON-1 gene was determined by the ratio of the PON/ PON-arylesterase activity, which was completely identical to the PON-1 genotype measured by the polymerase chain reactionrestriction fragment length polymorphism method as previously reported [23] and showed a clear separation among all three PON-1 phenotypes without overlapping between the groups in this study (data not shown). 2.4. Statistical methods The results for normally-distributed continuous variables are expressed as mean  SD, and continuous variables that did not have a normal distribution are presented as median and corresponding interquartile ranges. The ManneWhitney U test was used to compare two groups, and the nonparametric KruskaleWallis test was used to compare three groups. If a significant difference was found with the KruskaleWallis test, then the ManneWhitney U test was used to compare each pair of groups. Categorical variables were compared by use of c2 test. Pearson correlation coefficients were calculated to assess the relationships between plasma MPO levels and serum HDL cholesterol, PON-1 concentration, PON activity and PON-arylesterase activity. All analyses used two-sided tests with a significant level of P < 0.05. Data were analyzed using SPSS version 16.0 for Windows (SPSS Inc., Chicago, IL, USA). 3. Results 3.1. Characteristics of CAD patients and control subjects Characteristics of CAD patients and control subjects are shown in Table 1. Regarding risk factors, the frequency of multivessel disease, and the presence of arteriosclerosis obliterans, old cerebral infarction, there were no significant differences between patients with SAP and UAP. Regarding inflammatory markers, leukocyte counts (P < 0.005), neutrophil counts (P < 0.005) and hs-CRP levels (P < 0.01) in patients with UAP were significantly higher than those in SAP patients. Table 2 shows gender differences in patient characteristics, coronary risk factors and biomarkers. Mean age at presentation was significantly older in women than in men with SAP (P < 0.05) and UAP (P < 0.01) patients. Moreover, HDL cholesterol levels in women were significantly higher than those in men with control group and SAP (control, P < 0.05; SAP, P < 0.01), however, gender differences in HDL cholesterol levels were not found in patients with UAP. 3.2. Plasma MPO and serum HDL-associated PON-1 levels in SAP and UAP patients As shown in Fig. 1A, plasma MPO levels in UAP patients were significantly higher than those in SAP patients (P < 0.01) or in control subjects (P < 0.0001). The levels of plasma MPO in SAP patients were also significantly higher than those in control subjects (P < 0.0001) (UAP, 21.6[16.7e44.6]; SAP, 19.3[15.7e29.1]; control, 15.9[14.7e18.7] ng/mL). Gender differences in MPO levels were not found among groups in this study (Table 2). Among the 151 patients with UAP, plasma MPO levels in patients with Braunwald class III were significantly higher than those in patients with class I (P < 0.001) (class I, 18.4[15.6e27.2]; class II, 18.0[15.5e38.0]; class III, 28.0[18.0e57.7] ng/mL). Fig. 1B shows that HDL cholesterol levels in patients with UAP or SAP were significantly lower than those in control subjects (P < 0.0001, respectively). As shown in Fig. 1C, serum PON-1

Table 1 Baseline clinical characteristics. Control (n ¼ 99)

UAP (n ¼ 151)

66  10 174/52(77/23)

65  11 109/42(72/28)

23.9  3.2 152(67) 142(63)

24.1  3.1 107(71) 91(60)

0.053 <0.0001 0.0006

88(39) 141(62) 0.89  0.31 186  35

54(36) 88(58) 0.90  0.38 189  39

<0.0001 <0.0001 <0.0001 0.07

44  12 105  32 152  93 6169  1711 3677  1324 0.09(0.05 e0.20) 19.3(15.7 e29.1) 55.0(46.9 e64.9) 278(216e366)

43  12 103  39 124  72 6959  2266 4284  1970 0.16(0.06 e0.36) 21.6(16.7 e44.6) 55.6(45.9 e69.7) 258(192e338)

<0.0001 0.16 0.0003 0.0017 0.0036 0.0054

131(107e153)

124(102e152)

0.0497

e e e

e 113(50) 44(19)

42(28) 76(50) 31(21)

e

20(9)

15(10)

0.72

e e e e e e

167(74) 77(34) 67(30) 108(48) 77(34) 87(38)

83(55) 50(33) 44(29) 79(52) 48(32) 36(24)

0.0001 0.85 0.92 0.39 0.71 0.003

e

86(38)

34(23)

0.0015

64  10 71/28(72/ 28) Body mass index, kg/m2 23.2  2.1 Hypertension, (%) 25(25) Hypercholesterolemia, 40(40) (%) Diabetes mellitus, (%) 0(0) Smoking, (%) 34(34) Creatinine, mg/dL 0.75  0.15 Total cholesterol, mg/ 193  36 dL HDL cholesterol, mg/dL 54  16 LDL cholesterol, mg/dL 107  28 TG, mg/dL 113  50 Leukocyte count,/mm3 e 3 Neutrophil count,/mm e Hs-CRP, mg/dL e

PON-1 concentration, mg/mL PON activity, nmol/ min/mL PON-arylesterase activity, mmol/min/ mL TnT > 0.1 mg/L, (%) Multivessel disease, (%) Arteriosclerosis obliterans, (%) Old cerebral infarction, (%) Medications on admission, (%) Antiplatelets ACE-I/ARB b-blockers Ca antagonists Nitrates Lipid-lowering agents (statin, fibrate, and probucol) Statin

P

SAP (n ¼ 226)

Age, yrs Men/Women, (%)

MPO, ng/mL

CAD (n ¼ 377)

15.9(14.7 e18.7) 62.5(51.1 e78.8) 294(225 e385) 132(113 e167)

0.41 0.46

<0.0001 0.0002 0.08

e 0.95 0.80

Data are expressed as mean  SD, median (interquartile range), or n(%). CAD, coronary artery disease; SAP, stable angina pectoris; UAP, unstable angina pectoris; HDL, high-density lipoprotein; LDL, low-density lipoprotein; TG, triglyceride; HsCRP, high-sensitivity C-reactive protein; MPO, myeloperoxidase; PON, paraoxonase; TnT, troponin T; ACE-I, angiotensin converting enzyme inhibitors; ARB, angiotensin Ⅱ type 1 receptor blockers.

concentrations were significantly decreased in patients with UAP (P < 0.005) or SAP (P < 0.0001) compared with those in control subjects (UAP, 55.6[45.9e69.7]; SAP, 55.0[46.9e64.9]; control, 62.5 [51.1e78.8] mg/mL). Moreover, as shown in Table 1, serum PONarylesterase activity in patients with UAP was also significantly lower than that in control subjects (P < 0.05) (UAP, 124[102e152]; SAP, 131[107e153]; control, 132[113e167] mmol/min/mL). Serum PON activity in UAP patients tended to be lower than that in SAP patients or in control subjects, but was not significantly different (P ¼ 0.08) (UAP, 258[192e338]; SAP, 278[216e366]; control, 294 [225e385] nmol/min/mL). However, in women, serum PON activity in UAP patients was significantly lower than that in control subjects (P < 0.05) (UAP, 238[188e324]; SAP, 256[202e366]; control, 347 [274e395] nmol/min/mL) (Table 2). Gender differences in PON-1 concentrations and activities were not found except for serum PON activity in women with control group (P < 0.05), but PON-1

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3.3. Influence of inflammation, myocardial damage and medication status on plasma MPO and serum HDL-associated PON-1 levels

Table 2 Baseline clinical characteristics stratified by gender lines. Control (n ¼ 99)

CAD (n ¼ 377) SAP (n ¼ 226)

Age, yrs Men 64  10 65  10 Women 66  10 69  10* Hypertension, (%) Men 20(28) 107(62) Women 5(18) 45(87)z Hypercholesterolemia, (%) Men 24(34) 105(60) Women 16(57) 37(71) Diabetes mellitus, (%) Men 0(0) 69(40) Women 0(0) 19(37) Smoking, (%) Men 28(39) 127(73) Women 6(21) 14(27)x Total cholesterol, mg/dL Men 185  37 185  36 Women 211  26z 191  32 HDL cholesterol, mg/dL Men 51  15 43  11 Women 59  16* 48  12y LDL cholesterol, mg/dL Men 102  28 106  31 Women 117  26* 101  35 TG, mg/dL Men 119  53 150  88 Women 99  39 160  107 MPO, ng/mL Men 15.6(14.5e18.7) 19.1(15.3e29.3) Women 16.9(15.1e19.4) 19.7(16.5e28.1) PON-1 concentration, mg/mL Men 62.0(50.4e73.0) 54.2(46.3e63.8) Women 67.9(51.6e87.5) 58.6(49.7e68.8) PON activity, nmol/min/mL Men 265(203e385) 285(217e369) Women 347(274e395)* 256(202e366) PON-arylesterase activity, mmol/min/mL Men 130(111e157) 128(104e153) Women 145(122e186) 133(115e154)

311

P UAP (n ¼ 151) 63  11 69  8y

0.47 0.34

71(65) 36(86)*

<0.0001 <0.0001

63(58) 28(67)

0.0005 0.45

43(39) 12(29)

<0.0001 0.0004

74(68) 14(33)x

<0.0001 0.54

184  35 201  45y 43  12 43  12

0.51 0.03 <0.0001 <0.0001

99  38 113  41*

0.047 0.03

122  69 130  80

0.0039 0.02

21.6(16.7e51.5) 22.4(16.9e41.6)

<0.0001 0.0077

55.7(45.7e70.8) 55.2(48.6e68.1)

0.0056 0.02

262(192e352) 238(188e324)

0.41 0.01

121(98.5e152) 130(110e159)

0.17 0.23

Data are expressed as mean  SD, median (interquartile range), or n(%). Abbreviations are as in Table 1. *p < 0.05, yp < 0.01, zp < 0.005, xp < 0.0001, men vs. women at each groups.

concentrations in women with control group (P ¼ 0.08) and SAP (P ¼ 0.09) tended to be higher than those in men (Table 2). There were no significant differences in the frequency of the genetic polymorphism of Q192R among groups and between men and women (see Supplement Table).

Significant inverse correlations between hs-CRP levels and serum HDL cholesterol, PON-1 concentrations/PON-arylesterase activity were observed between SAP and UAP patients, especially among women than men (see Supplement Fig. 1AeC). Of the 151 patients with UAP, 109 patients (72%) had negative TnT levels (<0.1 mg/L). There were no significant differences in plasma MPO and serum HDL cholesterol/PON-1 levels between UAP patients with positive and negative TnT and between men and women with positive and negative TnT (see Supplement Fig. 2). Relationships between medication status on admission and plasma MPO, serum PON-1 levels were shown in Supplement Fig. 3AeC. 3.4. Correlation between plasma MPO, serum HDL cholesterol and PON-1 levels Fig. 2A shows the relationships between plasma MPO levels and serum HDL cholesterol, PON-1 concentrations, PON activity, and PON-arylesterase activity in patients with SAP and UAP and in control subjects. Plasma MPO levels showed a weak inverse correlation with serum HDL cholesterol (R ¼ 0.222, P < 0.0001), PON1 concentrations (R ¼ 0.163, P ¼ 0.0004), PON activity (R ¼ 0.129, P ¼ 0.0047), and PON-arylesterase activity (R ¼ 0.174, P ¼ 0.0001). Fig. 2B shows the relationships between serum HDL cholesterol levels and PON-1 concentrations, PON activity, and PON-arylesterase activity in patients with SAP and UAP and in control subjects. HDL cholesterol levels showed a significant positive correlation with serum PON-1 concentrations (R ¼ 0.410, P < 0.0001), PON activity (R ¼ 0.249, P < 0.0001), and PONarylesterase activity (R ¼ 0.373, P < 0.0001). Fig. 3 shows the gender-specific relationships between plasma MPO levels and serum PON-1 levels in control subjects and in SAP and UAP patients. In women, plasma MPO levels showed a significant inverse correlation with serum PON-1 concentrations and PON-arylesterase activity in SAP (PON-1 concentration: R ¼ 0.537, P < 0.0001; PON-arylesterase activity: R ¼ 0.469, P < 0.001) and UAP (PON-1 concentration: R ¼ 0.340, P < 0.05; PON-arylesterase activity: R ¼ 0.350, P < 0.05) patients. However, in men, plasma MPO levels were not correlated with serum PON-1 concentrations and PON-arylesterase activity in patients with SAP and UAP.

Fig. 1. A, C: Box plot shows median (horizontal lines), 25th to 75th percentiles (boxes), and 10th to 90th percentiles (whiskers) of plasma MPO levels (A) and serum PON-1 concentrations (C) in SAP and UAP patients, and in control subjects. B: Serum HDL cholesterol levels in SAP and UAP patients, and in control subjects. N indicates number of patients and controls analyzed.

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Fig. 2. A: Relationships between plasma MPO levels and serum HDL cholesterol levels, PON-1 concentrations, PON activity, and PON-arylesterase activity in all populations (control þ SAP þ UAP). B: Relationships between serum HDL cholesterol levels and serum PON-1 concentrations, PON activity, and PON-arylesterase activity in all populations (control þ SAP þ UAP).

Fig. 3. A: Relationships between plasma MPO levels and serum PON-1 concentrations in women of control subjects, SAP and UAP patients. B: Relationships between plasma MPO levels and serum PON-arylesterase activity in women of control subjects, SAP and UAP patients.

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4. Discussion To the best of our knowledge, the present study is the first to demonstrate the relationship between MPO levels and PON-1 concentration and activity in the blood in SAP and UAP patients and in control subjects. The most striking data found in our study is that plasma MPO levels showed a significant inverse correlation with serum PON-1 levels, especially in women with SAP and UAP. There is accumulating evidence that MPO may play a pivotal role in plaque instability. Our previous immunohistochemical study clearly demonstrated that most MPO-positive cells present at coronary culprit lesions of ACS patients were neutrophils, and that the number of neutrophils was significantly higher in the culprit lesions of UAP patients than in those of SAP patients [4]. MPO release has been shown to be a sign of neutrophil activation [2], and it has been reported that an intense and short-lasting burst of neutrophil activation occurs in ACS patients [24] and such widespread neutrophil activation occurs across the coronary vascular bed [25]. Therefore, these findings suggest that increased MPO levels may be a marker of plaque vulnerability and instability. Indeed, our previous data and the present study showed that circulating MPO concentrations are significantly higher in ACS patients than in SAP patients [7,8]. Hence, one could hypothesize that neutrophil infiltration and activation and MPO release at sites of inflammatory reactions and thrombosis in unstable plaques could contribute to an increase in MPO levels in the blood of UAP patients. Recent studies have focused on the balance between prooxidants and anti-oxidants in human blood. PON-1 has been suggested to be a primary determinant of the anti-oxidant capacity of HDL [10e12]. In the present study, PON-1 concentrations were significantly decreased in SAP and UAP patients compared with those in control subjects. Moreover, PON-arylesterase activity was also significantly lower in UAP patients compared with those in control subjects. These findings are in agreement with recent reports [11]. However, in the present study, PON activity using paraoxon as substrate, did not show such an association. This discrepancy between PON activity levels and PON-arylesterase activity levels may be explained, at least in part, by the effects of the polymorphism of PON-1 gene, as reported previously [11,26e 28]. In this study, significant inverse correlations between hs-CRP levels and serum HDL cholesterol, PON-1 concentrations/PONarylesterase activity were observed, and several previous studies have shown the linkage between HDL cholesterol and CRP [29,30]. We also found that plasma MPO levels were inversely correlated with HDL cholesterol and serum PON-1 concentrations and activities. Mediators of inflammation have been shown to induce major changes in HDL levels and HDL apolipoprotein content, so that PON-1 levels decrease, thus reducing the antioxidant properties of HDL and transforming normal HDL into dysfunctional form [31e 33]. Jornayvaz et al. reported recently that MPO is an independent, negative determinant of PON-1 activity in type 2 diabetic patients [34]. This raises the possibility that an interaction of MPO on HDL in the circulation may decrease serum PON-1 levels, and speculates that a balance between pro-oxidant stimuli and antioxidant defences may be disrupted in the blood of unstable patients. Moreover, these data support the hypothesis that both increased MPO levels and decreased PON-1 levels contribute to the development of ACS, and that an imbalance between pro-oxidants and anti-oxidants may play an important role in the progression of coronary plaque instability in humans. Recently, an evolving knowledge about fundamental physiopathologic differences between men and women regarding cardiovascular risk factors has emerged. Actually, gender differences in the presentation, prevalence, and clinical outcomes of CAD have been revealed and, in the classical cardiovascular risk factors, HDL

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cholesterol in women has particularly been demonstrated to be higher than in men throughout all ages. Therefore, it is necessary to take into account gender differences in the features of cardiovascular risk and treatment between men and women [35], and cardioprotective effect of sex hormones has been proposed as one of several mechanisms associated with gender differences. An epidemiologic study by Trevisan et al. demonstrated that postmenopausal status in women was associated with increased oxidative stress and reduced antioxidant potentials [36]. In fact, several studies have shown that serum oxidative stress markers increase and anti-oxidative markers decrease significantly after menopause [37] or bilateral ovariectomy [38]. Very recently, Vassalle et al. further reported that the oxidative stress index, reflecting the oxidant/anti-oxidant balance, was a powerful predictor of CAD in postmenopausal women [39]. Hormone replacement therapy (HRT) has been shown to have a preventive effect on an oxidative/anti-oxidative balance, and some studies actually demonstrated that HRT reduces MPO levels and increases PON-1 levels [40,41]. These results might be partially due to the fact that estradiol has recently been identified as a potential endogenous substrate for MPO in plasma [42]. Thus, there is a possibility that plasma MPO levels may relate to gender-specific differences in cardiovascular risks, and Hazen reported that plasma MPO levels showed a tendency toward being a stronger predictor of cardiac events in women than in men [43]. The pathophysiological mechanism responsible for this gender-specific difference has not yet been fully clarified, but these data may support our present finding of significant inverse relationship between plasma MPO and serum PON-1 levels in women with SAP and UAP. Moreover, these data led us to hypothesize that the depletion of estrogen in postmenopause could enhance an imbalance between pro-oxidants and antioxidants, and that plasma MPO and serum HDL-associated PON-1 are potential biomarkers associated with the gender-specific differences in the pathophysiology of ACS in humans. 4.1. Study limitation First, in our study, there were no DM patients in the control group. Since DM status greatly influences on inflammation and oxidative/anti-oxidative stress status irrespective of the presence of CAD, there was a certain degree of selection bias, which might affect our present results. Second, there were 3 common PON-1 polymorphism: Q192R, L55M, and the promoter polymorphism T(-107)C and these polymorphism were well known to strongly affect PON-1 activity and concentrations [44]. Especially, some, but not all, investigators reported that the glutamine isotype at codon 192 (QQ) and methionine at codon 55 (MM), which had been related to lower PON activity, were associated with increased CAD risk [27,45]. In the present study, the frequency of Q192R polymorphism was not different among the 3 groups. Moreover, the number of carriers of the 55M homozygote is very small (<10%) in Japanese. Therefore, these genetic polymorphism of PON-1 do not greatly influence on our conclusion regarding the inverse correlation between plasma MPO and serum PON-1 levels in SAP and UAP patients. 5. Conclusions The present study demonstrated for the first time that plasma MPO levels have a significant inverse correlation with serum PON-1 concentrations and PON-arylesterase activity in SAP and UAP patients, especially in women. Our results may help understanding not only of gender-specific differences in the pathophysiology of ACS, but also of availability of the biomarkers for oxidative and antioxidative stress in the clinical setting. Furthermore, by revealing

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