CLINICAL RESEARCH www.jasn.org

HDL , , and Cardiovascular Risk in Hemodialysis Patients

†‡ | Günther Silbernagel,* Bernd Genser, § Christiane Drechsler,§ Hubert Scharnagl,¶ † | | Tanja B. Grammer, Tatjana Stojakovic,¶ Vera Krane,§ Eberhard Ritz,** Christoph Wanner,§ ††‡‡ and Winfried März¶

*Department of Angiology, Swiss Cardiovascular Center, Inselspital, University of Bern, Bern, Switzerland; †Mannheim Institute of Public Health, Social and Preventive Medicine, and ††Medical Clinic V (Nephrology, Hypertensiology, Endocrinology, Diabetology, Rheumatology), Department of Internal Medicine, Mannheim Medical Faculty, University of Heidelberg, Mannheim, Germany; ‡Institute of Public Health, Federal University of Bahia, Salvador, Brazil; §Division of Nephrology, Department of Medicine I, and |Comprehensive Heart Failure Centre, University of Würzburg, Würzburg, Germany; ¶Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Graz, Graz, Austria; **Division of Nephrology, Department of Internal Medicine, University of Heidelberg, Heidelberg, Germany; and ‡‡Synlab Academy, Synlab Services LLC, Mannheim, Germany

ABSTRACT High concentrations of HDL cholesterol are considered to indicate efficient reverse cholesterol transport and to protect from atherosclerosis. However, HDL has been suggested to be dysfunctional in ESRD. Hence, our main objective was to investigate the effect of HDL cholesterol on outcomes in maintenance hemodialysis patients with diabetes. Moreover, we investigated the associations between the major protein components of HDL (apoA1, apoA2, and apoC3) and end points. We performed an exploratory, post hoc analysis with 1255 participants (677 men and 578 women) of the German Diabetes Dialysis study. The mean age was 66.3 years and the mean body mass index was 28.0 kg/m2.Theprimary end point was a composite of cardiac death, myocardial infarction, and stroke. The secondary end point included all-cause mortality. The mean duration of follow-up was 3.9 years. A total of 31.3% of the study participants reached the primary end point and 49.1% died from any cause. HDL cholesterol and apoA1 and apoC3 quartiles were not related to end points. However, there was a trend toward an inverse association between apoA2 and all-cause mortality. The hazard ratio for death from any cause in the fourth quartile compared with the first quartile of apoA2 was 0.63 (95% confidence interval, 0.40 to 0.89). The lack of an association between HDL cholesterol and cardiovascular risk may support the concept of dysfunctional HDL in hemodialysis. The possible beneficial effect of apoA2 on survival requires confir- mation in future studies.

J Am Soc Nephrol 26: 484–492, 2015. doi: 10.1681/ASN.2013080816

Reverse cholesterol transport is considered to pro- Received August 1, 2013. Accepted June 1, 2014. 1,2 tect from atherosclerosis. HDLs represent the G.S. and B.G. contributed equally to this work. major vehicle of reverse cholesterol transport.1,2 They primarily consist of phospholipids, choles- Published online ahead of print. Publication date available at www.jasn.org. terol, and proteins.1,2 The two major protein con- stituents of HDL are apoA1 and apoA2.1,2 ApoC3 Correspondence: Dr. Günther Silbernagel, Department of Angiology, Swiss Cardiovascular Center, Inselspital, University 3,4 forms another component of HDL. Whereas of Bern, 3010 Bern, Switzerland. Email: guenther.silbernagel@ apoA1 is regarded as beneficial to cardiovascular insel.ch health, apoC3 is rather regarded as proathero- – genic.3 6 The role of apoA2 has not been sufficiently Copyright © 2015 by the American Society of Nephrology

484 ISSN : 1046-6673/2602-484 J Am Soc Nephrol 26: 484–492, 2015 www.jasn.org CLINICAL RESEARCH examined.7,8 Nevertheless, recent data tend to support anti- RESULTS atherogenic effects.7–9 High concentrations of HDL cholesterol are generally Patient Characteristics at Baseline thought to reflect efficient reverse cholesterol transport.2,5 The 1255 (677 men and 578 women) participants of the 4D However, HDL composition and function are impaired in study had a mean age of 66.3 years and a mean body mass index CKD.10,11 In addition, dysfunctional HDL has been observed of 28.0 kg/m2. in patients on hemodialysis.12 Hence, high HDL cholesterol Tables 1–4 show the baseline characteristics of the study does not necessarily indicate efficient reverse cholesterol participants according to the quartiles of HDL cholesterol and transport in renal disease. apoA1, apoA2, and apoC3. Supplemental Table 1 shows the We conducted an exploratory, post hoc analysis of the baseline characteristics of the subgroups with HDL cholesterol German Diabetes Dialysis (4D) study to investigate whether levels #50 mg/dl and .50 mg/dl within the fourth quartile of HDL cholesterol predicts mortality and cardiovascular risk in HDL cholesterol. Supplemental Table 2 shows the baseline patients on maintenance hemodialysis treatment.13,14 More- characteristics of the propensity score–matched cohort. over, we sought to analyze the relationships of the major pro- In the entire cohort, the HDL cholesterol quartiles were tein components of HDL, namely apoA1, apoA2, and apoC3, inversely related to the proportion of men, to body mass index, with outcomes. , apoC3, and C-reactive protein and they were

Table 1. Baseline patient characteristics according to quartiles of HDL cholesterol Quartile of HDL Cholesterol Characteristic P Value Q1 Q2 Q3 Q4 Participants (n) 312 308 317 318 Range (mg/dl) 17.3–33.4 33.5–39.3 39.4–46.9 47.0–115.5 Age (yr) 65.3 (8.3) 66.0 (8.6) 67.2 (8.1) 66.7 (8.0) 0.03 Men (%) 205 (66) 185 (60) 156 (49) 131 (41) ,0.001 Ever smoking, n (%) 146 (47) 123 (40) 120 (38) 118 (37) 0.06 Body mass index (kg/m2) 30.0 (4.7) 28.2 (5.0) 27.7 (5.0) 26.2 (4.3) ,0.001 BP (mmHg) Systolic 145 (22) 147 (22) 144 (21) 147 (22) 0.19 Diastolic 75 (11) 76 (12) 76 (11) 77 (11) 0.13 Time receiving dialysis (mo) 8.9 (6.7) 8.3 (7.2) 8.1 (7.0) 7.8 (6.6) 0.24 History of disease, n (%)a Arrhythmia 76 (24) 52 (17) 49 (15) 59 (19) 0.03 MI, CABG, PCI, or CHD 103 (33) 94 (31) 79 (25) 93 (29) 0.16 Congestive heart failureb 132 (42) 107 (35) 102 (32) 103 (32) 0.03 Stroke or TIA 57 (18) 63 (20) 55 (17) 49 (15) 0.42 Peripheral vascular disease 148 (47) 147 (48) 125 (39) 140 (44) 0.13 Hemoglobin (g/dl) 10.8 (1.4) 11.0 (1.3) 10.8 (1.3) 11.0 (1.4) 0.08 Glycated hemoglobin (%) 6.84 (1.31) 6.72 (1.24) 6.61 (1.27) 6.72 (1.21) 0.15 Phosphate (mg/dl) 5.98 (1.72) 6.07 (1.56) 5.97 (1.51) 6.09 (1.63) 0.71 Albumin (g/dl) 3.83 (0.3) 3.81 (0.26) 3.80 (0.32) 3.81 (0.31) 0.47 Cholesterol (mg/dl) LDL 119.4 (26.9) 125.0 (30.2) 128.3 (30.4) 129.5 (40.0) ,0.001 HDL 29.6 (3.0) 36.1 (1.7) 43.0 (2.2) 56.9 (9.6) ,0.001 (mg/dl) A1 107.9 (14.4) 118.5 (16.8) 129.0 (16.1) 149.5 (23.2) ,0.001 A2 24.5 (4.4) 27.1 (4.5) 29.1 (4.9) 32.2 (6.1) ,0.001 C3 22.3 (10.6) 20.8 (10.0) 19.5 (8.8) 19.1 (8.1) ,0.001 Triglycerides (mg/dl) 349.0 (200.7) 291.6 (170.1) 236.2 (134.1) 181.4 (95.6) ,0.001 C-reactive protein (mg/L) 11.0 (4.3–13.6) 6.6 (2.8–12.0) 5.7 (2.5–11.7) 4.5 (1.9–11.1) ,0.001 NT-pro-BNP (ng/L) 3585 (1492–9949) 3117 (1416–6842) 2698 (1246–8375) 3953 (1686–11,115) 0.03 Data are presented as the mean (SD) or median (25th percentile to 75th percentile), unless otherwise specified. P values for comparison of groups were derived from an analysis of covariance model (for continuous variables) or logistic regression model (for categorical variables), both adjusted for age and sex. Q, quartile; MI, myocardial infarction; CABG, coronary artery bypass grafting surgery; PCI, percutaneous coronary intervention; CHD, coronary heart disease, documented by coronary angiography; TIA, transitory ischemic attack; NT-pro-BNP, N-terminal pro-brain natriuretic peptide. aTypes of disease and intervention are not mutually exclusive. bPredominantly New York Heart Association II.

J Am Soc Nephrol 26: 484–492, 2015 HDL Cholesterol and Outcomes in 4D 485 CLINICAL RESEARCH www.jasn.org positively related to LDL cholesterol and apoA1 and apoA2 different end points according to HDL cholesterol and apolipo- (Table 1). The apoA1 quartiles were inversely related to the protein quartiles are shown in Supplemental Tables 3–6. The proportion of men, triglycerides, and C-reactive protein and interaction terms between components of HDL and randomi- they were positively associated with albumin, LDL cholesterol, zation group were not significant for any end point in pooled and apopliproteins (Table 2). The apoA2 quartiles were inversely analyses. These findings were corroborated by analyses stratified related to C-reactive protein, N-terminal pro-brain natriuretic by randomization group (data not shown). peptide, congestive heart failure, and arrhythmia and they were Table 5 shows a summary of the pooled time-to-event anal- positively associated with hemoglobin, albumin, and LDL cho- yses. The quartiles of HDL cholesterol were not associated lesterol (Table 3). The apoC3 quartiles were inversely related to with any end point (Table 5). Further stratification of the age and N-terminal pro-brain natriuretic peptide and they were fourth quartile in patients with HDL cholesterol #50 mg/dl positively associated with body mass index, glycated hemoglo- and in those with HDL cholesterol .50 mg/dl did not reveal bin, LDL cholesterol, and triglycerides (Table 4). significant associations (Supplemental Table 7). Adjustment for HDL cholesterol measurements during the follow-up did Effect of Baseline HDL Cholesterol and Apolipoprotein not change the results (Supplemental Table 8). The cubic Concentrations on Outcomes spline models corroborated the absence of any significant ef- A total of 31.3% of the study participants reached the com- fect of HDL cholesterol on both cardiovascular risk and all- posite vascular end point and 49.1% died from any cause. The cause death (Figure 1).

Table 2. Baseline patient characteristics according to quartiles of apoA1 Quartile of ApoA1 at Baseline Characteristic P Value Q1 Q2 Q3 Q4 Participants (n) 312 311 301 327 Range (mg/dl) 74–109 110–123 124–139 140–271 Age (yr) 65.6 (8.6) 66.3 (7.9) 67.1 (7.7) 66.2 (8.6) 0.13 Men (%) 208 (67) 188 (60) 147 (48) 134 (41) ,0.001 Ever smoking, n (%) 145 (46) 130 (42) 115 (38) 117 (36) 0.03 Body mass index (kg/m2) 27.5 (4.9) 27.7 (4.7) 27.6 (4.6) 27.3 (5.1) 0.66 BP (mmHg) Systolic 146 (22) 145 (23) 146 (22) 145 (21) 0.80 Diastolic 75 (10) 76 (11) 77 (11) 76 (11) 0.31 Time receiving dialysis (mo) 8.5 (7.1) 8.2 (6.5) 8.4 (7.2) 8.1 (6.8) 0.88 History of disease, n (%)a Arrhythmia 73 (23) 63 (20) 45 (15) 55 (17) 0.03 MI, CABG, PCI, or CHD 110 (35) 88 (28) 81 (27) 90 (28) 0.07 Congestive heart failureb 126 (40) 120 (39) 104 (34) 94 (29) 0.01 Stroke or TIA 61 (20) 48 (15) 59 (19) 56 (17) 0.49 Peripheral vascular disease 156 (50) 140 (45) 136 (45) 128 (39) 0.05 Hemoglobin (g/dl) 10.8 (1.5) 10.9 (1.3) 10.9 (1.3) 11.0 (1.4) 0.13 Glycated hemoglobin (%) 6.8 (1.3) 6.7 (1.2) 6.6 (1.2) 6.9 (1.3) 0.06 Phosphate (mg/dl) 5.98 (1.70) 5.96 (1.60) 6.02 (1.53) 6.16 (1.60) 0.37 Albumin (g/dl) 3.76 (0.29) 3.81 (0.31) 3.86 (0.30) 3.84 (0.29) ,0.001 Cholesterol (mg/dl) LDL 117 (26) 124 (26) 129 (30) 132 (31) ,0.001 HDL 26 (6) 32 (7) 38 (9) 49 (15) ,0.001 Apolipoprotein (mg/dl) A1 99.8 (6.8) 116.5 (4.0) 130.6 (4.8) 157.1 (18.4) ,0.001 A2 23.3 (3.6) 26.7 (3.5) 29.4 (4.1) 33.4 (5.8) ,0.001 C3 18.6 (9.7) 20.3 (9.4) 20.9 (9.2) 21.8 (9.5) ,0.001 Triglycerides (mg/dl) 292 (190) 268 (155) 263 (159) 235 (158) ,0.001 C-reactive protein (mg/L) 10.0 (3.7–15.8) 6.8 (3.1–11.2) 6.1 (2.6–11.1) 4.6 (1.9–11.1) ,0.001 NT-pro-BNP (ng/L) 4051 (1641–11,625) 2926 (1460–8322) 3076 (1351–7553) 2975 (1288–8040) 0.02 Data are presented as the mean (SD) or median (25th percentile to 75th percentile), unless otherwise specified. P values for comparison of groups were derived from an analysis of covariance model (for continuous variables) or logistic regression model (for categorical variables), both adjusted for age and sex. Q, quartile; MI, myocardial infarction; CABG, coronary artery bypass grafting surgery; PCI, percutaneous coronary intervention; CHD, coronary heart disease, documented by coronary angiography; TIA, transitory ischemic attack; NT-pro-BNP, N-terminal pro-brain natriuretic peptide. aTypes of disease and intervention are not mutually exclusive. bPredominantly New York Heart Association II.

486 Journal of the American Society of Nephrology J Am Soc Nephrol 26: 484–492, 2015 www.jasn.org CLINICAL RESEARCH

Table 3. Baseline patient characteristics according to quartiles of apoA2 Quartile of ApoA2 at Baseline Characteristic P Value Q1 Q2 Q3 Q4 Participants (n) 232 314 338 367 Range (mg/dl) 12–23 24–26 28–30 31–54 Age (yr) 66.3 (7.8) 66.7 (8.4) 66.4 (8.0) 65.9 (8.6) 0.58 Men (%) 133 (57) 176 (56) 172 (51) 193 (53) 0.37 Ever smoking, n (%) 98 (42) 131 (42) 135 (40) 140 (38) 0.72 Body mass index (kg/m2) 27.4 (4.7) 27.7 (4.8) 27.6 (5.0) 27.4 (4.6) 0.74 BP (mmHg) Systolic 144 (23) 147 (22) 145 (21) 146 (22) 0.51 Diastolic 75 (11) 75 (11) 77 (11) 76 (11) 0.17 Time receiving dialysis (mo) 8.4 (7.1) 8.3 (6.5) 8.7 (7.6) 7.8 (6.4) 0.47 History of disease, n (%)a Arrhythmia 64 (28) 66 (21) 48 (14) 57 (16) ,0.001 MI, CABG, PCI, or CHD 80 (34) 99 (31) 90 (27) 100 (27) 0.13 Congestive heart failureb 113 (49) 125 (40) 102 (30) 102 (28) ,0.001 Stroke or TIA 42 (18) 62 (20) 53 (16) 67 (18) 0.59 Peripheral vascular disease 121 (52) 151 (48) 146 (43) 141 (38) 0.01 Hemoglobin (g/dl) 10.6 (1.4) 10.9 (1.4) 11.0 (1.3) 11.1 (1.3) ,0.001 Glycated hemoglobin (%) 6.8 (1.2) 6.8 (1.3) 6.7 (1.2) 6.7 (1.2) 0.77 Phosphate (mg/dl) 6.00 (1.83) 6.00 (1.57) 6.09 (1.51) 6.02 (1.60) 0.86 Albumin (g/dl) 3.73 (0.31) 3.81 (0.30) 3.83 (0.29) 3.86 (0.30) ,0.001 Cholesterol (mg/dl) LDL 117 (29) 126 (27) 126 (29) 130 (32) ,0.001 HDL 28 (9) 32 (10) 37 (12) 44 (15) ,0.001 Apolipoprotein (mg/dl) A1 105.8 (13.6) 115.9 (15.6) 127.9 (16.9) 146.8 (23.4) ,0.001 A2 20.8 (2.1) 25.2 (1.0) 28.7 (1.0) 35.2 (4.1) ,0.001 C3 17.6 (10.2) 19.8 (9.3) 20.8 (9.1) 22.4 (9.1) ,0.001 Triglycerides (mg/dl) 291 (209) 271 (162) 264 (157) 241 (147) 0.004 C-reactive protein (mg/L) 11.1 (5.8–18.9) 8.4 (3.5–12.9) 5.1 (2.1–11.1) 4.4 (1.9–11.1) ,0.001 NT-pro-BNP (ng/L) 5713 (2558–13,735) 3415 (1402–8652) 2510 (1118–6649) 2742 (1310–6473) ,0.001 Data are presented as the mean (SD) or median (25th percentile to 75th percentile), unless otherwise specified. P values for comparison of groups were derived from an analysis of covariance model (for continuous variables) or logistic regression model (for categorical variables), both adjusted for age and sex. Q, quartile; MI, myocardial infarction; CABG, coronary artery bypass grafting surgery; PCI, percutaneous coronary intervention; CHD, coronary heart disease, documented by coronary angiography; TIA, transitory ischemic attack; NT-pro-BNP, N-terminal pro-brain natriuretic peptide. aTypes of disease and intervention are not mutually exclusive. bPredominantly New York Heart Association II.

The quartiles of apoA1 and apoC3 were not associated with variable either (hazard ratio, 0.91; 95% confidence interval, end points (Table 5). By contrast, the multivariate adjusted 0.82 to 1.01; P=0.09). analyses conducted in the entire cohort showed that partici- pants in the fourth versus first quartile of apoA2 had decreased the risk of death from any cause (Table 5). The cubic spline DISCUSSION model corroborated the inverse association between apoA2 and the risk of death from any cause (P,0.001) (Figure 2). The main finding of this study was that the baseline concen- In the propensity score–matched subgroup, the hazard ratios trations of HDL cholesterol were not predictive of all-cause for all-cause mortality comparing the fourth, third, and sec- mortality and cardiovascular end points. The baseline con- ond with the first apoA2 quartile were similar to those centrations of apoA1 and apoC3 were also not associated with obtained in the entire cohort (see Supplemental Table 9). Be- outcomes. However, there was a trend toward an inverse causeofthelowersamplesize,theP value comparing the association between baseline apoA2 concentrations and death fourth with the first apoA2 quartile did not reach statistical from any cause. significance after Bonferroni correction in the subgroup anal- LDL lowering was demonstrated to reduce cardiovascular ysis. The association of apoA2 with the risk of death from any risk and mortality in the Study of Heart and Renal Protection cause did not reach statistical significance when apoA2 was study and in a post hoc analysis of the 4D study.15,16 Yet included in the Cox model as a standardized continuous lowering does not seem to be as effective in preventing

J Am Soc Nephrol 26: 484–492, 2015 HDL Cholesterol and Outcomes in 4D 487 CLINICAL RESEARCH www.jasn.org

Table 4. Baseline patient characteristics according to quartiles of apoC3 Quartile of Baseline ApoC3 Characteristic P Value Q1 Q2 Q3 Q4 Participants (n) 321 306 307 317 Range (mg/dl) 2–13 14–18 19–26 26–49 Age (yr) 67.1 (7.8) 67.7 (7.9) 65.9 (8.4) 64.6 (8.6) ,0.001 Men (%) 195 (61) 169 (55) 154 (50) 159 (50) 0.02 Ever smoking, n (%) 130 (41) 132 (43) 112 (36) 133 (41) 0.38 Body mass index (kg/m2) 26.4 (4.5) 26.6 (4.3) 28.2 (4.7) 29.0 (5.1) ,0.001 BP (mmHg) Systolic 146 (23) 146 (21) 147 (22) 143 (22) 0.25 Diastolic 76 (11) 75 (10) 76 (11) 75 (12) 0.14 Time receiving dialysis (mo) 7.7 (7.1) 7.8 (6.2) 9.1 (7.2) 8.6 (6.8) 0.03 History of disease, n (%)a Arrhythmia 68 (21) 63 (21) 53 (17) 52 (16) 0.29 MI, CABG, PCI, or CHD 98 (31) 82 (27) 91 (30) 98 (31) 0.71 Congestive heart failureb 123 (38) 114 (37) 99 (32) 108 (34) 0.33 Stroke or TIA 45 (14) 68 (22) 54 (16) 57 (18) 0.07 Peripheral vascular disease 146 (45) 145 (47) 139 (45) 130 (41) 0.35 Hemoglobin (g/dl) 10.8 (1.3) 11.0 (1.4) 11.0 (1.4) 10.8 (1.3) 0.12 Glycated hemoglobin (%) 6.6 (1.2) 6.5 (1.2) 6.8 (1.1) 7.0 (1.4) ,0.001 Phosphate (mg/dl) 5.75 (1.51) 6.04 (1.53) 6.19 (1.74) 6.15 (1.63) 0.002 Albumin (g/dl) 3.76 (0.32) 3.83 (0.27) 3.84 (0.30) 3.85 (0.31) 0.002 Cholesterol (mg/dl) LDL 114 (26) 126 (28) 133 (29) 130 (33) ,0.001 HDL 38 (13) 39 (12) 36 (14) 32 (133) ,0.001 Apolipoprotein (mg/dl) A1 120.5 (23.1) 127.7 (21.6) 127.6 (23.1) 129.6 (25.7) ,0.001 A2 26.1 (5.5) 28.8 (5.2) 28.9 (5.5) 29.3 (6.2) ,0.001 C3 10.1 (2.3) 15.9 (1.4) 21.9 (2.2) 33.9 (5.6) ,0.001 Triglycerides (mg/dl) 146.0 (61.0) 201.7 (77.7) 265.2 (102.1) 440.1 (202.1) ,0.001 C-reactive protein (mg/l) 6.8 (2.7–11.8) 5.0 (2.4–11.1) 7.1 (3.0–11.1) 7.4 (2.9–11.9) 0.02 NT-pro-BNP (ng/l) 5325 (1974–138,794) 3187 (1451–9503) 2659 (1171–7670) 2629 (1289–5207) ,0.001 Data are presented as the mean (SD) or median (25th percentile to 75th percentile), unless otherwise specified. P values for comparison of groups were derived from an analysis of covariance model (for continuous variables) or logistic regression model (for categorical variables), both adjusted for age and sex. Q, quartile; MI, myocardial infarction; CABG, coronary artery bypass grafting surgery; PCI, percutaneous coronary intervention; CHD, coronary heart disease, documented by coronary angiography; TIA, transitory ischemic attack; NT-pro-BNP, N-terminal pro-brain natriuretic peptide. aTypes of disease and intervention are not mutually exclusive. bPredominantly New York Heart Association II. cardiovascular events in hemodialysis patients as in the general disease in another cohort of 607 hemodialysis patients.24 In population.14,17 However, patients on maintenance hemo- summary, the association of HDL cholesterol with outcomes dialysis treatment carry a very high cardiovascular risk.18 is at least weakened in ESRD. The data may therefore sup- Raising low levels of HDL cholesterol may reduce this risk.19 port the idea that HDL become dysfunctional in chronic The aforementioned hypothesis is also based on the consis- hemodialysis.25 tent relationship between low HDL cholesterol and increased Surprisingly, high apoA2, unlike apoA1, tended to be cardiovascular risk in the general population.5,20,21 However, associated with a lower risk of death from any cause in the this association did not hold true in these analyses. Few stud- 4D study. This observation may reflect differences in the ies have previously investigated the association between HDL metabolism of apoA1 and apoA2 in ESRD.26 Moreover, the as- cholesterol and apolipoproteins with outcomes in patients sociations of apoA2 with death from any cause may suggest with ESRD.22–24 Consistent with our results, HDL choles- potentially protective effects of this protein.7–9 In contrast terol was not associated with survival in a cohort of 15,859 with the lack of an association between apoA1 and end points hemodialysis patients from the DaVita dialysis clinics in the in our study, a previous study showed that induction of apoA1 United States.22 Oxidized HDL cholesterol was even demon- may improve HDL functionality in hemodialysis patients.27 strated to positively correlate with future cardiovascular Hence, future studies are needed to further elucidate the rela- events in 412 hemodialysis patients.23 By contrast, HDL cho- tionships of apoA1 and apoA2 with cardiovascular end points lesterol was inversely related to prevalent cardiovascular in ESRD.

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Table 5. Prognostic value of baseline lipid concentrations on risk of end point occurrence obtained by recurrent events time- to-event analysis HDL Cholesterol ApoA1 ApoA2 ApoC3 End point P P P P HR (95% CI)a HR (95% CI)a HR (95% CI)a HR (95% CI)a Valueb Valueb Valueb Valueb Combined vascular end pointc Q2 1.02 (0.80 to 1.31) 0.85 1.14 (0.90 to 1.44) 0.29 0.78 (0.61 to 1.00) 0.05 0.94 (0.74 to 1.19) 0.60 Q3 0.96 (0.75 to 1.23) 0.75 0.93 (0.72 to 1.20) 0.58 0.94 (0.74 to 1.20) 0.61 0.98 (0.77 to 1.24) 0.87 Q4 1.06 (0.83 to 1.35) 0.65 0.93 (0.73 to 1.19) 0.57 0.74 (0.58 to 0.96) 0.02 0.93 (0.73 to 1.19) 0.58 Overalld 0.88 0.33 0.05 0.93 Cardiac death Q2 0.99 (0.71 to 1.39) 0.97 1.16 (0.84 to 1.62) 0.37 0.76 (0.54 to 1.07) 0.12 0.95 (0.67 to 1.35) 0.78 Q3 0.82 (0.58 to 1.18) 0.29 0.96 (0.67 to 1.31) 0.80 0.84 (0.59 to 1.18) 0.31 1.21 (0.87 to 1.70) 0.26 Q4 1.03 (0.74 to 1.43) 0.88 0.92 (0.65 to 1.31) 0.65 0.64 (0.45 to 0.93) 0.02 0.85 (0.59 to 1.23) 0.39 Overall 0.62 0.54 0.11 0.21 Sudden cardiac death Q2 0.92 (0.59 to 1.43) 0.71 0.86 (0.55 to 1.34) 0.50 0.60 (0.39 to 0.94) 0.02 0.95 (0.60 to 1.50) 0.82 Q3 0.78 (0.49 to 1.25) 0.30 0.99 (0.63 to 1.55) 0.98 0.69 (0.44 to 1.07) 0.10 1.23 (0.79 to 1.90) 0.36 Q4 1.13 (0.74 to 1.73) 0.56 0.95 (0.61 to 1.47) 0.82 0.61 (0.39 to 0.97) 0.04 0.84 (0.52 to 1.35) 0.46 Overall 0.43 0.91 0.09 0.36 Nonfatal MI Q2 1.16 (0.74 to 1.82) 0.51 1.13 (0.73 to 1.75) 0.58 0.82 (0.51 to 1.32) 0.42 0.92 (0.57 to 1.47) 0.72 Q3 1.17 (0.75 to 1.83) 0.49 1.01 (0.64 to 1.60) 0.96 1.12 (0.71 to 1.76) 0.64 1.01 (0.64 to 1.60) 0.97 Q4 1.11 (0.71 to 1.74) 0.65 0.98 (0.63 to 1.52) 0.91 0.84 (0.53 to 1.35) 0.48 1.25 (0.81 to 1.93) 0.32 Overall 0.90 0.91 0.42 0.54 All cardiac events Q2 1.21 (0.96 to 1.52) 0.10 1.36 (1.09 to 1.71) 0.01 0.83 (0.65 to 1.05) 0.13 0.86 (0.68 to 1.10) 0.24 Q3 1.02 (0.80 to 1.30) 0.89 1.07 (0.84 to 1.37) 0.59 0.94 (0.74 to 1.19) 0.61 1.06 (0.84 to 1.34) 0.61 Q4 1.10 (0.87 to 1.39) 0.42 0.98 (0.77 to 1.25) 0.88 0.73 (0.57 to 0.94) 0.02 0.94 (0.74 to 1.19) 0.60 Overall 0.33 0.01 0.01 0.34 All cerebrovascular events Q2 0.98 (0.63 to 1.52) 0.91 0.96 (0.62 to 1.47) 0.84 0.99 (0.64 to 1.53) 0.95 0.86 (0.57 to 1.29) 0.46 Q3 1.05 (0.68 to 1.62) 0.82 0.79 (0.50 to 1.24) 0.31 0.83 (0.52 to 1.32) 0.43 0.65 (0.42 to 1.07) 0.05 Q4 1.06 (0.69 to 1.62) 0.80 1.01 (0.67 to 1.53) 0.96 0.91 (0.58 to 1.42) 0.68 0.76 (0.49 to 1.17) 0.22 Overall 0.98 0.68 0.84 0.27 Death from all causes Q2 0.93 (0.74 to 1.16) 0.50 0.97 (0.77 to 1.21) 0.78 0.77 (0.62 to 0.96) 0.02 0.84 (0.67 to 1.05) 0.13 Q3 0.79 (0.63 to 0.99) 0.04 0.95 (0.76 to 1.20) 0.68 0.75 (0.59 to 0.94) 0.01 0.93 (0.74 to 1.16) 0.50 Q4 0.94 (0.76 to 1.17) 0.60 0.89(0.71 to 1.20) 0.32 0.63 (0.49 to 0.80) ,0.001 0.78 (0.61 to 0.98) 0.04 Overall 0.23 0.80 0.002 0.15 HR, hazard ratio; 95% CI, 95% confidence interval; Q, quartile; MI, myocardial infarction. aHR calculated with multivariate Andersen–Gill model; group of patients within the first quarter of the distribution used as the reference group; atorvastatin and placebo group pooled; multiple events considered. bP value of the Wald test statistic. cCombined vascular end point consisting of MI, cardiac death, and stroke. dP value of the multivariate Wald test statistic investigating whether there was any difference between any two quartiles.

This study has several strengths. First, the study participants and propensity score–matched subgroup analyses for apoA2. underwent very detailed clinical and laboratory examinations. Fifth, the sample size of the cohort including .1000 patients Second, there was a precise follow-up for all-cause mortality on maintenance hemodialysis treatment was very large. and various vascular end points. Third, laboratory testing was One limitation of these analyses is that they were secondary, repeated after 4 weeks and then every 6 months. In contrast exploratory, and thus not predefined. Consequently, the with previous studies, this enabled us to control for longitu- results, especially those for apoA2, require confirmation in dinal measurements of HDL cholesterol during the follow-up. future studies. We also do not present data on HDL function- Fourth, we performed very detailed statistical analyses of the ality. However, previous mechanistic studies addressed this associations of HDL cholesterol and apolipoproteins with issue in detail.10–12 HDL cholesterol levels were not considered outcomes including stratified analyses, cubic spline modeling, for the decision to include or exclude a patient. Nevertheless,

J Am Soc Nephrol 26: 484–492, 2015 HDL Cholesterol and Outcomes in 4D 489 CLINICAL RESEARCH www.jasn.org

Figure 1. No association of HDL cholesterol with end points. Figure 2. Trend toward inverse relationship of apoA2 with death Spline fit of the association between HDL cholesterol and the from any cause. Spline fit of the association between apoA2 and primary end point (A) and all-cause death (B). HR, hazard ratio. CB, the primary end point (A) and all-cause death (B). HR, hazard ratio; confidence band. CB, confidence band.

implicit confounding conferred by the use of exclusion criteria blood sample was taken and information about any suspected (e.g., exclusion of several outliers for LDL cholesterol) cannot end point or serious adverse event was obtained. The study was ap- be totally ruled out.13 proved by the local ethics committee and performed in accordance In summary, HDL cholesterol does not predict all-cause with the Declaration of Helsinki. Informed consent was obtained mortality and vascular complications in patients on mainte- from all participants. nance hemodialysis treatment. A possible beneficial effect of apoA2 on survival will require confirmation in future studies. Outcome Measures The 4D study end points were centrally adjudicated by three mem- bers of the end point committee blinded to study treatment and CONCISE METHODS according to predefined criteria. The primary end point of the 4D study was defined as a composite of cardiac death, nonfatal Study Design, Setting, and Participants myocardial infarction, and stroke, whichever occurred first (com- The design of the 4D study was previously reported in detail.13,14 bined cardiovascular events). Sudden cardiac death was considered Briefly, the 4D study is a prospective, randomized, multicenter trial as previously described.13,14 Myocardial infarction was diagnosed including 1255 patients with type 2 diabetes mellitus who were aged when at least two of three criteria were met, including typical symp- 18–80 years and were on hemodialysis for ,2 years. Recruitment toms, elevated levels of cardiac enzymes, or diagnostic changes in started in March 1998 and ended in October 2002. Patients were the electrocardiogram. Stroke was defined as a neurologic deficit randomly assigned to receive double-blinded treatment with either lasting longer than 24 hours. Computed tomographic or magnetic 20 mg of atorvastatin (n= 619) or placebo (n=636) once daily. The resonance imaging results were available in all but 16 patients. participants were reexamined at 4 weeks and then every 6 months We evaluated the following end points in this analysis: combined after randomization until March 2004. At each follow-up visit, a primary end point, cardiac death, sudden cardiac death, nonfatal

490 Journal of the American Society of Nephrology J Am Soc Nephrol 26: 484–492, 2015 www.jasn.org CLINICAL RESEARCH myocardialinfarction,allcardiacevents combined, all cerebrovascular algorithm without Caliper implemented in the STATA modul events, and all-cause death.14 psmatch2. Nonmatched individuals were excluded from the reanal- ysis. Finally, we calculated a Cox model for the association of apoA2 Laboratory Procedures as a standardized continuous variable with all-cause mortality (haz- The standard laboratory procedures have been performed in identical ard ratio refers to the change in hazard per 1-SD increase of apoA2). fashion as previously described.13,14 HDLcholesterolwasmeasuredwith All statistical tests were two sided. P values were corrected for multiple the homogenous assay from Roche Diagnostics (Mannheim, Germany). testing (4 parameters37 end points=28) according to the Bonferroni were separated by a combined ultracentrifugation- correction. Thus, P values ,0.002 (0.05/28) were considered signifi- precipitation method (b quantification). Cholesterol was measured cant. All statistical analyses were conducted using the Stata Statistical with enzymatic reagents from WAKO (Neuss, Germany) on a Software package (release 12; StataCorp., College Station, TX). WAKO R30 or Olympus AU640 analyzer. We measured apoA1, apoA2, and apoC3 by turbidimetry with reagents from Greiner (Flacht, Germany). ACKNOWLEDGMENTS

Statistical Analyses The 4D study was supported by Pfizer Inc. This work was also sup- Quartiles of HDL (subgroup analyses within the fourth HDL quartile ported by the German Federal Ministry of Education and Research for patients with HDL cholesterol #50 mg/dl and .50 mg/dl) cho- (BMBF01 EO1004). lesterol and apoA1, apoA2, and apoC3 were formed. The baseline Parts of this article were orally presented in the late-breaking clini- characteristics were presented for the quartiles of the aforementioned cal trials session at the 50th European Renal Association–European parameters. Means and SDs or medians with interquartile ranges Dialysis and Transplant Association Congress, held May 18–21, 2013, were presented for continuous variables and frequencies with per- in Istanbul, Turkey. centages for categorical variables. P values for differences in the base- line characteristics across the quartiles were calculated with analysis of covariance for continuous variables and logistic regression for DISCLOSURES categorical variables. All analyses were adjusted for age and sex. An fi extended Cox regression approach (Andersen–Gill model, which al- W.M. received consulting fees, lecture fees, and research grants from P zer. fi lows adjustment for multiple events) was used to evaluate the prog- B.G. received grant support from P zer for post hoc analyses of the original 4D study. Synlab offers testing for HDL cholesterol and apolipoproteins. nostic effect of HDL cholesterol (four quartiles and subgroup analysis within the fourth quartile) and apolipoproteins (four quartiles) on 28 fi cardiovascular events and mortality. Hazard ratios (95% con dence REFERENCES intervals) and P values adjusted for all potential confounding varia- bles were calculated across quartiles of HDL cholesterol and apoli- 1. 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492 Journal of the American Society of Nephrology J Am Soc Nephrol 26: 484–492, 2015 Supplementary table 1

Title: Baseline patient characteristics in subgroups of the 4th HDL cholesterol quartile

Q4a Q4b N 72 246 Range (mg/dl) 47.0-50.0 50.1-115.5 Age [years] 67.1 (6.8) 66.6 (8.2) Gender [males (%)] 38 (53) 93 (38) Ever smoking [n (%)] 26 (36) 92 (37) Body mass index [ kg/m2] 26.3 (3.9) 26.2 (4.4) Systolic blood pressure [mm Hg] 148 (23) 147 (22) Diastolic blood pressure [mm Hg] 77 (11) 77 (11) Time receiving dialysis [months] 7.5 (6.0) 7.9 (6.8) History of* Arrhythmia [n (%)] 17 (24) 42 (17) Myocardial infarction, CABG†, PCI‡, CHD§ [n(%)] 27 (38) 66 (27) Congestive heart failure|| [n (%)] 27 (38) 76 (31) Stroke or TIA# [n (%)] 9 (13) 40 (16) Peripheral vascular disease [n (%)] 34 (47) 106 (43) Hemoglobin [g/dl] 11.1 (1.3) 11.0 (1.4) Glycated hemoglobin [%] 6.7 (1.2) 6.7 (1.2) Phosphate [mg/l] 6.02 (1.48) 6.12 (1.68) Albumin [g/dl] 3.84 (0.27) 3.80 (0.30) LDL cholesterol [mg/dl] 131 (33) 129 (30) HDL cholesterol [mg/dl] 48.3 (0.9) 59.4 (9.5) [mg/dl] 138.6 (17.2) 152.8 (23.8) Apolipoprotein A2[mg/dl] 31.2 (5.4) 32.6 (6.3) Apolipoprotein C3[mg/dl] 20.2 (8.8) 18.7 (7.9) Triglycerides [mg/dl] 210 (116) 173 (87) C-reactive protein [mg/l, median (25th -75thperc.)] 4.4 (2.1-10.4) 4.6 (1.9-11.1) NT-pro-BNP [ng/l, median(25th -75thperc.)] 2776 (1351-7587) 4355 (1785-12337)

Legend: Data shown are mean (standard deviation), if not otherwise mentioned; Q4a HDL cholesterol levels ≤50 mg/dl; Q4b HDL cholesterol levels >50 mg/dl; *Types of disease and intervention are not mutually exclusive, † coronary artery bypass grafting surgery; ‡percutaneous coronary intervention; §coronary heart disease, documented by coronary angiography; ||predominantly New York Heart Association II; #transitory ischemic attack.

Supplementary table 2

Title: Baseline patient characteristics according to quartiles of apolipoprotein A2 in the propensity score matched subgroup

Q1 Q2 Q3 Q4 P-value1

N 188 269 286 312 Range (mg/dl) 12.5-22.5 23.8-26.3 27.5-30.0 31.3-53.8 Age [years] 66.6 (7.8) 66.9 (8.6) 66.7 (8.1) 66.1 (8.8) 0.705 Gender [males (%)] 57.5 53.5 49.7 51.6 0.394 Ever smoking [n (%)] 43.6 40,9 38.1 36.9 0.632 Body mass index [ kg/m2] 27.5 (4.7) 27.8 (4.9) 27.5 (4.9) 27.4 (4.6) 0.734 Systolic blood pressure [mm Hg] 145.3 (23.6) 146.1 (22.7) 144.2 (21.1) 147.0 (22.5) 0.472 Diastolic blood pressure [mm Hg] 75.1 (10.8) 75.3 (11.2) 76.7 (11.3) 76.0 (11.1) 0.358 Time receiving dialysis [months] 18.6 (8.1) 17.5 (8.5) 17.8 (8.7) 18.0 (8.3) 0.551 History of* Arrhythmia [n (%)] 27.7 21.6 20.0 19.1 0.465 Myocardial infarction, CABG†, 36.2 29.7 26.6 27.2 0.113 PCI‡, CHD§ [n(%)] Congestive heart failure|| [n (%)] 40.0 39.4 31.1 29.8 0.147 Stroke or TIA# [n (%)] 17.6 19.0 15.7 18.0 0.788 Peripheral vascular disease [n (%)] 47.1 46.8 42.7 41.8 0.440 Hemoglobin [g/dl] 10.6 (1.4) 10.9 (1.4) 11.0 (1.3) 11.2 (1.3) 0.186 Glycated hemoglobin [%] 6.7 (1.2) 6.7 (1.4) 6.7 (1.2) 6.6 (1.2) 0.539 Phosphate [mg/dl] 6.0 (1.8) 6.0 (1.6) 6.0 (1.5) 5.9 (1.6) 0.977 Albumin [g/dl] 3.7 (0.3) 3.8 (0.3) 3.8 (0.3) 3.8 (0.3) 0.589 LDL cholesterol [mg/dl] 116.5 (30.0) 125.1 (27.2) 124.0 (29.7) 128.3 (32.8) 0.001 HDL cholesterol [mg/dl] 28.1 (8.7) 32.1 (9.9) 36.4 (11.0) 44.1 (15.6) 0.001 Apolipoprotein A1[mg/dl] 105.2 (13.1) 115.1 (14.7) 127.1 (16.8) 147.4 (24.1) 0.001 Apolipoprotein A2[mg/dl] 20.7 (2.1) 25.2 (1.0) 28.7 (1.0) 35.4 (4.2) 0.001 Apolipoprotein C3[mg/dl] 17.6 (10.1) 20.0 (9.4) 20.6 (9.1) 22.2 (9.2) <0.001 Triglycerides [mg/dl] 288.1 (203.6) 276.0 (168.8) 258.9 (154.5) 239.7 (147.0) 0.006 C-reactive protein [mg/l, median 6.8 (10.1) 5.9 (11.1) 3.8 (8.0) 3.5 (5.2) 0.245 (25th -75thperc.)] NT-pro-BNP [ng/l, median (25th - 2712 (5254) 2415 (5253) 2509 (5531) 2742 (5163) 0.458 75thperc.)]

Legend: Data shown are mean (standard deviation), if not otherwise mentioned; Q1-Q4 quartile 1-4; 1 P-values for comparison of groups were derived from an analysis of covariance model (for continuous variables) or logistic regression model (for categorical variables), both adjusted for age and gender. *Types of disease and intervention are not mutually exclusive, † coronary artery bypass grafting surgery; ‡percutaneous coronary intervention; §coronary heart disease, documented by coronary angiography; ||predominantly New York Heart Association II; #transitory ischemic attack Supplementary table 3

Title: Endpoints according to quartiles of HDL cholesterol

Combined vascular endpoint n first events following events events total person-years Q1 312 121 16 137 876 Q2 308 117 16 133 851 Q3 317 108 20 128 917 Q4 318 123 27 150 910 Total 1255 469 79 548 3554

Cardiac death n first events following events events total person-years Q1 312 75 0 75 876 Q2 308 67 0 67 851 Q3 317 55 0 55 917 Q4 318 73 0 73 910 Total 1255 270 0 270 3554

Sudden cardiac death n first events following events events total person-years Q1 312 43 0 43 876 Q2 308 38 0 38 851 Q3 317 32 0 32 917 Q4 318 47 0 47 910 Total 1255 160 0 160 3554

Non-fatal MI n first events following events events total person-years Q1 312 37 1 38 876 Q2 308 38 4 42 851 Q3 317 37 6 43 917 Q4 318 37 6 43 910 Total 1255 149 17 166 3554

All cardiac events n first events following events events total person-years Q1 312 118 27 145 876 Q2 308 121 38 159 851 Q3 317 99 31 130 917 Q4 318 113 38 151 910 Total 1255 451 134 585 3554

All cerebrovascular events n first events following events events total person-years Q1 312 35 5 40 876 Q2 308 35 5 40 851 Q3 317 40 8 48 917 Q4 318 39 9 48 910 Total 1255 149 27 176 3554

Death from all causes n first events following events events total person-years Q1 312 167 0 167 876 Q2 308 149 0 149 851 Q3 317 137 0 137 917 Q4 318 164 0 164 910 Total 1255 617 0 617 3554

Legend: Q1-Q4 quartile 1-4

Supplementary table 4

Title: Endpoints according to quartiles of apolipoprotein A1

Combined vascular endpoint n first events following events events total person-years Q1 312 123 15 138 833 Q2 311 126 19 145 861 Q3 305 101 22 123 881 Q4 327 119 23 142 979 Total 1255 469 79 548 3554

Cardiac death n first events following events events total person-years Q1 312 71 0 71 833 Q2 311 74 0 74 861 Q3 305 60 0 60 881 Q4 327 65 0 65 979 Total 1255 270 0 270 3554

Sudden cardiac death n first events following events events total person-years Q1 312 46 0 46 833 Q2 311 35 0 35 861 Q3 305 38 0 38 881 Q4 327 41 0 41 979 Total 1255 160 0 160 3554

Non-fatal MI n first events following events events total person-years Q1 312 36 4 40 833 Q2 311 41 2 43 861 Q3 305 33 6 39 881 Q4 327 39 5 44 979 Total 1255 149 17 166 3554

All cardiac events n first events following events events total person-years Q1 312 113 26 139 833 Q2 311 126 47 173 861 Q3 305 104 29 133 881 Q4 327 108 32 140 979 Total 1255 451 134 585 3554

All cerebrovascular events n first events following events events total person-years Q1 312 39 5 44 833 Q2 311 35 7 42 861 Q3 305 32 5 37 881 Q4 327 43 10 53 979 Total 1255 149 27 176 3554

Death from all causes n first events following events events total person-years Q1 312 166 0 166 833 Q2 311 149 0 149 861 Q3 305 149 0 149 881 Q4 327 153 0 153 979 Total 1255 617 0 617 3554

Legend: Q1-Q4 quartile 1-4 Supplementary table 5

Title: Endpoints according to quartiles of apolipoprotein A2

Combined vascular endpoint n first events following events events total person-years Q1 232 111 12 123 602 Q2 314 109 22 131 882 Q3 338 130 24 154 948 Q4 367 119 21 140 1107 Total 1251 469 79 548 3539

Cardiac death n first events following events events total person-years Q1 232 67 0 67 602 Q2 314 69 0 69 882 Q3 338 72 0 72 948 Q4 367 62 0 62 1107 Total 1251 270 0 270 3539

Sudden cardiac death n first events following events events total person-years Q1 232 46 0 46 602 Q2 314 37 0 37 882 Q3 338 39 0 39 948 Q4 367 38 0 38 1107 Total 1251 160 0 160 3539

Non-fatal MI n first events following events events total person-years Q1 232 29 3 32 602 Q2 314 35 2 37 882 Q3 338 43 8 51 948 Q4 367 42 4 46 1107 Total 1251 149 17 166 3539

All cardiac events n first events following events events total person-years Q1 232 101 25 126 602 Q2 314 113 33 146 882 Q3 338 121 42 163 948 Q4 367 115 34 149 1107 Total 1251 450 134 584 3539

All cerebrovascular events n first events following events events total person-years Q1 232 32 4 36 602 Q2 314 39 9 48 882 Q3 338 35 6 41 948 Q4 367 43 8 51 1107 Total 1251 149 27 176 3539

Death from all causes n first events following events events total person-years Q1 232 156 0 156 602 Q2 314 163 0 163 882 Q3 338 153 0 153 948 Q4 367 144 0 144 1107 Total 1251 616 0 616 3539

Legend: Q1-Q4 quartile 1-4 Supplementary table 6

Title: Endpoints according to quartiles of apolipoprotein C3

Combined vascular endpoint n first events following events events total person-years Q1 321 123 16 139 884 Q2 306 112 17 129 847 Q3 307 119 24 143 882 Q4 321 115 22 137 941 Total 1255 469 79 548 3554

Cardiac death n first events following events events total person-years Q1 321 67 0 67 884 Q2 306 63 0 63 847 Q3 307 82 0 82 882 Q4 321 58 0 58 941 Total 1255 270 0 270 3554

Sudden cardiac death n first events following events events total person-years Q1 321 40 0 40 884 Q2 306 36 0 36 847 Q3 307 49 0 49 882 Q4 321 35 0 35 941 Total 1255 160 0 160 3554

Non-fatal MI n first events following events events total person-years Q1 321 35 3 38 884 Q2 306 32 3 35 847 Q3 307 36 5 41 882 Q4 321 46 6 52 941 Total 1255 149 17 166 3554

All cardiac events n first events following events events total person-years Q1 321 111 36 147 884 Q2 306 98 28 126 847 Q3 307 126 37 163 882 Q4 321 116 33 149 941 Total 1255 451 134 585 3554

All cerebrovascular events n first events following events events total person-years Q1 321 41 10 51 884 Q2 306 42 4 46 847 Q3 307 30 7 37 882 Q4 321 36 6 42 941 Total 1255 149 27 176 3554

Death from all causes n first events following events events total person-years Q1 321 171 0 171 884 Q2 306 146 0 146 847 Q3 307 164 0 164 882 Q4 321 136 0 136 941 Total 1255 617 0 617 3554

Legend: Q1-Q4 quartile 1-4 Supplementary table 7

Title: Prognostic value of HDL cholesterol on risk of endpoint occurrence in subgroups of the 4th HDL cholesterol quartile

Endpoint Quartile HR1 (95% CI) P-value2

Combined vascular endpoint Q4a 1.18 (0.81-1.70) 0.389 Q4b 1.02 (0.79-1.33) 0.876

Cardiac death Q4a 0.72 (0.38-1.36) 0.306 Q4b 1.21 (0.84-1.72) 0.305

Sudden cardiac death Q4a 0.61 (0.26-1.44) 0.259 Q4b 1.20 (0.76-1.88) 0.439

Nonfatal myocardial Q4a 1.65 (0.89-3.07) 0.114 infarction Q4b 1.00 (0.60-1.65) 0.990

All cardiac events Q4a 1.22 (0.85-1.75) 0.272 Q4b 1.10 (0.85-1.43) 0.452

All cerebrovascular events Q4a 0.98 (0.63-1.52) 0.914 Q4b 1.05 (0.68-1.62) 0.819

Death from all causes Q4a 1.10 (0.76-1.59) 0.619 Q4b 0.98 (0.76-1.26) 0.871

Legend: 1 hazard ratio vs. quartile 1; 2P-value calculated with Wald test; Q4a HDL cholesterol levels ≤50 mg/dl; Q4b HDL cholesterol levels >50 mg/dl. Supplementary table 8

Title: Associations of HDL cholesterol with endpoints adjusted for longitudinal measurements

Endpoint Quartile HR1 (95% CI) P-value2

Combined vascular endpoint Q2 1.00 (0.79-1.28) 0.982 Q3 1.02 (0.86-1.36) 0.874 Q4 1.10 (0.81-1.31) 0.312 Q4a 1.18 (0.81-1.70) 0.389 Q4b 1.02 (0.79-1.33) 0.876 Overall 0.865

Cardiac death Q2 0.98 (0.70-1.38) 0.910 Q3 0.87 (0.60-1.19) 0.447 Q4 1.06 (0.75-1.49) 0.748 Q4a 0.78 (0.44-1.42) 0.694 Q4b 1.12 (0.75-1.63 0.227 Overall 0.601

Sudden cardiac death Q2 1.15 (0.74-1.79) 0.540 Q3 0.88 (0.41-1.90) 0.105 Q4 1.33 (1.06-1.66) 0.219 Q4a 0.66 (0.31-1.49) 0.413 Q4b 1.27 (0.83-1.95) 0.356 Overall 0.389

Nonfatal myocardial infarction Q2 0.99 (0.79-1.24) 0.507 Q3 1.05 (0.83-1.32) 0.846 Q4 1.18 (0.77-1.81) 0.444 Q4a 1.56 (0.51-1.56) 0.179 Q4b 1.06 (0.66-1.71) 0.843 Overall 0.632

All cardiac events Q2 0.92 (0.73-1.16) 0.497 Q3 1.15 (0.92-1.45) 0.220 Q4 1.04 (0.82-1.32) 0.738 Q4a 1.15 (0.78-1.68) 0.251 Q4b 1.19 (0.94-1.52 0.573 Overall 0.307

All cerebrovascular events Q2 0.96 (0.64-1.53) 0.945 Q3 1.11 (1.05-1.15) 0.789 Q4 1.19 (0.85-1.66) 0.698 Q4a 1.63 (0.58-1.63) 0.111 Q4b 1.10 (0.70-1.75) 0.873 Overall 0.896

Death from all causes Q2 1.05 (0.85-1.32) 0.636 Q3 1.14 (0.91-1.43) 0.251 Q4 1.02 (0.81-1.28) 0.885 Q4a 1.20 (0.86-1.69) 0.723 Q4b 0.91 (0.69-1.19) 0.652 Overall 0.668

Legend: 1HR hazard ratio vs. quartile 1; 2P-value of global test; Q4a HDL cholesterol levels ≤50 mg/dl; Q4b HDL cholesterol levels >50 mg/dl. Supplementary table 9

Title: Endpoints according to quartiles of ApoA2 in the propensity score matched subgroup

Endpoint n events Quartile1 HR2 (95% CI) P-value3 Combined vascular endpoint 188 100 Q1 1.00 269 109 Q2 0.71 (0.52-0.97) 0.034 286 129 Q3 0.90 (0.67-1.20) 0.467 312 114 Q4 0.71 (0.50-0.99) 0.046 Overall - 0.086

Cardiac death 188 55 Q1 1.00 269 57 Q2 0.71 (0.46-1.09) 0.120 286 59 Q3 0.81 (0.55-1.20) 0.293 320 52 Q4 0.64 (0.40-1.01) 0.053 Overall - 0.234

Sudden cardiac death 188 38 Q1 1.00 269 32 Q2 0.54 (0.31-0.92) 0.022 286 33 Q3 0.61 (0.40-0.94) 0.024 312 34 Q4 0.58 0.34-0.99) 0.046 Overall - 0.065

Nonfatal myocardial infarction 188 31 Q1 1.00 269 31 Q2 0.62 (0.37-1.03) 0.066 286 45 Q3 0.92 (0.58-1.46) 0.722 312 34 Q4 0.60 (0.35-1.06) 0.080 Overall - 0.104

All cardiac events 188 109 Q1 1.00 269 119 Q2 0.71 (0.54-0.94) 0.020 286 140 Q3 0.92 (0.71-1.18) 0.493 312 121 Q4 0.70 (0.50-0.96) 0.029 Overall - 0.053

All cerebrovascular events 188 23 Q1 1.00 269 43 Q2 1.08 (0.64-1.82) 0.782 286 34 Q3 0.77 (0.41-1.43) 0.406 312 42 Q4 0.87 (0.52-1.46) 0.604 Overall - 0.632

Death from all causes 188 123 Q1 1.00 269 130 Q2 0.79 (0.61-1.03) 0.086 286 119 Q3 0.78 (0.59-1.03) 0.076 312 115 Q4 0.71 (0.52-0.96) 0.027 Overall - 0.139

Legend: 1 Q1-Q4 quartile 1-4, 2 HR hazard ratio vs. quartile 1; 3 P-value of global test;