Serum Malondialdehyde-Modified Low-Density Lipoprotein Is a Risk Factor for Central Arterial Stiffness in Maintenance Hemodialysis Patients

Circulating malondialdehyde-modified low-density lipoprotein (MDA-LDL) acts as a marker of oxidative stress and is associated with atherosclerotic cardiovascular disease. The relationship between serum MDA-LDL levels and aortic stiffness (AS) in patients with hemodialysis (HD) was evaluated. There were 155 HD patients enrolled in this study. Carotid-femoral pulse wave velocity (cfPWV) was measured by a validated tonometry system. Patients with cfPWV >10 m/s were used to define the AS group, while those with values of ≤10 m/s were regarded as the control group. Serum MDA-LDL levels were measured using a commercial enzyme-linked immunosorbent assay. Sixty-eight patients (43.9%) who were defined as AS sufferers, and were older, had a higher percentage of diabetes and hypertension and higher systolic blood pressure and serum MDA-LDL level compared to subjects in the control group. After adjusting for factors significantly associated with AS by multivariable logistic regression analysis, it was revealed that serum MDA-LDL levels, diabetes, and hypertension were independent predictors of AS in HD patients. Multivariable forward stepwise linear regression analysis also showed that a logarithmically transformed MDA-LDL level was significantly correlated with cfPWV values in HD patients. In HD patients, a high serum MDA-LDL level was positively associated with cfPWV values and was a significant predictor of the development of high AS.


Introduction
Cardiovascular disease (CVD), for which traditional risk factors include diabetes mellitus (DM) and hypertension (HTN), as well as chronic kidney disease (CKD)-specific risk factors, has long been known as the main cause of adverse long-term outcomes in patients with end-stage renal disease (ESRD) receiving maintenance hemodialysis (HD) [1]. Evidence has shown that the stiffening of vascular walls caused by deregulation of elastin and collagen, oxidative stress, disordered mineral metabolisms, and low-grade inflammation, may result in increased myocardial pre-load, and a decrease in perfusion pressure of the coronary artery and future CVD in CKD patients [2,3]. As arterial stiffening increases, there is abnormal pressure amplification with increased reflection wave and earlier backward

Determinations of Carotid-Femoral Pulse Wave Velocity
Carotid-femoral PWV (cfPWV) was measured by applanation tonometry (SphygmoCor system, AtCor Medical, West Ryde, Australia), as previous studies have also done [20]. Measurements were performed in a supine position after a minimum of 10 min rest in a quiet and temperature-controlled room. Records were taken simultaneously with electrocardiogram signals, which provided an R-timing reference. Pulse wave recordings were performed consecutively at two points on the superficial arteries (carotid-femoral segment). Integral software was used to process each set of pulse waves and electrocardiogram data to calculate the mean time difference between pulse wave and R-wave on a beat-to-beat basis, with an average of 10 consecutive cardiac cycles. The cfPWV was calculated using the difference in elapsed time and distance between the two recorded artery sites. Quality indices included in the software were set to ensure uniformity of data. A cfPWV > 10 m/s defined the high central arterial stiffness (AS) group and ≤10 m/s defined the control group, according to the European Society of Cardiology and the European Society of Hypertension guidelines [21].

Biochemical Determination
Blood samples were collected before patients received HD. Blood samples (approximately 5 mL) were immediately centrifuged at 3000× g for 10 min. The serum samples were stored at 4 • C and used for biochemical analyses within 1 h of collection. Serum values of blood urea nitrogen, creatinine, glucose, total cholesterol, triglyceride, total calcium, and phosphorus were measured using an autoanalyzer (Siemens Advia 1800, Siemens Healthcare GmbH, Henkestr, Germany). The fractional clearance index for urea (Kt/V) and urea reduction ratio were measured before dialysis and immediately afterwards using a formal, single-compartment dialysis urea kinetic model. The serum values of intact parathyroid hormone (iPTH) (Diagnostic Systems Laboratories, Webster, TX, USA) and MDA-LDL (Sekisui Diagnostics GmbH, Kaplaneigasse, Pfungstadt, Germany) were measured using commercially available, enzyme-linked, immunosorbent assays.

Statistical Analysis
Continuous variables were tested for normal distribution by the Kolmogorov-Smirnov test. Data have been expressed as the mean ± standard deviation or median with interquartile range (IQR), depending on normal distribution. Comparisons between the high-AS and control group were performed by the Student's independent t-test or Mann-Whitney U test (two-tailed), accordingly. Categorical data were analyzed by the χ 2 test and represented as a number and percentage. Nonnormally distributed continuous variables were logarithmically transformed when applied to linear regression analysis. Multivariate logistic and linear regression analyses were used to analyze the relationship between all variables and cfPWV and the risk factors for developing high AS in HD patients. A receiver operating characteristic (ROC) curve was used to calculate the area under the curve (AUC) to identify a cut-off value of MDA-LDL to predict high AS in HD patients. A p-value < 0.05 was considered statistically significant. Data were analyzed by SPSS for Windows (version 19.0; SPSS Inc., Chicago, IL, USA).

Discussion
The major findings of this study are that, in addition to DM and HTN, a high serum MDA-LDL level was associated with high cfPWV values and could predict the development of AS in HD patients.
Arterial stiffness, which was caused by multiple risk factors, resulted in irreversible changes of vascular wall structures with increased pulse pressure to low impedance circulation, along with exposure to high blood pressure (BP) and mechanical strain, which resulted in CVD, renal dysfunction, and mortality [2,3]. In a cross-sectional study of 4336 healthy subjects, there was a progressively and significantly increased baPWV with age [22]. Evidence has shown that aging might additionally induce vascular structural and functional changes in CKD patients, besides abnormal mineral metabolism, with the results of elastin fragmentation and medial layer calcification [2,23]. In ESRD patients, there was marked aging-related AS indicated by an age-associated increase in aortic PWV and decrease in aortic taper, as well as lower brachial/aortic stiffness gradient [23]. Progression of AS reduced the vascular lumen and led to a return of the reflected wave in late systole prematurely, resulting in increased pulse pressure accompanied with increased SBP and decreased DBP [24]. Studies have shown that cfPWV is positively correlated with SBP, along with MetS and waist circumference in HTN and DM patients [25,26]. Moreover, a systemic review that included 26,970 subjects underscored that, in addition to traditional risk factors, such as gender, dyslipidemia, smoking, and BMI, BP elevation and aging were independently associated with cfPWV [27]. In addition, impaired glucose tolerance was independently related to impaired arterial compliance, decreased carotid-femoral transit time, and increased aortic augmentation index in a cross-sectional population study [28]. In DM patients, there was an increase in the production of advanced glycation end products, which showed significant association with cfPWV independent of age, gender, BP, or fasting sugar in a community-dwelling population [29]. Moreover, increased cfPWV was associated with longer DM duration, independent

Discussion
The major findings of this study are that, in addition to DM and HTN, a high serum MDA-LDL level was associated with high cfPWV values and could predict the development of AS in HD patients.
Arterial stiffness, which was caused by multiple risk factors, resulted in irreversible changes of vascular wall structures with increased pulse pressure to low impedance circulation, along with exposure to high blood pressure (BP) and mechanical strain, which resulted in CVD, renal dysfunction, and mortality [2,3]. In a cross-sectional study of 4336 healthy subjects, there was a progressively and significantly increased baPWV with age [22]. Evidence has shown that aging might additionally induce vascular structural and functional changes in CKD patients, besides abnormal mineral metabolism, with the results of elastin fragmentation and medial layer calcification [2,23]. In ESRD patients, there was marked aging-related AS indicated by an age-associated increase in aortic PWV and decrease in aortic taper, as well as lower brachial/aortic stiffness gradient [23]. Progression of AS reduced the vascular lumen and led to a return of the reflected wave in late systole prematurely, resulting in increased pulse pressure accompanied with increased SBP and decreased DBP [24]. Studies have shown that cfPWV is positively correlated with SBP, along with MetS and waist circumference in HTN and DM patients [25,26]. Moreover, a systemic review that included 26,970 subjects underscored that, in addition to traditional risk factors, such as gender, dyslipidemia, smoking, and BMI, BP elevation and aging were independently associated with cfPWV [27]. In addition, impaired glucose tolerance was independently related to impaired arterial compliance, decreased carotid-femoral transit time, and increased aortic augmentation index in a cross-sectional population study [28]. In DM patients, there was an increase in the production of advanced glycation end products, which showed significant association with cfPWV independent of age, gender, BP, or fasting sugar in a community-dwelling population [29]. Moreover, increased cfPWV was associated with longer DM duration, independent of age, gender, BP, or renal function [30]. Our previous study revealed that CAD patients in the high-AS group had a higher percentage of DM, with higher fasting sugar and serum levels of insulin and HOMA-IR, compared to patients with low AS [31]. One meta-analysis, which enrolled 1222 DM patients, showed a significant association between DM incidence and AS measured by cfPWV [32]. Taken together, age and BP, as well as DM, were consistently and independently associated with cfPWV. Similarly, we found that being older, or having DM or HTN, is associated with a higher degree of cfPWV. Furthermore, we similarly found that DM and HTN are independently significant predictors for the development of high AS, after adjusting the confounders of HD patients.
The onset and acceleration of atherosclerotic lesions is related to oxidative modification, such as the oxygen and peroxyl radical synthesis, of LDL cholesterol [33]. Oxidized LDL is directly chemotactic for monocytes and could modulate the progression of arteriosclerosis after being incorporated via scavenger receptors, which induces monocytes transformed into macrophages and the proliferation of vascular smooth muscle cells, to increase foam cell formation in atherosclerotic lesions, endothelial injury, and plaque formation [34,35]. Oxidized LDL could exert a number of adverse effects on vasculature and CVD [7,8,22,36]. In a longitudinal study of 417 subjects, it was shown that vascular stiffness measured by cfPWV correlated with oxidized LDL in subjects with normal renal function and CKD stage II [36]. In participants who received health exams, a progressively and significantly increased baPWV and plasma oxidized LDL were found in those who were older than 45 years [22]. In addition, circulating oxidized LDL was reported to be associated with baPWV in patients with metabolic syndrome or pre-HTN [7,8].
As a marker of oxidative stress, MDA-LDL is a major end product of LDL oxidation and evidence has shown that MDA-LDL, which reflects endothelial injury or plaque instability, together with troponin I, could discriminate between stable CAD and acute myocardial infarction with high sensitivity and specificity [37]. In addition, MDA-LDL is a predictor for major CV events or vulnerable coronary arterial plaque in coronary artery disease (CAD) and DM patients [13][14][15]. Moreover, MDA-LDL was negatively associated with endothelial function measured by flow-mediated dilatation of the brachial artery [18] and positively correlated to levels of LDL, triglyceride and the severity of albuminuria, which was associated with high AS in the Framingham Study [22,38]. A cross-sectional study of HD patients revealed that there was significant increased baPWV and MDA-LDL, as well as C-reactive protein and reactive oxygen species, in those with intradialytic hypotension, which indicated that AS of this population was related to oxidative stress [39]. The MDA-LDL/LDL ratio, which indicated the extent of MDA modification per LDL particle, was a strong marker for CVD [18] and was significantly associated with coronary artery calcification in HD patients [19]. Lipid-lowering therapy with antioxidant effects has been reportedly associated with decreased values of MDA-LDL and MDA-LDL/LDL ratio, along with improved vascular function measured by baPWV, resistance index, and flow-mediated dilatation in hypercholesterolemia patients [16,17]. However, there was no significant difference in levels of MDA-LDL between those who used medications and those who did not (no statin, 90.88 mg/dL IQR 32.14-447.97 mg/dL; statin, 88.06 mg/dL, IQR 42.59-656.13 mg/dL; p = 0.443; data not shown) in this study. Taken together, other than these traditional and CKD-specific risk factors, there might be a role for MDA-LDL in the development of high AS in HD patients, but the definite mechanism and therapies need to be studied. This study is limited by being cross-sectional and conducted at a single center with a limited number of HD patients. The other limitation was that we only measured MDA-LDL, but evidence had shown that measuring MDA-LDL was correlated negatively with LDL size as well as being located in the dense LDL fraction and could be a useful marker of progression of atherosclerosis in CAD patients [12]. Therefore, the association of serum MDA-LDL or other types of LDL with AS, as well as the role of the development of AS in HD patients, should be confirmed by further longitudinal studies before a cause-effect can be established.

Conclusions
In addition to DM and HTN, serum MDA-LDL greater than 80.33 mg/dL was shown to be related to AS in HD patients in this study. The positive relationship between MDA-LDL and AS is both promising and provoking, as both were shown to be individually correlated with future CV events. These findings indicate that MDA-LDL may play a role in the pathogenesis of high AS and may also become an alternative therapeutic target to prevent the development of AS in HD patients, but the mechanism and feasibility remain to be further elucidated.