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Oxidized low-density lipoprotein (oxLDL) plasma levels and oxLDL to LDL ratio — Are they real oxidative stress markers in dialyzed patients?
Life Sciences 92 (2013) 253–258
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Oxidized low-density lipoprotein (oxLDL) plasma levels and oxLDL to LDL ratio — Are they real oxidative stress markers in dialyzed patients? Krystyna Pawlak a, Michal Mysliwiec b, Dariusz Pawlak c,⁎ a b c
Department of Monitored Pharmacotherapy, Medical University, Bialystok, Poland Department of Nephrology and Clinical Transplantation, Medical University, Bialystok, Poland Department of Pharmacodynamics, Medical University, Bialystok, Poland
a r t i c l e
i n f o
Article history: Received 9 March 2012 Accepted 13 December 2012 Keywords: Oxidized LDL oxLDL/LDL ratio Dialysis treatment Oxidative stress markers
a b s t r a c t Aims: Dyslipidemia and oxidative stress are commonly present in patients during maintenance dialysis treatment. However, the significance of oxidized LDL (oxLDL) as a marker of oxidative stress in uremia is still unresolved. The aim of this study was to establish the role of oxLDL and oxLDL/LDL ratio as markers of lipoprotein abnormalities and oxidative stress in the dialyzed patients. Main methods: Plasma oxLDL level was measured by ELISA, and oxLDL/LDL ratio was calculated in 106 dialyzed patients and 20 controls. The linkages between oxLDL, oxLDL/LDL ratio and lipid profile and oxidative stress markers malondialdehyde (MDA) and Cu/Zn superoxide dismutase (Cu/Zn SOD) levels were also analyzed. Key findings: OxLDL levels and oxLDL/LDL ratio were similar in hemodialyzed patients and controls, whereas these parameters were lower in peritoneally dialyzed patients when compared to healthy individuals. In contrast, both MDA and Cu/Zn SOD levels were significantly higher in uremics than in controls. oxLDL and oxLDL/LDL ratio positively correlated with lipid profile (except of HDL), whereas there were no positive associations between these parameters and both MDA and Cu/Zn SOD. Multiple regression analysis confirmed that increased oxLDL/HDL and TC/HDL ratios and total cholesterol levels are the parameters which independently predicted oxLDL in dialyzed patients. In the case of oxLDL/LDL ratio, the independent variables were oxLDL/HDL ratio, total cholesterol and HDL levels. Significance: oxLDL levels and oxLDL/LDL ratio seem to be the markers of lipoprotein abnormalities rather than the markers of oxidative stress in the population of dialyzed patients. © 2013 Elsevier Inc. All rights reserved.
Introduction Oxidative stress (SOX), dyslipidemia and accelerated atherosclerosis are commonly present in patients on maintenance dialysis who constitute the population with higher risk for cardiovascular disease (Usberti et al., 2002; Kaysen and Eiserich, 2004; Chen et al., 2011). Uremic patients are subjected to enhanced SOX as a result of impaired antioxidant defense system and increased pro-oxidant activity that are associated with a state of chronic inflammation, high frequency of diabetes, the uremic syndrome per se and bio-incompatibility of dialyzer membranes and solutions (Locatelli et al., 2003). Renal dyslipidemia is characterized by an abnormal apolipoprotein profile, including decreased levels of apoA-containing lipoproteins and increased levels of apoB-containing lipoproteins. Serum triglyceride levels are increased in the majority of uremic patients, whereas total cholesterol levels are generally normal or lower. In the same group of patients high-density lipoprotein (HDL) cholesterol is reduced, in a ⁎ Corresponding author at: Department of Pharmacodynamics, Mickiewicza 2C str, 15-089 Bialystok, Poland. Tel./fax: +48 85 7485622. E-mail address: [email protected] (D. Pawlak). 0024-3205/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.lfs.2012.12.002
contrast to increased levels of low-density lipoproteins (LDL) (Attman et al., 1999; Senti et al., 1992). Furthermore, the characteristic features of renal dyslipidemia remain essentially unchanged during long-term hemodialysis (HD) treatment (Attman et al., 1999), whereas peritoneal dialysis (PD) is often associated with both hypercholesterolemia and hypertriglyceridemia (Johansson et al., 2000). LDL plays a principal role in oxidative stress due to its oxidative modification to oxidized LDL (oxLDL). Formed oxLDL acts as chemoattractant for monocytes and induces inflammatory reactions within the arterial wall, which are implicated in all phases of the atherosclerotic plaque development (Itabe, 2009). Recently, we demonstrated that oxLDL to autoantibodies against oxLDL ratio can be a new biomarker associated with carotid atherosclerosis and cardiovascular complications in dialyzed patients (Pawlak et al., 2012). However, the significance of oxLDL as a marker of SOX in uremia is still unclear. Some studies reported significant differences between oxLDL levels in uremic patients and the healthy people (Takenaka et al., 2002; Samouilidou et al., 2010, 2012), but others did not show such differences (Diepeveen et al., 2004; Nissel et al., 2008; Johnson-Davis et al., 2011). Kuchta et al. (2011), demonstrated that levels of oxLDL, although higher in uremic patients when compared to the controls, were not altered in patients with different
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stages of uremia and treated with HD. oxLDL to LDL (oxLDL/LDL) ratio, an accurate estimation of in vivo LDL oxidation (Scheffer et al., 2003), was measured only during the solitary study of An et al. (2009) in HD patients with and without vascular calcification. There is limited evidence concerning the correlation between plasma level of oxLDL and lipid profile in patients on dialysis treatment (Samouilidou et al., 2010, 2012). We have also found a few controversial reports regarding the relationship of oxLDL and other oxidative stress markers in dialyzed patients (Nissel et al., 2008; Johnson-Davis et al., 2011; Kuchta et al., 2011). With respect to conflicting data about oxLDL and its role as a biomarker, the aim of this study was to establish the significance of oxLDL and oxLDL/LDL ratio as the markers of lipoprotein abnormalities and oxidative stress in the population of dialyzed patients. We focused on the association between oxLDL and oxLDL/LDL ratio with atherogenic lipoprotein: triglycerides, LDL, total cholesterol to HDL (TC/HDL) ratio, LDL to HDL (LDL/HDL) ratio, and oxLDL to HDL (oxLDL/HDL) ratio, which is the new and more potent biomarker than standard lipid measurements for discriminating between subjects with coronary artery disease and healthy subjects (Johnston et al., 2006; Lankin et al., 2011). Moreover, we analyzed other oxidative stress markers: plasma malondialdehyde (MDA) concentration as a commonly used biomarker of lipid peroxidation in uremia (Kuchta et al., 2011), and plasma Cu/Zn superoxide dismutase (Cu/Zn SOD) levels, which have been previously reported by us (Pawlak et al., 2005, 2012) and by another author (Washio et al., 2008) as a useful oxidative stress marker in uremic patients. Materials and methods Subjects One hundred and six patients were enrolled in this cross-sectional study and divided into 2 groups: 52 patients on PD and 54 on maintenance HD. All patients were in stable clinical conditions and without the features of active infections, autoimmune diseases or thrombotic complications. None of the patients received immunosuppressive treatment, lipid-lowering agents and non-steroidal anti-inflammatory drugs. Vitamin C, E or allopurinol is expected to have an impact on oxLDL levels, and for this reason the patients were asked not to take these medications 2 weeks before and at the time of the study. Body mass index (BMI) was calculated by dividing the weight in kilograms by the square of the height in meters. CVD was defined as documented in the medical record a history of myocardial infarction, ischaemic stroke, coronary revascularization procedures, angina pectoris, typical changes in coronary angiography, typical ischaemic electrocardiographic changes, peripheral artery surgery (not including the arteriovenous fistula), intermittent claudication or pain at rest. Patients who had smoked daily or occasionally within the 30 days preceding the study were defined as “smokers”. Antihypertensive drug and erythropoietin usage was recorded in the case of each participant, and it was presented as the percent of patients treated with expected drug. The group of hemodialysis patients received conventional 4-h HD, three times weekly, (the dialysate was endotoxin-free). Dialysis prescription was aimed to reach a value of Kt/V ≥ 1.2. All PD patients were performing four 2-liter exchanges a day using either the Baxter TwinBag system or the Fresenius Andy Plus system. Dwell times generally averaged 4–6 h during the day and 8 h overnight. The glucose concentration ranged from 1.36 to 3.86%. The osmotic pressure of PD dialysis fluid was adjusted in accordance to the extent of ultrafiltration required for each patient. Dialysis adequacy was assessed by measuring the value of Kt/v (mean Kt/v = 2.25 ± 0.47). Twenty of sex- and age-matched healthy subjects who were receiving no drugs or vitamin supplements during the study volunteered as controls. All of them had a regular diet and no history of hypertension, diabetes mellitus or renal disease. Detailed characteristics of the controls and dialyzed patients are presented in Table 1.
Table 1 Biochemical and clinical characteristics of controls and end-stage renal disease patients on peritoneal dialysis (PD) and hemodialysis (HD).
Sex, M/F Age, years BMI, kg/m2 Haematocrit, % White blood cells, ×103 μl Total protein, g/dl Albumin, g/dl Glucose, mg/dl Hs CRP, μg/ml SBP, mm Hg DBP, mm Hg Smokers, % ESRD etiology Glomerulonephritis, n Interstitial nephritis, n Polycystic kidney disease, n Diabetes mellitus, n Hypertensive nephropathy, n Secondary amyloidosis, n Unknown, n Vintage of dialysis treatment, months EPO treatment, % EPO dose, U/kg body weight/week Cardiovascular disease, % ACEI, % Calcium channel antagonists, % β-blockers, % α-blockers, % Nitrates, %
Controls
PD
HD
12/8 53.21 ± 10.79 26.04 ± 3.74 42.07 ± 3.05 5.98 ± 1.01
25/27 52.80 ± 12.43 25.50 ± 4.15 36.44 ± 4.79⁎⁎⁎ 6.68 ± 2.03
32/22 57.68 ± 13.39 24.99 ± 4.24 35.42 ± 4.11⁎⁎⁎ 5.82 ± 1.57^
6.74 ± 0.36 4.62 ± 1.40 85 (70–95) 0.98 (0.2–11.0) 128.89 ± 10.51 81.11 ± 6.74 20
6.56 ± 0.61 3.48 ± 0.48⁎⁎⁎ 103 (66–469)⁎ 3.04 (0.1–46.0)⁎⁎⁎ 131.80 ± 22.06 82.15 ± 12.23 25
6.76 ± 0.53 3.84 ± 0.43⁎⁎,^ 101 (36–334)⁎ 4.86 (0.1–49)⁎⁎⁎,^^ 135.47 ± 24.27 83.73 ± 11.73 29
N/A N/A N/A
15 4 10
17 9 7
N/A N/A
13 4
8 6
N/A
4
3
N/A N/A
2 15.0 (3.0–134.0)
4 34.0 (3.0–241.0)^^
N/A N/A
52 51.3 (26.3–160.0)
83^ 87.9 (18.7–161.6)^
N/A
50
72^
N/A N/A
54 54
61 63
N/A N/A N/A
44 10 12
46 14 20^
⁎pb 0.05, ⁎⁎pb 0.01, ⁎⁎⁎pb 0.001 controls versus patients; ^pb 0.05, ^^pb 0.01 PD versus HD. Data are shown as mean ± SD or median (full range) depending on their normal or skewed distribution. BMI = body mass index, SBP = systolic blood pressure, DBP = diastolic blood pressure, EPO = erythropoietin, ACEI = angiotensin-converting enzyme inhibitors, N/A = not applicable.
The study protocol was approved by our institutional ethical board, and informed consent was obtained from all patients and controls. Blood sampling and laboratory measurements Investigations were performed on the morning under fasting conditions. Blood samples were taken directly from the arteriovenous fistula immediately before the beginning of a routine 4-h HD session. Blood was drawn from controls and PD patients from the antecubital vein into the EDTA-containing vials. The plasma samples were prepared in a conventional manner, aliquoted and stored at − 70 °C until further assay. Serum lipids Routine blood chemistry and levels of lipids (total cholesterol, HDL and triglycerides) were analyzed using fresh blood samples according to established enzymatic methods in the local laboratories. LDL levels were calculated using Friedewald's formula (Friedewald et al., 1972). In vivo oxidized LDL Plasma level of oxidized LDL in each sample was measured in duplicate by a commercially available sandwich ELISA (Mercodia,
K. Pawlak et al. / Life Sciences 92 (2013) 253–258
Uppsala, Sweden). This assay is based on a murine monoclonal antibody (mAb-4E6) directed against a conformational epitope in the apoB-100 moiety of LDL, which is generated as a consequence of reaction of lysine residues with aldehydes (Holvoet et al., 1998). The intra-assay and interassay coefficients of variation were 5.0% and 5.5%, respectively. To obtain the percentage of oxidized LDL particles, the ratio of oxidized LDL to LDL (oxLDL/LDL) was also calculated. Therefore, results were expressed as U/l and U/mmol LDL, respectively. Oxidative stress markers and inflammatory state Plasma Cu/Zn SOD levels were measured by ELISA (Bender MedSystems, Vienna, Austria), according to the manufacturer's instruction. Malondialdehyde concentration was determined by highperformance liquid chromatography (HPLC) according to Londero and Lo Greco (1996). The reversed-phase HPLC system consisted of a Waters Sherisorb S3 ODS2 150× 2.1 mm column (USA) and HP 1050 series pump (Germany). Rheodyne injection valve was fitted to a sample loop (5 μl). The column effluent was monitored using a programmable fluorescence detector HP 1046A (Germany). The optimized conditions were determined by recording fluorescence spectra with a stop-flow technique. Excitation and emission wavelengths were set at 532/553 nm. The output of the detector was connected to a single instrument LC-2D ChemStation (Germany). The mobile phase was pumped at a flow-rate of 0.1 ml/min consisted of 40:60 (v/v) 0.05 M methanol–potassium phosphate buffer with pH 6.8. Chromatography was carried out at 25 °C. Plasma C-reactive protein levels were measured by high sensitivity ELISA (Imuclone CRP (hs) ELISA, American Diagnostica Inc., Greenwich, USA). Statistical analysis The Shapiro–Wilk test for normality was used for data distribution analysis. The normally distributed data were expressed as mean ± SD. The non-Gaussian data were presented as median (full range), depending on their distribution. Multiple group comparisons were performed by one-way analysis of variance (ANOVA), and significant differences between groups were assessed by Tukey–Kramer's test or non-parametric Mann– Whitney's U-test. The correlations between study variables were calculated by Spearman's rank correlation coefficients (Spearman's rho). Multiple regression analysis was performed using a stepwise model to determine which variables predict oxLDL and oxLDL/LDL ratio. A two-tailed p value b 0.05 was considered to be statistically significant. Computations were performed using Statistica ver.6 computer software (StatSoft, Tulsa, OK, USA).
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Results Basal characteristics of patients and controls are summarized in Table 1. Haematocrit and albumin levels were significantly decreased in PD and HD patients than in controls; furthermore, albumin level was significantly lower in PD group than in HD group. Glucose and hs CRP levels were significantly elevated in both groups of dialyzed patients when compared to the controls; hs CRP was significantly increased in HD but not in PD patients. White blood cell count was significantly lower in HD patients when compared to PD patients and controls. The median time of dialysis treatment was significantly prolonged in HD group than in PD group. Moreover, HD patients treated with erythropoietin were of greater percentage when compared to PD patients. The prevalence of cardiovascular complications was significantly increased in HD patients, and more subjects from this group received nitrates when compared to PD group. According to results of lipid profile (Table 2), dialyzed patients tended to have higher levels of total cholesterol and LDL when compared to controls. In contrast, HDL tended to be decreased in PD patients, while statistical significance was reached in HD group when compared to PD and controls. In both groups of dialyzed patients TC/HDL and LDL/HDL ratios and triglycerides were significantly increased than in controls. Peritoneally dialyzed patients had lower oxLDL levels and oxLDL/LDL and oxLDL/HDL ratios than HD and controls. Regarding oxidative stress markers, both Cu/Zn SOD and MDA plasma concentrations were significantly higher in dialyzed patients (particularly in those on HD treatment) than in controls (Table 2). As can be seen in Fig. 1, a strong positive correlation was between oxLDL and oxLDL/HDL ratio. oxLDL also positively correlated with other lipid parameters, such as total cholesterol, LDL, TC/HDL, LDL/ HDL and oxLDL/LDL ratios. Similarly, increased oxLDL/LDL ratio was associated with increased oxLDL/HDL ratio (rho = 0.721, p b 0.001) and with other lipid parameters, as presented in Table 3. In contrast, there was a tendency to inverse correlation between oxLDL and HDL levels in dialyzed patients, whereas the statistical significance was reached when the value of oxLDL was divided by LDL. Moreover, oxLDL was positively associated with haematocrit value and glucose concentration. There was a strong positive relationship between oxLDL/LDL ratio and oxLDL/HDL ratio, as well as oxLDL/LDL ratio was inversely associated with total cholesterol and LDL levels. MDA, another marker of lipid peroxidation, was inversely correlated with oxLDL (Fig. 2), and it was also inversely associated with oxLDL/LDL ratio (rho= −0.314, p = 0.009). Moreover, there was no relationship between Cu/Zn SOD and oxLDL as well as oxLDL/LDL ratio. In a contrast, MDA was positively associated with Cu/Zn SOD (rho= 0.464, p b 0.001). Neither MDA nor Cu/Zn SOD was correlated with lipid profile, although a tendency to inverse correlation was observed between Cu/Zn SOD and HDL (rho= −0.186, p = 0.056). Cu/Zn SOD was positively correlated
Table 2 Lipid profiles and oxidative stress markers in the healthy controls and end-stage renal disease patients on peritoneal dialysis (PD) and hemodialysis (HD).
Total cholesterol, mmol/l HDL, mmol/l LDL, mmol/l TC/HDL LDL/HDL Triglycerides, mmol/l oxLDL, U/l oxLDL/LDL, U/mmol oxLDL/HDL, U/mmol Cu/Zn SOD, ng/ml MDA, μM
Controls
PD
HD
4.86 ± 0.76 1.52 ± 0.30 2.95 ± 0.74 3.34 ± 0.94 2.05 ± 0.79 0.77 (0.43–1.70) 38.33 (17.87–106.26) 14.21 (5.96–33.96) 27.08 (14.53–79.48) 58 (24–86) 0.66 (0.35–1.60)
5.59 ± 1.18 1.44 ± 0.45 3.41 ± 1.02 4.13 ± 1.18⁎⁎ 2.55 ± 1.01⁎ 1.53 (0.64–4.11)⁎⁎⁎ 27.30 (8.33–81.73)⁎ 8.10 (2.32–35.64)⁎ 19.27 (5.22–63.6)⁎⁎ 179 (46–474)⁎⁎⁎ 0.89 (0.36–3.60)⁎
5.33 ± 1.27 1.17 ± 0.33⁎⁎,^^ 3.41 ± 1.16 4.82 ± 1.48⁎⁎⁎,^^ 3.09 ± 1.24⁎⁎,^ 1.43 (0.35–5.43)⁎⁎ 30.76 (6.77–104.07) 9.57 (3.88–56.55) 28.89 95.95–114.87)^^ 362 (146–560)⁎⁎⁎,^^^ 1.68 (0.50–3.25)⁎⁎⁎,^^
⁎p b 0.05, ⁎⁎p b 0.01, ⁎⁎⁎p b 0.001 controls versus patients; ^p b 0.05, ^^p b 0.01, ^^^p b 0.001 PD versus HD. Data are shown as mean ± SD or median (full range) depending on their normal or skewed distribution. HDL = high-density lipoproteins cholesterol, LDL = low-density lipoproteins cholesterol, TC/HDL = total cholesterol to HDL (TC/HDL) ratio, LDL/HDL = LDL to HDL ratio, oxLDL = oxidized LDL, oxLDL/LDL = oxLDL to LDL ratio, oxLDL/HDL= oxidized LDL to HDL ratio, Cu/Zn SOD = Cu/Zn superoxide dismutase, MDA = malondialdehyde.
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rho = 0.885, p<0.001
rho = -0.396, p = 0.004
120
4,0 3,5
100
2,5
MDA
oxLDL/HDL
3,0 80 60
2,0 1,5
40
1,0 20 0
0,5 0
20
40
60
80
100
120
0,0
0
20
40
60
80
100
120
140
160
oxLDL
oxLDL Fig. 1. Scatterplot showing the relationship between oxidized LDL (oxLDL) and oxLDL/ HDL ratio in the whole dialyzed group. Correlation was evaluated by Spearman's rank correlation test (rho).
Fig. 2. Scatterplot showing the relationship between oxidized LDL (oxLDL) and malondialdehyde (MDA) in the whole dialyzed group. Correlation was evaluated by Spearman's rank correlation test (rho).
with vintage of dialysis treatment (rho= 0.506, p b 0.001) and with albumin levels (rho= 0.293, p = 0.003), whereas it was inversely related to haematocrit (rho= −0.264, p = 0.007) and BMI (rho= −0.224, p = 0.026). MDA was positively associated with hs CRP (rho= 0.332, p = 0.005), and it was inversely associated with haematocrit (rho= −0.304, p =0.011). There were no statistical differences between oxLDL levels in males [27.64 (6.77–95.06 U/l)] when compared to females [29.24 (8.33–104.07 U/l)], p = 0.468 with use of the Mann–Whitney U test. oxLDL levels were significantly increased in patients treated with α-adrenergic receptor blockers [43.64 (21.87–72.07 U/l) versus 27.30 (6.77–104.07 U/l, p = 0.018], and a tendency to increase of this parameter was also observed in patients treated with β-adrenergic receptor blockers [31.23 (11.33–95.06 U/l) versus 26.53 (6.77–104.07 U/l, p = 0.055]. The median plasma oxLDL levels were not affected by the type of dialysis membrane used, smoking status, type of antihypertensive medication (except mentioned above), nitrates and erythropoietin treatment in all dialyzed patients. To establish determinants of oxLDL and oxLDL/LDL ratio in dialyzed patients, the variables significantly associated with their levels (Table 3), except LDL, which is expected to be a covariate, were included in a multiple regression analysis. oxLDL/HDL, total cholesterol and TC/HDL ratios were independently associated with oxLDL,
accounting for 91% of the variance in oxLDL levels. In the case of oxLDL/LDL ratio, the independent variables were oxLDL/HDL ratio, total cholesterol and HDL levels. The variables in the best subset model explained 72% of the oxLDL/LDL ratio variance (Table 4).
Table 3 The correlations of oxidized LDL (oxLDL) and oxLDL to LDL ratio (oxLDL/LDL) with age, lipid profile, oxidative stress markers and some biochemical parameters in the whole group of dialyzed patients. oxLDL
Age Total cholesterol HDL LDL TC/HDL LDL/HDL oxLDL/LDL Triglycerides Cu/Zn SOD Haematocrit Glucose
oxLDL/LDL
rho
p
rho
p
0.188 0.334 −0.178 0.409 0.426 0.401 0.749 0.139 −0.014 0.227 0.223
0.055 b0.001 0.068 b0.001 b0.001 b0.001 b0.001 0.155 0.886 0.020 0.027
0.181 −0.244 −0.198 −0.207 0.038 −0.058
0.064 0.012 0.043 0.034 0.701 0.559
0.053 0.010 0.041 0.087
0.591 0.913 0.678 0.395
The correlations were calculated by Spearman's rank correlation coefficients (Spearman's rho). HDL = high-density lipoproteins cholesterol, LDL = low-density lipoproteins cholesterol, TC/HDL = total cholesterol to HDL ratio, LDL/HDL = LDL to HDL ratio, oxLDL = oxidized LDL, oxLDL/LDL = oxLDL to LDL ratio, Cu/Zn SOD = Cu/Zn superoxide dismutase.
Discussion In the present study we found that median plasma oxLDL levels and oxLDL/LDL ratio, an accurate estimation of LDL oxidation in vivo (Scheffer et al., 2003), were similar in HD patients and controls, whereas their values were even lower in PD patients when compared to controls. oxLDL levels were not affected by gender, age, type of dialysis membrane used, smoking status, nitrates and erythropoietin treatment in all dialyzed patients; while using of some antihypertensive drugs, such as adrenergic receptor blockers, seems to affect its plasma levels. oxLDL levels in dialyzed patients were positively correlated with lipid profile, whereas the inverse relationship was found between oxidized LDL and MDA, a commonly used biomarker of lipid peroxidation in uremia (Kuchta et al., 2011). Moreover, no correlation has been found between plasma oxLDL and Cu/Zn SOD — the enzyme, which increased levels may represent a compensatory response to oxidative stress in uremia (Pawlak et al., 2005; Washio et al., 2008). The significance of oxLDL as an oxidative stress marker in uremia seems to be still unresolved. Although the reality of oxidative stress has been well established in these patients (Usberti et al., 2002; Kaysen and Eiserich, 2004; Locatelli et al., 2003; Diepeveen et al., 2004), the results concerning oxLDL levels in uremia are still controversial. As mentioned below, there are many discrepancies regarding oxLDL levels between uremics and the healthy people (Takenaka et al., 2002; Samouilidou et al.,
Table 4 Variables predicting oxidized LDL (oxLDL) and oxLDL to LDL ratio (oxLDL/LDL) in multiple regression analysis.
oxLDL
oxLDL/LDL*
Independent variables
Regression coefficient
Standard error
p value
oxLDL/HDL Total cholesterol TC/HDL oxLDL/HDL Total cholesterol HDL
1.113 0.489 −0.762 0.895 −0.658 0.586
0.050 0.048 0.199 0.077 0.073 0.083
b0.001 b0.001 b0.001 b0.001 b0.001 b0.001
Multiple r for variables in the model = 0.959 (*0.860), multiple r2 = 0.921 (*0.739), adjusted r2 = 0.914 (*0.723), both p b 0.001. oxLDL/HDL = oxidized LDL to HDL ratio, TC/HDL = total cholesterol to HDL ratio.
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2010, 2012; Diepeveen et al., 2004; Nissel et al., 2008; Johnson-Davis et al., 2011; Kuchta et al., 2011). The concentration of oxLDL depends not only on the degree of oxidative stress but also on the amount of substrate for oxidation (i.e., the number of LDL particles). In this study, the levels of LDL and total cholesterol were similar in uremic subjects and controls. Moreover, we demonstrated the positive correlations between oxLDL levels, oxLDL/LDL and lipid profile (except for HDL), which is in line with previous studies conducted on the general population (Johnston et al., 2006; Lankin et al., 2011; Alves et al., 2010) as well as on dialysis patients (Samouilidou et al., 2010, 2012; Nissel et al., 2008). Recently, Chen et al. (2011) demonstrated that LDL apheresis reduced LDL, LDL/ HDL ratio and oxLDL levels in uremic patients with hyperlipidemia, supporting this hypothesis. The multiple regression analysis confirmed increased oxLDL/HDL, total cholesterol and TC/HDL ratios as the parameters independently and significantly predicted oxLDL in dialyzed patients. This is a novel observation which suggests that oxLDL levels in peripheral circulation might be associated with reduced formation of serum HDL. In fact, we observed that peritoneally dialyzed patients, who had higher HDL levels than those on HD treatment had also particularly lower oxLDL levels. Moreover, the inverse association was observed between oxLDL and HDL levels in the whole group of dialyzed patients. Our results are in line with recent observation of Samouilidou et al. (2010) who demonstrated that oxLDL was inversely related to HDL and especially HDL2 subclass concentration in HD patients. According to Moradi et al. (2009), chronic kidney failure affects not only quantitative alteration of HDL but also its composition and antioxidant activity. Under the condition of systemic inflammation and oxidative stress, which are characteristic feature of uremia, antioxidant enzymes can be inactivated and oxidized lipids and proteins can accumulate within HDL, which becomes a proinflammatory agent. This dysfunctional HDL, estimated by oxidized HDL, is associated with protein-energy wasting (Honda et al., 2010) and with increased risk of cardiovascular-related mortality in HD patients (Honda et al., 2012). Paraoxonase is an enzyme mostly carried by HDL, which plays a critical role in its antioxidant action by reducing oxLDL (Kaysen and Eiserich, 2004; Kuchta et al., 2011; Moradi et al., 2009). Kuchta et al. (2011) noticed decreased paraoxonase-1 activity in each stage of chronic kidney disease when compared to the control, although the differences between its particular stages did not reach statistical significance. Paradoxically, in the same study, the total antioxidative status (TAS) was higher in the uremic patients than in the controls. The total cholesterol was found to be independent predictor of oxLDL in our analysis. However, total cholesterol also comprises HDL. Zelzer et al. (2011) replaced total cholesterol by a calculated variable “non-HDL-cholesterol”, and this variable remained as an independent predictor for increased oxLDL levels irrespective of gender, age, obesity, inflammatory or metabolic biomarkers in a large cohort of obese people, since the “non-HDL-cholesterol” contains all atherogenic lipoproteins, in contrast to atheroprotective HDL. In the present study, we have also shown that in contrast to the controls, dialyzed patients had higher levels of MDA and Cu/Zn SOD — two different oxidative stress markers. Moreover, we noticed the positive association between them. These markers can be split into two categories: formation of modified molecules by radical oxygen species, such as MDA and induction of antioxidative defense such as Cu/Zn SOD. Their increased values are confirmed by elevated oxidative stress in dialyzed patients. MDA is a lipid peroxidation product generated from polyunsaturated fatty acids during oxidative modification of LDL. MDA easily binds to the ε-amino group of lysine residues to form adducts such as Schiff's base. Another aldehyde, such as 4-hydroxynonenal readily reacts with histidine and cysteine residues. Thus, it is hard to define oxLDL structurally, since it is a mixture of heterogeneously modified lipoprotein particles (Itabe, 2009). In this study, we used oxLDL-measuring kit based on 4E6 mAb commercially available from Mercodia Inc., which is directed against a
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conformational epitope in the apolipoprotein B-100 moiety of LDL (Holvoet et al., 1998). Since only one mAb is used, it should be noted that the antigen detected may not necessarily be an oxidatively modified apoB, and it could partly explain the lack of differences in oxLDL levels in uremic patients and controls. However, we also have not found the positive association between oxLDL, oxLDL/LDL ratio and other oxidative stress markers determined in this study. This observation suggests that oxLDL and oxLDL/LDL ratio may be the questionable markers of oxidative stress in the population of dialyzed patients. Our study had certain limitations. The cross-sectional design did not clearly elucidate the cause-and-effect on the results. In addition, the residual confounders that may affect the oxidization of lipoproteins, but were not included in our present study, should also be considered. Another limitation may be that Mercodia ELISA applied for the analysis of oxLDL levels uses the monoclonal antibodies. Conclusions oxLDL levels and oxLDL/LDL ratio were not raised in uremic patients during dialysis treatment in comparison to controls. Both oxLDL and oxLDL/LDL ratio were independently correlated with lipid profile but not with other oxidative stress markers. These results suggest that oxLDL levels and oxLDL/LDL ratio can serve as the markers of lipoprotein abnormalities rather than the markers of oxidative stress in the population of dialyzed patients. Conflict of interest statement The authors declare that there are no conflicts of interest.
Acknowledgments This work is supported by a grant no 123-28854F from the Medical University, Bialystok, Poland. References Alves MIB, Plaza FA, Tomas RM, Sanchez-Campillo M, Larque E, Perez-Llamas F, et al. Oxidized LDL and its correlation with lipid profile and oxidative stress biomarkers in young healthy Spanish subjects. J Physiol Biochem 2010;66:221–7. An WS, Kim SE, Kim KH, Bae HR, Rha SH. Associations between oxidized LDL to LDL ratio, HDL and vascular calcification in the feet of hemodialysis patients. J Korean Med Sci 2009;24(Suppl. 1):S115–20. Attman PO, Samuelsson OG, Moberly J, Johansson AC, Ljungman S, Weiss LG, et al. Apolipoprotein B-containing lipoproteins in renal failure: the relation to mode of dialysis. Kidney Int 1999;55:1536–42. Chen T-S, Liou S-Y, Wu H-C, Tsai F-J, Tsai C-H, Huang C-Y, et al. Low density lipoprotein (LDL) apheresis reduces atherogenic and oxidative markers in uremic patients with hyperlipidemia. Int Urol Nephrol 2011;43:471–4. Diepeveen SH, Verhoeven GH, van der Palen J, Dikkeschei BL, van Tits LJ, Kolsters G, et al. Oxidative stress in patients with end-stage renal disease prior to the start of renal replacement therapy. Nephron Clin Pract 2004;98:c3–7. Friedewald WT, Levy RI, Fredrickson DS. Estimation of the concentration of low-density lipoprotein cholesterol in the plasma, without use of the preparative ultracentrifuge. Clin Chem 1972;18:499–502. Holvoet P, Vanhaecke J, Janssens S, Van de Werf F, Collen D. Oxidized LDL and malondialdehyde-modified LDL in patients with acute coronary syndromes and stable coronary artery disease. Circulation 1998;98:1487–94. Honda H, Ueda M, Kojima S, Mashiba S, Suzuki H, Hosaka N, et al. Oxidized high-density lipoproteins is associated with protein-energy wasting in maintenance hemodialysis patients. Clin J Am Soc Nephrol 2010;5:1021–8. Honda H, Ueda M, Kojima S, Mashiba S, Michihata T, Takahashi K, et al. Oxidized high-density lipoprotein as a risk factor for cardiovascular events in prevalent hemodialysis patients. Atherosclerosis 2012;220:493–501. Itabe H. Oxidative modification of LDL: its pathological role in atherosclerosis. Clin Rev Allergy Immunol 2009;37:4-11. Johansson AC, Samuelsson O, Attman PO, Haraldsson B, Moberly J, Knight-Gibson C, et al. Dyslipidemia in peritoneal dialysis — relation to dialytic variables. Perit Dial Int 2000;20:306–14. Johnson –Davis KL, Fernelius C, Eliason NB, Wilson A, Beddhu S, Roberts WL. Blood enzymes and oxidative stress in chronic kidney disease: a cross sectional study. Ann Clin Lab Sci 2011;41:331–9.
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Johnston N, Jernberg T, Lagerqvist Bo, Siegbahn A, Wallentin L. Improved identification of patients with coronary artery disease by the use of new lipid and lipoprotein biomarkers. Am J Cardiol 2006;97:640–5. Kaysen GA, Eiserich JP. The role of oxidative stress-altered lipoprotein structure and function and microinflammation on cardiovascular risk in patients with minor renal dysfunction. J Am Soc Nephrol 2004;15:538–48. Kuchta A, Pacanis A, Kortas-Stempak B, Cwiklinska A, Zietkiewicz M, Renke M, et al. Estimation of oxidative stress markers in chronic kidney disease. Kidney Blood Press Res 2011;34:12–9. Lankin V, Viigima M, Tikhaze A, Kumskova E, Konovalova G, Abina J, et al. Cholesterol-rich low density lipoproteins are also more oxidized. Moll Cell Biochem 2011;355:187–91. Locatelli F, Canaud B, Eckardt KU, Stenvinkel P, Wanner C, Zoccali C. Oxidative stress in end-stage renal disease: an emerging treat to patient outcome. Nephrol Dial Transplant 2003;18:1272–80. Londero D, Lo Greco P. Automated high-performance liquid chromatographic separation with spectrofluorometric detection of a malondialdehyde-tiobarbituric acid adduct in plasma. J Chromatogr A 1996;729:207–10. Moradi H, Pahl M, Elahimehr R, Vaziri N. Impaired antioxidant activity of high-density lipoprotein in chronic kidney disease. Transl Res 2009;153:77–85. Nissel R, Faraj S, Sommer K, Henning L, van der Giet M, Querfeld U. Oxidative stress markers in young hemodialysis patients — a pilot study. Clin Nephrol 2008;70: 135–43. Pawlak K, Pawlak D, Mysliwiec M. Cu/Zn superoxide dismutase plasma levels as a new useful clinical biomarker of oxidative stress in patients with end-stage renal disease. Clin Biochem 2005;38:700–5. Pawlak K, Pawlak D, Mysliwiec M. Oxidized LDL to autoantibodies against oxLDL ratio — the new biomarker associated with carotid atherosclerosis and cardiovascular complications in dialyzed patients. Atherosclerosis 2012;224:252–7.
Pawlak K, Pawlak D, Mysliwiec M. The alteration in Cu/Zn superoxide dismutase and adhesion molecules concentrations in diabetic patients with chronic kidney disease: The effect of dialysis treatment. Diabetes Res Clin Pract 2012;98:264–70. Samouilidou E, Karpouza A, Grapsa E, Tzanatou-Exarchou H. Serum oxidized LDL is inversely associated with HDL-2 cholesterol subclass in renal failure patients on hemodialysis. Nephron Clin Pract 2010;115:c289–94. Samouilidou EC, Karpouza AP, Kostopoulos V, Bakirtzi T, Pantelias K, Petras D, et al. Lipid abnormalities and oxidized LDL in chronic kidney disease patients on hemodialysis and peritoneal dialysis. Ren Fail 2012;34:160–4. Scheffer PG, Bos G, Volwater FGFM, Dekker JM, Heine RJ, Teerlink T. Associations of LDL size with in vitro oxidizability and plasma levels of in vivo oxidized LDL in type 2 diabetic patients. Diabet Med 2003;20:563–7. Senti M, Romero R, Pedro-Botet J, Pelegri A, Nogues X, Rubies-Prat J. Lipoprotein abnormalities in hyperlipidemic and normolipidemic men on hemodialysis with chronic renal failure. Kidney Int 1992;41:1394–9. Takenaka T, Takahashi K, Kobayashi T, Oshima E, Iwasaki S, Suzuki H. Oxidized low density lipoprotein (Ox-LDL) as a marker of atherosclerosis in hemodialysis (HD) patients. Clin Nephrol 2002;58:33–7. Usberti M, Gerardi GM, Gazzotti RM, Benedini S, Archetti S, Sugherini L, et al. Oxidative stress and cardiovascular disease in dialyzed patients. Nephron 2002;91:25–33. Washio K, Inagaki M, Tsuji M, Morio Y, Akiyama S, Gotoh H, et al. Oral vitamin C supplementation in hemodialysis patients and its effect on the plasma level of oxidized ascorbic acid and Cu/Zn superoxide dismutase, an oxidative stress marker. Nephron Clin Pract 2008;109:c49–54. Zelzer S, Fuchs N, Almer G, Raggam RB, Pr ller F, Truschnig-Wilders M, et al. High-density lipoprotein cholesterol level is a robust predictor of lipid peroxidation irrespectively of gender, age, obesity, and inflammatory biomarkers. Clin Chim Acta 2011;412: 1345–9.
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Oxidized low-density lipoprotein (oxLDL) plasma levels and oxLDL to LDL ratio — Are they real oxidative stress markers in dialyzed patients? Krystyna Pawlak a, Michal Mysliwiec b, Dariusz Pawlak c,⁎ a b c
Department of Monitored Pharmacotherapy, Medical University, Bialystok, Poland Department of Nephrology and Clinical Transplantation, Medical University, Bialystok, Poland Department of Pharmacodynamics, Medical University, Bialystok, Poland
a r t i c l e
i n f o
Article history: Received 9 March 2012 Accepted 13 December 2012 Keywords: Oxidized LDL oxLDL/LDL ratio Dialysis treatment Oxidative stress markers
a b s t r a c t Aims: Dyslipidemia and oxidative stress are commonly present in patients during maintenance dialysis treatment. However, the significance of oxidized LDL (oxLDL) as a marker of oxidative stress in uremia is still unresolved. The aim of this study was to establish the role of oxLDL and oxLDL/LDL ratio as markers of lipoprotein abnormalities and oxidative stress in the dialyzed patients. Main methods: Plasma oxLDL level was measured by ELISA, and oxLDL/LDL ratio was calculated in 106 dialyzed patients and 20 controls. The linkages between oxLDL, oxLDL/LDL ratio and lipid profile and oxidative stress markers malondialdehyde (MDA) and Cu/Zn superoxide dismutase (Cu/Zn SOD) levels were also analyzed. Key findings: OxLDL levels and oxLDL/LDL ratio were similar in hemodialyzed patients and controls, whereas these parameters were lower in peritoneally dialyzed patients when compared to healthy individuals. In contrast, both MDA and Cu/Zn SOD levels were significantly higher in uremics than in controls. oxLDL and oxLDL/LDL ratio positively correlated with lipid profile (except of HDL), whereas there were no positive associations between these parameters and both MDA and Cu/Zn SOD. Multiple regression analysis confirmed that increased oxLDL/HDL and TC/HDL ratios and total cholesterol levels are the parameters which independently predicted oxLDL in dialyzed patients. In the case of oxLDL/LDL ratio, the independent variables were oxLDL/HDL ratio, total cholesterol and HDL levels. Significance: oxLDL levels and oxLDL/LDL ratio seem to be the markers of lipoprotein abnormalities rather than the markers of oxidative stress in the population of dialyzed patients. © 2013 Elsevier Inc. All rights reserved.
Introduction Oxidative stress (SOX), dyslipidemia and accelerated atherosclerosis are commonly present in patients on maintenance dialysis who constitute the population with higher risk for cardiovascular disease (Usberti et al., 2002; Kaysen and Eiserich, 2004; Chen et al., 2011). Uremic patients are subjected to enhanced SOX as a result of impaired antioxidant defense system and increased pro-oxidant activity that are associated with a state of chronic inflammation, high frequency of diabetes, the uremic syndrome per se and bio-incompatibility of dialyzer membranes and solutions (Locatelli et al., 2003). Renal dyslipidemia is characterized by an abnormal apolipoprotein profile, including decreased levels of apoA-containing lipoproteins and increased levels of apoB-containing lipoproteins. Serum triglyceride levels are increased in the majority of uremic patients, whereas total cholesterol levels are generally normal or lower. In the same group of patients high-density lipoprotein (HDL) cholesterol is reduced, in a ⁎ Corresponding author at: Department of Pharmacodynamics, Mickiewicza 2C str, 15-089 Bialystok, Poland. Tel./fax: +48 85 7485622. E-mail address: [email protected] (D. Pawlak). 0024-3205/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.lfs.2012.12.002
contrast to increased levels of low-density lipoproteins (LDL) (Attman et al., 1999; Senti et al., 1992). Furthermore, the characteristic features of renal dyslipidemia remain essentially unchanged during long-term hemodialysis (HD) treatment (Attman et al., 1999), whereas peritoneal dialysis (PD) is often associated with both hypercholesterolemia and hypertriglyceridemia (Johansson et al., 2000). LDL plays a principal role in oxidative stress due to its oxidative modification to oxidized LDL (oxLDL). Formed oxLDL acts as chemoattractant for monocytes and induces inflammatory reactions within the arterial wall, which are implicated in all phases of the atherosclerotic plaque development (Itabe, 2009). Recently, we demonstrated that oxLDL to autoantibodies against oxLDL ratio can be a new biomarker associated with carotid atherosclerosis and cardiovascular complications in dialyzed patients (Pawlak et al., 2012). However, the significance of oxLDL as a marker of SOX in uremia is still unclear. Some studies reported significant differences between oxLDL levels in uremic patients and the healthy people (Takenaka et al., 2002; Samouilidou et al., 2010, 2012), but others did not show such differences (Diepeveen et al., 2004; Nissel et al., 2008; Johnson-Davis et al., 2011). Kuchta et al. (2011), demonstrated that levels of oxLDL, although higher in uremic patients when compared to the controls, were not altered in patients with different
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stages of uremia and treated with HD. oxLDL to LDL (oxLDL/LDL) ratio, an accurate estimation of in vivo LDL oxidation (Scheffer et al., 2003), was measured only during the solitary study of An et al. (2009) in HD patients with and without vascular calcification. There is limited evidence concerning the correlation between plasma level of oxLDL and lipid profile in patients on dialysis treatment (Samouilidou et al., 2010, 2012). We have also found a few controversial reports regarding the relationship of oxLDL and other oxidative stress markers in dialyzed patients (Nissel et al., 2008; Johnson-Davis et al., 2011; Kuchta et al., 2011). With respect to conflicting data about oxLDL and its role as a biomarker, the aim of this study was to establish the significance of oxLDL and oxLDL/LDL ratio as the markers of lipoprotein abnormalities and oxidative stress in the population of dialyzed patients. We focused on the association between oxLDL and oxLDL/LDL ratio with atherogenic lipoprotein: triglycerides, LDL, total cholesterol to HDL (TC/HDL) ratio, LDL to HDL (LDL/HDL) ratio, and oxLDL to HDL (oxLDL/HDL) ratio, which is the new and more potent biomarker than standard lipid measurements for discriminating between subjects with coronary artery disease and healthy subjects (Johnston et al., 2006; Lankin et al., 2011). Moreover, we analyzed other oxidative stress markers: plasma malondialdehyde (MDA) concentration as a commonly used biomarker of lipid peroxidation in uremia (Kuchta et al., 2011), and plasma Cu/Zn superoxide dismutase (Cu/Zn SOD) levels, which have been previously reported by us (Pawlak et al., 2005, 2012) and by another author (Washio et al., 2008) as a useful oxidative stress marker in uremic patients. Materials and methods Subjects One hundred and six patients were enrolled in this cross-sectional study and divided into 2 groups: 52 patients on PD and 54 on maintenance HD. All patients were in stable clinical conditions and without the features of active infections, autoimmune diseases or thrombotic complications. None of the patients received immunosuppressive treatment, lipid-lowering agents and non-steroidal anti-inflammatory drugs. Vitamin C, E or allopurinol is expected to have an impact on oxLDL levels, and for this reason the patients were asked not to take these medications 2 weeks before and at the time of the study. Body mass index (BMI) was calculated by dividing the weight in kilograms by the square of the height in meters. CVD was defined as documented in the medical record a history of myocardial infarction, ischaemic stroke, coronary revascularization procedures, angina pectoris, typical changes in coronary angiography, typical ischaemic electrocardiographic changes, peripheral artery surgery (not including the arteriovenous fistula), intermittent claudication or pain at rest. Patients who had smoked daily or occasionally within the 30 days preceding the study were defined as “smokers”. Antihypertensive drug and erythropoietin usage was recorded in the case of each participant, and it was presented as the percent of patients treated with expected drug. The group of hemodialysis patients received conventional 4-h HD, three times weekly, (the dialysate was endotoxin-free). Dialysis prescription was aimed to reach a value of Kt/V ≥ 1.2. All PD patients were performing four 2-liter exchanges a day using either the Baxter TwinBag system or the Fresenius Andy Plus system. Dwell times generally averaged 4–6 h during the day and 8 h overnight. The glucose concentration ranged from 1.36 to 3.86%. The osmotic pressure of PD dialysis fluid was adjusted in accordance to the extent of ultrafiltration required for each patient. Dialysis adequacy was assessed by measuring the value of Kt/v (mean Kt/v = 2.25 ± 0.47). Twenty of sex- and age-matched healthy subjects who were receiving no drugs or vitamin supplements during the study volunteered as controls. All of them had a regular diet and no history of hypertension, diabetes mellitus or renal disease. Detailed characteristics of the controls and dialyzed patients are presented in Table 1.
Table 1 Biochemical and clinical characteristics of controls and end-stage renal disease patients on peritoneal dialysis (PD) and hemodialysis (HD).
Sex, M/F Age, years BMI, kg/m2 Haematocrit, % White blood cells, ×103 μl Total protein, g/dl Albumin, g/dl Glucose, mg/dl Hs CRP, μg/ml SBP, mm Hg DBP, mm Hg Smokers, % ESRD etiology Glomerulonephritis, n Interstitial nephritis, n Polycystic kidney disease, n Diabetes mellitus, n Hypertensive nephropathy, n Secondary amyloidosis, n Unknown, n Vintage of dialysis treatment, months EPO treatment, % EPO dose, U/kg body weight/week Cardiovascular disease, % ACEI, % Calcium channel antagonists, % β-blockers, % α-blockers, % Nitrates, %
Controls
PD
HD
12/8 53.21 ± 10.79 26.04 ± 3.74 42.07 ± 3.05 5.98 ± 1.01
25/27 52.80 ± 12.43 25.50 ± 4.15 36.44 ± 4.79⁎⁎⁎ 6.68 ± 2.03
32/22 57.68 ± 13.39 24.99 ± 4.24 35.42 ± 4.11⁎⁎⁎ 5.82 ± 1.57^
6.74 ± 0.36 4.62 ± 1.40 85 (70–95) 0.98 (0.2–11.0) 128.89 ± 10.51 81.11 ± 6.74 20
6.56 ± 0.61 3.48 ± 0.48⁎⁎⁎ 103 (66–469)⁎ 3.04 (0.1–46.0)⁎⁎⁎ 131.80 ± 22.06 82.15 ± 12.23 25
6.76 ± 0.53 3.84 ± 0.43⁎⁎,^ 101 (36–334)⁎ 4.86 (0.1–49)⁎⁎⁎,^^ 135.47 ± 24.27 83.73 ± 11.73 29
N/A N/A N/A
15 4 10
17 9 7
N/A N/A
13 4
8 6
N/A
4
3
N/A N/A
2 15.0 (3.0–134.0)
4 34.0 (3.0–241.0)^^
N/A N/A
52 51.3 (26.3–160.0)
83^ 87.9 (18.7–161.6)^
N/A
50
72^
N/A N/A
54 54
61 63
N/A N/A N/A
44 10 12
46 14 20^
⁎pb 0.05, ⁎⁎pb 0.01, ⁎⁎⁎pb 0.001 controls versus patients; ^pb 0.05, ^^pb 0.01 PD versus HD. Data are shown as mean ± SD or median (full range) depending on their normal or skewed distribution. BMI = body mass index, SBP = systolic blood pressure, DBP = diastolic blood pressure, EPO = erythropoietin, ACEI = angiotensin-converting enzyme inhibitors, N/A = not applicable.
The study protocol was approved by our institutional ethical board, and informed consent was obtained from all patients and controls. Blood sampling and laboratory measurements Investigations were performed on the morning under fasting conditions. Blood samples were taken directly from the arteriovenous fistula immediately before the beginning of a routine 4-h HD session. Blood was drawn from controls and PD patients from the antecubital vein into the EDTA-containing vials. The plasma samples were prepared in a conventional manner, aliquoted and stored at − 70 °C until further assay. Serum lipids Routine blood chemistry and levels of lipids (total cholesterol, HDL and triglycerides) were analyzed using fresh blood samples according to established enzymatic methods in the local laboratories. LDL levels were calculated using Friedewald's formula (Friedewald et al., 1972). In vivo oxidized LDL Plasma level of oxidized LDL in each sample was measured in duplicate by a commercially available sandwich ELISA (Mercodia,
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Uppsala, Sweden). This assay is based on a murine monoclonal antibody (mAb-4E6) directed against a conformational epitope in the apoB-100 moiety of LDL, which is generated as a consequence of reaction of lysine residues with aldehydes (Holvoet et al., 1998). The intra-assay and interassay coefficients of variation were 5.0% and 5.5%, respectively. To obtain the percentage of oxidized LDL particles, the ratio of oxidized LDL to LDL (oxLDL/LDL) was also calculated. Therefore, results were expressed as U/l and U/mmol LDL, respectively. Oxidative stress markers and inflammatory state Plasma Cu/Zn SOD levels were measured by ELISA (Bender MedSystems, Vienna, Austria), according to the manufacturer's instruction. Malondialdehyde concentration was determined by highperformance liquid chromatography (HPLC) according to Londero and Lo Greco (1996). The reversed-phase HPLC system consisted of a Waters Sherisorb S3 ODS2 150× 2.1 mm column (USA) and HP 1050 series pump (Germany). Rheodyne injection valve was fitted to a sample loop (5 μl). The column effluent was monitored using a programmable fluorescence detector HP 1046A (Germany). The optimized conditions were determined by recording fluorescence spectra with a stop-flow technique. Excitation and emission wavelengths were set at 532/553 nm. The output of the detector was connected to a single instrument LC-2D ChemStation (Germany). The mobile phase was pumped at a flow-rate of 0.1 ml/min consisted of 40:60 (v/v) 0.05 M methanol–potassium phosphate buffer with pH 6.8. Chromatography was carried out at 25 °C. Plasma C-reactive protein levels were measured by high sensitivity ELISA (Imuclone CRP (hs) ELISA, American Diagnostica Inc., Greenwich, USA). Statistical analysis The Shapiro–Wilk test for normality was used for data distribution analysis. The normally distributed data were expressed as mean ± SD. The non-Gaussian data were presented as median (full range), depending on their distribution. Multiple group comparisons were performed by one-way analysis of variance (ANOVA), and significant differences between groups were assessed by Tukey–Kramer's test or non-parametric Mann– Whitney's U-test. The correlations between study variables were calculated by Spearman's rank correlation coefficients (Spearman's rho). Multiple regression analysis was performed using a stepwise model to determine which variables predict oxLDL and oxLDL/LDL ratio. A two-tailed p value b 0.05 was considered to be statistically significant. Computations were performed using Statistica ver.6 computer software (StatSoft, Tulsa, OK, USA).
255
Results Basal characteristics of patients and controls are summarized in Table 1. Haematocrit and albumin levels were significantly decreased in PD and HD patients than in controls; furthermore, albumin level was significantly lower in PD group than in HD group. Glucose and hs CRP levels were significantly elevated in both groups of dialyzed patients when compared to the controls; hs CRP was significantly increased in HD but not in PD patients. White blood cell count was significantly lower in HD patients when compared to PD patients and controls. The median time of dialysis treatment was significantly prolonged in HD group than in PD group. Moreover, HD patients treated with erythropoietin were of greater percentage when compared to PD patients. The prevalence of cardiovascular complications was significantly increased in HD patients, and more subjects from this group received nitrates when compared to PD group. According to results of lipid profile (Table 2), dialyzed patients tended to have higher levels of total cholesterol and LDL when compared to controls. In contrast, HDL tended to be decreased in PD patients, while statistical significance was reached in HD group when compared to PD and controls. In both groups of dialyzed patients TC/HDL and LDL/HDL ratios and triglycerides were significantly increased than in controls. Peritoneally dialyzed patients had lower oxLDL levels and oxLDL/LDL and oxLDL/HDL ratios than HD and controls. Regarding oxidative stress markers, both Cu/Zn SOD and MDA plasma concentrations were significantly higher in dialyzed patients (particularly in those on HD treatment) than in controls (Table 2). As can be seen in Fig. 1, a strong positive correlation was between oxLDL and oxLDL/HDL ratio. oxLDL also positively correlated with other lipid parameters, such as total cholesterol, LDL, TC/HDL, LDL/ HDL and oxLDL/LDL ratios. Similarly, increased oxLDL/LDL ratio was associated with increased oxLDL/HDL ratio (rho = 0.721, p b 0.001) and with other lipid parameters, as presented in Table 3. In contrast, there was a tendency to inverse correlation between oxLDL and HDL levels in dialyzed patients, whereas the statistical significance was reached when the value of oxLDL was divided by LDL. Moreover, oxLDL was positively associated with haematocrit value and glucose concentration. There was a strong positive relationship between oxLDL/LDL ratio and oxLDL/HDL ratio, as well as oxLDL/LDL ratio was inversely associated with total cholesterol and LDL levels. MDA, another marker of lipid peroxidation, was inversely correlated with oxLDL (Fig. 2), and it was also inversely associated with oxLDL/LDL ratio (rho= −0.314, p = 0.009). Moreover, there was no relationship between Cu/Zn SOD and oxLDL as well as oxLDL/LDL ratio. In a contrast, MDA was positively associated with Cu/Zn SOD (rho= 0.464, p b 0.001). Neither MDA nor Cu/Zn SOD was correlated with lipid profile, although a tendency to inverse correlation was observed between Cu/Zn SOD and HDL (rho= −0.186, p = 0.056). Cu/Zn SOD was positively correlated
Table 2 Lipid profiles and oxidative stress markers in the healthy controls and end-stage renal disease patients on peritoneal dialysis (PD) and hemodialysis (HD).
Total cholesterol, mmol/l HDL, mmol/l LDL, mmol/l TC/HDL LDL/HDL Triglycerides, mmol/l oxLDL, U/l oxLDL/LDL, U/mmol oxLDL/HDL, U/mmol Cu/Zn SOD, ng/ml MDA, μM
Controls
PD
HD
4.86 ± 0.76 1.52 ± 0.30 2.95 ± 0.74 3.34 ± 0.94 2.05 ± 0.79 0.77 (0.43–1.70) 38.33 (17.87–106.26) 14.21 (5.96–33.96) 27.08 (14.53–79.48) 58 (24–86) 0.66 (0.35–1.60)
5.59 ± 1.18 1.44 ± 0.45 3.41 ± 1.02 4.13 ± 1.18⁎⁎ 2.55 ± 1.01⁎ 1.53 (0.64–4.11)⁎⁎⁎ 27.30 (8.33–81.73)⁎ 8.10 (2.32–35.64)⁎ 19.27 (5.22–63.6)⁎⁎ 179 (46–474)⁎⁎⁎ 0.89 (0.36–3.60)⁎
5.33 ± 1.27 1.17 ± 0.33⁎⁎,^^ 3.41 ± 1.16 4.82 ± 1.48⁎⁎⁎,^^ 3.09 ± 1.24⁎⁎,^ 1.43 (0.35–5.43)⁎⁎ 30.76 (6.77–104.07) 9.57 (3.88–56.55) 28.89 95.95–114.87)^^ 362 (146–560)⁎⁎⁎,^^^ 1.68 (0.50–3.25)⁎⁎⁎,^^
⁎p b 0.05, ⁎⁎p b 0.01, ⁎⁎⁎p b 0.001 controls versus patients; ^p b 0.05, ^^p b 0.01, ^^^p b 0.001 PD versus HD. Data are shown as mean ± SD or median (full range) depending on their normal or skewed distribution. HDL = high-density lipoproteins cholesterol, LDL = low-density lipoproteins cholesterol, TC/HDL = total cholesterol to HDL (TC/HDL) ratio, LDL/HDL = LDL to HDL ratio, oxLDL = oxidized LDL, oxLDL/LDL = oxLDL to LDL ratio, oxLDL/HDL= oxidized LDL to HDL ratio, Cu/Zn SOD = Cu/Zn superoxide dismutase, MDA = malondialdehyde.
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rho = 0.885, p<0.001
rho = -0.396, p = 0.004
120
4,0 3,5
100
2,5
MDA
oxLDL/HDL
3,0 80 60
2,0 1,5
40
1,0 20 0
0,5 0
20
40
60
80
100
120
0,0
0
20
40
60
80
100
120
140
160
oxLDL
oxLDL Fig. 1. Scatterplot showing the relationship between oxidized LDL (oxLDL) and oxLDL/ HDL ratio in the whole dialyzed group. Correlation was evaluated by Spearman's rank correlation test (rho).
Fig. 2. Scatterplot showing the relationship between oxidized LDL (oxLDL) and malondialdehyde (MDA) in the whole dialyzed group. Correlation was evaluated by Spearman's rank correlation test (rho).
with vintage of dialysis treatment (rho= 0.506, p b 0.001) and with albumin levels (rho= 0.293, p = 0.003), whereas it was inversely related to haematocrit (rho= −0.264, p = 0.007) and BMI (rho= −0.224, p = 0.026). MDA was positively associated with hs CRP (rho= 0.332, p = 0.005), and it was inversely associated with haematocrit (rho= −0.304, p =0.011). There were no statistical differences between oxLDL levels in males [27.64 (6.77–95.06 U/l)] when compared to females [29.24 (8.33–104.07 U/l)], p = 0.468 with use of the Mann–Whitney U test. oxLDL levels were significantly increased in patients treated with α-adrenergic receptor blockers [43.64 (21.87–72.07 U/l) versus 27.30 (6.77–104.07 U/l, p = 0.018], and a tendency to increase of this parameter was also observed in patients treated with β-adrenergic receptor blockers [31.23 (11.33–95.06 U/l) versus 26.53 (6.77–104.07 U/l, p = 0.055]. The median plasma oxLDL levels were not affected by the type of dialysis membrane used, smoking status, type of antihypertensive medication (except mentioned above), nitrates and erythropoietin treatment in all dialyzed patients. To establish determinants of oxLDL and oxLDL/LDL ratio in dialyzed patients, the variables significantly associated with their levels (Table 3), except LDL, which is expected to be a covariate, were included in a multiple regression analysis. oxLDL/HDL, total cholesterol and TC/HDL ratios were independently associated with oxLDL,
accounting for 91% of the variance in oxLDL levels. In the case of oxLDL/LDL ratio, the independent variables were oxLDL/HDL ratio, total cholesterol and HDL levels. The variables in the best subset model explained 72% of the oxLDL/LDL ratio variance (Table 4).
Table 3 The correlations of oxidized LDL (oxLDL) and oxLDL to LDL ratio (oxLDL/LDL) with age, lipid profile, oxidative stress markers and some biochemical parameters in the whole group of dialyzed patients. oxLDL
Age Total cholesterol HDL LDL TC/HDL LDL/HDL oxLDL/LDL Triglycerides Cu/Zn SOD Haematocrit Glucose
oxLDL/LDL
rho
p
rho
p
0.188 0.334 −0.178 0.409 0.426 0.401 0.749 0.139 −0.014 0.227 0.223
0.055 b0.001 0.068 b0.001 b0.001 b0.001 b0.001 0.155 0.886 0.020 0.027
0.181 −0.244 −0.198 −0.207 0.038 −0.058
0.064 0.012 0.043 0.034 0.701 0.559
0.053 0.010 0.041 0.087
0.591 0.913 0.678 0.395
The correlations were calculated by Spearman's rank correlation coefficients (Spearman's rho). HDL = high-density lipoproteins cholesterol, LDL = low-density lipoproteins cholesterol, TC/HDL = total cholesterol to HDL ratio, LDL/HDL = LDL to HDL ratio, oxLDL = oxidized LDL, oxLDL/LDL = oxLDL to LDL ratio, Cu/Zn SOD = Cu/Zn superoxide dismutase.
Discussion In the present study we found that median plasma oxLDL levels and oxLDL/LDL ratio, an accurate estimation of LDL oxidation in vivo (Scheffer et al., 2003), were similar in HD patients and controls, whereas their values were even lower in PD patients when compared to controls. oxLDL levels were not affected by gender, age, type of dialysis membrane used, smoking status, nitrates and erythropoietin treatment in all dialyzed patients; while using of some antihypertensive drugs, such as adrenergic receptor blockers, seems to affect its plasma levels. oxLDL levels in dialyzed patients were positively correlated with lipid profile, whereas the inverse relationship was found between oxidized LDL and MDA, a commonly used biomarker of lipid peroxidation in uremia (Kuchta et al., 2011). Moreover, no correlation has been found between plasma oxLDL and Cu/Zn SOD — the enzyme, which increased levels may represent a compensatory response to oxidative stress in uremia (Pawlak et al., 2005; Washio et al., 2008). The significance of oxLDL as an oxidative stress marker in uremia seems to be still unresolved. Although the reality of oxidative stress has been well established in these patients (Usberti et al., 2002; Kaysen and Eiserich, 2004; Locatelli et al., 2003; Diepeveen et al., 2004), the results concerning oxLDL levels in uremia are still controversial. As mentioned below, there are many discrepancies regarding oxLDL levels between uremics and the healthy people (Takenaka et al., 2002; Samouilidou et al.,
Table 4 Variables predicting oxidized LDL (oxLDL) and oxLDL to LDL ratio (oxLDL/LDL) in multiple regression analysis.
oxLDL
oxLDL/LDL*
Independent variables
Regression coefficient
Standard error
p value
oxLDL/HDL Total cholesterol TC/HDL oxLDL/HDL Total cholesterol HDL
1.113 0.489 −0.762 0.895 −0.658 0.586
0.050 0.048 0.199 0.077 0.073 0.083
b0.001 b0.001 b0.001 b0.001 b0.001 b0.001
Multiple r for variables in the model = 0.959 (*0.860), multiple r2 = 0.921 (*0.739), adjusted r2 = 0.914 (*0.723), both p b 0.001. oxLDL/HDL = oxidized LDL to HDL ratio, TC/HDL = total cholesterol to HDL ratio.
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2010, 2012; Diepeveen et al., 2004; Nissel et al., 2008; Johnson-Davis et al., 2011; Kuchta et al., 2011). The concentration of oxLDL depends not only on the degree of oxidative stress but also on the amount of substrate for oxidation (i.e., the number of LDL particles). In this study, the levels of LDL and total cholesterol were similar in uremic subjects and controls. Moreover, we demonstrated the positive correlations between oxLDL levels, oxLDL/LDL and lipid profile (except for HDL), which is in line with previous studies conducted on the general population (Johnston et al., 2006; Lankin et al., 2011; Alves et al., 2010) as well as on dialysis patients (Samouilidou et al., 2010, 2012; Nissel et al., 2008). Recently, Chen et al. (2011) demonstrated that LDL apheresis reduced LDL, LDL/ HDL ratio and oxLDL levels in uremic patients with hyperlipidemia, supporting this hypothesis. The multiple regression analysis confirmed increased oxLDL/HDL, total cholesterol and TC/HDL ratios as the parameters independently and significantly predicted oxLDL in dialyzed patients. This is a novel observation which suggests that oxLDL levels in peripheral circulation might be associated with reduced formation of serum HDL. In fact, we observed that peritoneally dialyzed patients, who had higher HDL levels than those on HD treatment had also particularly lower oxLDL levels. Moreover, the inverse association was observed between oxLDL and HDL levels in the whole group of dialyzed patients. Our results are in line with recent observation of Samouilidou et al. (2010) who demonstrated that oxLDL was inversely related to HDL and especially HDL2 subclass concentration in HD patients. According to Moradi et al. (2009), chronic kidney failure affects not only quantitative alteration of HDL but also its composition and antioxidant activity. Under the condition of systemic inflammation and oxidative stress, which are characteristic feature of uremia, antioxidant enzymes can be inactivated and oxidized lipids and proteins can accumulate within HDL, which becomes a proinflammatory agent. This dysfunctional HDL, estimated by oxidized HDL, is associated with protein-energy wasting (Honda et al., 2010) and with increased risk of cardiovascular-related mortality in HD patients (Honda et al., 2012). Paraoxonase is an enzyme mostly carried by HDL, which plays a critical role in its antioxidant action by reducing oxLDL (Kaysen and Eiserich, 2004; Kuchta et al., 2011; Moradi et al., 2009). Kuchta et al. (2011) noticed decreased paraoxonase-1 activity in each stage of chronic kidney disease when compared to the control, although the differences between its particular stages did not reach statistical significance. Paradoxically, in the same study, the total antioxidative status (TAS) was higher in the uremic patients than in the controls. The total cholesterol was found to be independent predictor of oxLDL in our analysis. However, total cholesterol also comprises HDL. Zelzer et al. (2011) replaced total cholesterol by a calculated variable “non-HDL-cholesterol”, and this variable remained as an independent predictor for increased oxLDL levels irrespective of gender, age, obesity, inflammatory or metabolic biomarkers in a large cohort of obese people, since the “non-HDL-cholesterol” contains all atherogenic lipoproteins, in contrast to atheroprotective HDL. In the present study, we have also shown that in contrast to the controls, dialyzed patients had higher levels of MDA and Cu/Zn SOD — two different oxidative stress markers. Moreover, we noticed the positive association between them. These markers can be split into two categories: formation of modified molecules by radical oxygen species, such as MDA and induction of antioxidative defense such as Cu/Zn SOD. Their increased values are confirmed by elevated oxidative stress in dialyzed patients. MDA is a lipid peroxidation product generated from polyunsaturated fatty acids during oxidative modification of LDL. MDA easily binds to the ε-amino group of lysine residues to form adducts such as Schiff's base. Another aldehyde, such as 4-hydroxynonenal readily reacts with histidine and cysteine residues. Thus, it is hard to define oxLDL structurally, since it is a mixture of heterogeneously modified lipoprotein particles (Itabe, 2009). In this study, we used oxLDL-measuring kit based on 4E6 mAb commercially available from Mercodia Inc., which is directed against a
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conformational epitope in the apolipoprotein B-100 moiety of LDL (Holvoet et al., 1998). Since only one mAb is used, it should be noted that the antigen detected may not necessarily be an oxidatively modified apoB, and it could partly explain the lack of differences in oxLDL levels in uremic patients and controls. However, we also have not found the positive association between oxLDL, oxLDL/LDL ratio and other oxidative stress markers determined in this study. This observation suggests that oxLDL and oxLDL/LDL ratio may be the questionable markers of oxidative stress in the population of dialyzed patients. Our study had certain limitations. The cross-sectional design did not clearly elucidate the cause-and-effect on the results. In addition, the residual confounders that may affect the oxidization of lipoproteins, but were not included in our present study, should also be considered. Another limitation may be that Mercodia ELISA applied for the analysis of oxLDL levels uses the monoclonal antibodies. Conclusions oxLDL levels and oxLDL/LDL ratio were not raised in uremic patients during dialysis treatment in comparison to controls. Both oxLDL and oxLDL/LDL ratio were independently correlated with lipid profile but not with other oxidative stress markers. These results suggest that oxLDL levels and oxLDL/LDL ratio can serve as the markers of lipoprotein abnormalities rather than the markers of oxidative stress in the population of dialyzed patients. Conflict of interest statement The authors declare that there are no conflicts of interest.
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