Original Article

Oxidized low-density lipoprotein in postmenopausal women Felix Kork a, Vera Jankowski b, Alexander R. Just c, Johannes Pfeilschifter d, Martin Tepel e, Walter Zidek b, and Joachim Jankowski b

Background: Oxidized low-density lipoprotein (oxLDL) leads to atherosclerosis and cardiovascular disease, the most frequent causes of death worldwide. After menopause, lipid and lipoprotein metabolism changes and women are at greater risk of cardiovascular disease compared to fertile women. The aim of this study was to determine the prevalence of serum oxLDL in postmenopausal women and to identify possible associations of clinical and laboratory features with oxLDL in these patients. Method: After clinical examination and completing a clinical questionnaire, an ultrasound examination of both carotid arteries was conducted and blood was drawn from 533 postmenopausal women. oxLDL concentration was determined using proton NMR spectroscopy. Results: Oxidized LDL was detected in 12.4% (95% confidence interval 9.7–15.5) of postmenopausal women with a median of 0.18 mg/dl (interquartile range 0.10– 0.43). Although intima-media thickness did not differ, postmenopausal women with serous oxLDL had more often atherosclerotic plaques compared to women without oxLDL (6/66 vs. 0/467; P < 0.01). Higher concentrations of high-density lipoprotein, impaired glucose intolerance, and DBP were independently associated with the occurrence of oxLDL. If oxLDL was present, higher high-density lipoprotein and glucose intolerance were associated with higher concentrations of oxLDL. In contrast, higher blood urea concentrations were associated with lower concentrations of oxLDL. Conclusion: This study presents the prevalence and concentration of oxLDL in postmenopausal women and demonstrates that oxLDL concentration can be quantified by proton NMR spectroscopy in large patient samples. The data suggest that oxLDL may be a biomarker for incipient atherosclerotic changes in postmenopausal women. In contrary to the association of dyslipoproteinemia and diabetes, higher blood urea concentrations were associated with lower concentrations of oxLDL. Keywords: hypertension, low-density lipoprotein, oxidation, oxidative stress, uremia Abbreviations: CVD, cardiovascular disease; HDL, highdensity lipoprotein; 1H-NMR, proton nuclear magnetic resonance; IMT, intima–media thickness; LDL, low-density lipoprotein; oxLDL, oxidized low-density lipoprotein

INTRODUCTION

T

he WHO reports that cardiovascular disease (CVD) is the most frequent cause of death worldwide [1]. Despite tremendous efforts in implementing prevention programs, finding new treatment options, and investing billions of dollars in research, cardiovascular deaths are still representing 30% of all global deaths. The WHO projects that about 25 million people will die from CVD in 2030 [1]. Oxidized low-density lipoprotein (oxLDL) plays a major role in the pathogenesis of atherosclerosis which leads to CVD. All major cardiovascular risk factors cause oxidative stress: diabetes [2], hypertension [3], dyslipoproteinemia [4], and smoking [5], whereas oxidative stress induces the oxidation of LDL. OxLDL transverses the endothelium via the LOX-1 receptor and is phagocytized by macrophages. Subsequently, these macrophages transform into foam cells and accumulate to fatty streaks. Fatty streaks are converted via the formation of a fibrous capsule into atheromas and atherosclerotic plaques [6]. Women after menopause are at greater risk for CVD than fertile women [7]. Lipid and lipoprotein metabolism changes and postmenopausal women are exposed to more oxidative stress than fertile women [8]. In fact, few studies with small sample sizes have shown that postmenopausal women have elevated serum concentrations of oxLDL [8,9]. Therefore, the primary aim of this study was to determine the prevalence of serum oxLDL in a large sample of postmenopausal women. Secondary aim of the study was to identify possible associations of clinical and laboratory features with serum oxLDL in postmenopausal women.

Journal of Hypertension 2014, 32:1444–1449 a Klinik fu¨r Ana¨sthesiologie mit Schwerpunkt orperative Intensiv medizin, Campus Virchow Klinikum und Campus Charite´Mitte, Charite´ – Universita¨tsmedizin Berlin, b Medizinische Klinik IV, Campus Benjamin Franklin, Charite´ – Universita¨tsmedizin Berlin, Berlin, Germany, cKlinik fu¨r Plastiche Chirurgie und Dermatologie, Artemedic AG, Olten, Switzerland, dKlinik fu¨r Innere Medizin III, Alfred Krupp Krankenhaus Steele, Essen, Germany and eKlinisk Institut Nefrologi, Odense Univeristetshospital, Odense, Denmark

Correspondence to Professor Dr. rer. nat. Joachim Jankowski, Charite´ – Campus Benjamin Franklin, Medizinische Klinik IV, Hindenburgdamm 30, 12200 Berlin, Germany. Tel: +49 30 450 525 567; fax: +49 30 450 525 923; e-mail: Joachim. [email protected] Received 30 October 2013 Revised 5 March 2014 Accepted 5 March 2014 J Hypertens 32:1444–1449 ß 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins. DOI:10.1097/HJH.0000000000000197

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Volume 32  Number 7  July 2014

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Oxidized low-density lipoprotein in postmenopausal women

METHODS Patients The Ethics Board of the Ruhr University of Bochum (Germany) approved the study (registration number 1343). Patients were recruited through a random sample of 6289 postmenopausal women of the population-based cohort from the ‘Bochumer Postmenopausal Study’ [10]. Women were considered postmenopausal if they indicated to have stopped menstruating for at least 1 year. Patients were excluded if menopause occurred at least 10 years before questioning or were under hormone replacement therapy or corticoid medication. Five hundred and fortynine patients were consecutively enrolled in this substudy, agreeing to give a blood sample after written informed consent. Of the 549 included patients, 16 were not included for missing data, leaving the data of 533 patients to be analyzed.

Clinical questionnaire and anthropometric measures Patients filled out a questionnaire regarding atherosclerosis risk factors including age, weight, height, history of myocardial infarction, stroke, diabetes, hypertension, hypercholesterolemia, peripheral arterial disease, kidney dysfunction, medication, smoking and eating habits. After completion of the questionnaire, patients’ body weight was measured to the nearest kilogram and patients’ height to the nearest centimeter. BMI was calculated as weight/height2 (kg/m2). Blood pressure was measured with a mercury sphingomanometer after 5 min in the sitting position. Patients were than drawn blood for biochemical analysis, an oral glucose tolerance test (OGTT) was administered, and an ultrasound of the carotid arteries was conducted.

Biochemical analyses The blood sample was taken between 0700 and 0900 h after 12 h of fasting. The blood sample was used to determine a full blood cell count and the concentrations of fasting glucose, homocystein, total cholesterol, LDL, high-density lipoprotein (HDL), triglycerides, urea, creatinine, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, electrolytes, total protein and albumin. Additionally, an aliquot of a whole blood sample was immediately centrifuged for 20 min at 5000 r.p.m. to obtain serum for proton NMR (1H-NMR) spectroscopic analysis prepared immediately for 1H-NMR spectroscopy as described below. Patients subsequently performed an OGTT according to WHO standards and another blood glucose was measured 120 min after ingestion of the 75 g carbohydrates solution (Dextro OGT, Hoffmann-LaRoche, Germany). Glomerular filtration rate (GFR) was estimated using the ‘Modification of Diet in Renal Disease’ (MDRD) formula.

Scanning for intima–media thickness and artheroslerotic plaques The intima–media thickness (IMT) was scanned using a 7.5 MHz linear-array imaging probe on a Toshiba CoreVision Pro SSA 350A (Japan). IMT was measured on both common carotid arteries at 1.0, 1.5 and 2.0 cm and proximal Journal of Hypertension

of the carotid bifurcation and averaged as mean for each side. The maximum of both sides (IMTmax) was used in the multivariate model. In addition, the physician scanned the right and left common carotid arteries, the carotid bulbs, and the internal carotid arteries for atherosclerotic plaques.

Proton NMR spectroscopic measurement of oxidized low-density lipoprotein Proton NMR spectroscopy was used for measuring oxLDL concentrations because it allows a fast sample processing and sensitive and accurate measurements. 1H-NMR spectroscopy was conducted as previously described [11] using a serial dilution to obtain calibration measurements. We separated lipids from serum [12] and consecutively isolated chylomicrons, very-low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL) and LDL through a discontinuous potassium bromide gradient as described by Havel et al. [13]. Protein concentration was determined according to the method described by Lowry et al. [14]. After sodium perchlorate dialysis, the isolated and purified LDL was incubated with copper sulfate (CuSO4) for 24 h at 48C, resulting in oxLDL at a defined concentration. A 10level serial dilution was fabricated and 1H-NMR spectra were obtained. The three characteristic resonances of oxLDL correlated significantly with the concentration of oxLDL, the relative resonance intensity at 1.17 ppm correlated best with the concentration of oxLDL (r2 ¼ 0.983, P < 0.01). Thus, the regression line’s slope represents the concentration of oxLDL per arbitrary unit in relation to the standard 3-trimethylsilyl-[2,2,3,3,-2H4]-propionate at 0 ppm.

Statistics Continuous variables were tested for normal distribution with the Kolmogorov–Smirnov test. Normally distributed variables are presented as mean  SD, not normally distributed values as median and quartiles; categorical variables are presented in percentage and 95% confidence interval (CI). The probability of a type I error below 0.05 was considered to be statistically significant. Univariate testing was done using the Mann–Whitney Utest and Fisher’s exact test. For multivariate testing, we calculated two models. The first model was a binary logistic model to assess associations of covariates with the occurrence of oxLDL in patients’ serum; the second model was a linear model to assess the covariates’ influence on the concentration of oxLDL. Covariates were selected for inclusion in each of the two final models if they were selected in either a forward stepwise inclusion process with a cut-off at P less than 0.05 or a backward elimination model with a cut-off at P less than 0.10.

RESULTS Using 1H-NMR spectroscopy, we detected oxLDL in 66 of 533 (12.4%, 95% CI 9.7–15.5) sera of postmenopausal women with a median of 0.18 mg/dl [interquartile range (IQR) 0.10–0.43] and a range of 0.02–18.10 mg/dl. Basic demographic and laboratory data of the examined collective are shown in Table 1. Figure 1 shows a spectrum of LDL www.jhypertension.com

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Kork et al. TABLE 1. Patient characteristics and laboratory data No oxLDL (n ¼ 467)

oxLDL (n ¼ 66)

P

54.7 (51.0–59.5) 26.1 (23.3–29.8) 3.9% (n ¼ 18) 0.2% (n ¼ 1) 1.3% (n ¼ 6) 30.8% (n ¼ 144) 0.8 (0.7–0.8) 14 (12–17) 10 (8–11) 110 (75–149) 235 (215–265) 152.8  36.4 63.0  15.9 2.44 (1.89–3.10) 96 (90–104) 90 (77–110) 4 (18–11) 8.1% (n ¼ 38) 5.1% (n ¼ 24) 3.6% (n ¼ 17) 5.3 (5.1–5.6)

56.4 (50.2–60.7) 26.0 (21.7–30.5) 9.1% (n ¼ 6) 0.0% (n ¼ 0) 0.0% (n ¼ 0) 31.8% (n ¼ 21) 0.8 (0.6–0.9) 14 (11–17) 9 (8–12) 90 (67–153) 232 (212–261) 146.7  37.5 69.8  21.3 2.26 [1.65 – 2.71] 95 [87–100] 93 (78–116) 1 (18–23) 6.0% (n ¼ 4) 4.5% (n ¼ 3) 4.5% (n ¼ 3) 5.2 (5.0–5.7)

0.36 0.24 0.10 0.71 0.35 0.89 0.98

Oxidized low-density lipoprotein in postmenopausal women.

Oxidized low-density lipoprotein (oxLDL) leads to atherosclerosis and cardiovascular disease, the most frequent causes of death worldwide. After menop...
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