Circulating malondialdehyde-modified LDL and atherogenic lipoprotein profiles measured by nuclear magnetic resonance spectroscopy in patients with coronary artery disease

Circulating malondialdehyde-modified LDL and atherogenic lipoprotein profiles measured by nuclear magnetic resonance spectroscopy in patients with coronary artery disease

Atherosclerosis 179 (2005) 139–145 Circulating malondialdehyde-modified LDL and atherogenic lipoprotein profiles measured by nuclear magnetic resonan...

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Atherosclerosis 179 (2005) 139–145

Circulating malondialdehyde-modified LDL and atherogenic lipoprotein profiles measured by nuclear magnetic resonance spectroscopy in patients with coronary artery disease Tetsuro Miyazakia , Kazunori Shimadaa,∗ , Osamu Satob , Kazuo Kotanib , Atsumi Kumea , Katsuhiko Sumiyoshia , Yayoi Satoa , Hirotoshi Ohmuraa , Yoshiro Watanabea , Hiroshi Mokunoa , Hiroyuki Daidaa a

Department of Cardiology, Juntendo University School of Medicine, 2-1-1 Hongo Bunkyo-ku, Tokyo 113-8421, Japan b R&D Division, Daiichi Pure Chemicals, Iwate, Japan Received 28 April 2004; received in revised form 11 August 2004; accepted 16 September 2004 Available online 13 November 2004

Abstract Objective: Recent studies have suggested that circulating malondialdehyde-modified low-density lipoprotein (MDA-LDL) is a useful marker for the identification of patients with coronary artery disease (CAD). However, the role of MDA-LDL in atherogenic mechanisms has not yet been fully determined. Method and Results: We investigated lipoprotein profiles measured by nuclear magnetic resonance (NMR) spectroscopy and circulating MDA-LDL levels measured by ELISA in 25 male patients with CAD and 15 age-matched male controls. We selected subjects who had a serum LDL cholesterol < 160 mg/dL. The MDA-LDL levels were significantly higher in the CAD group than in the control group (P = 0.01) even though there was no significant difference in the LDL cholesterol levels between the two groups. NMR analysis demonstrated that the MDA-LDL levels were positively correlated with large and intermediate very low-density lipoprotein triglyceride and LDL particle concentrations, and negatively correlated with LDL diameter and large high-density lipoprotein cholesterol. The MDA-LDL levels were negatively correlated with flow-mediated dilatation of the brachial artery. Conclusions: The high concentrations of circulating MDA-LDL derived from the atherogenic lipoprotein profiles, which induce the exaggerated production of small dense LDL. The circulating MDA-LDL may impair endothelial function and play an important role in the pathogenesis of atherosclerosis. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Oxidized low-density lipoprotein; Endothelial function; Nuclear magnetic resonance spectroscopy; Metabolic syndrome; Coronary artery disease

1. Introduction Numerous studies have shown that oxidative modification of low-density lipoprotein (LDL) plays an important role in the pathogenesis of atherosclerosis [1,2]. Oxidized LDL is incorporated into macrophages by scavenger receptors and modulates various vascular functions, including the inhibi∗

Corresponding author. Tel.: +81 3 5802 1056; fax: +81 3 5689 0627. E-mail address: [email protected] (K. Shimada).

0021-9150/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2004.09.013

tion of nitric oxide (NO) generation, induction of endothelial apoptosis, proliferation of smooth muscle cells, and activation of proinflammatory molecule production in endothelial and smooth muscle cells [3]. Until recently, autoantibody titers against oxidized LDL [4] or oxidative susceptibility of LDL [5,6] has been measured to estimate the levels of LDL oxidation; however, these methods have been limited by the characteristics of their indirect estimation of oxidized LDL. Recently, direct assay system of circulating malondialdehyde-modified LDL (MDA-LDL), which is one of the

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candidates of oxidized LDL, was established. It has been suggested that levels of circulating MDA-LDL could be a useful indicator for the identification of patients with coronary artery disease (CAD) [7,8]. However, the relationship between circulating MDA-LDL and the detailed lipoprotein profiles, and the role of MDA-LDL in atherogenic mechanisms have not yet been fully determined. Nuclear magnetic resonance (NMR) spectroscopy is a newly developed method of measuring lipoprotein levels. It enables to measure the lipoprotein profiles by means of quantification based not on the cholesterol or triglyceride content, but on the detected amplitudes of spectral signals emitted by lipoprotein subclasses of different sizes. Furthermore, it can provide the detailed information of atherogenic lipoprotein profiles beyond merely an increased level of increased LDL cholesterol, by including high levels of LDL particles and large very low-density lipoprotein (VLDL) triglyceride and low levels of large HDL cholesterol [9–11]. To clarify the role of MDA-LDL in the pathogenesis of atherosclerosis and metabolic defects, which are associated with high levels of circulating MDA-LDL, we investigated the relationship between circulating MDA-LDL levels determined by enzyme-linked immunosorbent assay (ELISA) and the lipoprotein profiles measured by NMR spectroscopy in patients with normal or borderline-high levels of LDL cholesterol with CAD and in the controls. Furthermore, we examined the correlation of serum levels of MDA-LDL with endothelial dysfunction analyzed from flowmediated endothelium-dependent dilatation (FMD) of the brachial artery.

2. Material and method 2.1. Subjects We studied 25 male patients who had angiographic documentation of CAD and 15 age-adjusted healthy male volunteers. Angiograms of the CAD group showed more than 50% stenosis of at least one major coronary artery, and the control group had no clinical evidence of CAD. We selected subjects who had a serum LDL cholesterol concentration of less than 160 mg/dL that was defined as a normal (optimal and near optimal) or borderline-high level by the National Cholesterol Education Program (NCEP) [12]. Patients with acute coronary syndrome and/or ongoing congestive heart failure were excluded. Subjects who had liver and/or renal dysfunction, or were taking medications, including insulin, lipid-lowering drugs and vitamin E were also excluded. All subjects gave informed consent and the study was approved by the local ethical committee. 2.2. Blood sampling and biochemical analysis Whole blood samples were drawn after an overnight fasting. Serum levels of total cholesterol, triglycerides and HDL

cholesterol were measured by standard enzymatic methods (Kainos, Tokyo, Japan) and LDL cholesterol values were calculated by the Friedewald’ formula [13]. Plasma glucose concentrations were determined by the glucose oxidase method (Kainos, Tokyo, Japan). Glycosylated hemoglobin (HbA1c) was measured by high-performance liquid chromatography with a normal range of 4.3–5.8% (TOHSOH, Tokyo, Japan). 2.3. Measurement of MDA-LDL The MDA-LDL levels were measured by ELISA, as previously reported [14]. Briefly, serum samples were diluted 2400-fold in a dilution buffer containing sodium dodecyl sulfate. Duplicate 100 ␮L portions of diluted sample were added to the wells of the plates, which were coated with monoclonal antibody against MDA-LDL (ML25). The reaction was allowed to stand for 2 h at room temperature, and the plates were washed. Next, ß-galactosidase-conjugated monoclonal antibody against apoB (AB16) was added and the reaction was allowed to stand for 1 h at room temperature. Excess enzyme-labeled antibody was removed by washing and 100 ␮L of 10 mmol/L o-nitrophenyl-galactopyranoside as substrate was pipetted into the wells. After 2 h, the reaction was stopped by adding 100 ␮L of 0.2 mol/L sodium carbonate (pH 12). Absorbance in the wells was determined at 415 nm with an MPR-4A microplate reader (Tosho). Primary standard was used with preparative MDA-LDL, in which 15% of the total amino groups were modified. We tentatively defined 1 U/L MDA-LDL as the absorbance obtained with the primary standard at a concentration of 1 mg/L. A calibration curve was prepared by diluting reference serum as a secondary standard from 300- to 9600fold with a dilution buffer and calculating the amount of MDA-LDL in the samples. Reference sera were prepared from pooled sera obtained from healthy volunteers. The intraand inter-assay coefficients of variation were 6.5 and 9.0%, respectively. 2.4. Analysis of lipoprotein profiles measured by NMR Lipoprotein subclass profiles were measured by proton NMR spectroscopy [15]. Aliquots (1.0 mL) of EDTA plasma stored at −80 ◦ C at our laboratory were shipped to LipoScience, Inc. In brief, the NMR method uses the characteristic signals broadcast by lipoprotein subclasses of different size as the basis of their quantification. Each subclass signal emanates from the aggregate number of terminal methyl groups on the lipids contained within the particles. Cholesterol esters and triglycerides in the particle core each contribute three methyl groups, and phospholipids and unesterified cholesterol in the surface shell each contribute two methyl groups. Each measurement produces the concentrations of six subclasses in VLDL, four subclasses in LDL (including intermediate-density lipoprotein (IDL)), and five subclasses in HDL. The levels of VLDL subclasses

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are expressed in mass concentration units of milligrams per deciliter triglyceride, and the levels of LDL and HDL subclasses are expressed as milligrams per deciliter cholesterol. LDL particle sizes (nanometer diameter) and LDL particle concentration (nanomolar) are calculated from the subclass levels. To simplify the data analysis, we grouped some of the subclasses to yield the following 10 subclass categories: large VLDL (60–200 nm), intermediate VLDL (35–60 nm), small VLDL (27–35 nm), IDL (23–27 nm), large LDL (21.3–23 nm), intermediate LDL (19.8–21.2 nm), small LDL (18.3–19.7 nm), large HDL (8.8–13.0 nm), intermediate HDL (8.2–8.8 nm), and small HDL (7.3– 8.2 nm). 2.5. Flow mediated dilatation FMD and nitroglycerin-induced dilatation of the brachial artery were performed in 19 male patients with CAD and 11 male control subjects. Measurements were performed as previously reported [16]. Examinations were made in the morning after at least 12 h fasting and in a quiet temperaturecontrolled (22–24 ◦ C) room. Subjects were kept in bed for at least 15 min to stabilize their condition before the examination. The diameter of the artery was measured by high resolution, two-dimensional ultrasonography (Sonos 2500; Hewlet Packard) with a 7.5 MHz linear array transducer. The left brachial artery was scanned over a longitudinal section 3–5 cm above the elbow, and the arm was kept in the same position throughout the study. A pneumatic tourniquet was placed around the forearm distal to the target artery and inflated to 250 mmHg for 5 min, and then deflated suddenly. Reactive hyperemia was observed after the sudden deflation. After 15 min, a sublingual NTG spray (300 mg; Myocol Spray, Toa Eiyo Co.) was administered for measurement of the NTG-induced dilatation. Scanning was performed four times (before inflation, from 45 to 120 s after deflation, just before NTG administration and 5 min after NTG administration). The ultrasound images were recorded on S-VHS videotape. The diameter of the brachial artery was measured from the anterior to the posterior interface and synchronized with R-wave peaks on the ECG. The diameter changes caused by FMD and NTG were expressed as the % change relative to the diameter on the initial resting image. Three investigators blinded to the subjects’ profile subsequently analyzed the images. This technique has an intra-observer variability of 0.093 mm and inter-observer variability of 0.226 mm. 2.6. Statistical analysis Data are presented as mean ± S.D. Statistical differences between the groups were analyzed by one-way ANOVA and the chi-square test. Correlation between the two parameters was determined by simple linear regression analysis. Values with P < 0.05 were considered to be statistically significant.

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Table 1 Characteristics of the study subjects

Age (years) Body mass index (kg/m2 ) Waist circumference (cm) Hypertension Diabetes mellitus Cigarette smoking Family history Total cholesterol (mg/dL) Triglyceride (mg/dL) HDL cholesterol (mg/dL) LDL cholesterol (mg/dL) HbA1c (%) Fasting blood sugar (mg/dL)

Control (n = 15)

CAD (n = 25)

P value

59 ± 12 23.3 ± 2.6 83.2 ± 7.8 5 (33%) 1 (7%) 7 (47%) 4 (27%) 196 ± 20 100 ± 29 53 ± 16 120 ± 19 5.1 ± 0.5 96 ± 14

60 ± 7 25.9 ± 3.4 91.3 ± 8.4 19 (76%) 9 (36%) 8 (32%) 5 (21%) 202 ± 25 158 ± 81 42 ± 11 124 ± 22 6.0 ± 1.2 114 ± 33

NS 0.02 0.006 0.008 0.04 NS NS NS 0.01 0.02 NS 0.007 NS

Data are mean ± S.D. unless otherwise stated. CAD—coronary artery disease; HDL—high-density lipoprotein; LDL—low-density lipoprotein.

3. Results 3.1. Characteristics of the study subjects The characteristics of the subjects in the present study are shown in Table 1. Compared with the control group, the CAD group had significantly higher levels of body mass index (P = 0.02) and waist circumference (P = 0.006). The CAD group more often had a history of hypertension (P = 0.008) and diabetes mellitus (P = 0.04). The CAD group had significantly higher levels of triglyceride (P = 0.01) and lower levels of HDL cholesterol (P = 0.02) than the control group. The levels of HbA1c in the CAD group were significantly higher than in the control group (P = 0.007). Total cholesterol and LDL cholesterol were not significantly different between the two groups. 3.2. MDA-LDL, clinical characteristics and lipid profiles As shown in Fig. 1A and B, the CAD group had significantly higher levels of MDA-LDL and the ratio of MDA-LDL and LDL cholesterol than the control group (95.4 ± 29.9 versus 73.0 ± 14.5 IU/L, P = 0.01, 0.76 ± 0.17 versus 0.62 ± 0.14, P = 0.008, respectively). The serum MDA-LDL levels were positively correlated with total cholesterol (r = 0.52, P = 0.006), LDL cholesterol (r = 0.56, P = 0.002), triglyceride (r = 0.36, P = 0.02), and waist circumference (r = 0.34, P = 0.04). However, there was no significantly correlation of serum MDA-LDL levels with age, BMI, fasting blood sugar and HbA1c. 3.3. MDA-LDL and lipoprotein profiles from NMR spectroscopy Table 2 shows the lipoprotein subclass profiles detected by NMR spectroscopy of the subjects in this study. NMR analysis demonstrated that the values of LDL particle concentration (P = 0.02), small LDL (18.3–19.7 nm) cholesterol

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Fig. 1. Comparison of serum MDA-LDL levels between the CAD and control groups. Bar graph shows the serum (A) MDA-LDL levels or (B) the ratio of MDA-LDL and LDL cholesterol of the CAD group and the control group, respectively. Data are presented as mean ± S.D.

both size and concentration of LDL particle may represent the result of significant positive correlation between the MDA-LDL levels and small LDL cholesterol (r = 0.53, P < 0.001).

(P = 0.004), intermediate VLDL (35–60 nm) triglyceride (P = 0.002), and large and intermediate VLDL (35–200 nm) triglyceride (P = 0.005) were significantly higher, and large LDL (21.3–23 nm) cholesterol (P = 0.03) and large HDL (8.8–13.0 nm) cholesterol (P = 0.03) were significantly lower in the CAD group than in the control group. The CAD group had a significantly smaller LDL particle size than the control group (20.5 ± 0.6 versus 21.2 ± 0.7 nm, P = 0.002). As shown in Fig. 2, the MDA-LDL levels were negatively correlated with LDL particle size (r = −0.52, P < 0.001) and large HDL cholesterol (r = −0.38, P = 0.02), and positively correlated with LDL particle concentration (r = 0.53, P < 0.001) and large and intermediate VLDL triglyceride (r = 0.34, P = 0.03). Among three LDL subclasses, small LDL cholesterol also had a negative correlation with LDL particle diameter (r = 0.88, P < 0.001) and a positive correlation with LDL particle concentration (r = 0.79, P < 0.001) Therefore, these associations of the MDA-LDL levels with

3.4. Association between MDA-LDL and endothelial function from FMD FMD of the brachial artery after reactive hyperemia was significantly impaired in the subjects with CAD compared to the subjects in the control group (4.1 ± 3.8 versus 9.4 ± 4.1%, P = 0.001). No significant changes in nitroglycerin-induced dilatation occurred in either group of subjects (14.9 ± 7.8 versus 20.3 ± 7.1%). A significant negative correlation was observed between the % changes of FMD and the serum MDA-LDL levels (r = −0.42, P = 0.02) (Fig. 3). An analysis of the control subjects showed a similar negative correlation (r = 0.56, P = 0.07).

Table 2 Lipoprotein subclass profiles detected by NMR spectroscopy Control (n = 15)

CAD (n = 25)

P value

LDL particle concentration (nmol/L) LDL size (nm) Large LDL cholesterol (mg/dL) Intermediate LDL cholesterol (mg/dL) Small LDL cholesterol (mg/dL) IDL cholesterol (mg/dL)

1257 21.2 76.8 32.2 12.5 1.1

± ± ± ± ± ±

343 0.7 41.2 27.1 29.5 1.3

1501 20.5 51.7 32.3 42.3 1.4

± ± ± ± ± ±

270 0.6 27.6 19.6 29.8 3.8

0.02 0.002 0.03 NS 0.004 NS

Chylomicron triglyceride (mg/dL) Large VLDL triglyceride (mg/dL) Intermediate VLDL triglyceride (mg/dL) Small VLDL triglyceride (mg/dL) Large + intermediate VLDL triglyceride (mg/dL)

0.4 8.1 49.8 9.2 58.0

± ± ± ± ±

0.5 13.7 22.4 5.8 26.3

0.5 22.2 92.1 11.1 114.2

± ± ± ± ±

0.7 44.1 44.9 7.6 70.2

NS NS 0.002 NS 0.005

Large HDL cholesterol (mg/dL) Intermediate HDL cholesterol (mg/dL) Small HDL cholesterol (mg/dL)

29.3 ± 15.7 3.5 ± 3.6 17.2 ± 4.7

20.0 ± 10.7 3.2 ± 3.2 19.1 ± 3.9

0.03 NS NS

Data are mean ± S.D. unless otherwise stated. NMR—nuclear magnetic resonance; CAD—coronary artery disease; LDL—low-density lipoprotein; IDL—intermediate-density lipoprotein; VLDL—very low-density lipoprotein; HDL—high-density lipoprotein.

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Fig. 2. Correlation of serum levels of MDA-LDL with (A) LDL particle size, (B) LDL particle concentrations, (C) large HDL cholesterol or (D) large and intermediate VLDL triglyceride.

4. Discussion The present study showed that the increased serum levels of MDA-LDL and the ratio of MDA-LDL and LDL cholesterol in patients with stable CAD compared with control subjects without CAD as previously described [8]. The further analysis of the lipoprotein fractions by NMR method demonstrated that MDA-LDL levels were significantly associated with LDL particle size, LDL particle number, large VLDL triglycerides and large HDL cholesterol in the athero-

Fig. 3. Correlation of the % changes in flow-mediated dilatation of the brachial artery with serum levels of MDA-LDL.

genic metabolic defects beyond LDL cholesterol. In addition, we firstly indicated increased circulating levels of MDA-LDL participate in endothelial dysfunction predisposing to initiation and progression of atherosclerosis. These results suggest that circulating MDA-LDL may be an alternative therapeutic target to prevent the development of CAD in subjects with normal or borderline-high levels of LDL cholesterol. Metabolic syndrome is regarded as a secondary target of risk reduction therapy for CAD after the improvement of the high level of LDL cholesterol as the primary target. The patients with metabolic syndrome fundamentally have increased visceral fat accumulation and subsequent atherogenic dyslipidemia, which induce an increase in small dense LDL [12]. Our present study showed a significant positive correlation between the levels of MDA-LDL and waist circumference, which is reflecting visceral fat accumulation [17]. The further analysis of lipoprotein fractions by NMR spectroscopy demonstrated that the serum levels of MDA-LDL, as well as small LDL cholesterol, were negatively associated with LDL particle diameter and positively associated with LDL particle concentration. These correlations indicate that small LDL subclass is strongly correlated with the serum MDA-LDL levels. In this study, the serum MDALDL levels had a positive association with particularly small LDL cholesterol among three LDL subclasses, significantly. We speculate that high susceptibility of small dense LDL to oxidation resulted in the increased levels of MDA-LDL in metabolic syndrome [6,18,19]. In addition to LDL fractions, it has been reported that MDA-LDL levels were pos-

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itively correlated with increased levels of triglycerides and decreased levels of HDL cholesterol [8]. However, we could demonstrate not only plasma lipid levels, but also the various correlations of MDA-LDL levels with detailed lipoprotein profiles estimated by NMR method. Indeed the present study showed that MDA-LDL levels were positively correlated with the large and intermediate VLDL subclass containing abundant triglycerides, and negatively correlated with the large HDL subclass, which has been shown to be the most cardioprotective HDL component in several studies [9,20,21]. Since exaggerated production of small dense LDL in patients with metabolic syndrome is strongly associated with increase in large VLDL subclass and decrease in large HDL subclass as a result of lipid metabolic interaction [9], these changes of lipoprotein profile may attribute to the increase in serum MDA-LDL levels by increase in its source of small LDL. Therefore, our results indicate that high levels of circulating MDA-LDL in patients with normal or borderlinehigh levels of LDL cholesterol may play an important role for the atherogenic lipoprotein in patients with metabolic syndrome. Recently, increased circulating MDA-LDL levels in subjects with CAD and a positive correlation of the MDA-LDL level with intima-media thickness in carotid arteries have been reported [8]. An association between elevated plasma levels of MDA-LDL and plaque instability in patients with acute coronary syndrome has also been noted [7]. We also demonstrated the increased serum levels of MDA-LDL and the ratio of MDA-LDL and LDL cholesterol in patients with stable CAD compared with control subjects without CAD even though there was no significant difference in LDL cholesterol between the groups. Focusing on the endothelial function, we showed that FMD of the brachial artery after reactive hyperemia was significantly impaired in subjects with CAD compared to subjects in the control group [24]. In addition, a significant negative correlation was newly observed between the % changes of FMD and the serum MDA-LDL levels. It has been reported that oxidative stress, such as smoking, hyperglycemia and hyperlipidemia in the postprandial state, is associated with impaired FMD of the brachial artery [25–27] and oxidized LDL affects endothelium-dependent vascular tone through their interaction with nitric oxides in vitro [28,29]. These results suggest that the high concentrations of MDA-LDL impair endothelial function and induce subsequent coronary artery disease. To our knowledge, this is the first clinical study to indicate an association of circulating oxidized LDL with endothelial dysfunction. In the recent studies, the effects of both statin and fibrate on reducing circulating MDALDL concentration have been reported [22,23]. If decreased serum levels of MDA-LDL may improve endothelial function by the medication, circulating MDA-LDL may become a new therapeutic target to prevent the development of atherosclerosis. This study had a small sample size. We need further studies with large population. Since our investigation was a cross-

sectional study, we could not fully evaluate the mechanisms of the production of circulating MDA-LDL. We need to clarify the effects of each lipoprotein disorders on circulating MDA-LDL by intervention with a variety of risk reduction therapies. In conclusion, the high concentrations of circulating MDA-LDL in the atherogenic lipoprotein profiles, which are strongly associated with the exaggerated production of small dense LDL, may impair endothelial function. We suggest that circulating MDA-LDL plays an important role in the pathogenesis of atherosclerosis and might become a new therapeutic target to prevent the development of CAD in patients with metabolic syndrome.

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