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Oxidized high-density lipoprotein as a risk factor for cardiovascular events in prevalent hemodialysis patients
Atherosclerosis 220 (2012) 493–501
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Oxidized high-density lipoprotein as a risk factor for cardiovascular events in prevalent hemodialysis patients Hirokazu Honda a,∗ , Masashi Ueda b , Shiho Kojima b , Shinichi Mashiba b , Tetsuo Michihata c , Keiko Takahashi d , Kanji Shishido d , Tadao Akizawa a a
Division of Nephrology, Department of Medicine, Showa University School of Medicine, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8666, Japan Ikagaku Co. Ltd., Kyoto, Japan c Ebara Clinic, Tokyo, Japan d Kawasaki Clinic, Kawasaki, Japan b
a r t i c l e
i n f o
Article history: Received 1 June 2011 Received in revised form 13 October 2011 Accepted 25 October 2011 Available online 3 November 2011 Keywords: Oxidized high-density lipoprotein Interleukin-6 Cardiovascular disease
a b s t r a c t Background and objectives: Here, we assessed the impact of oxidized high-density lipoprotein (oxHDL), dysfunctional HDL, on mortality and cardiovascular disease (CVD) events in prevalent HD patients and compared oxHDL to interleukin-6 (IL-6), a strong predictor of CVD events in HD patients. Design, setting, participants, and measurements: This prospective study examined a cohort of prevalent HD patients (n = 412). Blood samples were obtained at baseline to measure lipids, high-sensitive C-reactive protein (hsCRP), IL-6, oxidized low-density lipoprotein, N-terminal pro B-type natriuretic peptide, intercellular adhesion molecule 1 (ICAM-1), myeloperoxidase, adiponectin, and oxHDL. Carotid intima-media thickness (CIMT) was assessed at baseline and 3-year follow-up. Nutritional status was assessed by subjective global assessment (SGA), body mass index, and geriatric nutritional risk index (GNRI). After the baseline assessment, study patients were prospectively followed up (mean observational period, 40 months). Results: At baseline, patients with high oxHDL had a worse nutritional state and higher HDL-cholesterol (HDL-chol), ICAM-1, and adiponectin levels and a higher oxHDL/HDL-chol ratio than low oxHDL patients. A combination of high oxHDL and high IL-6 was significantly associated with increased CIMT at baseline and a larger increase in CIMT at 3-year follow-up. High oxHDL did not predict all-cause mortality; however, it was significantly associated with CVD-related mortality and composite CVD events, particularly with concomitant high IL-6. These associations were confirmed in multivariate Cox hazard models adjusted with confounding variables. Conclusions: High oxHDL, particularly with concomitant high IL-6, may be associated with an increased risk of CVD events and CVD-related mortality in prevalent HD patients. © 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction High-density lipoprotein (HDL) has the capacity to promote cholesterol efflux from monocytes and macrophages, and it has antioxidant and anti-inflammatory activities in its interactions with circulating cells. HDL inhibits both leukocyte and platelet activation and thus exerts further systemic anti-inflammatory actions [1–5]. These anti-inflammatory effects may contribute to protection from cardiovascular disease (CVD) events [6,7], although how HDL activities other than lipid transport may contribute to protection against atherosclerosis is unclear [8,9].
∗ Corresponding author. Tel.: +81 3 3784 8533; fax: +81 3 3784 5934. E-mail address: [email protected] (H. Honda). 0021-9150/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2011.10.038
In the presence of systemic inflammation and oxidative stress, antioxidant enzymes can be inactivated and HDL can accumulate oxidized lipids and proteins and thus become proinflammatory. Under these conditions, the most abundant protein in HDL, apolipoprotein (apo) A1, can be modified by reactive oxygen species. Alterations of HDL by oxidation impair its anti-inflammatory effects, resulting in a dysfunctional HDL with pro-inflammatory effects [5–7]. In fact, dysfunctional HDL is often present in patients with advanced chronic kidney disease [5,7]. Kalantar-Zadeh et al. demonstrated that dysfunctional HDL is significantly associated with more co-morbid conditions and a lower quality of life in prevalent hemodialysis (HD) patients; moreover, HD patients with dysfunctional HDL had significantly worse outcomes than patients with anti-inflammatory HDL [10]. A recent study in prevalent HD patients demonstrated that dysfunctional HDL, estimated by oxidized HDL (oxHDL), is strongly associated with protein-energy wasting (PEW), particularly in patients with
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inflammation [11]. Thus, dysfunctional HDL in patients on HD is a risk factor for the development and advancement of the PEW state, although factors associated with uremia per se may contribute to PEW [12]. Therefore, oxHDL is thought to represent a risk factor for mortality and morbidity in prevalent HD patients. However, associations between dysfunctional HDL and atherosclerosis or CVD events have not been fully evaluated. Moreover, whether dysfunctional HDL levels can be used as a biomarker for risk of CVD events and replace currently used biomarkers in prevalent HD patients is unclear. The present study of prevalent HD patients investigated (1) associations between oxHDL and CVD-related biomarkers, (2) the impact of oxHDL on atherosclerosis and CVD events and mortality, and (3) the potential of oxHDL together with interleukin-6 (IL-6), which alone is a strong predictor of CVD, PEW, and mortality in end-stage renal disease (ESRD) patients [13], as a combination biomarker for risk of CVD events. 2. Materials and methods 2.1. Patients This study was performed as part of an ongoing prospective cohort study that recruited 412 prevalent HD patients treated at two hemodialysis clinics. Patients who did not provide blood samples, had an anticipated life expectancy of <6 months, or presented with clinical signs of overt infection, acute vasculitis, or liver disease at the time of recruitment were excluded from the study. Participants were outpatients who had been undergoing HD for ≥6 months and were ≥20 years old. Written informed consent was obtained from all patients. The study protocol was approved by the ethics committee of our institute. Medical treatments used to manage the condition of all patients were similar, and the medication for each patient was prescribed according to Kidney Disease Outcomes Quality Initiative (K/DOQI) guidelines [14]. Patients were managed similarly in terms of protocols for HD, including prescription of dialysis dose. Basically, all recruited patients underwent routine hemodialysis 3 times/week (3–4 h/session) using conventional bicarbonate or acetate-free dialysate and standard high-flux cellulose triacetate, polysulfone, or other dialysis membranes. Patients were instructed to maintain a daily protein intake >1.0–1.2 g/kg body weight. Dialysis dosage was calculated using second-generation Kt/V, which was estimated using the Daugirdas formula. 2.2. Study design This cohort study was designed as a prospective study with a cross-sectional analysis at baseline. The baseline analysis assessed (1) the associations between oxHDL and biomarkers associated with mortality and CVD events and (2) the impact of oxHDL on nutritional state and atherosclerosis estimated by carotid intimamedia thickness (CIMT). After the baseline study, patients were followed up within 48 months to assess the impact of oxHDL on change in CIMT, number of CVD events, and mortality; the influence of inflammation, as determined by IL-6 levels, on these associations was also assessed. The following clinicopathological factors were recorded at baseline: cause of ESRD, presence of diabetes mellitus (DM), history of CVD, and smoking history. History of CVD was defined by medical history, clinical symptoms, or findings indicating cerebrovascular (stroke) and/or peripheral vascular disease. Non-fasting venous blood samples were drawn 3 days after a hemodialysis session and immediately before the next hemodialysis session. Routine biochemical parameters, lipid markers, and
high-sensitivity C-reactive protein (hsCRP) were measured in the blood samples. Serum was isolated by centrifugation and maintained at −80 ◦ C until use; oxidized low-density lipoprotein (oxLDL), oxHDL, IL-6, N-terminal pro B-type natriuretic peptide (NT-proBNP), myeloperoxidase (MPO), and adiponectin were measured at baseline. In the survival analysis, the time to death from all causes or from a major CVD-related event (mean follow-up period: 40 months) was determined. A major CVD-related death was defined as a nonfatal myocardial infarction (MI), non-fatal stroke, or death from CVD causes. In the analysis of composite CVD events, the time to each event was determined, and composite CVD events consisted of non-fatal CVD events, fatal MI, angina pectoris (AP), fatal cerebral infarction, or arterial or peripheral artery disease. The fatal CVD events were defined by electrocardiography and cardiac angiography for MI or AP, brain CT or MRI for cerebral infarction, and enhanced CT scan or arterial or peripheral angiography for arterial or peripheral artery disease. Change in CIMT was estimated by carotid ultrasound at baseline and at follow up (within 48 months). 2.3. Assessment of nutritional status PEW Nutritional status was assessed by subjective global assessment (SGA) and geriatric nutritional risk index (GNRI) [15]. Fully trained physicians assessed SGA according to K/DOQI recommendations; 4 items (weight loss during the preceding 6 months; anorexia; subcutaneous fat; and muscle mass) were scored on a 7-point Likert scale [16]. Actual body mass index was calculated as weight (kg)/height (m)2 . Normalized protein catabolic rate (nPCR) was also calculated. 2.4. Carotid ultrasound A B-mode ultrasound scanner equipped with a 14-MHz linear probe was used to examine transversal and longitudinal views of carotid artery walls in all 412 participants at the baseline assessment and in 248 participants at 3-year follow-up. Left and right carotid arteries were examined, and CIMT was measured in both arteries and expressed as the mean CIMT of both side. The method used to estimate CIMT was described in a previous report [17]. The inter-observer technical error of measurement was <5.5% [17]. 2.5. Biochemical methods The immunonephelometric method was used to measure hsCRP and LDL-cholesterol (chol). Creatinine, urea, and albumin (bromocresol purple) (Alb) were measured using routine procedures. HDL-cholesterol was analyzed after precipitation of apoB-containing lipoprotein using phosphotungstic acid. Several proteins were measured using an enzyme-linked immunosorbent assay (ELISA) and commercially available ELISA kits. Serum oxLDL levels were measured using the Oxidized LDL ELISA Kit (Mercodia, Uppsala, Sweden); the sensitivity of this kit was 1 mU/L with an intra-assay coefficient of variation (intra-CV) <2.5% and an interassay coefficient of variation (inter-CV) <9.0%. Levels of IL-6 were measured using the QuantiGlo Human IL-6 ELISA Kit (R&D Systems Inc., Minneapolis, MN, USA); the sensitivity of this kit was 0.16 pg/mL, with an intra-CV <4.8% and an inter-CV <7.8%. Serum MPO levels were measured using the MPO ELISA Kit (Immundiagnostik AG, Bensheim, Germany); the sensitivity of this kit was 0.35 ng/mL, with an intra-CV <3.4% and an inter-CV <5.5%. Serum adiponectin levels were measured using the Human Adiponectin ELISA Kit (Circulex, Nagano, Japan); the sensitivity of this kit was 1.46 ng/mL, with an intra-CV <5.0% and an inter-CV <4.4%. NT-proBNP levels were measured by fully automated electrochemiluminescence [18], with an intra-CV <2.8% and an inter-CV <2.2%.
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Analysis of oxHDL was performed by ELISA using anti-oxidized ApoA1 antibody according to the methods described in our previous study [11,19]. The sensitivity of the oxHDL measurements was 1.5 ng/mL, with an intra-CV <8.2% and an inter-CV <10.0%. 2.6. Statistical analyses Data are presented as mean ± standard deviation or median (range) unless otherwise noted, and P values <0.05 were considered to indicate statistical significance. Comparisons between two groups for normally distributed variables were performed using Student’s t-test, and the Wilcoxon rank-sums test was used for comparisons between non-normally distributed variables. For nominal variables, Fisher’s exact test was used for comparisons between two groups, and the 2 test was used for comparisons among more than two groups. To assess the relationships between the oxHDL levels and the diseases that caused death in nonsurviving patients, oxHDL data were log transformed, and the relationships were assessed using a Dunnett analysis. Correlations were calculated by the Spearman rank test () for non-parametric data. Multivariate analysis of variance (MANOVA) was conducted to estimate the degree of correlation among continuous variables and oxHDL, and IL-6; oxHDL and IL-6 data were log transformed for this analysis. Independent associations between one dependent variable and more than two independent variables were assessed using multiple linear regression analysis. Survival was assessed by the Kaplan–Meier method. Cox proportional hazard model was used to determine independent predictors of all-cause, CVD-related mortality, and composite CVD events, and interaction of two independent variables to CVD-related mortality or to composite CVD events was estimated. Data were analyzed using JMP version 9.0.2 (SAS Institute, Cary, NC, USA) and STASA 12.0 (StataCorp, Lakeway, TX, USA). 3. Results Of the 412 enrolled patients, 131 had DM, and 6 patients with DM did not present with diabetic nephropathy. Nearly half of the patients (n = 194) had a history of one or more CVDs: 50 patients presented with MI or clinical signs of ischemic heart disease (i.e., AP), 99 presented with peripheral artery disease, 29 had a history of stroke or cerebral bleeding, 28 had a history of valvular disease, and 5 had a history of aortic aneurysm. Causes of ESRD were chronic glomerulonephritis (n = 148, 36%), diabetic nephropathy (n = 144, 35%; type 1 DM, n = 3), nephrosclerosis (n = 58, 14%), polycystic kidney disease (n = 16, 4%), other diseases (n = 15, 4%), and unknown (n = 31, 7%). No patients were newly diagnosed with DM during follow-up examinations. Five patients were taking vitamin C and E supplements. The patients were grouped according to median value of serum oxHDL level at baseline, and patient characteristics within each group are shown in Table 1. The percentage of male patients was higher in the low oxHDL group than in the high oxHDL group, and DM was more prevalent in patients with low oxHDL than patients with high oxHDL. Nutritional status based on PEW and GNRI was worse in patients with high HDL than patients with low oxHDL. Patients with high oxHDL had a higher oxHDL/HDL-chol ratio and higher serum HDL-chol, ICAM-1, and adiponectin levels; in contrast, serum hsCRP levels were lower in the high oxHDL group (Table 1). The relationships between oxHDL and selected variables were assessed by univariate correlation analysis; serum oxHDL levels were positively associated with the oxHDL/HDL-chol ratio and serum creatinine, HDL-chol, and adiponectin levels (Table 1). Serum oxHDL levels were not associated with serum MPO levels and mean CIMT (Table 1).
Fig. 1. Kaplan–Meier curves for time to all-cause mortality (A), cardiovascular disease-related mortality (B), and composite cardiovascular events (C) in patients with high oxidized HDL (oxHDL) versus low oxHDL levels. Patients were grouped into high oxHDL and low oxHDL based on the median value of oxHDL at the baseline measurements.
3.1. Associations of oxHDL with all-cause and CVD-related mortality and with composite CVD events During the follow-up period, 78 patients (19%) died, and 28 of these patients (36% of all deaths) died of a CVD-related disease. As expected, high IL-6 was a predictor for all-cause mortality, CVDrelated mortality, and composite CVD events (data not shown). Low HDL-chol also predicted composite CVD events; however, low HDL-chol was not significantly associated with mortality (data not shown). The occurrence of all-cause mortality was similar in the high oxHDL and low oxHDL groups (Fig. 1 A), and high oxHDL was inversely associated with non-CVD related mortality. However, the occurrence of CVD-related death and of composite CVD events was higher in the high oxHDL group than the low oxHDL group (Fig. 1B and C). Crude and adjusted Cox hazard ratios (HRs) for CVD-related death and composite CVD events were significantly elevated for patients with high oxHDL (Table 2). Causes of death are shown in Table 3. Oxidized HDL levels (reported as log oxHDL) in participants that died due to a
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Table 1 Patient characteristics and laboratory data at baseline according to the group based on oxidized HDL median value and correlation of oxidized HDL to each biomarker. High oxidized HDL (n = 206) Oxidized HDL (U/mL) Age (years) Gender (male, %) Diabetes (%) CVD (%) BMI (kg/m2 ) PEW (%) GNRI Normalized PCRe (g/kg/day) HD duration (months) Kt/V ACE-I (%) ARB (%) Statins (%) Hemoglobin (g/dL) Albumin (g/dL) Creatinine (mg/dL) HDL-cholesterol (mg/dL) LDL-cholesterol (mg/dL) Oxidized LDL (U/L) Oxidized HDL/HDL-cholesterol NT-proBNP (pg/mL)f High-sensitivity CRP (mg/dL) Interleukin-6 (pg/mL) ICAM-1 (ng/mL)g Myeloperoxidase (ng/mL) Adiponectin (g/mL) Mean CIMT (mm)h a b c d e f g h * ** ***
c
162.2 (126.5, 1239.0) 63 ± 13d 52 27 47 20.7 ± 3.1 31 94.8 ± 6.3 1.00 ± 0.20 133 (6–489) 1.47 ± 0.29 4 27 16 9.9 ± 1.0 3.8 ± 0.3 11.6 ± 2.8 51.2 ± 16.0 79.1 ± 22.0 62.3 ± 32.8 4.2 ± 2.8 3815 (205, 89,900) 0.07 (0.008–8.16) 3.45 (0.84, 83.0) 226.5 ± 79.8 55.6 (8.0, 375.5) 19.3 ± 10.0 0.84 ± 0.10
Low oxidized HDL (n = 206)
pa
b
97.2 (43.2, 126.1) 62 ± 14 72 40 50 22.0 ± 3.4 23 96.5 ± 6.2 0.96 ± 0.21 87 (6–489) 1.37 ± 0.27 8 29 19 10.0 ± 1.0 3.9 ± 0.4 11.4 ± 3.0 42.3 ± 12.6 80.0 ± 23.5 65.3 ± 31.3 2.4 ± 0.7 3080 (143, 168,000) 0.14 (0.005–10.39) 3.91 (0.28, 78.69) 208.5 ± 66.5 54.2 (8.0, 436.0) 16.0 ± 12.1 0.81 ± 0.12
<0.0001 0.88 <0.0001 0.007 0.33 0.0001 0.05 0.01 0.006 0.002 0.0002 0.08 0.65 0.49 0.52 0.60 0.24 <0.0001 0.87 0.17 <0.0001 0.42 0.002 0.34 0.03 0.56 <0.0001 0.28
– −0.06 – – – −0.16** − −0.05 0.11* 0.15 0.23*** – – – 0.01 0.03 0.12* 0.36*** −0.04 −0.05 0.67*** −0.003 −0.16** −0.03 0.09 0.002 0.24*** −0.012
P value means the significance in variables between high oxidized HDL and low oxidized HDL groups. Spearman rank correlation test between oxidized HDL and each variables. Median (range). Mean ± SD. Normalized protein catabolic rate. N-terminal pro B-type natriuretic peptide. Inter-Cellular Adhesion Molecule 1. Mean carotid intima-media thickness. <0.05. <0.001. <0.0001.
non-CVD-related disease, such as cancer or infectious disease, were significantly lower than in participants that died due to CVD-related diseases, but the oxHDL/HDL ratio did not differ significantly between these two groups (Table 4). Table 2 Hazard ratio of oxidized HDL to composite CVD events, all-cause and CVD-related mortality. HR (95%CI) Composite end points Crude Model 1 Model 2 (model 1 + interleukin-6) Model 3 (model 1 + HDL)
2.1 (1.4, 3.0) 1.9 (1.3, 2.9) 1.7 (1.2, 2.7) 2.1 (1.4, 3.3)
P <0.0001 0.002 0.008 0.0006
All cause mortality Crude
0.8 (0.5, 1.4)
0.40
CVD-related mortality Crude Model 4 Model 5 (model 4 + interleukin-6) Model 6 (model 4 + HDL)
– 2.9 (1.3, 5.6) 4.1 (1.9, 8.5) 4.1 (1.9, 8.1) 3.8 (1.6, 8.1)
– 0.007 0.0004 0.0004 0.002
Crude model included log oxidized HDL as an independent variable. Model 1 included log oxidized HDL, age (years), gender (male vs. female), diabetes mellitus (yes vs. no), history of cardiovascular disease state (present vs. absent), body mass index (kg/m2 ), subjective global assessment status (malnourished vs. nourished), HD vintage (months) as independent variables. Model 4 includes age (years), gender (male vs. female), diabetes mellitus (yes vs. no), HD vintage (months) as independent variables.
3.2. Patient characteristics according to groups based on median values of oxidized HDL and interleukin-6 To estimate the association of oxHDL with inflammation, patients were grouped according to the median values of serum oxHDL and IL-6. Clinical and biochemical characteristics were compared among four groups of patients: high oxHDL and high IL-6, high oxHDL and low IL-6, low oxHDL and high IL-6, low oxHDL and low IL-6. The comparisons were evaluated using MANOVA (Table 5). Patients with high oxHDL and high IL-6 had higher oxHDL/HDL-chol ratios, higher adiponectin, and higher ICAM-1 than patients in the other groups (Table 5). While prevalence of all-cause mortality was higher in the high IL-6 group than in the low IL-6 group (Table 5), the prevalence of CVDrelated and composite CVD events was significantly higher in the high oxHDL and high IL-6 group than in the other groups (Table 5). Mean CIMT in the high oxHDL and high IL-6 group was significantly higher than the means for CIMT in the other groups (Fig. 2 A), and there was a significant interaction between high oxHDL and high IL-6 for increased mean CIMT (Table 5). High oxHDL with high IL-6 was an independent contributor to increased CIMT based on multivariate models adjusted with confounders, specifically HDL-chol, oxLDL, NT-proBNP, hsCRP, ICAM-1, myeloperoxidase, and adiponectin (Table 6).
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Table 3 Individual cause for mortality classified by oxidized HDL level. High oxidized HDL (n = 206)
Low oxidized HDL (n = 206)
CVD-related death (n = 28) Myocardial infarction (n = 13) Cerebral infarction (n = 3) Other CVD-related disease (n = 12)
21 (10.2%)a 9 3 9
Non-CVD-related death (n = 50) Infection (n = 21) Cancer disease (n = 10) Hemorrhage (n = 2) Others (n = 8) Unknown or not determined (n = 9)
16 (7.8%) 6 1 – 3 6
34 (16.5%) 15 9 2 5 3
All-cause death (n = 78)
37 (17.9%)
41 (19.9%)
7 (3.3%) 4 – 3
Patients were grouped according to the median value of oxidized HDL. a Number of patient (percentage). Prevalence of causative diseases was incrementally higher across between the groups that was assessed by 2 test (P = 0.0005 for all-cause death and P = 0.007 for CVD-related death). Table 4 Oxidized HDL levels and oxidized HDL/HDL ratio among non-survivor patients. CVD-related (n = 28)
Log oxidized-HDL Oxidized HDL/HDL a
5.03 ± 0.09 4.54 ± 5.83
Non-CVD-related (n = 50)
P value
Cancer (n = 10)
Infection (n = 21)
Other (n = 8)
Unknown (n = 9)
4.46 ± 0.15a 2.39 ± 0.65
4.65 ± 0.10a 2.71 ± 1.24
4.70 ± 0.16 3.67 ± 1.21
5.01 ± 0.16 3.15 ± 1.12
0.0012 0.09
Log oxidized-HDL levels in non-survivor patient by cancer or infection disease were significantly decreased compared with by CVD-related disease (Dunnett analysis).
3.3. Association of oxHDL and interleukin-6 with mortality and with composite CVD events The effects of an interaction between oxHDL and IL-6 on allcause mortality, on CVD-related mortality, or on composite CVD events were estimated. An interaction between oxHDL and IL-6 was significantly associated with composite CVD events (Table 7). All-cause mortality was similar in patients belonging to the high oxHDL and high IL-6 group and the low oxHDL and high IL-6 group (Fig. 3 A). However, the occurrence of CVD-related death and composite CVD events were significantly higher for patients in the high oxHDL and high IL-6 group than for patients in the other groups (Fig. 3B and C). Crude and adjusted Cox HRs for CVD-related mortality and composite CVD events in patients with high oxHDL and high IL-6 were significantly elevated (Table 8). Crude and adjusted Cox HRs for all-cause mortality in patients with high IL-6 were elevated; however, these associations did not depend on serum oxHDL levels (data not shown).
3.4. Prospective change in mean CIMT according to groups based on the median values of oxidized HDL and interleukin-6 The influence of the high oxHDL and IL-6 combination on change in CIMT at 3 years was estimated using four groups that were based on median values of serum oxHDL and serum IL-6. Follow-up data on CIMT were collected from 248 patients (60% of the enrolled patients); the other patients died, moved, transferred to another hospital, or did not undergo a follow-up cervical ultrasound test. Mean increases in CIMT in patients with high oxHDL and high IL-6 were larger than in the mean increases in CIMT patients with low oxHDL and low IL-6 over the 3-year study period (Fig. 2B).
4. Discussion Fig. 2. Baseline carotid intima-media thickness (CIMT) in four groups based on the median values of oxidized HDL and interleukin-6 (A) and change in CIMT at 3 years in those groups (B).
The present study demonstrated that oxHDL could be a risk factor for CVD events in prevalent HD patients. Oxidation renders HDL dysfunctional, and oxHDL probably does not have the cardioprotective effects of HDL.
498
Table 5 Patient characteristics according to four groups classified by the median values of oxidized HDL and interleukin-6. High oxHDL low IL-6 (n = 106)
Low oxHDL high IL-6 (n = 110)
Low oxHDL low IL-6 (n = 96)
MANOVAa
64 ± 13 48 33 56 39 20.9 ± 3.2 94.6 ± 6.5 1.00 ± 0.21 126 (6, 426) 1.46 ± 0.32 3.8 ± 0.3 11.6 ± 2.7 48.7 ± 17.2 64.6 ± 32.1 4.6 ± 3.4 5020 (662, 89,900) 0.12 (0.012–8.16) 238.5 ± 90.1 58.6 (19.0, 370.5) 18.2 (4.3, 74.4) 0.88 ± 0.16 27 20 46
61 ± 12 54 22 35 23 20.6 ± 3.0 95.0 ± 6.4 1.03 ± 0.19 137 (6, 489) 1.47 ± 0.25 3.8 ± 0.4 11.7 ± 2.8 53.6 ± 14.6 60.3 ± 33.5 3.8 ± 2.1 3270 (205, 49,700) 0.05 (0.008–0.47) 215.2 ± 68.6 52.7 (8.0, 375.5) 15.6 (7.1, 51.9) 0.80 ± 0.15 9 3 18
67 ± 12 69 42 57 30 21.8 ± 3.6 96.0 ± 6.6 0.95 ± 0.20 94 (6, 489) 1.38 ± 0.26 3.8 ± 0.3 11.5 ± 3.2 39.8 ± 12.8 70.5 ± 30.3 2.5 ± 0.8 4340 (509, 168,000) 0.23 (0.005–10.39) 216.0 ± 71.9 60.1 (24.6, 436.0) 13.5 (6.5, 38.2) 0.83 ± 0.12 28 3 25
59 ± 14 75 34 43 15 21.9 ± 3.1 97.0 ± 5.9 0.96 ± 0.21 80 (6, 424) 1.34 ± 0.28 3.9 ± 0.4 11.3 ± 2.8 44.9 ± 11.9 59.8 ± 31.7 2.3 ± 0.7 2650 (143, 104,000) 0.07 (0.013–1.3) 200.3 ± 58.9 49.1 (8.0, 416.0) 13.3 (3.2, 133.7) 0.80 ± 0.11 10 2 20
IL-6 0.0002b 0.02 0.003 0.0007 oxHDL NS oxHDL oxHDL oxHDL NS NS oxHDL, IL-6 IL-6 oxHDL, IL-6 IL-6 IL-6 oxHDL, IL-6 IL-6 oxHDL IL-6, oxHDL × IL-6 <0.0001 <0.0001 <0.0001
a MANOVA (multivariable analysis of variance) was performed to assess associations among continuous variables, log oxidized HDL and log interleukin-6; b Associations among nominal variables in 4 groups by combination with oxidized HDL and interleukin-6 were assessed by 2 test. Abbreviation: see Table 1.
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Age (year) Gender (male, %) DM (%) CVD (%) SGA (%) BMI (kg/m2 ) GNRI nPCRd (g/kg/day) HD vintage (months) Kt/V Albumin (g/dL) Creatinine (mg/dL) HDL-chol (mg/dL) Oxidized LDL (U/L) Oxidized HDL/HDL-chol NT-proBNP (pg/mL) Hs-CRP (mg/dL) ICAM-1 (ng/mL) Myeloperoxidase (ng/mL) Adiponectin (g/mL) Mean CIMT (mm) All-cause mortality (%) CVD-related mortality (%) Composite CVD events (%)
High oxHDL high IL-6 (n = 100)
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Table 6 Association among oxidized HDL, interleukin-6, and mean carotid intima-media thickness. Model 1 (adjusted R2 = 0.39) ˇ, SE Intercept High oxHDL, high IL-6 High oxHDL, low IL-6 Low oxHDL, high IL-6 Low oxHDL, low IL-6
Model 2 (adjusted R2 = 0.43) ˇ, SE
P
0.44, 0.15 0.04, 0.02 −0.001, 0.02 −0.02, 0.02 Ref.
<0.0001 0.03 0.94 0.20 –
0.58, 0.12 0.04, 0.02 −0.007, 0.02 −0.01, 0.02 Ref.
0.0002 0.02 0.67 0.40 –
Association among oxidized HDL, interleukin-6, and mean carotid intima-media thickness (CIMT) were assessed according to four groups classified by the median values of oxidized HDL and interleukin-6. Models included mean CIMT as a dependent factor. Model 1 included age (years), gender (male vs. female), body mass index (kg/m2 ), diabetes mellitus (yes vs. no), cardiovascular state (yes vs. no), subjective global assessment status (positive vs. negative), HD vintage (months), Smoking (yes vs. no), geriatric nutritional risk index. Model 2 included variables in model 1 and normalized protein catabolic rate (g/kg/day), Kt/V, HDL-cholesterol (mg/dL), oxLDL (U/L), log NT-proBNP, log high-sensitivity CRP, ICAM-1 (ng/mL), log myeloperoxidase, log adiponectin as independent variables.
Table 7 Impact of interaction between oxidized HDL and interleukin-6 on CVD events. Crude
Composite CVD events (n = 112) CVD-related mortality (n = 28)
Model 1
Model 2
Coefficient, SE
P value
Coefficient, SE
P value
Coefficient, SE
P value
0.97, 0.41 0.86, 1.27
0.01 0.49
1.10, 0.41 –
0.008 –
1.10, 0.42 –
0.008 –
Model 1 included age (years), gender (male vs. female), body mass index (kg/m2 ), diabetes mellitus (yes vs. no), cardiovascular state (yes vs. no), subjective global assessment status (positive vs. negative), HD vintage (months), Smoking (yes vs. no). Model 2 included variables in model 1 and HDL-cholesterol (mg/dL) as independent variables.
Navab et al. reported that dysfunctional HDL could appear without any change in HDL-chol levels [20]. This association between dysfunctional HDL and HDL-chol were verified in a study of dysfunctional HDL in maintenance HD patients [7]. The protective effect of HDL against CVD seen in large population studies is lost by individuals with impaired glucose metabolism or diabetes, even when HDL-chol levels are not affected [4]. Here, we demonstrated that high HDL-chol was significantly associated with low prevalence of CVD events, and serum HDL-chol levels in patients presenting high oxHDL was higher than in those presenting low oxHDL (Table 1). However, the oxHDL/HDL-chol ratio was significantly higher in patients with high oxHDL (Table 1). Therefore, excess oxidative stress may have yielded dysfunctional HDL in patients on HD, and patients with high HDL-chol under excess oxidative stress may have enriched oxHDL. That is, whereas serum HDL-chol levels are inversely associated with CVD risk in large epidemiologic studies [4,21,22], HDL-chol measurements alone fail to provide an accurate assessment of the protective versus dysfunctional nature of HDL particles [22,23]; this shortcoming of HDL-chol measurements is of particular concern for patients undergoing HD. Activities of HDL are compromised in many pathological states associated with inflammation and oxidative stress [4–6]. Oxidized
HDL was independently associated with CVD events and with CVDrelated mortality (Table 2). However, high oxHDL with low IL-6 was not associated with CVD events, but high oxHDL with high IL-6 was significantly associated with CVD events in the present study (Fig. 3, Tables 5 and 8). The effects of an interaction between oxHDL and IL-6 on CVD-related mortality were not significant; however, this interaction was associated with high prevalence of composite CVD events including non-fatal CVD (Table 7). This finding indicated that the predictive value of oxHDL (or IL-6) for CVD events depends on IL-6 (or oxHDL). High oxHDL with high IL-6 was significantly associated with CVD-related mortality based on the Kaplan–Meier analysis (Fig. 3), but the effect of interaction between oxHDL and IL6 on CVD-related mortality was not statistically significant based on the model (Table 7); this difference between analysis may be due to a statistical power issue. The oxHDL/HDL-chol ratio in the high oxHDL and low IL-6 group was higher than that in the low oxHDL and low IL-6 group (Table 5). Even the imbalance between functional and dysfunctional HDL may be associated with the CVD events. However, oxHDL values may be more predictive of CVD events in patients that are experiencing inflammation. Dysfunctional HDL is associated with CVD events and atherosclerosis in non-uremic patients [20,24], but the impact of dysfunctional HDL
Table 8 Association among oxHDL, interleukin-6, and CVD-related mortality, and composite CVD events. Crude
Composite CVD events High oxHDL, high IL-6 High oxHDL, low IL-6 Low oxHDL, high IL-6 Low oxHDL, lowIL-6 CVD-related mortality High oxHDL, high IL-6 High oxHDL, low IL-6 Low oxHDL, high IL-6 Low oxHDL, low IL-6
Model 1
Model 2
HR (95% CI)
P value
HR (95% CI)
P value
HR (95% CI)
P value
3.1 (1.8, 5.4) 0.9 (0.5, 1.7) 1.5 (0.7, 2.8) Ref.
<0.0001 0.69 0.14 –
2.5 (1.4, 4.5) 0.8 (0.4, 1.5) 1.2 (0.6, 2.2) Ref.
0.001 0.49 0.59 –
2.5 (1.4, 4.5) 0.8 (0.4, 1.6) 1.1 (0.6, 2.1) Ref.
0.001 0.53 0.70 –
11.1 (3.2, 69.5) 1.4 (0.2, 10.3) 2.0 (0.4, 14.7) Ref.
<0.0001 0.73 0.40 –
10.3 (3.0, 65.8) 1.6 (0.3, 12.1) 1.5 (0.3, 10.8) Ref.
<0.0001 0.61 0.64 –
9.5 (2.7, 60.6) 1.5 (0.2, 11.6) 1.7 (0.3, 12.8) Ref.
<0.0001 0.61 0.64 –
Associations among oxidized HDL, interleukin-6, and CVD events were assessed according to four groups classified by the median values of oxidized HDL and interleukin-6. Model 1 included age (years), gender (male vs. female), body mass index (kg/m2 ), diabetes mellitus (yes vs. no), cardiovascular state (yes vs. no), subjective global assessment status (positive vs. negative), HD vintage (months), smoking (yes vs. no), geriatric nutritional risk index, mean carotid intima-media thickness (mm) as independent variables. Model 2 included variables in model 1 and, HDL-cholesterol (mg/dL), as independent variables. Model 3 included age (years), gender (male vs. female), diabetes mellitus (yes vs. no), and cardiovascular state (yes vs. no) as independent variables. Model 4 included all variables in model 1 and HDL-cholesterol (mg/dL) as independent variables.
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died due to cancer or infection disease (Table 4). At baseline, nutritional status was worse in patients with high oxHDL than in patients with low oxHDL (Table 1). Recently, we reported the impact of oxHDL on PEW in HD patients; that study had a longitudinal design and indicated that increased oxHDL may be associated with a high prevalence of PEW [11]. Therefore, elevated oxHDL levels may be associated with worsening of nutritional state in prevalent HD patients; however, high oxHDL was not predictive of non-CVDrelated mortality in those patients. The results of our study must be considered with the following caveats. The number of patients was relatively small, and we could only evaluate the findings for prevalent HD patients. Additionally, the single-point measurement of oxHDL and IL-6 does not permit accurate evaluation of these biomarkers [32]. Finally, we could not assess a specific association between oxHDL and atherosclerosis because atherosclerosis was only assessed by increases or changes in CIMT. Moreover, change in CIMT, as a quantitative assessment of advanced atherosclerosis, was based on measurements at just two time points and was only performed in patients that survived; therefore, patients with severe atherosclerotic change may have been excluded because they died before the 3-year follow-up examination. Thus, a large prospective study with more accurate quantitative assessment of atherosclerosis is required to clarify the reliability of oxHDL values as a predictor for progression of atherosclerosis in prevalent HD patients. 5. Conclusion In conclusion, oxHDL seems to be an important, contributing factor for CVD events and CVD-related mortality in prevalent HD patients, especially when high oxHDL is concomitant with high IL-6. Disclosure None. Fig. 3. Kaplan–Meier curves for time to all-cause mortality (A), cardiovascular disease-related mortality (B), and composite cardiovascular events (C). Patients were separated into four groups based the median values of oxidized HDL and interleukin-6; each curve represents data from one of the four groups.
on CVD may be significant in HD patients with chronic inflammation. Evidence that IL-6 is a strong risk factor for CVD events has accumulated [13,25], and high IL-6 is closely associated with mortality [13,25–27] and increased CIMT in HD patients [28,29]. On another front, dysfunctional HDL is also associated with increased CIMT [30,31]. We did not find a significant association between oxHDL and CIMT at baseline (Table 1); however, increased mean CIMT at baseline was significantly associated with an interaction between oxHDL and IL-6, as well as with IL-6 (Table 5). High IL-6 together with high oxHDL was particularly associated with changes in CIMT over the 3-year study period (Fig. 2B). Moreover, the impact of IL-6 on CVD events was dependent upon the elevation of oxHDL levels (Fig. 3, Table 8). Taken together, these data indicated that the predictive value of high IL-6 for CVD events may be most accurate in prevalent HD patients who have elevated serum oxHDL levels. Oxidized HDL was not a predictor of all-cause mortality in the present study. Kalantar-Zadeh demonstrated that inflammatory (dysfunctional) HDL was significantly associated with worse outcomes for prevalent HD patients [10]. This discrepancy might be associated with differences in the methods used to measure dysfunctional HDL. In the present study, diseases causing death were obviously different between the high oxHDL and low oxHDL group (Table 3), and oxHDL was significantly lower in patients that had
Acknowledgments The authors wish to thank Ms. Naoko Nishiyama for her technical assistance. Dr HH was supported by the Kidney Foundation, Japan (grant no. JKF 06-1). References [1] Tabet F, Rye KA, High-density lipoproteins, inflammation and oxidative stress. Clin Sci (Lond) 2009;116:87–98. [2] Florentin M, Liberopoulos EN, Wierzbicki AS, et al. Multiple actions of highdensity lipoprotein. Curr Opin Cardiol 2008;23:370–8. [3] Francis GA. The complexity of HDL. Biochim Biophys Acta 2010;1801:1286–93. [4] Assinger A, Koller F, Schmid W, et al. Specific binding of hypochlorite-oxidized HDL to platelet CD36 triggers proinflammatory and procoagulant effects. Atherosclerosis 2010;212:153–60. [5] Vaziri ND, Navab M, Fogelman AM. HDL metabolism and activity in chronic kidney disease. Nat Rev Nephrol 2010;6:287–96. [6] Navab M, Reddy S, Van Lenten BJ, et al. Role of dysfunctional HDL in atherosclerosis. J Lipid Res 2008;50:S145–9. [7] Navab KD, Elboudwarej O, Gharif M, et al. Chronic inflammatory disorders and accelerated atherosclerosis: chronic kidney disease. Curr Pharm Des 2011;17:17–20. [8] Toth PP, Davidson MH. High-density lipoproteins: marker of cardiovascular risk and therapeutic target. J Clin Lipidol 2010;4:359–64. [9] Heinecke J. HDL and cardiovascular-disease risk—time for a new approach? N Engl J Med 2011;364:170–1. [10] Kalantar-Zadeh K, Kopple JD, Kamranpour N, et al. HDL-inflammatory index correlates with poor outcome in hemodialysis patients. Kidney Int 2007;72:1149–56. [11] Honda H, Ueda M, Kojima S, et al. Oxidized high-density lipoprotein is associated with protein-energy wasting in maintenance hemodialysis patients. Clin J Am Soc Nephrol 2010;5:1021–8. [12] Stenvinkel P, Heimbürger O, Lindholm B, et al. Are there two types of malnutrition in chronic renal failure? Evidence for relationships between malnutrition,
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inflammation and atherosclerosis (MIA syndrome). Nephrol Dial Transplant 2000;15:953–60. Honda H, Qureshi AR, Heimbüger O, et al. Serum albumin, C-reactive protein, interleukin 6, and fetuin a as predictors of malnutrition, cardiovascular disease, and mortality in patients with ESRD. Am J Kidney Dis 2006;47:139–48. Group KDOQIKD. K/DOQI clinical practice guidelines for management of dyslipidemias in patients with kidney disease. Am J Kidney Dis 2003;41(I–IV):S1–91. Yamada K, Furuya R, Takita T, et al. Simplified nutritional screening tools for patients on maintenance hemodialysis. Am J Clin Nutr 2008;87:106–13. Clinical practice guidelines for nutrition in chronic renal failure K/DOQI, National Kidney Foundation. Am J Kidney Dis 2000;35:S1–140. Honda H, Ueda M, Kojima S, et al. Assessment of myeloperoxidase and oxidative alpha1-antitrypsin in patients on hemodialysis. Clin J Am Soc Nephrol 2009;4:142–51. Prontera C, Emdin M, Zucchelli GC, et al. Analytical performance and diagnostic accuracy of a fully-automated electrochemiluminescent assay for the N-terminal fragment of the pro-peptide of brain natriuretic peptide in patients with cardiomyopathy: comparison with immunoradiometric assay methods for brain natriuretic peptide and atrial natriuretic peptide. Clin Chem Lab Med 2004;42:37–44. Ueda M, Hayase Y, Mashiba S. Establishment and evaluation of 2 monoclonal antibodies against oxidized apolipoprotein A-I (apoA-I) and its application to determine blood oxidized apoA-I levels. Clin Chim Acta 2007;378:105–11. Navab M, Anantharamaiah GM, Reddy ST, et al. HDL as a biomarker, potential therapeutic target, and therapy. Diabetes 2009;58:2711–7. Navab M, Reddy ST, Van Lenten B.J., et al. HDL and cardiovascular disease: atherogenic and atheroprotective mechanisms. Nat Rev Cardiol 2011;201:222–32.
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[22] Toth PP, Davidson MH. High-density lipoproteins: marker of cardiovascular risk and therapeutic target. J Clin Lipidol 2010;4:359–64. [23] Smith JD. Dysfunctional HDL as a diagnostic and therapeutic target. Arterioscler Thromb Vasc Biol 2010;30:151–5. [24] McMahon M, Grossman J, Skaggs B, et al. Dysfunctional proinflammatory highdensity lipoproteins confer increased risk of atherosclerosis in women with systemic lupus erythematosus. Arthritis Rheum 2009;60:2428–37. [25] Panichi V, Rizza GM, Paoletti S, et al. Chronic inflammation and mortality in haemodialysis: effect of different renal replacement therapies. Results from the RISCAVID study. Nephrol Dial Transplant 2008;23:2337–43. [26] Zoccali C, Tripepi G, Mallamaci F. Dissecting inflammation in ESRD: do cytokines and C-reactive protein have a complementary prognostic value for mortality in dialysis patients? J Am Soc Nephrol 2006;17:S169–73. [27] Panichi V, Maggiore U, Taccola D, et al. Interleukin-6 is a stronger predictor of total and cardiovascular mortality than C-reactive protein in haemodialysis patients. Nephrol Dial Transplant 2004;19:1154–60. [28] Stenvinkel P, Heimbürger O, Jogestrand T. Elevated interleukin-6 predicts progressive carotid artery atherosclerosis in dialysis patients: association with Chlamydia pneumoniae seropositivity. Am J Kidney Dis 2002;39:274–82. [29] Kato A, Odamaki M, Takita T, et al. Association between interleukin-6 and carotid atherosclerosis in hemodialysis patients. Kidney Int 2002;61:1143–52. [30] Dodani S, Kaur R, Reddy S, et al. Can dysfunctional HDL explain high coronary artery disease risk in South Asians? Int J Cardiol 2008;129:125–32. [31] van Leuven SI, Hezemans R, Levels JH, et al. Enhanced atherogenesis and altered high density lipoprotein in patients with Crohn’s disease. J Lipid Res 2007;48:2640–6. [32] Tsirpanlis G, Bagos P, Ioannou D, et al. The variability and accurate assessment of microinflammation in haemodialysis patients. Nephrol Dial Transplant 2004;19:150–7.
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Atherosclerosis journal homepage: www.elsevier.com/locate/atherosclerosis
Oxidized high-density lipoprotein as a risk factor for cardiovascular events in prevalent hemodialysis patients Hirokazu Honda a,∗ , Masashi Ueda b , Shiho Kojima b , Shinichi Mashiba b , Tetsuo Michihata c , Keiko Takahashi d , Kanji Shishido d , Tadao Akizawa a a
Division of Nephrology, Department of Medicine, Showa University School of Medicine, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8666, Japan Ikagaku Co. Ltd., Kyoto, Japan c Ebara Clinic, Tokyo, Japan d Kawasaki Clinic, Kawasaki, Japan b
a r t i c l e
i n f o
Article history: Received 1 June 2011 Received in revised form 13 October 2011 Accepted 25 October 2011 Available online 3 November 2011 Keywords: Oxidized high-density lipoprotein Interleukin-6 Cardiovascular disease
a b s t r a c t Background and objectives: Here, we assessed the impact of oxidized high-density lipoprotein (oxHDL), dysfunctional HDL, on mortality and cardiovascular disease (CVD) events in prevalent HD patients and compared oxHDL to interleukin-6 (IL-6), a strong predictor of CVD events in HD patients. Design, setting, participants, and measurements: This prospective study examined a cohort of prevalent HD patients (n = 412). Blood samples were obtained at baseline to measure lipids, high-sensitive C-reactive protein (hsCRP), IL-6, oxidized low-density lipoprotein, N-terminal pro B-type natriuretic peptide, intercellular adhesion molecule 1 (ICAM-1), myeloperoxidase, adiponectin, and oxHDL. Carotid intima-media thickness (CIMT) was assessed at baseline and 3-year follow-up. Nutritional status was assessed by subjective global assessment (SGA), body mass index, and geriatric nutritional risk index (GNRI). After the baseline assessment, study patients were prospectively followed up (mean observational period, 40 months). Results: At baseline, patients with high oxHDL had a worse nutritional state and higher HDL-cholesterol (HDL-chol), ICAM-1, and adiponectin levels and a higher oxHDL/HDL-chol ratio than low oxHDL patients. A combination of high oxHDL and high IL-6 was significantly associated with increased CIMT at baseline and a larger increase in CIMT at 3-year follow-up. High oxHDL did not predict all-cause mortality; however, it was significantly associated with CVD-related mortality and composite CVD events, particularly with concomitant high IL-6. These associations were confirmed in multivariate Cox hazard models adjusted with confounding variables. Conclusions: High oxHDL, particularly with concomitant high IL-6, may be associated with an increased risk of CVD events and CVD-related mortality in prevalent HD patients. © 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction High-density lipoprotein (HDL) has the capacity to promote cholesterol efflux from monocytes and macrophages, and it has antioxidant and anti-inflammatory activities in its interactions with circulating cells. HDL inhibits both leukocyte and platelet activation and thus exerts further systemic anti-inflammatory actions [1–5]. These anti-inflammatory effects may contribute to protection from cardiovascular disease (CVD) events [6,7], although how HDL activities other than lipid transport may contribute to protection against atherosclerosis is unclear [8,9].
∗ Corresponding author. Tel.: +81 3 3784 8533; fax: +81 3 3784 5934. E-mail address: [email protected] (H. Honda). 0021-9150/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2011.10.038
In the presence of systemic inflammation and oxidative stress, antioxidant enzymes can be inactivated and HDL can accumulate oxidized lipids and proteins and thus become proinflammatory. Under these conditions, the most abundant protein in HDL, apolipoprotein (apo) A1, can be modified by reactive oxygen species. Alterations of HDL by oxidation impair its anti-inflammatory effects, resulting in a dysfunctional HDL with pro-inflammatory effects [5–7]. In fact, dysfunctional HDL is often present in patients with advanced chronic kidney disease [5,7]. Kalantar-Zadeh et al. demonstrated that dysfunctional HDL is significantly associated with more co-morbid conditions and a lower quality of life in prevalent hemodialysis (HD) patients; moreover, HD patients with dysfunctional HDL had significantly worse outcomes than patients with anti-inflammatory HDL [10]. A recent study in prevalent HD patients demonstrated that dysfunctional HDL, estimated by oxidized HDL (oxHDL), is strongly associated with protein-energy wasting (PEW), particularly in patients with
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inflammation [11]. Thus, dysfunctional HDL in patients on HD is a risk factor for the development and advancement of the PEW state, although factors associated with uremia per se may contribute to PEW [12]. Therefore, oxHDL is thought to represent a risk factor for mortality and morbidity in prevalent HD patients. However, associations between dysfunctional HDL and atherosclerosis or CVD events have not been fully evaluated. Moreover, whether dysfunctional HDL levels can be used as a biomarker for risk of CVD events and replace currently used biomarkers in prevalent HD patients is unclear. The present study of prevalent HD patients investigated (1) associations between oxHDL and CVD-related biomarkers, (2) the impact of oxHDL on atherosclerosis and CVD events and mortality, and (3) the potential of oxHDL together with interleukin-6 (IL-6), which alone is a strong predictor of CVD, PEW, and mortality in end-stage renal disease (ESRD) patients [13], as a combination biomarker for risk of CVD events. 2. Materials and methods 2.1. Patients This study was performed as part of an ongoing prospective cohort study that recruited 412 prevalent HD patients treated at two hemodialysis clinics. Patients who did not provide blood samples, had an anticipated life expectancy of <6 months, or presented with clinical signs of overt infection, acute vasculitis, or liver disease at the time of recruitment were excluded from the study. Participants were outpatients who had been undergoing HD for ≥6 months and were ≥20 years old. Written informed consent was obtained from all patients. The study protocol was approved by the ethics committee of our institute. Medical treatments used to manage the condition of all patients were similar, and the medication for each patient was prescribed according to Kidney Disease Outcomes Quality Initiative (K/DOQI) guidelines [14]. Patients were managed similarly in terms of protocols for HD, including prescription of dialysis dose. Basically, all recruited patients underwent routine hemodialysis 3 times/week (3–4 h/session) using conventional bicarbonate or acetate-free dialysate and standard high-flux cellulose triacetate, polysulfone, or other dialysis membranes. Patients were instructed to maintain a daily protein intake >1.0–1.2 g/kg body weight. Dialysis dosage was calculated using second-generation Kt/V, which was estimated using the Daugirdas formula. 2.2. Study design This cohort study was designed as a prospective study with a cross-sectional analysis at baseline. The baseline analysis assessed (1) the associations between oxHDL and biomarkers associated with mortality and CVD events and (2) the impact of oxHDL on nutritional state and atherosclerosis estimated by carotid intimamedia thickness (CIMT). After the baseline study, patients were followed up within 48 months to assess the impact of oxHDL on change in CIMT, number of CVD events, and mortality; the influence of inflammation, as determined by IL-6 levels, on these associations was also assessed. The following clinicopathological factors were recorded at baseline: cause of ESRD, presence of diabetes mellitus (DM), history of CVD, and smoking history. History of CVD was defined by medical history, clinical symptoms, or findings indicating cerebrovascular (stroke) and/or peripheral vascular disease. Non-fasting venous blood samples were drawn 3 days after a hemodialysis session and immediately before the next hemodialysis session. Routine biochemical parameters, lipid markers, and
high-sensitivity C-reactive protein (hsCRP) were measured in the blood samples. Serum was isolated by centrifugation and maintained at −80 ◦ C until use; oxidized low-density lipoprotein (oxLDL), oxHDL, IL-6, N-terminal pro B-type natriuretic peptide (NT-proBNP), myeloperoxidase (MPO), and adiponectin were measured at baseline. In the survival analysis, the time to death from all causes or from a major CVD-related event (mean follow-up period: 40 months) was determined. A major CVD-related death was defined as a nonfatal myocardial infarction (MI), non-fatal stroke, or death from CVD causes. In the analysis of composite CVD events, the time to each event was determined, and composite CVD events consisted of non-fatal CVD events, fatal MI, angina pectoris (AP), fatal cerebral infarction, or arterial or peripheral artery disease. The fatal CVD events were defined by electrocardiography and cardiac angiography for MI or AP, brain CT or MRI for cerebral infarction, and enhanced CT scan or arterial or peripheral angiography for arterial or peripheral artery disease. Change in CIMT was estimated by carotid ultrasound at baseline and at follow up (within 48 months). 2.3. Assessment of nutritional status PEW Nutritional status was assessed by subjective global assessment (SGA) and geriatric nutritional risk index (GNRI) [15]. Fully trained physicians assessed SGA according to K/DOQI recommendations; 4 items (weight loss during the preceding 6 months; anorexia; subcutaneous fat; and muscle mass) were scored on a 7-point Likert scale [16]. Actual body mass index was calculated as weight (kg)/height (m)2 . Normalized protein catabolic rate (nPCR) was also calculated. 2.4. Carotid ultrasound A B-mode ultrasound scanner equipped with a 14-MHz linear probe was used to examine transversal and longitudinal views of carotid artery walls in all 412 participants at the baseline assessment and in 248 participants at 3-year follow-up. Left and right carotid arteries were examined, and CIMT was measured in both arteries and expressed as the mean CIMT of both side. The method used to estimate CIMT was described in a previous report [17]. The inter-observer technical error of measurement was <5.5% [17]. 2.5. Biochemical methods The immunonephelometric method was used to measure hsCRP and LDL-cholesterol (chol). Creatinine, urea, and albumin (bromocresol purple) (Alb) were measured using routine procedures. HDL-cholesterol was analyzed after precipitation of apoB-containing lipoprotein using phosphotungstic acid. Several proteins were measured using an enzyme-linked immunosorbent assay (ELISA) and commercially available ELISA kits. Serum oxLDL levels were measured using the Oxidized LDL ELISA Kit (Mercodia, Uppsala, Sweden); the sensitivity of this kit was 1 mU/L with an intra-assay coefficient of variation (intra-CV) <2.5% and an interassay coefficient of variation (inter-CV) <9.0%. Levels of IL-6 were measured using the QuantiGlo Human IL-6 ELISA Kit (R&D Systems Inc., Minneapolis, MN, USA); the sensitivity of this kit was 0.16 pg/mL, with an intra-CV <4.8% and an inter-CV <7.8%. Serum MPO levels were measured using the MPO ELISA Kit (Immundiagnostik AG, Bensheim, Germany); the sensitivity of this kit was 0.35 ng/mL, with an intra-CV <3.4% and an inter-CV <5.5%. Serum adiponectin levels were measured using the Human Adiponectin ELISA Kit (Circulex, Nagano, Japan); the sensitivity of this kit was 1.46 ng/mL, with an intra-CV <5.0% and an inter-CV <4.4%. NT-proBNP levels were measured by fully automated electrochemiluminescence [18], with an intra-CV <2.8% and an inter-CV <2.2%.
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Analysis of oxHDL was performed by ELISA using anti-oxidized ApoA1 antibody according to the methods described in our previous study [11,19]. The sensitivity of the oxHDL measurements was 1.5 ng/mL, with an intra-CV <8.2% and an inter-CV <10.0%. 2.6. Statistical analyses Data are presented as mean ± standard deviation or median (range) unless otherwise noted, and P values <0.05 were considered to indicate statistical significance. Comparisons between two groups for normally distributed variables were performed using Student’s t-test, and the Wilcoxon rank-sums test was used for comparisons between non-normally distributed variables. For nominal variables, Fisher’s exact test was used for comparisons between two groups, and the 2 test was used for comparisons among more than two groups. To assess the relationships between the oxHDL levels and the diseases that caused death in nonsurviving patients, oxHDL data were log transformed, and the relationships were assessed using a Dunnett analysis. Correlations were calculated by the Spearman rank test () for non-parametric data. Multivariate analysis of variance (MANOVA) was conducted to estimate the degree of correlation among continuous variables and oxHDL, and IL-6; oxHDL and IL-6 data were log transformed for this analysis. Independent associations between one dependent variable and more than two independent variables were assessed using multiple linear regression analysis. Survival was assessed by the Kaplan–Meier method. Cox proportional hazard model was used to determine independent predictors of all-cause, CVD-related mortality, and composite CVD events, and interaction of two independent variables to CVD-related mortality or to composite CVD events was estimated. Data were analyzed using JMP version 9.0.2 (SAS Institute, Cary, NC, USA) and STASA 12.0 (StataCorp, Lakeway, TX, USA). 3. Results Of the 412 enrolled patients, 131 had DM, and 6 patients with DM did not present with diabetic nephropathy. Nearly half of the patients (n = 194) had a history of one or more CVDs: 50 patients presented with MI or clinical signs of ischemic heart disease (i.e., AP), 99 presented with peripheral artery disease, 29 had a history of stroke or cerebral bleeding, 28 had a history of valvular disease, and 5 had a history of aortic aneurysm. Causes of ESRD were chronic glomerulonephritis (n = 148, 36%), diabetic nephropathy (n = 144, 35%; type 1 DM, n = 3), nephrosclerosis (n = 58, 14%), polycystic kidney disease (n = 16, 4%), other diseases (n = 15, 4%), and unknown (n = 31, 7%). No patients were newly diagnosed with DM during follow-up examinations. Five patients were taking vitamin C and E supplements. The patients were grouped according to median value of serum oxHDL level at baseline, and patient characteristics within each group are shown in Table 1. The percentage of male patients was higher in the low oxHDL group than in the high oxHDL group, and DM was more prevalent in patients with low oxHDL than patients with high oxHDL. Nutritional status based on PEW and GNRI was worse in patients with high HDL than patients with low oxHDL. Patients with high oxHDL had a higher oxHDL/HDL-chol ratio and higher serum HDL-chol, ICAM-1, and adiponectin levels; in contrast, serum hsCRP levels were lower in the high oxHDL group (Table 1). The relationships between oxHDL and selected variables were assessed by univariate correlation analysis; serum oxHDL levels were positively associated with the oxHDL/HDL-chol ratio and serum creatinine, HDL-chol, and adiponectin levels (Table 1). Serum oxHDL levels were not associated with serum MPO levels and mean CIMT (Table 1).
Fig. 1. Kaplan–Meier curves for time to all-cause mortality (A), cardiovascular disease-related mortality (B), and composite cardiovascular events (C) in patients with high oxidized HDL (oxHDL) versus low oxHDL levels. Patients were grouped into high oxHDL and low oxHDL based on the median value of oxHDL at the baseline measurements.
3.1. Associations of oxHDL with all-cause and CVD-related mortality and with composite CVD events During the follow-up period, 78 patients (19%) died, and 28 of these patients (36% of all deaths) died of a CVD-related disease. As expected, high IL-6 was a predictor for all-cause mortality, CVDrelated mortality, and composite CVD events (data not shown). Low HDL-chol also predicted composite CVD events; however, low HDL-chol was not significantly associated with mortality (data not shown). The occurrence of all-cause mortality was similar in the high oxHDL and low oxHDL groups (Fig. 1 A), and high oxHDL was inversely associated with non-CVD related mortality. However, the occurrence of CVD-related death and of composite CVD events was higher in the high oxHDL group than the low oxHDL group (Fig. 1B and C). Crude and adjusted Cox hazard ratios (HRs) for CVD-related death and composite CVD events were significantly elevated for patients with high oxHDL (Table 2). Causes of death are shown in Table 3. Oxidized HDL levels (reported as log oxHDL) in participants that died due to a
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Table 1 Patient characteristics and laboratory data at baseline according to the group based on oxidized HDL median value and correlation of oxidized HDL to each biomarker. High oxidized HDL (n = 206) Oxidized HDL (U/mL) Age (years) Gender (male, %) Diabetes (%) CVD (%) BMI (kg/m2 ) PEW (%) GNRI Normalized PCRe (g/kg/day) HD duration (months) Kt/V ACE-I (%) ARB (%) Statins (%) Hemoglobin (g/dL) Albumin (g/dL) Creatinine (mg/dL) HDL-cholesterol (mg/dL) LDL-cholesterol (mg/dL) Oxidized LDL (U/L) Oxidized HDL/HDL-cholesterol NT-proBNP (pg/mL)f High-sensitivity CRP (mg/dL) Interleukin-6 (pg/mL) ICAM-1 (ng/mL)g Myeloperoxidase (ng/mL) Adiponectin (g/mL) Mean CIMT (mm)h a b c d e f g h * ** ***
c
162.2 (126.5, 1239.0) 63 ± 13d 52 27 47 20.7 ± 3.1 31 94.8 ± 6.3 1.00 ± 0.20 133 (6–489) 1.47 ± 0.29 4 27 16 9.9 ± 1.0 3.8 ± 0.3 11.6 ± 2.8 51.2 ± 16.0 79.1 ± 22.0 62.3 ± 32.8 4.2 ± 2.8 3815 (205, 89,900) 0.07 (0.008–8.16) 3.45 (0.84, 83.0) 226.5 ± 79.8 55.6 (8.0, 375.5) 19.3 ± 10.0 0.84 ± 0.10
Low oxidized HDL (n = 206)
pa
b
97.2 (43.2, 126.1) 62 ± 14 72 40 50 22.0 ± 3.4 23 96.5 ± 6.2 0.96 ± 0.21 87 (6–489) 1.37 ± 0.27 8 29 19 10.0 ± 1.0 3.9 ± 0.4 11.4 ± 3.0 42.3 ± 12.6 80.0 ± 23.5 65.3 ± 31.3 2.4 ± 0.7 3080 (143, 168,000) 0.14 (0.005–10.39) 3.91 (0.28, 78.69) 208.5 ± 66.5 54.2 (8.0, 436.0) 16.0 ± 12.1 0.81 ± 0.12
<0.0001 0.88 <0.0001 0.007 0.33 0.0001 0.05 0.01 0.006 0.002 0.0002 0.08 0.65 0.49 0.52 0.60 0.24 <0.0001 0.87 0.17 <0.0001 0.42 0.002 0.34 0.03 0.56 <0.0001 0.28
– −0.06 – – – −0.16** − −0.05 0.11* 0.15 0.23*** – – – 0.01 0.03 0.12* 0.36*** −0.04 −0.05 0.67*** −0.003 −0.16** −0.03 0.09 0.002 0.24*** −0.012
P value means the significance in variables between high oxidized HDL and low oxidized HDL groups. Spearman rank correlation test between oxidized HDL and each variables. Median (range). Mean ± SD. Normalized protein catabolic rate. N-terminal pro B-type natriuretic peptide. Inter-Cellular Adhesion Molecule 1. Mean carotid intima-media thickness. <0.05. <0.001. <0.0001.
non-CVD-related disease, such as cancer or infectious disease, were significantly lower than in participants that died due to CVD-related diseases, but the oxHDL/HDL ratio did not differ significantly between these two groups (Table 4). Table 2 Hazard ratio of oxidized HDL to composite CVD events, all-cause and CVD-related mortality. HR (95%CI) Composite end points Crude Model 1 Model 2 (model 1 + interleukin-6) Model 3 (model 1 + HDL)
2.1 (1.4, 3.0) 1.9 (1.3, 2.9) 1.7 (1.2, 2.7) 2.1 (1.4, 3.3)
P <0.0001 0.002 0.008 0.0006
All cause mortality Crude
0.8 (0.5, 1.4)
0.40
CVD-related mortality Crude Model 4 Model 5 (model 4 + interleukin-6) Model 6 (model 4 + HDL)
– 2.9 (1.3, 5.6) 4.1 (1.9, 8.5) 4.1 (1.9, 8.1) 3.8 (1.6, 8.1)
– 0.007 0.0004 0.0004 0.002
Crude model included log oxidized HDL as an independent variable. Model 1 included log oxidized HDL, age (years), gender (male vs. female), diabetes mellitus (yes vs. no), history of cardiovascular disease state (present vs. absent), body mass index (kg/m2 ), subjective global assessment status (malnourished vs. nourished), HD vintage (months) as independent variables. Model 4 includes age (years), gender (male vs. female), diabetes mellitus (yes vs. no), HD vintage (months) as independent variables.
3.2. Patient characteristics according to groups based on median values of oxidized HDL and interleukin-6 To estimate the association of oxHDL with inflammation, patients were grouped according to the median values of serum oxHDL and IL-6. Clinical and biochemical characteristics were compared among four groups of patients: high oxHDL and high IL-6, high oxHDL and low IL-6, low oxHDL and high IL-6, low oxHDL and low IL-6. The comparisons were evaluated using MANOVA (Table 5). Patients with high oxHDL and high IL-6 had higher oxHDL/HDL-chol ratios, higher adiponectin, and higher ICAM-1 than patients in the other groups (Table 5). While prevalence of all-cause mortality was higher in the high IL-6 group than in the low IL-6 group (Table 5), the prevalence of CVDrelated and composite CVD events was significantly higher in the high oxHDL and high IL-6 group than in the other groups (Table 5). Mean CIMT in the high oxHDL and high IL-6 group was significantly higher than the means for CIMT in the other groups (Fig. 2 A), and there was a significant interaction between high oxHDL and high IL-6 for increased mean CIMT (Table 5). High oxHDL with high IL-6 was an independent contributor to increased CIMT based on multivariate models adjusted with confounders, specifically HDL-chol, oxLDL, NT-proBNP, hsCRP, ICAM-1, myeloperoxidase, and adiponectin (Table 6).
H. Honda et al. / Atherosclerosis 220 (2012) 493–501
497
Table 3 Individual cause for mortality classified by oxidized HDL level. High oxidized HDL (n = 206)
Low oxidized HDL (n = 206)
CVD-related death (n = 28) Myocardial infarction (n = 13) Cerebral infarction (n = 3) Other CVD-related disease (n = 12)
21 (10.2%)a 9 3 9
Non-CVD-related death (n = 50) Infection (n = 21) Cancer disease (n = 10) Hemorrhage (n = 2) Others (n = 8) Unknown or not determined (n = 9)
16 (7.8%) 6 1 – 3 6
34 (16.5%) 15 9 2 5 3
All-cause death (n = 78)
37 (17.9%)
41 (19.9%)
7 (3.3%) 4 – 3
Patients were grouped according to the median value of oxidized HDL. a Number of patient (percentage). Prevalence of causative diseases was incrementally higher across between the groups that was assessed by 2 test (P = 0.0005 for all-cause death and P = 0.007 for CVD-related death). Table 4 Oxidized HDL levels and oxidized HDL/HDL ratio among non-survivor patients. CVD-related (n = 28)
Log oxidized-HDL Oxidized HDL/HDL a
5.03 ± 0.09 4.54 ± 5.83
Non-CVD-related (n = 50)
P value
Cancer (n = 10)
Infection (n = 21)
Other (n = 8)
Unknown (n = 9)
4.46 ± 0.15a 2.39 ± 0.65
4.65 ± 0.10a 2.71 ± 1.24
4.70 ± 0.16 3.67 ± 1.21
5.01 ± 0.16 3.15 ± 1.12
0.0012 0.09
Log oxidized-HDL levels in non-survivor patient by cancer or infection disease were significantly decreased compared with by CVD-related disease (Dunnett analysis).
3.3. Association of oxHDL and interleukin-6 with mortality and with composite CVD events The effects of an interaction between oxHDL and IL-6 on allcause mortality, on CVD-related mortality, or on composite CVD events were estimated. An interaction between oxHDL and IL-6 was significantly associated with composite CVD events (Table 7). All-cause mortality was similar in patients belonging to the high oxHDL and high IL-6 group and the low oxHDL and high IL-6 group (Fig. 3 A). However, the occurrence of CVD-related death and composite CVD events were significantly higher for patients in the high oxHDL and high IL-6 group than for patients in the other groups (Fig. 3B and C). Crude and adjusted Cox HRs for CVD-related mortality and composite CVD events in patients with high oxHDL and high IL-6 were significantly elevated (Table 8). Crude and adjusted Cox HRs for all-cause mortality in patients with high IL-6 were elevated; however, these associations did not depend on serum oxHDL levels (data not shown).
3.4. Prospective change in mean CIMT according to groups based on the median values of oxidized HDL and interleukin-6 The influence of the high oxHDL and IL-6 combination on change in CIMT at 3 years was estimated using four groups that were based on median values of serum oxHDL and serum IL-6. Follow-up data on CIMT were collected from 248 patients (60% of the enrolled patients); the other patients died, moved, transferred to another hospital, or did not undergo a follow-up cervical ultrasound test. Mean increases in CIMT in patients with high oxHDL and high IL-6 were larger than in the mean increases in CIMT patients with low oxHDL and low IL-6 over the 3-year study period (Fig. 2B).
4. Discussion Fig. 2. Baseline carotid intima-media thickness (CIMT) in four groups based on the median values of oxidized HDL and interleukin-6 (A) and change in CIMT at 3 years in those groups (B).
The present study demonstrated that oxHDL could be a risk factor for CVD events in prevalent HD patients. Oxidation renders HDL dysfunctional, and oxHDL probably does not have the cardioprotective effects of HDL.
498
Table 5 Patient characteristics according to four groups classified by the median values of oxidized HDL and interleukin-6. High oxHDL low IL-6 (n = 106)
Low oxHDL high IL-6 (n = 110)
Low oxHDL low IL-6 (n = 96)
MANOVAa
64 ± 13 48 33 56 39 20.9 ± 3.2 94.6 ± 6.5 1.00 ± 0.21 126 (6, 426) 1.46 ± 0.32 3.8 ± 0.3 11.6 ± 2.7 48.7 ± 17.2 64.6 ± 32.1 4.6 ± 3.4 5020 (662, 89,900) 0.12 (0.012–8.16) 238.5 ± 90.1 58.6 (19.0, 370.5) 18.2 (4.3, 74.4) 0.88 ± 0.16 27 20 46
61 ± 12 54 22 35 23 20.6 ± 3.0 95.0 ± 6.4 1.03 ± 0.19 137 (6, 489) 1.47 ± 0.25 3.8 ± 0.4 11.7 ± 2.8 53.6 ± 14.6 60.3 ± 33.5 3.8 ± 2.1 3270 (205, 49,700) 0.05 (0.008–0.47) 215.2 ± 68.6 52.7 (8.0, 375.5) 15.6 (7.1, 51.9) 0.80 ± 0.15 9 3 18
67 ± 12 69 42 57 30 21.8 ± 3.6 96.0 ± 6.6 0.95 ± 0.20 94 (6, 489) 1.38 ± 0.26 3.8 ± 0.3 11.5 ± 3.2 39.8 ± 12.8 70.5 ± 30.3 2.5 ± 0.8 4340 (509, 168,000) 0.23 (0.005–10.39) 216.0 ± 71.9 60.1 (24.6, 436.0) 13.5 (6.5, 38.2) 0.83 ± 0.12 28 3 25
59 ± 14 75 34 43 15 21.9 ± 3.1 97.0 ± 5.9 0.96 ± 0.21 80 (6, 424) 1.34 ± 0.28 3.9 ± 0.4 11.3 ± 2.8 44.9 ± 11.9 59.8 ± 31.7 2.3 ± 0.7 2650 (143, 104,000) 0.07 (0.013–1.3) 200.3 ± 58.9 49.1 (8.0, 416.0) 13.3 (3.2, 133.7) 0.80 ± 0.11 10 2 20
IL-6 0.0002b 0.02 0.003 0.0007 oxHDL NS oxHDL oxHDL oxHDL NS NS oxHDL, IL-6 IL-6 oxHDL, IL-6 IL-6 IL-6 oxHDL, IL-6 IL-6 oxHDL IL-6, oxHDL × IL-6 <0.0001 <0.0001 <0.0001
a MANOVA (multivariable analysis of variance) was performed to assess associations among continuous variables, log oxidized HDL and log interleukin-6; b Associations among nominal variables in 4 groups by combination with oxidized HDL and interleukin-6 were assessed by 2 test. Abbreviation: see Table 1.
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Age (year) Gender (male, %) DM (%) CVD (%) SGA (%) BMI (kg/m2 ) GNRI nPCRd (g/kg/day) HD vintage (months) Kt/V Albumin (g/dL) Creatinine (mg/dL) HDL-chol (mg/dL) Oxidized LDL (U/L) Oxidized HDL/HDL-chol NT-proBNP (pg/mL) Hs-CRP (mg/dL) ICAM-1 (ng/mL) Myeloperoxidase (ng/mL) Adiponectin (g/mL) Mean CIMT (mm) All-cause mortality (%) CVD-related mortality (%) Composite CVD events (%)
High oxHDL high IL-6 (n = 100)
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499
Table 6 Association among oxidized HDL, interleukin-6, and mean carotid intima-media thickness. Model 1 (adjusted R2 = 0.39) ˇ, SE Intercept High oxHDL, high IL-6 High oxHDL, low IL-6 Low oxHDL, high IL-6 Low oxHDL, low IL-6
Model 2 (adjusted R2 = 0.43) ˇ, SE
P
0.44, 0.15 0.04, 0.02 −0.001, 0.02 −0.02, 0.02 Ref.
<0.0001 0.03 0.94 0.20 –
0.58, 0.12 0.04, 0.02 −0.007, 0.02 −0.01, 0.02 Ref.
0.0002 0.02 0.67 0.40 –
Association among oxidized HDL, interleukin-6, and mean carotid intima-media thickness (CIMT) were assessed according to four groups classified by the median values of oxidized HDL and interleukin-6. Models included mean CIMT as a dependent factor. Model 1 included age (years), gender (male vs. female), body mass index (kg/m2 ), diabetes mellitus (yes vs. no), cardiovascular state (yes vs. no), subjective global assessment status (positive vs. negative), HD vintage (months), Smoking (yes vs. no), geriatric nutritional risk index. Model 2 included variables in model 1 and normalized protein catabolic rate (g/kg/day), Kt/V, HDL-cholesterol (mg/dL), oxLDL (U/L), log NT-proBNP, log high-sensitivity CRP, ICAM-1 (ng/mL), log myeloperoxidase, log adiponectin as independent variables.
Table 7 Impact of interaction between oxidized HDL and interleukin-6 on CVD events. Crude
Composite CVD events (n = 112) CVD-related mortality (n = 28)
Model 1
Model 2
Coefficient, SE
P value
Coefficient, SE
P value
Coefficient, SE
P value
0.97, 0.41 0.86, 1.27
0.01 0.49
1.10, 0.41 –
0.008 –
1.10, 0.42 –
0.008 –
Model 1 included age (years), gender (male vs. female), body mass index (kg/m2 ), diabetes mellitus (yes vs. no), cardiovascular state (yes vs. no), subjective global assessment status (positive vs. negative), HD vintage (months), Smoking (yes vs. no). Model 2 included variables in model 1 and HDL-cholesterol (mg/dL) as independent variables.
Navab et al. reported that dysfunctional HDL could appear without any change in HDL-chol levels [20]. This association between dysfunctional HDL and HDL-chol were verified in a study of dysfunctional HDL in maintenance HD patients [7]. The protective effect of HDL against CVD seen in large population studies is lost by individuals with impaired glucose metabolism or diabetes, even when HDL-chol levels are not affected [4]. Here, we demonstrated that high HDL-chol was significantly associated with low prevalence of CVD events, and serum HDL-chol levels in patients presenting high oxHDL was higher than in those presenting low oxHDL (Table 1). However, the oxHDL/HDL-chol ratio was significantly higher in patients with high oxHDL (Table 1). Therefore, excess oxidative stress may have yielded dysfunctional HDL in patients on HD, and patients with high HDL-chol under excess oxidative stress may have enriched oxHDL. That is, whereas serum HDL-chol levels are inversely associated with CVD risk in large epidemiologic studies [4,21,22], HDL-chol measurements alone fail to provide an accurate assessment of the protective versus dysfunctional nature of HDL particles [22,23]; this shortcoming of HDL-chol measurements is of particular concern for patients undergoing HD. Activities of HDL are compromised in many pathological states associated with inflammation and oxidative stress [4–6]. Oxidized
HDL was independently associated with CVD events and with CVDrelated mortality (Table 2). However, high oxHDL with low IL-6 was not associated with CVD events, but high oxHDL with high IL-6 was significantly associated with CVD events in the present study (Fig. 3, Tables 5 and 8). The effects of an interaction between oxHDL and IL-6 on CVD-related mortality were not significant; however, this interaction was associated with high prevalence of composite CVD events including non-fatal CVD (Table 7). This finding indicated that the predictive value of oxHDL (or IL-6) for CVD events depends on IL-6 (or oxHDL). High oxHDL with high IL-6 was significantly associated with CVD-related mortality based on the Kaplan–Meier analysis (Fig. 3), but the effect of interaction between oxHDL and IL6 on CVD-related mortality was not statistically significant based on the model (Table 7); this difference between analysis may be due to a statistical power issue. The oxHDL/HDL-chol ratio in the high oxHDL and low IL-6 group was higher than that in the low oxHDL and low IL-6 group (Table 5). Even the imbalance between functional and dysfunctional HDL may be associated with the CVD events. However, oxHDL values may be more predictive of CVD events in patients that are experiencing inflammation. Dysfunctional HDL is associated with CVD events and atherosclerosis in non-uremic patients [20,24], but the impact of dysfunctional HDL
Table 8 Association among oxHDL, interleukin-6, and CVD-related mortality, and composite CVD events. Crude
Composite CVD events High oxHDL, high IL-6 High oxHDL, low IL-6 Low oxHDL, high IL-6 Low oxHDL, lowIL-6 CVD-related mortality High oxHDL, high IL-6 High oxHDL, low IL-6 Low oxHDL, high IL-6 Low oxHDL, low IL-6
Model 1
Model 2
HR (95% CI)
P value
HR (95% CI)
P value
HR (95% CI)
P value
3.1 (1.8, 5.4) 0.9 (0.5, 1.7) 1.5 (0.7, 2.8) Ref.
<0.0001 0.69 0.14 –
2.5 (1.4, 4.5) 0.8 (0.4, 1.5) 1.2 (0.6, 2.2) Ref.
0.001 0.49 0.59 –
2.5 (1.4, 4.5) 0.8 (0.4, 1.6) 1.1 (0.6, 2.1) Ref.
0.001 0.53 0.70 –
11.1 (3.2, 69.5) 1.4 (0.2, 10.3) 2.0 (0.4, 14.7) Ref.
<0.0001 0.73 0.40 –
10.3 (3.0, 65.8) 1.6 (0.3, 12.1) 1.5 (0.3, 10.8) Ref.
<0.0001 0.61 0.64 –
9.5 (2.7, 60.6) 1.5 (0.2, 11.6) 1.7 (0.3, 12.8) Ref.
<0.0001 0.61 0.64 –
Associations among oxidized HDL, interleukin-6, and CVD events were assessed according to four groups classified by the median values of oxidized HDL and interleukin-6. Model 1 included age (years), gender (male vs. female), body mass index (kg/m2 ), diabetes mellitus (yes vs. no), cardiovascular state (yes vs. no), subjective global assessment status (positive vs. negative), HD vintage (months), smoking (yes vs. no), geriatric nutritional risk index, mean carotid intima-media thickness (mm) as independent variables. Model 2 included variables in model 1 and, HDL-cholesterol (mg/dL), as independent variables. Model 3 included age (years), gender (male vs. female), diabetes mellitus (yes vs. no), and cardiovascular state (yes vs. no) as independent variables. Model 4 included all variables in model 1 and HDL-cholesterol (mg/dL) as independent variables.
500
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died due to cancer or infection disease (Table 4). At baseline, nutritional status was worse in patients with high oxHDL than in patients with low oxHDL (Table 1). Recently, we reported the impact of oxHDL on PEW in HD patients; that study had a longitudinal design and indicated that increased oxHDL may be associated with a high prevalence of PEW [11]. Therefore, elevated oxHDL levels may be associated with worsening of nutritional state in prevalent HD patients; however, high oxHDL was not predictive of non-CVDrelated mortality in those patients. The results of our study must be considered with the following caveats. The number of patients was relatively small, and we could only evaluate the findings for prevalent HD patients. Additionally, the single-point measurement of oxHDL and IL-6 does not permit accurate evaluation of these biomarkers [32]. Finally, we could not assess a specific association between oxHDL and atherosclerosis because atherosclerosis was only assessed by increases or changes in CIMT. Moreover, change in CIMT, as a quantitative assessment of advanced atherosclerosis, was based on measurements at just two time points and was only performed in patients that survived; therefore, patients with severe atherosclerotic change may have been excluded because they died before the 3-year follow-up examination. Thus, a large prospective study with more accurate quantitative assessment of atherosclerosis is required to clarify the reliability of oxHDL values as a predictor for progression of atherosclerosis in prevalent HD patients. 5. Conclusion In conclusion, oxHDL seems to be an important, contributing factor for CVD events and CVD-related mortality in prevalent HD patients, especially when high oxHDL is concomitant with high IL-6. Disclosure None. Fig. 3. Kaplan–Meier curves for time to all-cause mortality (A), cardiovascular disease-related mortality (B), and composite cardiovascular events (C). Patients were separated into four groups based the median values of oxidized HDL and interleukin-6; each curve represents data from one of the four groups.
on CVD may be significant in HD patients with chronic inflammation. Evidence that IL-6 is a strong risk factor for CVD events has accumulated [13,25], and high IL-6 is closely associated with mortality [13,25–27] and increased CIMT in HD patients [28,29]. On another front, dysfunctional HDL is also associated with increased CIMT [30,31]. We did not find a significant association between oxHDL and CIMT at baseline (Table 1); however, increased mean CIMT at baseline was significantly associated with an interaction between oxHDL and IL-6, as well as with IL-6 (Table 5). High IL-6 together with high oxHDL was particularly associated with changes in CIMT over the 3-year study period (Fig. 2B). Moreover, the impact of IL-6 on CVD events was dependent upon the elevation of oxHDL levels (Fig. 3, Table 8). Taken together, these data indicated that the predictive value of high IL-6 for CVD events may be most accurate in prevalent HD patients who have elevated serum oxHDL levels. Oxidized HDL was not a predictor of all-cause mortality in the present study. Kalantar-Zadeh demonstrated that inflammatory (dysfunctional) HDL was significantly associated with worse outcomes for prevalent HD patients [10]. This discrepancy might be associated with differences in the methods used to measure dysfunctional HDL. In the present study, diseases causing death were obviously different between the high oxHDL and low oxHDL group (Table 3), and oxHDL was significantly lower in patients that had
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