General characterization of the cohort and grouping
A total of 222 case-control matched participants (134 men and 88 women) were selected from the parental ESSE-RF cohort as described in the Materials and Methods section. The case subjects (N=111) included 48 cardiovascular deaths (due to fatal AMI accounting for 77% of deaths and due to other cardiovascular diseases accounting for 23% of deaths, 62.5% men) and 63 cases of nonfatal AMI (58.7% men) detected during follow up of 6.5 years. Cardiovascular deaths and nonfatal AMI were significantly associated with pre-existing cardiovascular diseases diagnosed during the initial examination, including pre-existing AMI, ischemic heart disease, arrhythmia, and other cardiovascular diseases (data not shown). These associations may directly or indirectly account for mortality and nonfatal AMI. A total of 111 essentially healthy control subjects were manually pairwise selected from the parental ESSE-RF cohort to match the baseline demographics and other parameters of the case subjects (Fig. 1). The events detected in the control groups were not associated with baseline diseases indicated for the case subjects thus indirectly confirming apparently healthy baseline conditions of the control groups. Baseline clinical and demographic characteristics of the groups are listed in Table 1. The age of the participants ranged from 36 to 65 years (a median of 57 years). Case-control matches were confirmed by identical values of demographic characteristics (Table 1).
Table 1. Baseline demographics and characteristics of the total cohort including the case-control groups. The data are shown as median (range).
Groups
|
N
|
Age (years)
|
Height (cm)
|
Weight (kg)
|
BMI (kg/m2)
|
SBP (mm Hg)
|
DBP (mm Hg)
|
Smoking (score)
|
Men
|
1, case, cardiovascular deaths
|
30
|
55 (36-64)
|
173 (159-191)
|
81.9 (54.9-131.6)
|
27.3 (19.5-41.0)
|
144 (107-240)
|
88 (68-128)
|
3 (1-3)
|
2, matched control to group 1
|
30
|
54 (36-63)
|
174 (158-191)
|
79.1 (49.0-112.1)
|
26.5 (19.3-41.1)
|
136 (110-174)
|
85 (68-109)
|
2 (1-3)
|
3, case, nonfatal AMI
|
37
|
57 (39-63)
|
174 (163-187)
|
82.2 (59.3-143)
|
27.8 (21.5-40.9)
|
140 (105-179)
|
83 (61-103)
|
2 (1-3)
|
4, matched control to group 3
|
37
|
57 (39-63)
|
175 (157-184)
|
82 (58-117)
|
27.7 (20.9-37.5)
|
144 (118-179)
|
87 (59-114)
|
2 (1-3)
|
Women
|
1, case, cardiovascular deaths
|
18
|
61 (36-65)
|
159 (153-174)
|
81.4 (59.0-106.7)
|
31.4 (24.0-43.6)
|
138 (103-167)
|
84 (65-94)
|
1 (1-3)
|
2, matched control to group 1
|
18
|
60 (35-64)
|
160 (151-172)
|
76.9 (54.5-113.3)
|
30.1 (19.3-40.2)
|
143 (121-182)
|
89 (65-98)
|
1 (1-3)
|
3, case, nonfatal AMI
|
26
|
57 (33-64)
|
161 (146-171)
|
79.1 (44.9-124.9)
|
29.7 (19.2-50.7)
|
153 (99-200)
|
82 (65-104)
|
1 (1-3)
|
4, matched control to group 3
|
26
|
58 (34-64)
|
159 (148-172)
|
71.3 (54.2-107.1)
|
27.6 (20.6-42.5)
|
142 (117-193)0
|
84 (64-101)
|
1 (1-3)
|
Men and women
|
1, case, cardiovascular deaths
|
48
|
57 (36-65)
|
168 (153-191)
|
81.9 (54.9-131.6)
|
29.1 (19.5-43.6)
|
144 (103-240)
|
85 (65-128)
|
2 (1-3)
|
2, matched control to group 1
|
48
|
56 (35-64)
|
169 (151-191)
|
79.1 (49.0-113.3)
|
27.7 (19.3-41.1)
|
140 (110-182)
|
88 (65-109)
|
2 (1-3)
|
3, case, nonfatal AMI
|
63
|
57 (33-64)
|
168 (146-187)
|
81.0 (44.9-143.0)
|
28.1 (19.2-50.7)
|
143 (99-200)
|
82 (61-104)
|
2 (1-3)
|
4, matched control to group 3
|
63
|
57 (34-64)
|
167 (148-184)
|
80.0 (54.2-117.4)
|
27.7 (20.6-42.5)
|
144 (117-193)
|
85 (59-114)
|
2 (1-3)
|
Total cohort
|
222
|
57 (33-65)
|
168 (146-191)
|
80.6 (44.9-143.0)
|
27.9 (19.2-50.7)
|
142 (99-240)
|
85 (59-128)
|
2 (1-3)
|
BMI, body mass index, SBP, systolic blood pressure, DBP, diastolic blood pressure, AMI, acute myocardial infarction. Smoking status: never smoked (1), quitted (2), actual smoker (3)
The cohort was used to evaluate the associations of 15 biomarkers with risks for cardiovascular death and nonfatal AMI. Seven biomarkers are routinely used in clinical practice and have been validated in multiple studies, including CRP, creatinine, total cholesterol, LDL cholesterol, HDL cholesterol, glucose, and insulin. Additional 8 biomarkers were considered experimental for the purposes of the present study, even though cTnI has been previously shown to be associated with mortality in a number of studies [18]. These experimental biomarkers included leptin, adiponectin, endothelin-1, PCSK9, galectin-3, ANGPTL3, NOx, and cTnI [19]. Inclusion of cTnI enabled validation of potential experimental markers by direct comparison and provided overall quality control of the results of regression analysis.
The whole study cohort was divided into 4 subgroups as shown in Table 2.
Table 2. Number of participants in the case and control groups used for model assessment.
Groups and events
|
Men, N
|
Women, N
|
Men and women, N
|
Main groups
|
1, case, cardiovascular death
|
30
|
18
|
48
|
2, control, matched with group 1
|
30
|
18
|
48
|
3, case, nonfatal AMI
|
37
|
26
|
63
|
4, control, matched with group 3
|
37
|
26
|
63
|
Additional groups
|
5, apparently healthy, groups 2 and 4 combined
|
67
|
44
|
111
|
6, alive at endpoint, groups 2, 3, and 4 combined
|
104
|
70
|
174
|
Total cohort
|
134
|
88
|
222
|
AMI, acute myocardial infarction.
The main groups included group 1 (cardiovascular deaths, case), group 2 (matched control to group 1), group 3 (nonfatal AMI, case), and group 4 (matched control to group 2). Two additional subgroups were defined to facilitate the comparison and provide for internal validation of the results of regression analysis. These additional subgroups included group 5 (apparently healthy participants of group 2 and group 4 combined) and group 6 (all participants alive at the endpoint of the follow up period of groups 2, 3, and 4 combined). The main approach to the optimization involved initial comparison of the case groups with the corresponding control groups (group 1 versus group 2 and group 3 versus group 4) with subsequent internal validation of identified associations by comparing group 1 versus group 6 and group 3 versus group 5. The workflow of the analysis is shown in Fig. 1.
Univariate associations of biomarkers with cardiovascular deaths
All 15 biomarkers were assayed in the samples of all participants, and the results are presented in Table 3, which also lists the corresponding reference values for routine biomarkers for moderate cardiovascular risks [19].
Table 3 Biochemical markers in the cohort.
Parameter
|
Leptin
|
Adiponectin
|
Endothelin-1
|
PCSK9
|
Galectin-3
|
ANGPTL3
|
NOx
|
cTnI
|
Total cholesterol
|
HDL cholesterol
|
LDL cholesterol
|
Glucose
|
Insulin
|
CRP
|
Creatinine
|
Units
|
ng/ml
|
µg/ml
|
pg/ml
|
ng/ml
|
ng/ml
|
ng/ml
|
µM
|
pg/ml
|
mM
|
mM
|
mM
|
mM
|
µIU/ml
|
mg/L
|
µM
|
Men
|
Group 3, N=37
|
Мedian
|
9.39
|
9.95
|
0.99
|
800.00
|
5.98
|
670.92
|
27.54
|
3.00
|
5.23
|
1.24
|
3.45
|
5.38
|
7.50
|
1.24
|
76.50
|
Мin
|
1.19
|
2.43
|
0.08
|
173.46
|
3.55
|
367.67
|
0.05
|
0.90
|
2.74
|
0.79
|
1.39
|
3.20
|
2.70
|
0.26
|
57.00
|
Max
|
49.26
|
52.31
|
2.25
|
800.00
|
12.48
|
1111.89
|
190.83
|
446.40
|
8.18
|
1.82
|
6.45
|
7.60
|
44.50
|
16.49
|
120.20
|
Group 4, N=37
|
Мedian
|
8.73
|
9.58
|
0.96
|
353.22
|
5.58
|
647.40
|
15.03
|
1.80
|
5.60
|
1.23
|
3.39
|
5.46
|
7.90
|
1.60
|
76.70
|
Мin
|
0.37
|
3.24
|
0.08
|
179.05
|
3.40
|
315.65
|
2.59
|
0.10
|
3.50
|
0.80
|
2.06
|
4.04
|
2.20
|
0.19
|
59.40
|
Max
|
61.51
|
114.87
|
2.10
|
800.00
|
16.33
|
1203.62
|
145.04
|
27.80
|
7.47
|
1.85
|
5.52
|
7.45
|
22.50
|
8.32
|
109.70
|
Group 1, N=30
|
Мedian
|
6.64
|
7.70
|
0.81
|
389.24
|
6.84
|
428.82
|
24.58
|
7.30
|
5.31
|
1.13
|
3.53
|
5.53
|
9.25
|
2.36
|
79.65
|
Мin
|
0.77
|
3.75
|
0.08
|
223.96
|
4.18
|
273.11
|
4.89
|
0.30
|
3.40
|
0.67
|
1.48
|
4.06
|
1.30
|
0.24
|
60.70
|
Max
|
30.27
|
62.79
|
1.39
|
800.00
|
9.63
|
709.65
|
148.98
|
147.00
|
10.00
|
2.44
|
7.58
|
28.00
|
23.70
|
84.56
|
286.20
|
Group 2, N=30
|
Мedian
|
7.86
|
7.82
|
0.75
|
800.00
|
4.97
|
417.72
|
18.55
|
1.40
|
5.16
|
1.31
|
3.25
|
5.56
|
8.25
|
1.13
|
76.35
|
Мin
|
0.07
|
3.97
|
0.08
|
184.20
|
2.46
|
256.95
|
1.96
|
0.30
|
3.14
|
0.80
|
1.56
|
4.14
|
3.30
|
0.23
|
60.00
|
Max
|
52.99
|
22.95
|
2.01
|
800.00
|
10.55
|
836.04
|
115.89
|
17.40
|
7.20
|
2.25
|
5.65
|
6.66
|
22.90
|
4.12
|
96.70
|
All male , N=134
|
Мedian
|
8.61
|
8.28
|
0.80
|
395.83
|
5.78
|
536.83
|
18.55
|
2.50
|
5.28
|
1.23
|
3.41
|
5.46
|
7.95
|
1.56
|
77.10
|
Мin
|
0.07
|
2.43
|
0.08
|
173.46
|
2.46
|
256.95
|
0.05
|
0.10
|
2.74
|
0.67
|
1.39
|
3.20
|
1.30
|
0.19
|
57.00
|
Max
|
61.51
|
114.87
|
2.25
|
800.00
|
16.33
|
1203.62
|
190.83
|
446.40
|
10.00
|
2.44
|
7.58
|
28.00
|
44.50
|
84.56
|
286.20
|
Women
|
Group 3, N=26
|
Мedian
|
30.98
|
8.07
|
1.07
|
800.00
|
7.85
|
794.96
|
28.46
|
2.60
|
6.66
|
1.50
|
4.01
|
5.52
|
12.55
|
2.16
|
69.05
|
Мin
|
2.05
|
2.88
|
0.08
|
271.92
|
3.26
|
521.99
|
3.36
|
0.40
|
4.87
|
1.07
|
2.28
|
3.86
|
3.40
|
0.27
|
52.10
|
Max
|
70.80
|
113.29
|
1.81
|
800.00
|
10.77
|
1226.66
|
99.23
|
18.40
|
9.62
|
2.44
|
6.97
|
10.32
|
54.50
|
12.82
|
254.90
|
Group 4, N=26
|
Мedian
|
22.79
|
10.89
|
0.69
|
800.00
|
7.24
|
760.59
|
10.62
|
1.85
|
5.65
|
1.49
|
3.61
|
5.08
|
6.30
|
1.73
|
62.70
|
Мin
|
5.04
|
3.44
|
0.08
|
277.02
|
0.03
|
310.68
|
0.05
|
0.10
|
4.16
|
1.03
|
0.56
|
4.37
|
3.30
|
0.16
|
51.70
|
Max
|
73.70
|
44.16
|
1.75
|
800.00
|
10.33
|
1275.31
|
56.52
|
4.20
|
8.49
|
2.67
|
5.91
|
20.80
|
53.50
|
11.60
|
86.10
|
Group 1, N=18
|
Мedian
|
27.56
|
10.27
|
0.77
|
587.05
|
6.04
|
495.98
|
20.25
|
3.85
|
5.52
|
1.35
|
3.30
|
5.53
|
10.80
|
2.32
|
70.55
|
Мin
|
0.49
|
3.29
|
0.08
|
240.82
|
4.05
|
247.14
|
6.50
|
0.50
|
2.94
|
0.75
|
1.76
|
4.36
|
3.20
|
0.73
|
57.20
|
Max
|
52.28
|
39.36
|
3.03
|
800.00
|
9.63
|
780.14
|
88.29
|
31.10
|
7.61
|
1.94
|
5.64
|
16.16
|
199.70
|
31.89
|
88.40
|
Group 2, N=18
|
Мedian
|
35.37
|
11.94
|
0.62
|
800.00
|
6.02
|
481.30
|
22.26
|
1.45
|
5.98
|
1.49
|
4.15
|
5.53
|
9.00
|
2.53
|
66.10
|
Мin
|
12.02
|
3.17
|
0.08
|
299.98
|
3.04
|
272.87
|
0.18
|
0.10
|
3.99
|
1.15
|
2.24
|
3.11
|
4.80
|
0.22
|
55.30
|
Max
|
58.52
|
26.04
|
1.37
|
800.00
|
11.07
|
975.69
|
60.87
|
10.00
|
7.65
|
2.10
|
5.17
|
6.09
|
14.90
|
20.20
|
78.00
|
All female, N=88
|
Мedian
|
28.70
|
10.09
|
0.71
|
800.00
|
6.91
|
645.62
|
20.54
|
2.00
|
6.11
|
1.46
|
3.85
|
5.48
|
9.40
|
2.17
|
66.30
|
Мin
|
0.49
|
2.88
|
0.08
|
240.82
|
0.03
|
247.14
|
0.05
|
0.10
|
2.94
|
0.75
|
0.56
|
3.11
|
3.20
|
0.16
|
51.70
|
Max
|
73.70
|
113.29
|
3.03
|
800.00
|
11.07
|
1275.31
|
99.23
|
31.10
|
9.62
|
2.67
|
6.97
|
20.80
|
199.70
|
31.89
|
254.90
|
Men and women
|
Group 3, N=63
|
Мedian
|
16.83
|
8.30
|
1.01
|
800.00
|
6.62
|
709.34
|
27.54
|
2.90
|
5.91
|
1.31
|
3.70
|
5.38
|
8.60
|
1.66
|
73.60
|
Мin
|
1.19
|
2.43
|
0.08
|
173.46
|
3.26
|
367.67
|
0.05
|
0.40
|
2.74
|
0.79
|
1.39
|
3.20
|
2.70
|
0.26
|
52.10
|
Max
|
70.80
|
113.29
|
2.25
|
800.00
|
12.48
|
1226.66
|
190.83
|
446.40
|
9.62
|
2.44
|
6.97
|
10.32
|
54.50
|
16.49
|
254.90
|
Group 4, N=63
|
Мedian
|
12.26
|
10.13
|
0.88
|
393.85
|
6.30
|
670.92
|
14.05
|
1.80
|
5.60
|
1.32
|
3.46
|
5.31
|
7.30
|
1.63
|
70.60
|
Мin
|
0.37
|
3.24
|
0.08
|
179.05
|
0.03
|
310.68
|
0.05
|
0.10
|
3.50
|
0.80
|
0.56
|
4.04
|
2.20
|
0.16
|
51.70
|
Max
|
73.70
|
114.87
|
2.10
|
800.00
|
16.33
|
1275.31
|
145.04
|
27.80
|
8.49
|
2.67
|
5.91
|
20.80
|
53.50
|
11.60
|
109.70
|
Group 1, N=48
|
Мedian
|
14.67
|
8.90
|
0.78
|
389.24
|
6.66
|
444.66
|
23.57
|
4.25
|
5.41
|
1.17
|
3.39
|
5.53
|
10.00
|
2.32
|
73.80
|
Мin
|
0.49
|
3.29
|
0.08
|
223.96
|
4.05
|
247.14
|
4.89
|
0.30
|
2.94
|
0.67
|
1.48
|
4.06
|
1.30
|
0.24
|
57.20
|
Max
|
52.28
|
62.79
|
3.03
|
800.00
|
9.63
|
780.14
|
148.98
|
147.00
|
10.00
|
2.44
|
7.58
|
28.00
|
199.70
|
84.56
|
286.20
|
Group 2, N=48
|
Мedian
|
15.25
|
8.74
|
0.69
|
800.00
|
5.52
|
429.86
|
19.87
|
1.40
|
5.43
|
1.37
|
3.56
|
5.56
|
8.40
|
1.23
|
72.85
|
Мin
|
0.07
|
3.17
|
0.08
|
184.20
|
2.46
|
256.95
|
0.18
|
0.10
|
3.14
|
0.80
|
1.56
|
3.11
|
3.30
|
0.22
|
55.30
|
Max
|
58.52
|
26.04
|
2.01
|
800.00
|
11.07
|
975.69
|
115.89
|
17.40
|
7.65
|
2.25
|
5.65
|
6.66
|
22.90
|
20.20
|
96.70
|
Total cohort, N=222
|
Мedian
|
14.60
|
8.83
|
0.79
|
800.00
|
6.25
|
578.63
|
19.73
|
2.30
|
5.56
|
1.31
|
3.51
|
5.47
|
8.40
|
1.68
|
72.95
|
Мin
|
0.07
|
2.43
|
0.08
|
173.46
|
0.03
|
247.14
|
0.05
|
0.10
|
2.74
|
0.67
|
0.56
|
3.11
|
1.30
|
0.16
|
51.70
|
Max
|
73.70
|
114.87
|
3.03
|
800.00
|
16.33
|
1275.31
|
190.83
|
446.40
|
10.00
|
2.67
|
7.58
|
28.00
|
199.70
|
84.56
|
286.20
|
Reference
|
|
|
|
|
|
|
|
26.2
|
<4.9
|
>1.0 men, >1.2 women
|
<3.0
|
<6.1
|
2-22
|
<3.0
|
74.3-107
|
PCSK9, proprotein convertase subtilisin/kexin type, ANGPTL3, angiopoietin-like protein 3, NOx, nitrate and nitrite, cTnI, cardiac troponin I, HDL, high density lipoproteins, LDL, low density lipoproteins, CRP, C-reactive protein.
However, experimental biomarkers do not have certified and validated reference values. All biochemical parameters were divided into terciles. Associations of the upper tercile (higher 33% values above cutoff shown in Table 4) with cardiovascular death and nonfatal AMI endpoints were compared with those for the two lower terciles combined. Initially, comparison was performed using univariate logistic regression for group 1 versus group 2, group 1 versus group 5, group 1 versus group 6, group 3 versus group 4, and group 3 versus group 5 (Fig1, Tables 5 and 6).
Table 4. Cutoff concentrations of biomarkers for upper tercile in the total cohort, in men, and in women.
N
|
Biomarker
|
Units
|
Men
|
Women
|
Men and women
|
1
|
cTnI
|
pg/ml
|
3.90
|
2.90
|
3.40
|
2
|
NOx
|
µM
|
29.42
|
29.67
|
29.42
|
3
|
ANGPTL3
|
ng/ml
|
630.99
|
759.63
|
670.92
|
4
|
PCSK9
|
ng/ml
|
800.00
|
800.00
|
800.00
|
5
|
Galectin-3
|
ng/ml
|
6.74
|
7.49
|
7.22
|
6
|
Leptin
|
ng/ml
|
13.71
|
38.02
|
22.59
|
7
|
Adiponectin
|
µg/ml
|
11.28
|
13.31
|
11.89
|
8
|
Endothelin
|
pg/ml
|
1.01
|
1.10
|
1.03
|
9
|
CRP
|
mg/ml
|
2.08
|
4.5
|
2.44
|
10
|
Creatinine
|
mg/ml
|
81.6
|
70.6
|
77.4
|
11
|
Total cholesterol
|
mM
|
5.85
|
6.7
|
6.22
|
12
|
LDL cholesterol
|
mM
|
3.86
|
4.51
|
4.12
|
13
|
HDL cholesterol
|
mM
|
1.34
|
1.61
|
1.46
|
14
|
Glucose
|
mM
|
5.75
|
5.73
|
5.75
|
15
|
Insulin
|
µIU/ml
|
9.70
|
11.70
|
10.80
|
PCSK9, proprotein convertase subtilisin/kexin type, ANGPTL3, angiopoietin-like protein 3, NOx, nitrate and nitrite, cTnI, cardiac troponin I, HDL, high density lipoproteins, LDL, low density lipoproteins, CRP, C-reactive protein.
The results of this univariate logistic analysis indicated that six biomarkers were associated with cardiovascular deaths, including 5 biomarkers associated with an increase in mortality (cTnI, galectin-3, NOx, CRP, and creatinine) and HDL cholesterol associated with a decrease in mortality. The levels of cTnI selected as a reference were clearly associated with mortality regardless of sex, and the associations remained significant in all three tested comparisons (Table 5). Galectin-3, СRP, and HDL cholesterol had significant associations with mortality in comparison of group 1 versus group 2 in men.
Table 5. Univariate logistic regression for associations of biomarkers with cardiovascular deaths using binary logit values of the highest tercile versus combined lower terciles and Fisher scoring.
Biomarker
|
men
|
women
|
|
men and women
|
|
OR
|
95% CI
|
ROC
|
Pr > ChiSq
|
OR
|
95% CI
|
ROC
|
Pr > ChiSq
|
OR
|
95% CI
|
ROC
|
Pr > ChiSq
|
Experimental
|
Group 1 versus group 2
|
Leptin
|
1.35
|
0.46
|
3.97
|
0.53
|
0.58
|
0.29
|
0.07
|
1.21
|
0.64
|
0.09
|
0.91
|
0.39
|
2.12
|
0.51
|
0.83
|
Adiponectin
|
1.46
|
0.44
|
4.86
|
0.53
|
0.54
|
0.79
|
0.20
|
3.07
|
0.53
|
0.73
|
1.11
|
0.46
|
2.70
|
0.51
|
0.82
|
Endothelin
|
1.98
|
0.51
|
7.64
|
0.55
|
0.32
|
1.00
|
0.17
|
5.77
|
|
1.00
|
1.54
|
0.53
|
4.46
|
0.53
|
0.42
|
PCSK9
|
0.59
|
0.21
|
1.62
|
0.57
|
0.30
|
0.39
|
0.10
|
1.54
|
0.61
|
0.18
|
0.51
|
0.23
|
1.15
|
0.58
|
0.10
|
Galectin-3
|
3.76
|
1.24
|
11.39
|
0.65
|
0.02
|
3.08
|
0.51
|
18.54
|
0.58
|
0.22
|
2.84
|
1.12
|
7.18
|
0.60
|
0.03
|
ANGPTL3
|
1.00
|
0.13
|
7.61
|
|
1.00
|
1.00
|
0.06
|
17.33
|
|
1.00
|
1.53
|
0.25
|
9.61
|
0.51
|
0.65
|
cTnI
|
5.68
|
1.84
|
17.49
|
0.70
|
0.01
|
6.25
|
1.33
|
29.43
|
0.69
|
0.02
|
5.13
|
2.11
|
12.47
|
0.69
|
0.01
|
NOx
|
2.10
|
0.71
|
6.22
|
0.58
|
0.18
|
2.08
|
0.52
|
8.34
|
0.58
|
0.30
|
2.09
|
0.89
|
4.92
|
0.58
|
0.09
|
Routine
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
CRP
|
6.54
|
1.97
|
21.74
|
0.70
|
0.01
|
0.77
|
0.19
|
3.19
|
0.53
|
0.72
|
2.28
|
0.97
|
5.35
|
0.59
|
0.06
|
Creatinine
|
2.10
|
0.71
|
6.22
|
0.58
|
0.18
|
2.60
|
0.65
|
10.38
|
0.61
|
0.18
|
1.71
|
0.74
|
3.95
|
0.56
|
0.21
|
Total cholesterol
|
2.67
|
0.84
|
8.46
|
0.60
|
0.10
|
0.57
|
0.13
|
2.51
|
0.56
|
0.46
|
1.85
|
0.75
|
4.52
|
0.56
|
0.18
|
LDL cholesterol
|
3.06
|
0.97
|
9.66
|
0.62
|
0.06
|
0.60
|
0.15
|
2.45
|
0.56
|
0.48
|
1.00
|
0.42
|
2.37
|
|
1.00
|
HDL cholesterol
|
0.25
|
0.08
|
0.79
|
0.65
|
0.02
|
0.74
|
0.16
|
3.38
|
0.53
|
0.70
|
0.50
|
0.20
|
1.21
|
0.57
|
0.12
|
Glucose
|
1.15
|
0.41
|
3.20
|
0.52
|
0.79
|
2.23
|
0.52
|
9.59
|
0.58
|
0.28
|
1.31
|
0.57
|
3.02
|
0.53
|
0.53
|
Insulin
|
2.04
|
0.71
|
5.90
|
0.58
|
0.19
|
3.18
|
0.67
|
15.15
|
0.61
|
0.15
|
1.74
|
0.74
|
4.05
|
0.56
|
0.20
|
Group 1 versus group 5
|
Leptin
|
1.27
|
0.51
|
3.13
|
0.53
|
0.61
|
0.45
|
0.13
|
1.61
|
0.58
|
0.22
|
1.04
|
0.51
|
2.14
|
0.50
|
0.91
|
Adiponectin
|
0.74
|
0.29
|
1.94
|
0.53
|
0.54
|
0.72
|
0.23
|
2.28
|
0.54
|
0.58
|
0.73
|
0.35
|
1.52
|
0.53
|
0.40
|
Endothelin
|
0.67
|
0.25
|
1.80
|
0.54
|
0.42
|
0.43
|
0.11
|
1.73
|
0.58
|
0.23
|
0.57
|
0.26
|
1.28
|
0.55
|
0.17
|
PCSK9
|
0.89
|
0.37
|
2.11
|
0.51
|
0.79
|
0.52
|
0.17
|
1.58
|
0.58
|
0.25
|
0.72
|
0.37
|
1.42
|
0.54
|
0.34
|
Galectin-3
|
3.11
|
1.27
|
7.64
|
0.63
|
0.01
|
1.03
|
0.30
|
3.50
|
0.50
|
0.97
|
1.62
|
0.80
|
3.29
|
0.55
|
0.18
|
ANGPTL3
|
0.15
|
0.03
|
0.67
|
0.63
|
0.01
|
0.11
|
0.01
|
0.94
|
0.64
|
0.04
|
0.15
|
0.04
|
0.52
|
0.62
|
0.01
|
cTnI
|
5.99
|
2.34
|
15.31
|
0.70
|
0.01
|
5.63
|
1.69
|
18.76
|
0.69
|
0.01
|
5.53
|
2.66
|
11.53
|
0.69
|
<.0001
|
NOx
|
2.44
|
0.98
|
6.09
|
0.60
|
0.06
|
3.11
|
0.95
|
10.16
|
0.62
|
0.06
|
2.68
|
1.30
|
5.52
|
0.61
|
0.01
|
Routine
|
|
CRP
|
3.85
|
1.55
|
9.54
|
0.66
|
0.01
|
0.92
|
0.27
|
3.10
|
0.51
|
0.89
|
2.09
|
1.04
|
4.21
|
0.59
|
0.04
|
Creatinine
|
2.08
|
0.85
|
5.13
|
0.58
|
0.11
|
3.40
|
1.06
|
10.87
|
0.64
|
0.04
|
1.92
|
0.95
|
3.88
|
0.57
|
0.07
|
Total cholesterol
|
1.46
|
0.60
|
3.57
|
0.54
|
0.41
|
0.68
|
0.19
|
2.47
|
0.54
|
0.56
|
1.30
|
0.63
|
2.66
|
0.53
|
0.48
|
LDL cholesterol
|
1.80
|
0.74
|
4.38
|
0.57
|
0.20
|
0.82
|
0.25
|
2.77
|
0.52
|
0.75
|
0.84
|
0.41
|
1.73
|
0.52
|
0.64
|
HDL cholesterol
|
0.35
|
0.13
|
0.96
|
0.61
|
0.04
|
0.50
|
0.14
|
1.78
|
0.57
|
0.28
|
0.47
|
0.22
|
1.02
|
0.58
|
0.06
|
Glucose
|
1.56
|
0.65
|
3.79
|
0.55
|
0.32
|
1.91
|
0.59
|
6.14
|
0.57
|
0.28
|
1.55
|
0.76
|
3.14
|
0.55
|
0.23
|
Insulin
|
1.92
|
0.79
|
4.64
|
0.58
|
0.15
|
2.48
|
0.75
|
8.20
|
0.59
|
0.14
|
1.76
|
0.87
|
3.57
|
0.56
|
0.12
|
Group 1 versus group 6
|
Leptin
|
1.14
|
0.49
|
2.66
|
0.52
|
0.76
|
0.48
|
0.14
|
1.63
|
0.57
|
0.24
|
0.93
|
0.47
|
1.82
|
0.51
|
0.82
|
Adiponectin
|
0.63
|
0.26
|
1.56
|
0.55
|
0.32
|
0.96
|
0.32
|
2.87
|
0.50
|
0.94
|
0.74
|
0.37
|
1.49
|
0.53
|
0.40
|
Endothelin
|
0.51
|
0.20
|
1.29
|
0.57
|
0.15
|
0.32
|
0.08
|
1.20
|
0.61
|
0.09
|
0.43
|
0.20
|
0.92
|
0.59
|
0.03
|
PCSK9
|
0.80
|
0.35
|
1.80
|
0.53
|
0.58
|
0.56
|
0.20
|
1.58
|
0.57
|
0.27
|
0.69
|
0.36
|
1.31
|
0.55
|
0.25
|
Galectin-3
|
2.82
|
1.23
|
6.49
|
0.62
|
0.01
|
0.65
|
0.21
|
2.04
|
0.55
|
0.46
|
1.35
|
0.70
|
2.60
|
0.53
|
0.38
|
ANGPTL3
|
0.10
|
0.02
|
0.43
|
0.68
|
0.01
|
0.08
|
0.01
|
0.66
|
0.68
|
0.02
|
0.09
|
0.03
|
0.30
|
0.68
|
<.0001
|
cTnI
|
4.93
|
2.08
|
11.67
|
0.69
|
0.01
|
3.13
|
1.08
|
9.06
|
0.63
|
0.04
|
4.01
|
2.06
|
7.81
|
0.66
|
<.0001
|
NOx
|
1.65
|
0.72
|
3.78
|
0.56
|
0.24
|
1.75
|
0.61
|
5.03
|
0.57
|
0.30
|
1.68
|
0.88
|
3.24
|
0.56
|
0.12
|
Routine
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
CRP
|
3.38
|
1.46
|
7.83
|
0.64
|
0.01
|
0.69
|
0.22
|
2.17
|
0.54
|
0.53
|
1.88
|
0.98
|
3.61
|
0.57
|
0.06
|
Creatinine
|
1.65
|
0.72
|
3.78
|
0.56
|
0.24
|
2.33
|
0.81
|
6.71
|
0.60
|
0.12
|
1.68
|
0.88
|
3.24
|
0.56
|
0.12
|
Total cholesterol
|
1.37
|
0.59
|
3.17
|
0.54
|
0.46
|
0.48
|
0.14
|
1.63
|
0.57
|
0.24
|
1.07
|
0.55
|
2.09
|
0.51
|
0.85
|
LDL cholesterol
|
1.65
|
0.72
|
3.78
|
0.56
|
0.24
|
0.65
|
0.21
|
2.04
|
0.55
|
0.46
|
0.84
|
0.42
|
1.67
|
0.52
|
0.62
|
HDL cholesterol
|
0.40
|
0.15
|
1.06
|
0.59
|
0.07
|
0.48
|
0.14
|
1.63
|
0.57
|
0.24
|
0.49
|
0.23
|
1.02
|
0.58
|
0.06
|
Glucose
|
1.58
|
0.69
|
3.61
|
0.55
|
0.28
|
1.30
|
0.45
|
3.79
|
0.53
|
0.63
|
1.35
|
0.70
|
2.60
|
0.53
|
0.38
|
Insulin
|
1.88
|
0.82
|
4.31
|
0.57
|
0.13
|
1.30
|
0.45
|
3.79
|
0.53
|
0.63
|
1.51
|
0.78
|
2.90
|
0.55
|
0.22
|
PCSK9, proprotein convertase subtilisin/kexin type, ANGPTL3, angiopoietin-like protein 3, NOx, nitrate and nitrite, cTnI, cardiac troponin I, HDL, high density lipoproteins, LDL, low density lipoproteins, CRP, C-reactive protein.
Table 6. Univariate logistic regression of associations of biomarkers with nonfatal AMI binary logit values of the highest tercile versus combined lower terciles and Fisher scoring.
|
men
|
women
|
men and women
|
|
OR
|
95% CI
|
ROC
|
Pr > ChiSq
|
OR
|
95% CI
|
ROC
|
Pr > ChiSq
|
OR
|
95% CI
|
ROC
|
Pr > ChiSq
|
Group 3 versus group 4
|
Leptin
|
1.27
|
0.49
|
3.30
|
0.53
|
0.63
|
1.19
|
0.37
|
3.80
|
0.52
|
0.77
|
1.52
|
0.73
|
3.19
|
0.55
|
0.26
|
Adiponectin
|
1.00
|
0.40
|
2.51
|
|
1.00
|
0.41
|
0.12
|
1.36
|
0.60
|
0.14
|
0.72
|
0.35
|
1.47
|
0.54
|
0.36
|
Endothelin
|
1.12
|
0.45
|
2.78
|
0.51
|
0.82
|
1.36
|
0.46
|
4.07
|
0.54
|
0.58
|
1.21
|
0.60
|
2.44
|
0.52
|
0.59
|
PCSK9
|
1.93
|
0.77
|
4.88
|
0.58
|
0.16
|
1.00
|
0.33
|
3.06
|
|
1.00
|
1.47
|
0.73
|
2.96
|
0.55
|
0.29
|
Galectin-3
|
1.13
|
0.42
|
3.04
|
0.51
|
0.80
|
1.87
|
0.62
|
5.63
|
0.58
|
0.27
|
1.14
|
0.56
|
2.35
|
0.52
|
0.71
|
ANGPTL3
|
1.25
|
0.50
|
3.13
|
0.53
|
0.64
|
1.00
|
0.34
|
2.98
|
|
1.00
|
1.69
|
0.83
|
3.43
|
0.56
|
0.15
|
cTnI
|
1.74
|
0.61
|
4.93
|
0.55
|
0.30
|
3.60
|
1.04
|
12.48
|
0.63
|
0.04
|
2.37
|
1.07
|
5.24
|
0.59
|
0.03
|
NOx
|
3.08
|
1.12
|
8.50
|
0.62
|
0.03
|
5.50
|
1.48
|
20.46
|
0.67
|
0.01
|
3.86
|
1.74
|
8.60
|
0.64
|
0.01
|
Routine
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
CRP
|
1.00
|
0.38
|
2.65
|
|
1.00
|
2.33
|
0.73
|
7.42
|
0.60
|
0.15
|
1.24
|
0.59
|
2.62
|
0.52
|
0.57
|
Creatinine
|
1.84
|
0.69
|
4.90
|
0.57
|
0.22
|
3.08
|
0.88
|
10.72
|
0.62
|
0.08
|
1.56
|
0.73
|
3.32
|
0.55
|
0.25
|
Total cholesterol
|
0.80
|
0.31
|
2.04
|
0.53
|
0.63
|
2.71
|
0.85
|
8.64
|
0.62
|
0.09
|
1.31
|
0.64
|
2.69
|
0.53
|
0.46
|
LDL cholesterol
|
0.89
|
0.35
|
2.29
|
0.51
|
0.81
|
2.33
|
0.73
|
7.42
|
0.60
|
0.15
|
0.87
|
0.42
|
1.80
|
0.52
|
0.71
|
HDL cholesterol
|
0.89
|
0.34
|
2.32
|
0.51
|
0.81
|
0.85
|
0.28
|
2.58
|
0.52
|
0.78
|
0.87
|
0.43
|
1.80
|
0.52
|
0.71
|
Glucose
|
1.30
|
0.48
|
3.52
|
0.53
|
0.61
|
2.33
|
0.73
|
7.42
|
0.60
|
0.15
|
1.67
|
0.78
|
3.54
|
0.56
|
0.18
|
Insulin
|
1.00
|
0.38
|
2.65
|
|
1.00
|
3.89
|
1.18
|
12.84
|
0.65
|
0.03
|
1.54
|
0.73
|
3.25
|
0.55
|
0.26
|
Group 3 versus group 5
|
Leptin
|
1.33
|
0.58
|
3.09
|
0.53
|
0.50
|
0.84
|
0.31
|
2.31
|
0.52
|
0.74
|
1.37
|
0.72
|
2.61
|
0.54
|
0.34
|
Adiponectin
|
1.56
|
0.68
|
3.56
|
0.55
|
0.29
|
0.43
|
0.15
|
1.29
|
0.59
|
0.13
|
0.95
|
0.50
|
1.82
|
0.51
|
0.88
|
Endothelin
|
2.08
|
0.91
|
4.74
|
0.59
|
0.08
|
2.14
|
0.79
|
5.81
|
0.59
|
0.13
|
2.10
|
1.11
|
3.97
|
0.59
|
0.02
|
PCSK9
|
1.37
|
0.61
|
3.06
|
0.54
|
0.45
|
0.83
|
0.30
|
2.26
|
0.52
|
0.71
|
1.13
|
0.61
|
2.11
|
0.52
|
0.69
|
Galectin-3
|
1.31
|
0.55
|
3.13
|
0.53
|
0.55
|
3.11
|
1.13
|
8.60
|
0.63
|
0.03
|
1.62
|
0.85
|
3.11
|
0.55
|
0.14
|
ANGPTL3
|
3.00
|
1.31
|
6.89
|
0.63
|
0.01
|
2.26
|
0.84
|
6.08
|
0.60
|
0.11
|
3.94
|
2.05
|
7.56
|
0.66
|
<.0001
|
cTnI
|
1.66
|
0.68
|
4.08
|
0.55
|
0.27
|
3.86
|
1.30
|
11.44
|
0.64
|
0.01
|
2.23
|
1.13
|
4.40
|
0.58
|
0.02
|
NOx
|
2.71
|
1.15
|
6.38
|
0.61
|
0.02
|
3.89
|
1.35
|
11.25
|
0.65
|
0.01
|
3.13
|
1.61
|
6.08
|
0.63
|
0.01
|
Routine
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
CRP
|
1.41
|
0.59
|
3.41
|
0.54
|
0.44
|
2.04
|
0.75
|
5.60
|
0.58
|
0.16
|
1.33
|
0.68
|
2.57
|
0.53
|
0.40
|
Creatinine
|
1.86
|
0.79
|
4.34
|
0.57
|
0.15
|
2.49
|
0.87
|
7.12
|
0.60
|
0.09
|
1.42
|
0.74
|
2.74
|
0.54
|
0.30
|
Total cholesterol
|
1.19
|
0.51
|
2.78
|
0.52
|
0.69
|
2.38
|
0.87
|
6.52
|
0.60
|
0.09
|
1.66
|
0.87
|
3.17
|
0.56
|
0.12
|
LDL cholesterol
|
1.27
|
0.54
|
2.99
|
0.53
|
0.58
|
1.84
|
0.68
|
4.99
|
0.57
|
0.23
|
0.99
|
0.52
|
1.89
|
0.50
|
0.98
|
HDL cholesterol
|
0.67
|
0.29
|
1.55
|
0.55
|
0.35
|
1.09
|
0.40
|
2.98
|
0.51
|
0.86
|
0.91
|
0.48
|
1.72
|
0.51
|
0.77
|
Glucose
|
0.98
|
0.42
|
2.31
|
0.50
|
0.97
|
2.57
|
0.92
|
7.20
|
0.61
|
0.07
|
1.46
|
0.76
|
2.79
|
0.54
|
0.26
|
Insulin
|
1.05
|
0.45
|
2.49
|
0.51
|
0.91
|
4.54
|
1.57
|
13.14
|
0.67
|
0.01
|
1.52
|
0.79
|
2.92
|
0.55
|
0.21
|
PCSK9, proprotein convertase subtilisin/kexin type, ANGPTL3, angiopoietin-like protein 3, NOx, nitrate and nitrite, cTnI, cardiac troponin I, HDL, high density lipoproteins, LDL, low density lipoproteins, CRP, C-reactive protein.
Internal validation by comparison of group 1 with group 5 indicated that these 3 biomarkers preserved their associations. Moreover, two additional associations were detected including ANGPTL3 и NOx, which were not sex-dependent similar to cTnI and unlike CRP, creatinine, and HDL cholesterol. Higher levels of ANGPTL3 were associated with a decrease in mortality similar to associations detected for HDL cholesterol.
Subsequent expansion of internal validation by comparison of group 1 versus group 6 indicated that NOx was no longer associated with mortality (Table 5) apparently due to the impact of NOx associations with nonfatal AMI (Table 6). However, ANGPTL3 and cTnI maintained the associations regardless of sex, and CRP maintained the association in men. Creatinine lost its association (Table 5) apparently due to normal glomerular filtration rate, which was within the reference range for all subjects.
Univariate associations of biomarkers with nonfatal AMI
Associations of biomarkers with nonfatal AMI were analyzed by comparisons of group 3 versus group 4 and group 3 versus group 5 (Fig. 1, Table 6). Case-control comparison of group 3 versus group 4 indicated significant and sex-independent associations with NOx and associations with cTnI and insulin only in female subjects, although cTnI preserved the associations in the combined male and female subgroups.
Internal validation of these associations by expanded comparison of group 3 versus group 5 confirmed sex-independent associations with NOx and association with cTnI in the female subgroup. This expanded comparison revealed sex-dependent associations of endothelin-1, galectin-3, and ANGPTL3.
Thus, the results of univariate analysis indicated that only elevated NOx was sex-independently associated with nonfatal AMI with an average odds ratio of approximately 3 (P<0.03), and elevated ANGPTL3 was associated with nonfatal AMI only in men (Table 6). Elevated levels of cTnI, galectin-3, and insulin were associated with a higher risk of nonfatal AMI predominantly in women. Moreover, cTnI, galectin-3 ANGPTL3, and NOx were also sex-independently associated with cardiovascular death.
Stepwise Cox regression analysis
Cox regression was used to expand the description of the associations of biomarkers by inclusion of the time variable. Stepwise analysis was used to select optimal models (Table 7).
Table 7. Stepwise Cox regression analysis of associations of biomarkers with cardiovascular death and nonfatal AMI.
|
Sex
|
Model
|
eHR
|
95% CI
|
Pr > ChiSq
|
Cardiovascular death
|
Group 1 versus group 2
|
Men
|
cTnI
|
3.01
|
1.42
|
6.37
|
0.01
|
|
|
CRP
|
3.19
|
1.53
|
6.64
|
0.01
|
|
Women
|
cTnI
|
3.00
|
1.17
|
7.68
|
0.02
|
|
Men and women
|
PCSK9
|
0.52
|
0.29
|
0.96
|
0.04
|
|
|
Galectin-3
|
1.90
|
1.06
|
3.42
|
0.03
|
|
|
cTnI
|
2.81
|
1.56
|
5.05
|
0.01
|
Group 1 versus group 5
|
Men
|
ANGPTL3
|
0.14
|
0.03
|
0.62
|
0.01
|
|
|
cTnI
|
4.34
|
2.04
|
9.24
|
0.01
|
|
|
CRP
|
4.13
|
1.94
|
8.81
|
0.01
|
|
Women
|
cTnI
|
3.44
|
1.35
|
8.74
|
0.01
|
|
Men and women
|
ANGPTL3
|
0.23
|
0.07
|
0.76
|
0.02
|
|
|
cTnI
|
3.35
|
1.87
|
6.00
|
<.0001
|
|
|
CRP
|
1.97
|
1.11
|
3.48
|
0.02
|
Group 1 versus group 6
|
Men
|
Endothelin
|
0.33
|
0.14
|
0.77
|
0.01
|
|
|
Galectin-3
|
2.12
|
1.01
|
4.46
|
0.05
|
|
|
ANGPTL3
|
0.10
|
0.02
|
0.42
|
0.01
|
|
|
cTnI
|
3.58
|
1.65
|
7.76
|
0.01
|
|
|
CRP
|
3.59
|
1.68
|
7.68
|
0.01
|
|
Women
|
ANGPTL3
|
0.10
|
0.01
|
0.72
|
0.02
|
|
|
cTnI
|
3.06
|
1.20
|
7.80
|
0.02
|
|
Men and women
|
Endothelin
|
0.48
|
0.23
|
0.97
|
0.04
|
|
|
ANGPTL3
|
0.13
|
0.04
|
0.42
|
0.00
|
|
|
cTnI
|
3.17
|
1.77
|
5.68
|
<0.0001
|
|
|
CRP
|
2.16
|
1.22
|
3.82
|
0.01
|
Nonfatal AMI
|
Group 3 versus group 4
|
Men
|
PCSK9
|
2.03
|
1.06
|
3.91
|
0.03
|
|
|
NOx
|
2.08
|
1.08
|
4.00
|
0.03
|
|
Women
|
NOx
|
3.26
|
1.47
|
7.23
|
0.01
|
|
|
Insulin
|
2.74
|
1.23
|
6.09
|
0.01
|
|
Men and women
|
PCSK9
|
1.67
|
1.01
|
2.76
|
0.05
|
|
|
NOx
|
2.60
|
1.57
|
4.31
|
<0.0001
|
Group 3 versus group 5
|
Men
|
ANGPTL3
|
2.91
|
1.49
|
5.68
|
<0.0001
|
|
Women
|
Leptin
|
0.24
|
0.09
|
0.66
|
0.01
|
|
|
cTnI
|
3.47
|
1.40
|
8.64
|
0.01
|
|
|
NOx
|
4.10
|
1.81
|
9.31
|
0.01
|
|
|
Insulin
|
5.52
|
2.00
|
15.29
|
0.01
|
|
|
Total cholesterol
|
2.55
|
1.05
|
6.17
|
0.04
|
|
Men and women
|
ANGPTL3
|
3.29
|
1.96
|
5.51
|
<0.0001
|
|
|
cTnI
|
1.79
|
1.08
|
2.98
|
0.03
|
|
|
NOx
|
2.41
|
1.47
|
3.96
|
0.01
|
PCSK9, proprotein convertase subtilisin/kexin type, ANGPTL3, angiopoietin-like protein 3, NOx, nitrate and nitrite, cTnI, cardiac troponin I, CRP, C-reactive protein, eHR, estimated hazard ratio.
The results indicated that ANGPTL3, cTnI, PCSK9, endothelin, galectin-3, and CRP were associated with cardiovascular death, and ANGPTL3, cTnI, PCSK9, NOx, leptin, insulin, and total cholesterol were associated with nonfatal AMI. ANGPTL3, cTnI, and PCSK9 were not specific and did not discriminate between mortality and nonfatal AMI. Galectin-3, CRP, and endothelin were specific toward cardiovascular death, and leptin, NOx, total cholesterol, and insulin were specific toward nonfatal AMI. CRP, endothelin-1, and galectin-3 were associated with mortality only in women and not in men. PCSK9 and ANGPTL3 were associated with nonfatal AMI in men, and HDL cholesterol, cTnI, insulin, and leptin were associated with nonfatal AMI in women (Table 7). The data are summarized in Table 8, which lists all models that included 11 specific factors with sex-dependent significant associations. Overall, cTnI appears to be the most impactful sex-independent marker of cardiovascular death (eHR of approximately 3.0), and NOx appeared to have the highest overall impact on nonfatal AMI (eHR of approximately 3.0) that was not associated with sex (Table 8).
Table 8. Summary of biomarker included in stepwise Cox regression analysis for associations with cardiovascular death and nonfatal AMI in men and women.
|
Number of models
|
Mean eHR for all models
|
95%CI (mean for all models)
|
Pr > ChiSq
|
Men
|
Women
|
Men and women
|
Cardiovascular death
|
cTnI
|
9
|
3.31
|
1.56
|
7.15
|
0.01
|
3
|
3
|
3
|
ANGPTL3
|
5
|
0.14
|
0.04
|
0.59
|
0.01
|
2
|
1
|
2
|
CRP
|
5
|
3.01
|
1.49
|
6.09
|
0.01
|
3
|
0
|
2
|
Endothelin
|
2
|
0.40
|
0.19
|
0.87
|
0.03
|
1
|
0
|
1
|
Galectin-3
|
2
|
2.01
|
1.03
|
3.94
|
0.04
|
1
|
0
|
1
|
PCSK9
|
1
|
0.52
|
0.29
|
0.96
|
0.04
|
0
|
0
|
0
|
Nonfatal AMI
|
NOx
|
5
|
2.89
|
1.48
|
5.76
|
0.01
|
1
|
2
|
2
|
ANGPTL3
|
2
|
3.10
|
1.73
|
5.60
|
0.00
|
1
|
0
|
1
|
cTnI
|
2
|
2.63
|
1.24
|
5.81
|
0.02
|
0
|
1
|
1
|
Insulin
|
2
|
4.13
|
1.61
|
10.69
|
0.01
|
0
|
2
|
0
|
PCSK9
|
2
|
1.85
|
1.03
|
3.34
|
0.04
|
1
|
0
|
1
|
Leptin
|
1
|
0.24
|
0.09
|
0.66
|
0.01
|
0
|
1
|
0
|
Total cholesterol
|
1
|
2.55
|
1.05
|
6.17
|
0.04
|
0
|
0
|
1
|
PCSK9, proprotein convertase subtilisin/kexin type, ANGPTL3, angiopoietin-like protein 3, NOx, nitrate and nitrite, cTnI, cardiac troponin I, CRP, C-reactive protein, eHR, estimated hazard ratio.
Multivariate Cox regression analysis
Ten markers that were associated with endpoints in stepwise Cox regression and logistic regression were input into multivariate Cox regression adjusted for age and sex (Table 9).
Table 9. Multivariate Cox regression analysis for associations of top 11 biomarkers with cardiovascular deaths and nonfatal AMI adjusted for sex and age
|
Biomarker
|
eHR
|
95% CI
|
Pr > ChiSq
|
Cardiovascular death
|
Group 1 versus group 2
|
cTnI
|
2.59
|
1.20
|
5.60
|
0.02
|
|
ANGPTL3
|
0.39
|
0.11
|
1.44
|
0.16
|
|
NOx
|
1.36
|
0.72
|
2.57
|
0.34
|
|
CRP
|
2.00
|
1.06
|
3.76
|
0.03
|
|
PCSK9
|
0.71
|
0.37
|
1.36
|
0.30
|
|
Endothelin-1
|
1.12
|
0.50
|
2.48
|
0.78
|
|
Galectin-3
|
1.61
|
0.84
|
3.10
|
0.15
|
|
Insulin
|
0.98
|
0.43
|
2.24
|
0.96
|
|
Total cholesterol
|
1.51
|
0.79
|
2.89
|
0.21
|
|
Leptin
|
1.04
|
0.46
|
2.35
|
0.92
|
Group 1 versus group 5
|
cTnI
|
3.44
|
1.71
|
6.93
|
0.00
|
|
ANGPTL3
|
0.16
|
0.05
|
0.55
|
0.00
|
|
NOx
|
1.79
|
0.96
|
3.35
|
0.07
|
|
CRP
|
2.24
|
1.23
|
4.10
|
0.01
|
|
PCSK9
|
0.89
|
0.47
|
1.65
|
0.70
|
|
Endothelin-1
|
0.65
|
0.30
|
1.42
|
0.28
|
|
Galectin3
|
1.62
|
0.86
|
3.06
|
0.13
|
|
Insulin
|
1.00
|
0.47
|
2.12
|
1.00
|
|
Total cholesterol
|
1.35
|
0.72
|
2.56
|
0.35
|
|
Leptin
|
1.28
|
0.59
|
2.76
|
0.53
|
Group 1 versus group 6
|
cTnI
|
3.47
|
1.77
|
6.81
|
0.00
|
|
ANGPTL3
|
0.10
|
0.03
|
0.33
|
0.00
|
|
NOx
|
1.50
|
0.83
|
2.71
|
0.18
|
|
CRP
|
2.27
|
1.25
|
4.14
|
0.01
|
|
PCSK9
|
0.88
|
0.48
|
1.64
|
0.69
|
|
Endothelin-1
|
0.42
|
0.20
|
0.91
|
0.03
|
|
Galectin3
|
1.56
|
0.86
|
2.85
|
0.15
|
|
Insulin
|
0.93
|
0.46
|
1.88
|
0.83
|
|
Total cholesterol
|
1.29
|
0.68
|
2.42
|
0.44
|
|
Leptin
|
1.28
|
0.62
|
2.65
|
0.51
|
Nonfatal AMI
|
Group 3 versus group 4
|
cTnI
|
1.97
|
1.11
|
3.50
|
0.02
|
|
ANGPTL3
|
0.92
|
0.52
|
1.63
|
0.78
|
|
NOx
|
3.05
|
1.75
|
5.30
|
0.00
|
|
CRP
|
1.12
|
0.63
|
2.00
|
0.69
|
|
PCSK9
|
1.72
|
0.98
|
3.02
|
0.06
|
|
Endothelin-1
|
0.84
|
0.46
|
1.54
|
0.58
|
|
Galectin3
|
0.94
|
0.53
|
1.66
|
0.83
|
|
Insulin
|
1.46
|
0.83
|
2.54
|
0.19
|
|
Total cholesterol
|
1.44
|
0.83
|
2.47
|
0.19
|
|
Leptin
|
0.88
|
0.48
|
1.63
|
0.69
|
Group 3 versus group 5
|
cTnI
|
1.84
|
1.02
|
3.33
|
0.04
|
|
ANGPTL3
|
1.89
|
1.08
|
3.28
|
0.02
|
|
NOx
|
2.76
|
1.61
|
4.73
|
0.00
|
|
CRP
|
1.06
|
0.57
|
1.97
|
0.84
|
|
PCSK9
|
1.21
|
0.70
|
2.10
|
0.49
|
|
Endothelin
|
1.30
|
0.70
|
2.40
|
0.40
|
|
Galectin-3
|
1.02
|
0.57
|
1.81
|
0.94
|
|
Insulin
|
1.41
|
0.78
|
2.54
|
0.26
|
|
Total cholesterol
|
1.59
|
0.92
|
2.73
|
0.09
|
|
Leptin
|
0.71
|
0.39
|
1.28
|
0.25
|
PCSK9, proprotein convertase subtilisin/kexin type, ANGPTL3, angiopoietin-like protein 3, NOx, nitrate and nitrite, cTnI, cardiac troponin I, CRP, C-reactive protein, eHR, estimated hazard ratio.
The results indicated significant associations of 4 out of top 6 biomarkers, which were associated with mortality according to stepwise regression (Table 8), including cTnI, ANGPTL3, CRP, and endothelin-1. Associations with nonfatal AMI were confirmed for 3 out of 7 top biomarkers, including NOx, ANGPTL3, and cTnI. The case-control design of the present study implied the lack of meaningful associations of age or sex with the endpoints (P>0.05), which was confirmed by Cox regression analysis. Thus, top 6 biomarkers associated with either one of the endpoints were included into another multivariate Cox regression adjusted for age and sex (Table 10).
Table 10. Multivariate Cox regression analysis for associations of top 6 biomarkers with cardiovascular death and nonfatal AMI adjusted for sex and age.
|
Biomarker
|
eHR
|
95% CI
|
Pr > ChiSq
|
Cardiovascular death
|
Group 1 versus group 2
|
cTnI
|
2.93
|
1.62
|
5.32
|
0.00
|
|
ANGPTL3
|
0.42
|
0.12
|
1.43
|
0.16
|
|
NOx
|
1.31
|
0.72
|
2.38
|
0.38
|
|
CRP
|
2.31
|
1.27
|
4.20
|
0.01
|
Group 1 versus group 5
|
cTnI
|
3.74
|
2.07
|
6.75
|
0.00
|
|
ANGPTL3
|
0.16
|
0.05
|
0.51
|
0.00
|
|
NOx
|
1.83
|
1.02
|
3.29
|
0.04
|
|
CRP
|
2.42
|
1.34
|
4.38
|
0.00
|
Group 1 versus group 6
|
cTnI
|
3.55
|
1.96
|
6.42
|
0.00
|
|
ANGPTL3
|
0.09
|
0.03
|
0.29
|
0.00
|
|
NOx
|
1.69
|
0.94
|
3.01
|
0.08
|
|
CRP
|
2.15
|
1.20
|
3.87
|
0.01
|
Nonfatal AMI
|
Group 3 versus group 4
|
cTnI
|
1.78
|
1.05
|
3.02
|
0.03
|
|
ANGPTL3
|
1.17
|
0.69
|
1.99
|
0.56
|
|
NOx
|
2.67
|
1.61
|
4.42
|
0.00
|
|
CRP
|
1.14
|
0.67
|
1.93
|
0.63
|
Group 3 versus group 5
|
cTnI
|
1.85
|
1.09
|
3.14
|
0.02
|
|
ANGPTL3
|
2.07
|
1.23
|
3.51
|
0.01
|
|
NOx
|
2.42
|
1.46
|
3.99
|
0.00
|
|
CRP
|
1.20
|
0.70
|
2.06
|
0.51
|
ANGPTL3, angiopoietin-like protein 3, NOx, nitrate and nitrite, cTnI, cardiac troponin I, CRP, C-reactive protein, eHR, estimated hazard ratio.
The results indicated independent associations of 4 biomarkers with cardiovascular death, including cTnI, NOx, CRP, and ANGPTL3. Nonfatal AMI was independently associated with cTnI, NOx, and ANGPTL3. Notably, higher levels of ANGPTL3 were associated with a lower risk of cardiovascular death and a higher risk of nonfatal AMI. ROC analysis was thus used to resolve this apparent controversy in the case of ANGPTL3 and further validate potential applicability of detected associations in actual patient stratification at baseline. The results of ROC analysis of associations of biomarkers with pre-existing manifestations of acute coronary syndrome at baseline are shown in Table 11, indicating that cTnI, NOx, creatinine, and HDL cholesterol had significant associations and ANGPTL3 was not associated with these manifestations.
Table 11. ROC analysis of associations of biomarkers with acute coronary syndrome according to baseline anamnesis (N=27) in the total cohort (N=222).
|
AUC
|
95%CI
|
P
|
cTnI
|
0.71
|
0.58
|
0.84
|
0.00
|
NOx
|
0.65
|
0.52
|
0.78
|
0.03
|
PCSK9
|
0.51
|
0.38
|
0.64
|
0.91
|
Galectin-3
|
0.48
|
0.36
|
0.60
|
0.75
|
ANGPTL3
|
0.47
|
0.34
|
0.60
|
0.63
|
Leptin
|
0.51
|
0.39
|
0.64
|
0.84
|
Adiponectin
|
0.50
|
0.37
|
0.62
|
0.96
|
Endothelin-1
|
0.57
|
0.44
|
0.69
|
0.33
|
Insulin
|
0.62
|
0.48
|
0.75
|
0.09
|
Glucose
|
0.51
|
0.36
|
0.67
|
0.84
|
CRP
|
0.61
|
0.47
|
0.75
|
0.10
|
Creatinine
|
0.70
|
0.50
|
0.80
|
0.01
|
Total cholesterol
|
0.40
|
0.30
|
0.60
|
0.31
|
HDL cholesterol
|
0.32
|
0.21
|
0.43
|
0.01
|
LDL cholesterol
|
0.45
|
0.30
|
0.59
|
0.42
|
PCSK9, proprotein convertase subtilisin/kexin type, ANGPTL3, angiopoietin-like protein 3, NOx, nitrate and nitrite, cTnI, cardiac troponin I, CRP, C-reactive protein, HDL, high density lipoproteins, LDL, low density lipoproteins, AUC, area under the curve.