European Heart Journal Advance Access published April 28, 2016

European Heart Journal CLINICAL RESEARCH

doi:10.1093/eurheartj/ehw148 Coronary artery disease

Plasma ceramides predict cardiovascular death

in patients with stable coronary artery disease
and acute coronary syndromes beyond
LDL-cholesterol
Reijo Laaksonen 1,2,3*, Kim Ekroos 1, Marko Sysi-Aho 1, Mika Hilvo1, Terhi Vihervaara 1,
Dimple Kauhanen 1, Matti Suoniemi 1, Reini Hurme 1, Winfried März 4,5,
Hubert Scharnagl 6, Tatjana Stojakovic6, Efthymia Vlachopoulou 7, Marja-Liisa Lokki 7,

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Markku S. Nieminen 7,8, Roland Klingenberg 9, Christian M. Matter 9,
Thorsten Hornemann 10, Peter Jüni 11, Nicolas Rodondi 12,13, Lorenz Räber 14,
Stephan Windecker14, Baris Gencer 15, Eva Ringdal Pedersen 16, Grethe S. Tell 17,
Ottar Nygård16,18†, Francois Mach15†, Juha Sinisalo 7,8†, and Thomas F. Lüscher 10†
1
Zora Biosciences, Espoo, Finland; 2Medical School, Tampere University, Tampere, Finland; 3Finnish Clinical Biobank Tampere, University Hospital of Tampere, Tampere, Finland;
4
Medical Clinic V (Nephrology, Hypertensiology, Rheumatology, Endocrinology, Diabetology), Medical Faculty Mannheim, University of Heidelberg, Heidelberg, Germany; 5synlab
Academy, synlab Holding Deutschland GmbH, Mannheim and Augsburg, Germany; 6Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University Graz, Graz,
Austria; 7Transplantation Laboratory, Haartman Institute, University of Helsinki, Helsinki, Finland; 8Heart and Lung Center, Helsinki University Hospital, Helsinki, Finland; 9Department
of Cardiology, University Heart Center, University Hospital Zürich and University of Zürich, Zürich, Switzerland; 10Institute of Clinical Chemistry, University Hospital, Zürich,
Switzerland; 11Applied Health Research Centre (AHRC), Li Ka Shing Knowledge Institute of St. Michael’s Hospital, and Department of Medicine, University of Toronto, Toronto,
Canada; 12Department of General Internal Medicine, University Hospital Bern, Bern, Switzerland; 13Department of Ambulatory Care and Community Medicine, University of Lausanne,
Lausanne, Switzerland; 14Cardiovascular Center, Department of Cardiology, University Hospital Bern, Bern, Switzerland; 15Cardiovascular Center, Department of Cardiology,
University Hospital Geneva, Geneva, Switzerland; 16Department of Clinical Science, University of Bergen, Bergen, Norway; 17Department of Global Public Health and Primary Care,
University of Bergen, Bergen, Norway; and 18Department of Heart Disease, Haukeland University Hospital, Bergen, Norway

Received 8 January 2016; revised 15 February 2016; accepted 17 March 2016

Aims The aim was to study the prognostic value of plasma ceramides (Cer) as cardiovascular death (CV death) markers in
three independent coronary artery disease (CAD) cohorts.
.....................................................................................................................................................................................
Methods Corogene study is a prospective Finnish cohort including stable CAD patients (n ¼ 160). Multiple lipid biomarkers and
and results C-reactive protein were measured in addition to plasma Cer(d18:1/16:0), Cer(d18:1/18:0), Cer(d18:1/24:0), and
Cer(d18:1/24:1). Subsequently, the association between high-risk ceramides and CV mortality was investigated in
the prospective Special Program University Medicine—Inflammation in Acute Coronary Syndromes (SPUM-ACS)
cohort (n ¼ 1637), conducted in four Swiss university hospitals. Finally, the results were validated in Bergen Coronary
Angiography Cohort (BECAC), a prospective Norwegian cohort study of stable CAD patients. Ceramides, especially
when used in ratios, were significantly associated with CV death in all studies, independent of other lipid markers and
C-reactive protein. Adjusted odds ratios per standard deviation for the Cer(d18:1/16:0)/Cer(d18:1/24:0) ratio were
4.49 (95% CI, 2.24– 8.98), 1.64 (1.29– 2.08), and 1.77 (1.41 –2.23) in the Corogene, SPUM-ACS, and BECAC studies,
respectively. The Cer(d18:1/16:0)/Cer(d18:1/24:0) ratio improved the predictive value of the GRACE score
(net reclassification improvement, NRI ¼ 0.17 and DAUC ¼ 0.09) in ACS and the predictive value of the Marschner
score in stable CAD (NRI ¼ 0.15 and DAUC ¼ 0.02).

* Corresponding author. Zora Biosciences Oy, Biologinkuja 1, 02150 Espoo, Finland. Tel: +358 40 724 077, Email: reijo.laaksonen@zora.fi
†
Equal contribution.
& The Author 2016. Published by Oxford University Press on behalf of the European Society of Cardiology.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted
reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
Page 2 of 10 R. Laaksonen et al.

.....................................................................................................................................................................................
Conclusions Distinct plasma ceramide ratios are significant predictors of CV death both in patients with stable CAD and ACS, over
and above currently used lipid markers. This may improve the identification of high-risk patients in need of more ag-
gressive therapeutic interventions.
-----------------------------------------------------------------------------------------------------------------------------------------------------------
Keywords Ceramide † Acute coronary syndrome † Coronary artery disease † Biomarker † LDL-cholesterol † Risk
prediction † Prognosis

death certificate registry. As cases, all patients who experienced coron-

Introduction ary death within an average follow-up of 212 years were selected.
Given the high prevalence of coronary artery disease (CAD) and Matched control patients had established CAD (.50% stenosis at least
associated mortality, prevention of fatal and non-fatal myocardial in one epicardial coronary artery), but remained alive during the follow-
infarctions (MI) in CAD patients remains an ongoing clinical chal- up period. Baseline characteristics of the Corogene subjects are shown
in Table 1 and Supplementary material online, Table S1.
lenge. Mortality rates among stable CAD patients range between
1% and 3%, while rates of non-fatal events are 1 – 2% annually.1 In Bergen Coronary Angiography Cohort cohort: patients
patients with acute coronary syndromes (ACS) who survive the with stable coronary artery disease
acute event, the rate of MI and death is markedly higher, particularly The Bergen Coronary Angiography Cohort (BECAC) includes 1580

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during the first year.2 However, at the individual level, the event risk adults referred to elective coronary angiography because of suspected
may vary considerably, which makes risk estimation tools necessary stable angina pectoris recruited at the Haukeland University Hospital in
Bergen, Norway between 2000 and 2004. Information on cardiovascu-
to improve patient management. Expedient risk stratification should
lar deaths was collected from the Cause of Death Registry at the Nor-
identify individuals at risk requiring more intensive therapy. Con-
wegian Institute of Public Health, and verified against hospital medical
versely, patients with a favorable prognosis should be identified to records whenever available. During a median follow-up of 4.6 years, a
avoid drug overuse and associated side effects.3 total of 81 patients died from cardiovascular disease. Baseline character-
Hypothesis free lipidomic analyses have revealed a handful of istics of the BECAC participants are reported in Table 1 and Supplemen-
lipids potentially qualifying as useful prognostic markers for tary material online, Table S1.
CAD.4 – 6 In our initial lipidomic study, distinct ceramide species
SPUM-ACS cohort: patients with acute coronary
were significantly associated with CVD among CAD patients.4 Mo-
syndromes
lecular lipid species, particularly ceramide(d18:1/16:0), were also as- Special Program University Medicine—Inflammation in Acute Coronary
sociated with necrotic core tissue type and lipid core burden in Syndromes (SPUM-ACS) is a prospective, multi-centre (Bern, Geneva,
coronary angiography, and were predictive for 1-year clinical out- Lausanne, and Zürich) cohort study. Patients with a primary diagnosis of
come in 581 ACS and stable CAD patients.7 In these studies, plasma ACS and referred for invasive management were enrolled at four Swiss
CVD risk-related ceramide molecules (Cer(d18:1/16:0), Cer(d18:1/ university hospitals. Baseline characteristics of the SPUM-ACS patients
18:0), and Cer(d18:1/24:1)), and their ratios with Cer(d18:1/24:0), are summarized in Table 1 and Supplementary material online, Table S1.
emerged as potential risk stratifiers for CAD patients.4 Ceramides At one-year follow-up, a total of 51 patients died from cardiac reasons.
are known to associate with many central processes of atheroscler-
osis development including lipoprotein uptake, inflammation, and Clinical laboratory analyses
apoptosis (Supplementary material online, Figure S1).8 Ceramide Standard lipids measurements were determined using standard methods
species are produced by six fatty acyl selective ceramide synthases available at each of the three study sites. In Corogene subjects, apolipo-
(CerSs; Supplementary material online, Figure S2), and it is becoming proteins (AI, AII, and B), lipoprotein (a), lipoprotein-associated phospho-
lipase A2 activity, and HDL and LDL particle numbers and sizes were
evident that individual ceramide species have specific physiological
measured as described in Supplementary material online, Methods.
functions.9 – 12 Thus, monitoring ratios of ceramides species may
provide insight into the metabolic regulation of atherosclerotic
Quantification of ceramides
events. In this study, we establish the suggested role of ceramides
The plasma levels of Cer(d18:1/16:0), Cer(d18:1/18:0), Cer(d18:1/24:0),
and their distinct ratios as risk predictors for CV death in patients
and Cer(d18:1/24:1) were quantified on a 5500 QTRAP (SCIEX, Fra-
with stable CAD and ACS. mingham, MA) mass spectrometer equipped with an Eksigent 100-XL
UHPLC system as described recently.13
Methods
More detailed method descriptions are available in Supplementary
Statistical analyses
material online. Wilcoxon’s rank sum test was applied for group comparisons. Odds ra-
tios (ORs) per standard deviation were estimated using logistic regres-
Study subjects sion. Hazard ratios were calculated using the Cox proportional hazard
Corogene study: stable coronary artery disease patients model. The GRACE14 risk score, consisting of Killip class, systolic blood
Corogene is a prospective, consecutive cohort study of Finnish patients pressure, heart rate, age, creatinine, cardiac arrest at admission,
referred for coronary angiography to the Helsinki University Central ST-segment deviation, and elevated cardiac enzyme levels (troponin,
Hospital between 2006 and 2008. A nested case control study was de- CK-MB), was used to calculate the risk of long-term mortality for
signed using the Corogene database and including data from the national ACS patients. The following Marschner score15 variables were used in
Plasma ceramides predict cardiovascular death Page 3 of 10

Table 1 Baseline characteristics of the subjects in Corogene, SPUM-ACS and BECAC studies

Characteristic COROGENE SPUM-ACS BECAC

........................................... ......................................... ...........................................
Cases Controls Cases Controls Cases Controls
...............................................................................................................................................................................
No of subjects 80 80 51 1586 81 1506
Gender
Male, n (%) 60 (75%) 60 (75%) 42 (82%) 1223 (77%) 55 (68%) 889 (59%)
...............................................................................................................................................................................
Age (years) 70.2 (62.6–77.1) 70 (63.4–76.9) 77.1 (69–83) 62.8 (53.9–72.9) 71 (64–78) 61 (54–70)
Body mass index 27.5 (23.6–30.9) 26 (24.1–29.5) 25.1 (23.1–28.3) 26.6 (24.3–29.4) 24 (22–28) 26 (23–28)
Time to death/follow-up time (days) 528 (134–739) 1955 (1669– 2173) 25 (7– 216) 365 (359– 365) 626 (196–1332) 1720 (1368– 2111)
Creatinine (mmol/L) 98 (84–134) 79 (69–90) 100 (79–134) 75 (65–88) 98 (87–115) 87 (79–97)
...............................................................................................................................................................................
Current smoker
Yes, n (%) 37 (46%) 37 (46%) 16 (31%) 663 (42%) 29 (36%) 355 (24%)
No, n (%) 43 (54%) 43 (54%) 33 (65%) 897 (57%) 50 (62%) 1146 (76%)
NA 2 (4%) 26 (2%) 2 (2%) 5 (0%)
...............................................................................................................................................................................

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Diabetes
Yes, n (%) 32 (40%) 32 (40%) 11 (22%) 266 (17%) 16 (20%) 159 (11%)
No, n (%) 48 (60%) 48 (60%) 40 (78%) 1320 (83%) 64 (79%) 1333 (89%)
NA 1 (1%) 14 (1%)
...............................................................................................................................................................................
Hypertension
Yes, n (%) 60 (75%) 60 (75%) 36 (71%) 912 (58%) 55 (68%) 674 (45%)
No, n (%) 20 (25%) 20 (25%) 15 (29%) 674 (42%) 26 (32%) 832 (55%)
...............................................................................................................................................................................
Lipid-lowering treatment
Yes, n (%) 61 (76%) 61 (76%) 14 (27%) 432 (27%) 61 (75%) 933 (62%)
No, n (%) 19 (24%) 19 (24%) 34 (67%) 1146 (72%) 20 (25%) 573 (38%)
NA 3 (6%)
...............................................................................................................................................................................
Previous AMI
Yes, n (%) 67 (84%) 0 (0%) 8 (16%) 210 (13%) 54 (67%) 482 (32%)
No, n (%) 13 (16%) 80 (100%) 43 (84%) 1374 (87%) 27 (33%) 1024 (68%)
NA 2 (0%)
...............................................................................................................................................................................
Previous stroke
Yes, n (%) 17 (21%) 10 (12%) 2 (4%) 37 (2%) 15 (19%) 108 (7%)
No, n (%) 63 (79%) 70 (88%) 49 (96%) 1549 (98%) 66 (81%) 1398 (93%)

the modeling of stable CAD patient data: total cholesterol, HDL-C, age, More details on statistical methods can be found in Supplementary
gender, smoking status, previous acute MI, diabetes, hypertension, and material online.
prior stroke.
Net reclassification improvement was estimated as described by
Pencina et al. 16 For the 1-year event risk of the secondary prevention Results
population in the SPUM-ACS study we categorized subjects to low
risk (,1% event probability), intermediate (1 – 5%) risk or high-risk Ceramide concentrations in high- and low-
(.5%) groups. For the BECAC study the same categorization was risk patients with coronary artery disease
used for 3-year risk.
In stable CAD patients of the Corogene study LDL-based markers
The ceramide risk score was calculated as follows: For each individual, all
such as LDL-C, LDL particle number (LDL-P), small dense LDL
three ceramide ratios and each concentration (apart from Cer(d18:1/24:0))
were compared with the whole study population. If the variable belonged (sdLDL), and apoB did not differ significantly between cases who ex-
to the 3rd quartile, the individual received +1 point, and if to the 4th quar- perienced coronary death and controls who remained alive, and nei-
tile, +2 points (Supplementary material online, Table S11). Thus, the score ther did Lp(a) nor Lp-PLA2. However, the HDL-related markers
ranges from 0 to 12 and based on the score, the subjects were split into HDL-C, HDL particle number (HDL-P), small dense HDL (sdHDL),
four risk categories (0–2, 3–6, 7–9, and 10–12). and ApoA1 were all significantly (P , 0.001) different between
Page 4 of 10 R. Laaksonen et al.

Table 2 Medians and inter quartile ranges of established lipid markers and ceramides in case and control groupsa

BECAC SPUM-ACS
.................................................................. .................................................................
Cases (n 5 81) Controls (n 5 1499) P-value Cases (n 5 51) Controls (n 5 1586) P-value
...............................................................................................................................................................................
Cer(d18:1/16:0)/Cer(d18:1/24:0) 0.121 (0.101– 0.145) 0.100 (0.085– 0.119) ,0.001 0.116 (0.099– 0.170) 0.093 (0.079– 0.113) ,0.001
Cer(d18:1/18:0)/Cer(d18:1/24:0) 0.046 (0.036– 0.059) 0.038 (0.031– 0.049) ,0.001 0.064 (0.044– 0.084) 0.047 (0.037– 0.060) ,0.001
Cer(d18:1/24:1)/Cer(d18:1/24:0) 0.498 (0.408– 0.624) 0.413 (0.337– 0.508) ,0.001 0.489 (0.415– 0.675) 0.394 (0.337– 0.474) ,0.001
Cer(d18:1/16:0) (mmol/L) 0.271 (0.235– 0.326) 0.253 (0.213– 0.300) 0.010 0.313 (0.255– 0.385) 0.292 (0.247– 0.346) 0.090
Cer(d18:1/18:0) (mmol/L) 0.108 (0.077– 0.143) 0.096 (0.076– 0.123) 0.097 0.161 (0.109– 0.234) 0.146 (0.112– 0.189) 0.163
Cer(d18:1/24:0) (mmol/L) 2.335 (1.843– 2.866) 2.548 (2.030– 3.098) 0.035 2.366 (2.112– 3.084) 3.107 (2.490– 3.826) ,0.001
Cer(d18:1/24:1) (mmol/L) 1.056 (0.927– 1.344) 1.028 (0.844– 1.257) 0.026 1.421 (1.012– 1.628) 1.229 (1.004– 1.484) 0.175
LDL-C (mg/dL) 110 (89–133) 116 (93–147) 0.087 101 (81–128) 121 (93–150) 0.001
HDL-C (mg/dL) 46 (35–58) 50 (41–62) 0.036 48 (36–58) 44 (36–53) 0.266
TC (mg/dL) 185 (158– 212) 193 (166– 224) 0.081 159 (147– 189) 189 (161– 221) ,0.001
TG (mg/dL) 135 (100– 169) 126 (92–182) 0.738 76 (54–108) 92 (61–142) 0.014
...............................................................................................................................................................................
COROGENE
..................................................................

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Cases (n 5 80) Controls (n 5 80) P-value
...............................................................................................................................................................................
Cer(d18:1/16:0)/Cer(d18:1/24:0) 0.132 (0.105– 0.175) 0.105 (0.090– 0.128) ,0.001
Cer(d18:1/18:0)/Cer(d18:1/24:0) 0.062 (0.047– 0.077) 0.046 (0.037– 0.062) ,0.001
Cer(d18:1/24:1)/Cer(d18:1/24:0) 0.703 (0.582– 0.846) 0.556 (0.483– 0.665) ,0.001
Cer(d18:1/16:0) (mmol/L) 0.275 (0.222– 0.326) 0.235 (0.212– 0.282) 0.007
Cer(d18:1/18:0) (mmol/L) 0.118 (0.094– 0.152) 0.107 (0.092– 0.137) 0.195
Cer(d18:1/24:0) (mmol/L) 1.923 (1.475– 2.511) 2.235 (1.993– 2.672) 0.008
Cer(d18:1/24:1) (mmol/L) 1.385 (1.189– 1.620) 1.245 (1.091– 1.427) 0.017
TC (mg/dL) 128 (111– 165) 139 (122– 163) 0.064
TG (mg/dL) 108 (86–140) 92 (75–139) 0.110
LDL-C (mg/dL) 69 (55–99) 75 (65–92) 0.251
LDL-P (nmol/L) 830 (694– 1110) 928 (712– 1175) 0.395
sdLDL (nmol/L) 533 (304– 659) 548 (376– 737) 0.265
ApoB (mg/dL) 67 (55–82) 68.5 (57–84) 0.997
HDL-C (mg/dL) 34 (29–40) 41 (33–51) ,0.001
HDL-P (mmol/L) 24 (21–27) 28 (24–31) ,0.001
sdHDL (mmol/L) 12.8 (9.3–15.6) 15.8 (13.1– 18.2) ,0.001
ApoA1 (mg/dL) 115 (101– 131) 132 (115– 150) ,0.001
Lp(a) (mg/dL) 7.2 (2 –35) 3.6 (1 –28) 0.319
Lp-PLA2 (nmol/min/ml) 138 (119– 166) 130 (115– 163) 0.354
C-reactive protein (mg/L) 3.1 (1.6–8.7) 1.1 (0.7–2.8) ,0.001

a
Cer, ceramide; TC, total cholesterol; TG, triacylglycerols, LDL-C low-density lipoprotein cholesterol, HDL-C high-density lipoprotein cholesterol, sdLDL small dense low-density
lipoprotein cholesterol, LDL-P low-density lipoprotein particle number, sdHDL small dense high-density lipoprotein cholesterol, HDL-P high-density lipoprotein particle number,
ApoB apolipoprotein B, ApoA1 apolipoprotein A1, Lp(a) lipoprotein (a), Lp-PLA2 lipoprotein-associated phospholipase A2. SI conversion factors: To convert cholesterol to
mmol/L, multiply values by 0.0259; to convert triacylglycerols to mmol/L, multiply values by 0.01129.

the groups with the medians being 217.1, 214.3, 219.0, and +10.3, and +11.2% higher than in controls, respectively). In con-
212.9% lower in cases, respectively. The differences between cases trast, the Cer(d18:1/24:0) behaved differently, with the median in
and controls in plasma ceramides and established lipid markers are cases being 214.9% lower than in controls (P , 0.001). Similarly
provided in Table 2 (the percentage of observations for each marker to our earlier observations,4 highly significant differences, were
is provided in Supplementary material online, Table S2). observed for the three predefined ceramide ratios, with the med-
In the Corogene study, the concentrations of Cer(d18:1/16:0), ians of cases ranging between +25.7 and +34.8% (P , 0.001)
Cer(d18:1/18:0), and Cer(d18:1/24:1) were significantly different relative to controls. The difference between stable CAD patients
(P , 0.001 for all) between cases who had a fatal MI during and controls is illustrated in Supplementary material online,
the follow-up period and controls (medians in cases +17.0%, Figure S3.
Plasma ceramides predict cardiovascular death Page 5 of 10

Ceramide ratios in coronary artery ceramide ratios were significantly higher in 81 patients who died fol-
disease patients lowing a CV event within 4.6-year follow-up compared with those
who did not die during follow-up (Table 2; Supplementary material
In the Corogene study, the most significant ORs for coronary death
online, Table S2). For comparability with the Corogene results, non-
were found for HDL markers and ceramide ratios. Ceramide ORs re-
adjusted and adjusted ORs for standard lipid markers and ceramides
mained predictive after adjustment for conventional lipid markers
are given in Supplementary material online, Table S3. The incremen-
LDL-C, HDL-C, total cholesterol, triacylglycerols, and C-reactive pro-
tal improvement of discrimination for CV death was further demon-
tein. Lp-PLA2 and Lp(a) had no significant association with CV
strated by calculating hazard ratios adjusted for standard lipids and
mortality.
the Marschner score variables (Table 3).
LDL-C and LDL particle number were inversely associated with
Adjustment for statin treatment did not have a major impact on the
risk (LDL-C unadjusted upper quartile OR 0.89 95% CI 0.37–2.14;
results (Supplementary material online, Table S4). The odds ratios
LDL-P upper quartile OR 0.67, 95%CI 0.29–1.59). For comparison,
were calculated for ceramides and LDL-C also in patients that were
the unadjusted upper quartile OR for the Cer(d18:1/16:0)/
on or not on statin treatment both at baseline and after 1-year of
Cer(d18:1/24:0) ratio was 10.33 (95% CI 3.69 – 28.97). Figure 1
follow-up (Supplementary material online, Table S5). Ceramides
shows non-adjusted and adjusted ORs for different lipid markers
were predictive in both instances, although in patients without statin
and ceramides in the Corogene study.
treatment the odds ratios were better. LDL-C did not show significant
predictive value, confirming that the lack of a direct association
Ceramides and risk in stable coronary between LDL-C and CV death was not caused by interference with

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artery disease patients statin treatment.
An independent assessment of ceramides was performed in a co- The incremental prognostic value of ceramides was tested by
hort (BECAC) of stable patients. We found that the predefined comparing the base model composed of the Marschner score

Figure 1 Cardiovascular death odds ratios for per standard deviation and 4th quartile for different lipid markers and ceramides in Corogene
study. Adjustment is made for total cholesterol, triacylglycerols, LDL-C, HDL-C, and C-reactive protein.
Page 6 of 10 R. Laaksonen et al.

Table 3 Association between ceramides and cardiovascular death in BECACa

Univariate model Multivariableb model 1b Multivariablec model 2c

.......................................... .......................................... ..........................................
Hazard ratiod (95% CI) P-value Hazard ratiod (95% CI) P-value Hazard ratiod (95% CI) P-value
...............................................................................................................................................................................
Cer(d18:1/16:0)/Cer(d18:1/24:0)e 1.77 (1.46–2.16) ,0.001 1.79 (1.45–2.20) ,0.001 1.52 (1.21– 1.92) ,0.001
Cer(d18:1/18:0)/Cer(d18:1/24:0)e 1.63 (1.31–2.04) ,0.001 1.58 (1.25–2.00) ,0.001 1.29 (1.01– 1.65) 0.039
Cer(d18:1/24:1)/Cer(d18:1/24:0)e 1.61 (1.30–1.98) ,0.001 1.58 (1.27–1.97) ,0.001 1.31 (1.03– 1.66) 0.028
Cer(d18:1/16:0)e 1.44 (1.17–1.77) ,0.001 2.09 (1.61–2.73) ,0.001 1.75 (1.30– 2.35) ,0.001
Cer(d18:1/18:0)e 1.33 (1.07–1.65) 0.011 1.54 (1.19–2.01) 0.001 1.27 (0.98– 1.66) 0.076
Cer(d18:1/24:0)e 0.83 (0.67–1.02) 0.081 0.82 (0.62–1.10) 0.182 0.91 (0.69– 1.21) 0.510
Cer(d18:1/24:1)e 1.39 (1.13–1.72) 0.002 1.74 (1.34–2.25) ,0.001 1.38 (1.04– 1.82) 0.023

Cer denotes ceramide.

a
CV death denotes death from MI, stroke, and heart failure.
b
The model was adjusted for TC, TG, HDL-C, and LDL-C.
c
The model was adjusted as for model 1 with additional adjustment for the following Marschner score variables: age, gender, smoking status, previous acute MI, diabetes,
hypertension, and prior stroke.
d
Hazard ratios are for 1 SD increase.

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e
Natural logarithm of the ceramides and ceramide ratio.

Table 4 Association between ceramides and cardiovascular death in SPUM-ACSa

Univariate model Multivariableb model 1b Multivariablec model 2c

........................................... ........................................... ...........................................
Hazard ratiod (95% CI) P-value Hazard ratiod (95% CI) P-value Hazard ratiod (95% CI) P-value
...............................................................................................................................................................................
Cer(d18:1/16:0)/Cer(d18:1/24:0) 1.81 (1.52–2.14) ,0.001 1.82 (1.51– 2.21) ,0.001 1.69 (1.39–2.06) ,0.001
Cer(d18:1/18:0)/Cer(d18:1/24:0) 1.66 (1.43–1.96) ,0.001 1.65 (1.39– 1.97) ,0.001 1.48 (1.24–1.76) ,0.001
Cer(d18:1/24:1)/Cer(d18:1/24:0) 1.74 (1.45–2.08) ,0.001 1.77 (1.44– 2.17) ,0.001 1.64 (1.32–2.03) ,0.001
Cer(d18:1/16:0)e 1.45 (1.10–1.93) 0.010 1.96 (1.45– 2.66) ,0.001 1.98 (1.49–2.62) ,0.001
Cer(d18:1/18:0)e 1.43 (1.07–1.90) 0.015 1.77 (1.31– 2.38) ,0.001 1.66 (1.26–2.20) ,0.001
Cer(d18:1/24:0)e 0.66 (0.51–0.87) 0.003 0.74 (0.52– 1.05) 0.090 0.91 (0.65–1.29) 0.609
Cer(d18:1/24:1)e 1.23 (0.93–1.63) 0.154 1.74 (1.25– 2.42) 0.001 1.73 (1.27–2.36) ,0.001

Cer denotes ceramide.

a
CV death denotes death from MI, stroke, and heart failure.
b
The model was adjusted for TC, TG, HDL-C, and LDL-C.
c
The model was adjusted as for model 1 with additional adjustment for the Grace score (Killip class, systolic blood pressure, heart rate, age, creatinine, cardiac arrest at admission,
ST-segment deviation, and elevated cardiac enzyme levels).
d
Hazard ratios are for one standard deviation increase.
e
Natural logarithm of the ceramides.

variables to a new model with the Marschner score variables survived during follow-up (Table 2; Supplementary material online,
combined with the Cer(d18:1/16:0)/Cer(d18:1/24:0) ratio. The Table S2). For comparability with the Corogene results, non-
ceramides increased the cross-validated c-statistics from 0.78 adjusted and adjusted ORs for standard lipid markers and ceramides
(0.75 – 0.80) to 0.80 (0.77 – 0.82). Further, the predicted probabil- are given in Supplementary material online, Table S3, and the effects
ities for a 1-year event risk by logistic regression yielded an NRI of of statin treatment are accounted for the results in Supplementary
0.15 (95% CI 0.06 – 0.25; 9.6% improvement for events and 5.8% material online, Table S4. The incremental improvement of discrim-
improvement for non-events). ination for cardiac death was further demonstrated by adjusting for
standard lipids and the GRACE score (Table 4), and also by taking
into account diabetes mellitus and smoking status (Supplementary
Ceramides and risk prediction in acute material online, Table S6).
coronary syndromes patients The incremental prognostic value of ceramides was tested by
Another independent assessment of ceramides was performed in comparing the base model composed of the GRACE score to a
the SPUM-ACS cohort enrolling ACS patients. In 51 patients who new model with the GRACE score and the Cer(d18:1/16:0)/
died following a cardiac event within one-year-follow-up the cera- Cer(d18:1/24:0) ratio on top. The ceramide ratio increased
mide ratios were significantly higher compared with those who the cross-validated c-statistics from 0.73 (0.70 – 0.77) to 0.82
Plasma ceramides predict cardiovascular death Page 7 of 10

Table 5 Ceramide score and risk for cardiovascular death

BECAC (5-year risk) SPUM-ACS (1-year risk)

..................................................................................... ....................................................................................
Score No death Death % Relative risk Score No death Death % Relative risk
...............................................................................................................................................................................
0 –2 534 15 2.7% 1.0 0– 2 566 9 1.6% 1.0
3 –6 572 29 4.8% 1.8 3– 6 595 16 2.6% 1.7
7 –9 268 20 6.9% 2.5 7– 9 261 9 3.3% 2.1
10– 12 132 17 11.4% 4.2 10– 12 164 17 9.4% 6.0

LDL-C (mg/dl) No death Death % Relative risk LDL-C (mg/dl) No death Death % Relative risk
...............................................................................................................................................................................
≤100 513 36 6.6% 1.0 ≤106 532 27 4.8% 1.0
100 –143 572 29 4.8% 0.7 106–145 576 17 2.9% 0.6
143 –175 278 10 3.5% 0.5 145–174 260 3 1.1% 0.2
≥175 142 6 4.1% 0.6 ≥174 174 2 1.1% 0.2

See Supplementary material online, Table S11 for information on Ceramide Score calculation. To compare Ceramide Score performance with that of LDL-C study, subjects were
sorted according to their LDL-C levels and split into four categories in the same proportion as for the ceramide risk score.

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(0.79 – 0.85). Furthermore, the predicted probabilities for a 1-year (low – moderate – increased – high) and both in the BECAC and
event risk obtained by logistic regression yielded an NRI of 0.17 SPUM-ACS studies the risk increased along with the increasing
(95% CI 0.07 – 0.27; 8.2% improvement for events and 9.1% score (Table 5). In the stable CAD and ACS patients 4.2- and
improvement for non-events). 6.0-fold relative risk increase was observed when comparing
The performance of the ceramide ratio Cer(d18:1/16:0)/ the high- to low-risk category, respectively. When subjects were
Cer(d18:1/24:0) in predicting non-fatal MI was also investigated by sorted according to their LDL-C concentrations and split into
calculating the hazard ratios both for Q-wave and non-Q wave four categories in the same proportion as for the ceramide risk
MIs. The ratio showed a significant result for Q-wave MI, while score the enrichment of high-risk patients was not observed along
no significant results were obtained for non-Q wave infarctions with increasing LDL-C concentration.
(Supplementary material online, Table S7).

Ceramides and C-reactive protein Discussion

In the Corogene and SPUM-ACS studies, ceramides associated sig-
The present results provide evidence that distinct ceramide species
nificantly with LDL-C and C-reactive protein. Particularly, the CV
serve as significant predictors for cardiovascular death beyond
mortality-related Cer(d18:1/16:0) and Cer(d18:1/18:0) were posi-
currently used lipid markers in two patient groups—patients with
tively correlated with C-reactive protein, while small negative corre-
stable CAD and higher risk ACS patients . Importantly, the predic-
lations were seen in both studies between C-reactive protein and
tion also works in patients who are already statin treated and is
Cer(d18:1/24:0). Furthermore, the ‘protective’ Cer(d18:1/24:0)
therefore a potential indicator of residual risk.
had the strongest associations with LDL-C. Correlation coefficients
Battes et al. 17 recently performed a systematic review of models
for associations of ceramides and ceramide ratios with LDL-C and
predicting outcome in patients with stable CAD. The authors
C-reactive protein are presented in Supplementary material online,
concluded that risk stratification should be improved to predict re-
Tables S8 and S9.
current coronary events and to optimize secondary prevention
Finally, the synergy of C-reactive protein and Cer(d18:1/16:0)/
strategies. Our data using ceramides address this unmet need and
Cer(d18:1/24:0) in risk stratification was investigated by calculating
show robust performance for predicting coronary death both in
event rates in different quartiles for both BECAC and SPUM-ACS.
stable CAD and ACS patients. The present results do not prove
Especially in the SPUM-ACS study the highest enrichment of events
causality. However, it is tempting to speculate that ceramides are
(11.4% 1-year mortality) was observed if both the ceramide ratio
associated with plaque vulnerability as they are known to fuel
and C-reactive protein were in the highest quartile of the whole
many central atherosclerosis processes including lipoprotein aggre-
population (Supplementary material online, Table S10).
gation and uptake, inflammation, superoxide anion production, and
apoptosis8,18 – 21 (Supplementary material online, Figure S1). Several
Ceramide score and risk for enzymes of the sphingolipid synthesis have already been tested as
cardiovascular death potential drug targets as inhibition of glycosphingolipid biosynthesis
We have developed a tentative risk score (Supplementary material has been shown to decrease atherosclerosis in mice.10,22 Evidence
online, Table S11) based on ceramide concentrations and their ra- is also accumulating on ceramide chain-length-specific functions.
tios to model the clinical use of these risk predictors. Based on In a recent study, the relative increase in long-chain species (C16)
the score, the patients were placed into four risk categories but not in very-long-chain (C24-24:1) species was shown to
Page 8 of 10 R. Laaksonen et al.

mediate insulin resistance in mice.11,12 In Caenorhabditis elegans, Ceramide measurement in high-throughput quality controlled
long-chain ceramides were pro-apoptotic, and very-long-chain cer- environments is straightforward and cost-efficient. Isotope labelled
amides were anti-apoptotic.9 Consistently in the present study, standards enable precise quantification and analytical stability. Most
long-chain species (d18:1/16:0 and d18:1/18:0) were more harmful clinical laboratories are equipped with robotized sample handling
than very-long-chain (d18:1/24:0) species. Altered ceramide com- systems and also house mass spectrometry equipment.
positions may partially be explained by CerS isoforms, providing a Thus, ceramide-based identification of coronary patients at high
putative biological explanation for the use of ceramide ratios, and cardiovascular risk will soon be possible. These high-risk patients
possibilities for medical intervention (Supplementary material on- should then benefit from treatments that extend beyond standard
line, Figure S2). Interestingly, Cer(d18:1/24:1) behaved differently care. The suggested actions could include more frequent follow-up
compared with Cer(d18:1/24:0), emphasizing that additional regula- visits and efficient life-style counselling as well as the consideration
tion also takes place. While the current study reveals an association for higher statin doses, ezetimibe combinations, or novel therapies
between ceramides and CV events, it will be a highly interesting such as PCSK9 inhibitors. In future, additional therapies may include
topic for future investigations to establish if ceramide composition other options, for example, ongoing randomized clinical trials are
can be influenced and how it might translate to cardiovascular looking into the effect of methotrexate and interleukin-1b inhibition
benefit. The response of ceramides to lipid-lowering treatments for treating cardiovascular risk.28 Indeed, ceramides are closely
such as statins has been documented in our previous study.4 We linked to inflammatory processes and recently CERS6 has been
have observed that PCSK9 knock-out mice have significantly identified as a target for methotrexate.29 It is hence plausible to
reduced plasma ceramide concentrations and that human PCSK9 think that ceramide testing could become increasingly relevant,

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loss-of-function mutations are associated with lower plasma cera- especially if the trials with anti-inflammatory compounds turn out
mide concentrations compared with individuals carrying the major positive. A health economic dimension of ceramide testing is its abil-
alleles.4,23 Study limitations in addition to the lack of causality data ity to target more intense, potentially more expensive treatments
include the limited number of events both in BECAC and such as PCSK9 inhibitors to those at the highest risk. Another aspect
SPUM-ACS. Thus, the ceramide risk score derived from these of ceramide testing is its potential for motivating patient’s adher-
studies should be further validated in sizeable cohorts in order to ence, whether for medication or life-style changes, due to its rather
fine-tune the relative risk estimates for different risk categories. direct linkage with CV mortality. It has been shown that over 40% of
Finally, it is likely that the careful one-to-one case– control matching the patients prescribed statins are non-adherent, which may trans-
in the Corogene study is leading to somewhat optimistic biomarker late to many avoidable additional events and hospitalizations.30
results compared with a real-life patient care situation where con- While the ceramide-based risk stratification extends beyond the
trolling for confounding factors is more difficult. current lipid-based diagnostics and addresses the unmet need for
The lack of a discernible relationship between LDL-related improved identification of high-risk CAD patients there are further
parameters and CV risk across the studies included here, even after scientific and clinical issues that need attention. There are two major
statin stratification, is thought-provoking but, in line with previous lines of future research concerning the present ceramide correl-
reports.24 – 26 For example, Sachdeva et al.24 analysed admission lipid ation and cardiovascular risk. On one hand it is of interest to pursue
levels in a broad population of 136,995 patients hospitalized for the biology of these molecules and work out their molecular mech-
CAD in 541 hospitals and observed that nearly half of the admission anism of action in cardiovascular disease. This will involve multi-
LDL-C concentrations were ,100 mg/dl although before admis- disciplinary efforts of cell biologists, biochemists, geneticists, and
sion only 21.1% patients were receiving lipid-lowering medications. clinicians developing appropriate cell and animal models. Efforts in
Furthermore, in the MIRACL trial, the plasma HDL-C, but not this regard are already being made for example in the scope of
LDL-C, measured in the initial stage of ACS predicted the risk of the EU funded ‘EUFP7-Atheroflux’ consortium. The other line to
recurrent cardiovascular events.25 The lower LDL-C in higher risk follow is to establish the utility of these markers in clinical practice.
patients may not be a phenomenon of ACS solely as in the Saturn In the USA, the ceramide testing is entering the clinic this year and
trial investigators observed that C-reactive protein, but not only this real-life evaluation will allow for a better judgement of the
LDL-C levels, were associated with coronary atheroma regression ceramide utility and will establish them as a new armament in the
and cardiovascular events after intensive statin therapy.26 Taken clinical diagnostic tool-kit.
together, it appears that the LDL-C concentrations may be similar
or even lower in CAD patients at high risk for future CV events
compared with patients with more favorable prognosis. This may
Supplementary material
be a phenomenon related to disease progression and culmination, Supplementary material is available at European Heart Journal online.
and should not detract from the value of applying LDL-C to gauge
the lifetime risk to develop atherosclerotic plaques. A potential ex-
planation for this is provided in Gierens et al. 27 who demonstrated
Authors’ contributions
that interleukin-6 (IL-6) activates LDL-receptor (LDLr) transcrip- M. S.-A., M. H., M. S. performed statistical analysis; R. L., R. H., M. N.,
tion and subsequently enhances LDLr activity in the liver leading J. S., O. N., T. L., W. M. handled funding and supervision; K. E., D. K.,
to an increased elimination of LDL-C from the circulation. Thus, H. S., T. S., E. V., M.-L. L., R. K., C. M., T. H., P. J., N. R., L. R., S. W.,
chronic, and in particular acute bursts of, inflammation in CAD B. G., E. R. P., G. S. T., F. M. acquired the data; R. L., R. H., J. S., T. L.,
patients may enhance LDL-C clearance, resulting in lowered blood F. M., O. N. conceived and designed the research; R. L., T. V. drafted
LDL-C concentrations and impaired risk prediction. the manuscript; K. E., M. S.-A., M. H., R. H., D. K., W. M., H. S., T. S.,
Plasma ceramides predict cardiovascular death Page 9 of 10

E. V., M. L. L., M. N., R. K., C. M., T. H., P. J., N. R., L. R., S. W., E. P., Senior R, Taggart DP, van der Wall EE, Vrints CJM, Zamorano JL,
Baumgartner H, Bax JJ, Bueno H, Dean V, Deaton C, Erol C, Fagard R, Ferrari R,
G. T., B. G., F. M., J. S., O. N., T. L. made critical revision of the manu-
Hasdai D, Hoes AW, Kirchhof P, Knuuti J, Kolh P, Lancellotti P, Linhart A,
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33CM30-124112), the Swiss Heart Foundation, both Bern Switzerland,
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Regional Health Authority (911570). The funders had no role in the plaques by radiofrequency intravascular ultrasound and cardiovascular outcome:
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MSD, Eli Lilly, and St. Jude Medical including speaker or consultant fees. Mauer J, Xu E, Hammerschmidt P, Brönneke HS, Trifunovic A, LoSasso G,
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