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SUPPLEMENTAL MATERIAL
Supplemental Methods
CDCS protocol
This study (The Acute Coronary Syndrome, ACS) is registered with the Australian and New
Zealand Clinical Trials Registry, ID ACTRN 12605000431628.
Briefly, inclusion criteria at index admission were: ischaemic discomfort plus one or more of
ECG changes (ST segment depression or elevation of at least 0.5mm, T-wave inversion of at
least 3mm in at least 3 leads, or left bundle branch block), elevated levels of cardiac markers,
a history of coronary disease, or age of at least 65 years in patients with diabetes or vascular
disease. Patients were excluded in the presence of any severe co-morbidity limiting life
expectancy to less than 3 years. Since numbers of suitable cases presenting each week
exceeded the capacity to process, cases were randomly selected weekly from a list of eligible
candidates, prior to obtaining informed consent. The overall aim of the CDCS was to assess
candidate predictive markers in the subacute to chronic phase of CAD. CDCS intentionally
recruited a “real life” post-ACS population with a broad spectrum of age, both genders and
significant numbers of subjects in the three distinct sub-groups (NSTEMI, STEMI and UA)
determined at the time of discharge As such, the CDCS represents the burden of expected
important antecedent risk factors and disease processes such as previous myocardial
infarction, heart failure, hypertension, diabetes and vascular disease (1-3).
Establishing outcomes: All events (hospital admissions and mortality) were documented by
scrutinising data obtained from i) outpatient attendance or phone calls with subjects, family
member or primary care physician; ii) Hospital Patient Management System (PMS); iii)
National Health Information Service records (case note examination) and iv) standardised
questionnaires mailed to participants at year 2 and year 3 post index discharge.
Echocardiography.
Standard M-mode measurements of LV dimensions and wall thickness and LA dimensions
were made according to the recommendations of the American Society of Echocardiography.
LV volume was measured by the Simpson modified biplane method and LA area and volume
were estimated by the biplane area-length method. LVEDV and LVESV were indexed to
BSA to derive the indexed volumes (LVEDVi and LVESVi) and LV ejection fraction
(LVEF). Mitral pulsed-wave Doppler velocities of early passive (E) and atrial (A) filling
were obtained from the apical 4-chamber view with a 5-mm sample volume placed between
the tips of the mitral leaflets. Tissue Doppler measurements of early diastolic myocardial
relaxation (e′) was recorded from the lateral mitral annulus in the apical 4-chamber view with
a 5-mm sample volume and with filters set to exclude high frequency signals and with gain
minimized.
Natriuretic Peptide assays.
Samples were stored frozen at minus 80 C and assayed within 6 months of receipt.
Circulating CNP (4), NT-proCNP (4), BNP (5), NT-proBNP (6), ANP and NT-proANP (7)
were assayed as previously described. Intra- and inter assay coefficients of variations were as
follows:- CNP (5.0% and 8.5% at 8.1 pmol/L); NTproCNP (6.6% and 8.1% at 45 pmol/L);
1
BNP (7.9% and 15.2% at 26 pmol/L); NT-proBNP (4.9% and 5.9% at 83 pmol/L); ANP
(4.5% and 5.7% at 25.5 pmol/L); NT-proANP (6.4% and 8.8% at 440 pmol/L).
Reference Intervals for Natriuretic Peptides
Normal reference values ( 95% confidence interval for adults (age 40-80yr) are:- CNP 0.4 –
1.0 pmol/L; NT proCNP 11 – 28 pmol/L; BNP 3 – 12 pmol/L; NTproBNP 2 – 50 pmol/L;
ANP 4 – 27 pmol/L; NTproANP 0.13 – 0.91 nmol/L. All NP measurements were performed
in the same laboratory (Endolab, Christchurch, New Zealand). To convert NP concentrations
to pg/ml, multiply pmol/L values by 2.20 (CNP), 4.98 (NT-proCNP), 3.46 (BNP), 8.47 (NTproBNP), 3.08 (ANP), 10.6 (NT-proANP).
Supplemental Figure 1
LVEF <40% NT-proCNP Tertiles
LVEF >40% NT-proCNP Tertiles
100
80
80
60
60
Cumulative Survival (%)
100
40
40
Low
Middle
High
20
20
0
0
0
2
4
6
8
0
LVEF <40% NT-proBNP Tertiles
100
80
80
60
60
40
40
20
20
Cumulative Survival (%)
4
6
8
LVEF >40% NT-proBNP Tertiles
100
0
2
0
0
2
4
6
8
0
2
Years
4
6
8
Years
Tertile
Numbers at risk
NT-proCNP
(pmol/L)
<16.5
16.5 - 21.9
>21.9
38
50
114
34
45
80
23
35
52
16
18
21
4
7
9
608
585
506
589
562
458
435
430
323
205
227
162
62
70
69
NT-proBNP
(pmol/L)
<49
49 -126
>126
9
34
164
9
34
121
6
25
84
5
12
40
2
4
14
636
613
482
627
596
418
476
451
282
267
233
106
84
86
34
Figure 1. Kaplan-Meier curves as tertiles of NT-proCNP, NT-proBNP at baseline for allcause mortality across all subjects stratified by LVEF.
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Supplemental Tables
Table 1. Summary of data collection in subjects with ACS
Baseline
5-56 days
after onset of
ACS
3-5
months
12-14
months
X
X
24-26
months
36-38
months
Check eligibility/exclusion criteria
X
Obtain informed consent
X
Physical exam *
X
Medical history
Family medical history
Admission details †
X
X
X
Medications on admission
X
Medications at discharge
X
Current medications
X
X
X
X
X
Adverse Events
X
X
X
X
X
ECG
X
X
X
Echocardiograph
X
X
X
Neurohormonal blood samples ‡
X
X
X
DNA blood sample
X
Questionnaire
X
X
X
NYHA, CCS Scores
X
X
X
X
X
*
i.e, height, weight, girth, BP, pulse.
†
Killip score, TIMI Risk Scores for STEMI and UA/NSTEMI, All ECGs CXR reports,
Angio reports, thrombolysis details, MUGA Scan report, serial biochemistry, haematology,
lipids, cardiac enzymes reports.
‡
NTproBNP, BNP, ANP, NTproANP, CNP and NTproCNP.
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Table 2. Spearman correlation coefficients showing the associations between clinical
variables and baseline concentrations of C-type Natriuretic Peptides.
Variable
CNP
NT-proCNP
r
r
Age (n=2083)
0.31 †
BMI (n=2055)
-0.13 *
0.00
Waist hip ratio (males, n=1471)
-0.03
0.07 *
Waist hip ratio (females, n=575)
0.07
0.07
Height (males, n=1483)
-0.18 †
-0.15 †
Height (females, n=576)
-0.15 †
-0.18 †
0.03
-0.02
-0.13 †
-0.09 †
Systolic BP (n=2040)
Diastolic BP (n =2040)
0.23 †
* P<0.01, † P<0.001
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Table 3. Median plasma CNP and NT-proCNP (interquartile range) at baseline according to
gender, comorbidity and drug use&.
CNP (pmol/L)
Variable
Male Sex (n=1496)
NT-proCNP (pmol/L)
Presence
Absence
Presence
Absence
0.54 (0.34 – 0.84) †
0.61 (39 – 0.93)
19.5 (16.1 – 24.9) *
17.2 (14.3 – 22.3)
‡
18.7 (15.5 – 23.6)
Diabetes (n=341)
0.61 (0.38 – 0.95) *
0.55 (0.35 – 0.85)
20.6 (15.8 – 28.0)
HT (n=1074)
0.60 (0.38 – 0.91) ‡
0.52 (0.33 – 0.80)
19.5 (15.8 -26.3) ‡
†
18.3 (15.2 – 18.3)
H-Cholesterol (n=1108)
0.55 (0.35 – 0.85)
0.56 (0.36 – 0.88)
19.1 (15.7 – 25.2)
PVD (n=185)
0.70 (0.48-1.04) ‡
0.55 (0.35-0.85)
23.8 (17.3-33.5) ‡
Statin use (n=949)
0.55 (0.35 – 0.85)
0.57 (0.35 – 0.87)
18.7 (15.5 – 24.2) ‡
19.1 (15.6 – 24.5)
†
18.8 (15.5 – 24.1)
Steroid use (n=218)
0.61 (0.37 – 0.92)
0.55 (0.35-0.86
19.4 (15.5 – 28.1)
18.5 (15.2 – 23.2)
18.6 (15.4-23.5)
Variable states were compared using 2-way ANOVA with gender as a fixed factor.
Abbreviations: HT, hypertension; H-cholesterol, hypercholesterolemia; PVD, peripheral
vascular disease.
&
To convert NP concentrations to pg/ml, multiply pmol/L values by 2.20 (CNP), 4.98 (NT-proCNP).
* P<0.05, † P<0.01, ‡ P<0.001
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Table 4. Multivariate regression analysis – baseline data.
Standardized
Coefficients (Beta)
t
Significance
(P)
Age
0.19
7. 8
0.000
Height
-0.05
-2.2
0.028
BMI
-0.04
-1.9
0.054
E/e’
0.23
10.1
0.000
Systolic blood pressure
-0.06
-2.8
0.006
Creatinine
0.17
7.6
0.000
Gender
0.11
4.4
0.000
Age
0.12
6.2
0.000
Height
-0.07
-2.9
0.004
BMI
0.04
2.1
0.038
Waist Hip ratio
0.04
1.8
0.079
E/e’
0.08
4.2
0.000
Systolic blood pressure
-0.05
-2.6
0.009
Creatinine
0.65
38.6
0.000
Model
Dependent Variable: Log10 CNP
Dependent Variable: Log10 NT-proCNP
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Table 5. Temporal changes in Natriuretic Peptides. Values are medians (IQR)&.
Baseline
4 Month visit
12 Month visit
(n=1075)
(n=1014)
(n=954)
ANP (pmol/L)
35.9 (22.7-56.8)
34.7 (22.2-54.3)
34.6 (21.7-53.4)
0.000
NT-proANP (nmol/L)
1.06 (0.68-1.82)
1.02 (0.64-1.70)
0.97 (0.64-1.63)
0.001
BNP (pmol/L)
16.6 (8.9-32.7)
13.4 (7.2-25.5)
12.1 (6.9-22.9)
0.000
NT-proBNP (pmol/L)
79.1 (35.9-172)
59.9 (26.0-142)
54.3 (24.9-125)
0.000
CNP (pmol/L)
0.53 (0.34-0.88)
0.49 (0.30-0.78)
0.47 (0.28-0.73)
0.000
NT-proCNP (pmol/L)
19.0 (15.5-25.4)
18.6 (15.5-24.8)
18.4 (15.3-24.0)
0.022
(n=479)
(n=458)
(n=447)
ANP (pmol/L)
33.9 (22.5-53.1)
30.8 (19.8-48.0)
29.7 (18.6-46.7)
0.000
NT-proANP (nmol/L)
0.95 (0.62-1.52)
0.86 (0.57-1.44)
0.82 (0.52-1.33)
0.000
BNP (pmol/L)
20.4 (11.3-34.3)
12.0 (7.0-20.9)
10.3 (6.2-18.3)
0.000
NT-proBNP (pmol/L)
95.9 (49.7-170)
56.3 (26.2-118)
43.8 (21.4-97.9)
0.000
CNP (pmol/L)
0.62 (0.42-0.90)
0.50 (0.33-0.74)
0.47 (0.30-0.66)
0.000
NT-proCNP (pmol/L)
18.6 (15.5-22.9)
18.8 (15.6-23.4)
18.0 (15.2-23.1)
0.41
Unstable Angina
(n=562)
(n=522)
(n=502)
ANP (pmol/L)
33.8 (21.4-52.6)
33.9 (21.9-49.2)
36.0 (22.8-55.0)
0.018
NT-proANP (nmol/L)
1.03 (0.66-1.63)
1.04 (0.63-1.54)
1.04 (0.64-1.72)
0.026
BNP (pmol/L)
13.4 (7.5-24.3)
12.4 (7.0-21.2)
12.8 (7.2-21.9)
0.10
NT-proBNP (pmol/L)
63.7 (27.4-124)
59.6 (23.9-107)
55.8 (27.0-121)
0.008
CNP (pmol/L)
0.53 (0.32-0.80)
0.48 (0.32-0.73)
0.50 (0.31-0.75)
0.42
NT-proCNP (pmol/L)
19.0 (15.5-25.4)
18.6 (15.5-24.8)
18.4 (15.3-24.0)
0.51
NSTEMI
STEMI
P*
* Analysis of the effect of time on NP by RM-ANOVA
&
To convert NP concentrations to pg/ml, multiply pmol/L values by 2.20 (CNP), 4.98 (NT-proCNP),
3.46 (BNP), 8.47 (NT-proBNP), 3.08 (ANP), 10.6 (NT-proANP).
7
Table 6. Spearman Correlation coefficients of cardiac and renal function indices with
Natriuretic Peptides.
ANP
NT-proANP
BNP
NT-proBNP
CNP
NT-proCNP
Baseline
LA Area (n=1967)
LVESVol (n=1578)
E/e’ (n=2005)
LVEF (n=1938)
Creatinine (n=2053)
0.33 †
0.21 †
0.40 †
-0.31 †
0.23 †
0.37 †
0.18 †
0.44 †
-0.30 †
0.37 †
0.37 †
0.27 †
0.43 †
-0.41 †
0.23 †
0.38 †
0.28 †
0.47 †
-0.41 †
0.29 †
0.30 †
0.21 †
0.37 †
-0.33 †
0.23 †
0.14 †
0.12 †
0.19 †
-0.19 †
0.63 †
4 Month visit
LA Area (n=1839)
LVESVol (n=1505)
E/e’ (n=2005)
LVEF (n=1901)
Creatinine (n=1952)
0.37 †
0.22 †
0.37 †
-0.28 †
0.27 †
0.38 †
0.17 †
0.40 †
-0.26 †
-0.40 †
0.39 †
0.23 †
0.41 †
-0.31 †
0.25 †
0.40 †
0.23 †
0.44 †
-0.32 †
0.30 †
0.35 †
0.14 †
0.36 †
-0.26 †
-0.21 †
0.12 †
0.12 †
0.16 †
-0.19 †
0.63 †
12 Month visit
LA Area (n=1760)
LVESVol (n=1433)
E/e’ (n=1827)
LVEF (n=1732)
Creatinine (n=1857)
0.33 †
0.20 †
0.37 †
-0.25 †
0.23 †
0.34 †
0.18 †
0.42 †
-0.25 †
0.37 †
0.38 †
0.23 †
0.41 †
-0.30 †
0.23 †
0.37 †
0.23 †
0.45 †
-0.32 †
0.29 †
0.32 †
0.17 †
0.38 †
-0.29 †
0.23 †
0.15 †
0.11 †
0.18 †
-0.18 †
0.61 †
†
P<0.001
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Table 7. Univariate hazard ratios, HR, (95% confidence intervals, CI) at baseline across
brackets of E/e’ (<8, 8-15, >15) and across tertiles of creatinine, eGFR and natriuretic
peptides for all-cause mortality over median 4 years follow up.
HR (CI)
E/e’
3.22 (2.72 – 3.80) *
Creatinine
1.78 (1.58 – 2.02) *
eGFR
0.43 (0.38-0.49) *
CNP
2.04 (1.80 – 2.32) *
NT-proCNP
1.94 (1.71 – 2.20) *
BNP
2.63 (2.30 – 3.02) *
NT-proBNP
2.98 (2.58 – 3.43) *
ANP
2.46 (2.16 – 2.81) *
NT-proANP
3.31 (2.86 – 3.84) *
* P <0.001
Table 8. Changes in aortic plasma CNP peptides after percutaneous coronary intervention
(PCI)
In 51 subjects with STEMI undergoing percutaneous intervention (8) paired aortic plasma
samples were drawn just prior to and immediately after the successful PCI and measured for
CNP and NTproCNP concentrations. Values (pmol/L) are mean (SE)&.
CNP
NTproCNP
PRE
POST
Delta % CHANGE
1.14 (0.08)
23.7 (1.5)
1.27 (0.07)
31.3 ( 1.1)
15.8 (3.8)% *
46.8 (7.5)% *
* P <0.001
&
To convert NP concentrations to pg/ml, multiply pmol/L values by 2.20 (CNP), 4.98 (NT-proCNP).
9
Supplemental References
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Doughty RN, Whalley GA, Ellis CJ, Troughton RW, Richards AM, Cameron VA.
Angiotensin-converting enzyme 2 A1075G polymorphism is associated with survival
in an acute coronary syndromes cohort. Am Heart J 2008;156(4):752-8.
2. Palmer BR, Frampton CM, Skelton L, Yandle TG, Doughty RN, Whalley GA, Ellis
CJ, Troughton RW, Richards AM, Cameron VA. KCNE5 polymorphism rs697829 is
associated with QT interval and survival in acute coronary syndromes patients.
Journal of cardiovascular electrophysiology 2012;23(3):319-24.
3. Earle NJ, Poppe KK, Pilbrow AP, Cameron VA, Troughton RW, Skinner JR, Love
DR, Shelling AN, Whalley GA, Ellis CJ, Richards AM, Doughty RN. Genetic
markers of repolarization and arrhythmic events after acute coronary syndromes. Am
Heart J 2015;169(4):579-86 e3.
4. Olney RC, Permuy JW, Prickett TC, Han JC, Espiner EA. Amino-terminal propeptide
of C-type natriuretic peptide (NTproCNP) predicts height velocity in healthy children.
Clin Endocrinol (Oxf) 2012;77:416-422.
5. Palmer SC, Prickett TC, Espiner EA, Yandle TG, Richards AM. Regional Release
and Clearance of C-Type Natriuretic Peptides in the Human Circulation and Relation
to Cardiac Function. Hypertension 2009;54:612-618.
6. Hunt PJ, Richards AM, Nicholls MG, Yandle TG, Doughty RN, Espiner EA.
Immunoreactive amino-terminal pro-brain natriuretic peptide (NT-PROBNP): a new
marker of cardiac impairment. Clinical Endocrinology 1997;47(3):287-296.
7. Yandle TG, Espiner EA, Nicholls MG, Duff H. Radioimmunoassay and
characterization of atrial natriuretic peptide in human plasma. Journal of Clinical
Endocrinology & Metabolism 1986;63(1):72-9.
8. Marshall CJ, Nallaratnam M, Mocatta T, Smyth D, Richards M, Elliott JM, Blake J,
Winterbourn CC, Kettle AJ, McClean DR. Factors influencing local and systemic
levels of plasma myeloperoxidase in ST-segment elevation acute myocardial
infarction. Am J Cardiol. 2010;106:316-22.
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