.....................................................................................................................................................................................
CLINICAL RESEARCH
Co ona y a e y disease
Plasma ce amides p edic ca dio ascula dea h
in pa ien s wi h s able co ona y a e y disease
and acu e co ona y synd omes beyond
LDL-choles e ol
Reijo Laaksonen1,2,3*, Kim Ek oos1, Ma ko Sysi-Aho1, Mika Hil o1, Te hi Vihe aa a1,
Dimple Kauhanen1, Ma i Suoniemi1, Reini Hu me1, Win ied Ma
¨ z4,5,
Hube Scha nagl6, Ta jana S ojako ic6, E hymia Vlachopoulou7, Ma ja-Liisa Lokki7,
Ma kku S. Nieminen7,8, Roland Klingenbe g9, Ch is ian M. Ma e 9,
Tho s en Ho nemann10, Pe e Ju
¨ni11, Nicolas Rodondi12,13, Lo enz Ra
¨be 14,
S ephan Windecke 14, Ba is Gence 15, E a Ringdal Pede sen16, G e he S. Tell17,
O a Nyga
˚ d16,18†, F ancois Mach15†, Juha Sinisalo7,8†, and Thomas F. Lu
¨sche 10†
1
Zo a Biosciences, Espoo, Finland;
2
Medical School, Tampe e Uni e si y, Tampe e, Finland;
3
Finnish Clinical Biobank Tampe e, Uni e si y Hospi al o Tampe e, Tampe e, Finland;
4
Medical Clinic V (Neph ology, Hype ensiology, Rheuma ology, Endoc inology, Diabe ology), Medical Facul y Mannheim, Uni e si y o Heidelbe g, Heidelbe g, Ge many;
5
synlab
Academy, synlab Holding Deu schland GmbH, Mannheim and Augsbu g, Ge many;
6
Clinical Ins i u e o Medical and Chemical Labo a o y Diagnos ics, Medical Uni e si y G az, G az,
Aus ia;
7
T ansplan a ion Labo a o y, Haa man Ins i u e, Uni e si y o Helsinki, Helsinki, Finland;
8
Hea and Lung Cen e , Helsinki Uni e si y Hospi al, Helsinki, Finland;
9
Depa men
o Ca diology, Uni e si y Hea Cen e , Uni e si y Hospi al Zu¨ ich and Uni e si y o Zu¨ ich, Zu¨ ich, Swi ze land;
10
Ins i u e o Clinical Chemis y, Uni e si y Hospi al, Zu¨ ich,
Swi ze land;
11
Applied Heal h Resea ch Cen e (AHRC), Li Ka Shing Knowledge Ins i u e o S . Michael’s Hospi al, and Depa men o Medicine, Uni e si y o To on o, To on o,
Canada;
12
Depa men o Gene al In e nal Medicine, Uni e si y Hospi al Be n, Be n, Swi ze land;
13
Depa men o Ambula o y Ca e and Communi y Medicine, Uni e si y o Lausanne,
Lausanne, Swi ze land;
14
Ca dio ascula Cen e , Depa men o Ca diology, Uni e si y Hospi al Be n, Be n, Swi ze land;
15
Ca dio ascula Cen e , Depa men o Ca diology,
Uni e si y Hospi al Gene a, Gene a, Swi ze land;
16
Depa men o Clinical Science, Uni e si y o Be gen, Be gen, No way;
17
Depa men o Global Public Heal h and P ima y Ca e,
Uni e si y o Be gen, Be gen, No way; and
18
Depa men o Hea Disease, Haukeland Uni e si y Hospi al, Be gen, No way
Recei ed 8 Janua y 2016; e ised 15 Feb ua y 2016; accep ed 17 Ma ch 2016; online publish-ahead-o -p in 28 Ap il 2016
Aims The aim was o s udy he p ognos ic alue o plasma ce amides (Ce ) as ca dio ascula dea h (CV dea h) ma ke s in
h ee independen co ona y a e y disease (CAD) coho s.
Me hods
and esul s
Co ogene s udy is a p ospec i e Finnish coho including s able CAD pa ien s (n¼160). Mul iple lipid bioma ke s and
C- eac i e p o ein we e measu ed in addi ion o plasma Ce (d18:1/16:0), Ce (d18:1/18:0), Ce (d18:1/24:0), and
Ce (d18:1/24:1). Subsequen ly, he associa ion be ween high- isk ce amides and CV mo ali y was in es iga ed in
he p ospec i e Special P og am Uni e si y Medicine—In lamma ion in Acu e Co ona y Synd omes (SPUM-ACS)
coho (n¼1637), conduc ed in ou Swiss uni e si y hospi als. Finally, he esul s we e alida ed in Be gen Co ona y
Angiog aphy Coho (BECAC), a p ospec i e No wegian coho s udy o s able CAD pa ien s. Ce amides, especially
when used in a ios, we e signi ican ly associa ed wi h CV dea h in all s udies, independen o o he lipid ma ke s and
C- eac i e p o ein. Adjus ed odds a ios pe s anda d de ia ion o he Ce (d18:1/16:0)/Ce (d18:1/24:0) a io we e
4.49 (95% CI, 2.24–8.98), 1.64 (1.29–2.08), and 1.77 (1.41–2.23) in he Co ogene, SPUM-ACS, and BECAC s udies,
espec i ely. The Ce (d18:1/16:0)/Ce (d18:1/24:0) a io imp o ed he p edic i e alue o he GRACE sco e
(ne eclassi ica ion imp o emen , NRI ¼0.17 and DAUC ¼0.09) in ACS and he p edic i e alue o he Ma schne
sco e in s able CAD (NRI ¼0.15 and DAUC ¼0.02).
*Co esponding au ho . Zo a Biosciences Oy, Biologinkuja 1, 02150 Espoo, Finland. Tel: +358 40 724 077, Email: [email p o ec ed]
†
Equal con ibu ion.
&The Au ho 2016. Published by Ox o d Uni e si y P ess on behal o he Eu opean Socie y o Ca diology.
This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License (h p://c ea i ecommons.o g/licenses/by/4.0/), which pe mi s un es ic ed
euse, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed.
Eu opean Hea Jou nal (2016) 37, 1967–1976
doi:10.1093/eu hea j/ehw148
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Conclusions Dis inc plasma ce amide a ios a e signi ican p edic o s o CV dea h bo h in pa ien s wi h s able CAD and ACS, o e
and abo e cu en ly used lipid ma ke s. This may imp o e he iden i ica ion o high- isk pa ien s in need o mo e ag-
g essi e he apeu ic in e en ions.
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Keywo ds Ce amide †Acu e co ona y synd ome †Co ona y a e y disease †Bioma ke †LDL-choles e ol †Risk
p edic ion †P ognosis
In oduc ion
Gi en he high p e alence o co ona y a e y disease (CAD) and
associa ed mo ali y, p e en ion o a al and non- a al myoca dial
in a c ions (MI) in CAD pa ien s emains an ongoing clinical chal-
lenge. Mo ali y a es among s able CAD pa ien s ange be ween
1% and 3%, while a es o non- a al e en s a e 1–2% annually.
1
In
pa ien s wi h acu e co ona y synd omes (ACS) who su i e he
acu e e en , he a e o MI and dea h is ma kedly highe , pa icula ly
du ing he i s yea .
2
Howe e , a he indi idual le el, he e en isk
may a y conside ably, which makes isk es ima ion ools necessa y
o imp o e pa ien managemen . Expedien isk s a i ica ion should
iden i y indi iduals a isk equi ing mo e in ensi e he apy. Con-
e sely, pa ien s wi h a a o able p ognosis should be iden i ied o
a oid d ug o e use and associa ed side e ec s.
3
Hypo hesis ee lipidomic analyses ha e e ealed a hand ul o
lipids po en ially quali ying as use ul p ognos ic ma ke s o
CAD.
4–6
In ou ini ial lipidomic s udy, dis inc ce amide species
we e signi ican ly associa ed wi h CVD among CAD pa ien s.
4
Mo-
lecula lipid species, pa icula ly ce amide(d18:1/16:0), we e also as-
socia ed wi h nec o ic co e issue ype and lipid co e bu den in
co ona y angiog aphy, and we e p edic i e o 1-yea clinical ou -
come in 581 ACS and s able CAD pa ien s.
7
In hese s udies, plasma
CVD isk- ela ed ce amide molecules (Ce (d18:1/16:0), Ce (d18:1/
18:0), and Ce (d18:1/24:1)), and hei a ios wi h Ce (d18:1/24:0),
eme ged as po en ial isk s a i ie s o CAD pa ien s.
4
Ce amides
a e known o associa e wi h many cen al p ocesses o a he oscle -
osis de elopmen including lipop o ein up ake, in lamma ion, and
apop osis (Supplemen a y ma e ial online, Figu e S1).
8
Ce amide
species a e p oduced by six a y acyl selec i e ce amide syn hases
(Ce Ss; Supplemen a y ma e ial online, Figu e S2), and i is becoming
e iden ha indi idual ce amide species ha e speci ic physiological
unc ions.
9–12
Thus, moni o ing a ios o ce amides species may
p o ide insigh in o he me abolic egula ion o a he oscle o ic
e en s. In his s udy, we es ablish he sugges ed ole o ce amides
and hei dis inc a ios as isk p edic o s o CV dea h in pa ien s
wi h s able CAD and ACS.
Me hods
Mo e de ailed me hod desc ip ions a e a ailable in Supplemen a y
ma e ial online.
S udy subjec s
Co ogene s udy: s able co ona y a e y disease pa ien s
Co ogene is a p ospec i e, consecu i e coho s udy o Finnish pa ien s
e e ed o co ona y angiog aphy o he Helsinki Uni e si y Cen al
Hospi al be ween 2006 and 2008. A nes ed case con ol s udy was de-
signed using he Co ogene da abase and including da a om he na ional
dea h ce i ica e egis y. As cases, all pa ien s who expe ienced co on-
a y dea h wi hin an a e age ollow-up o 21
2yea s we e selec ed.
Ma ched con ol pa ien s had es ablished CAD (.50% s enosis a leas
in one epica dial co ona y a e y), bu emained ali e du ing he ollow-
up pe iod. Baseline cha ac e is ics o he Co ogene subjec s a e shown
in Table 1and Supplemen a y ma e ial online, Table S1.
Be gen Co ona y Angiog aphy Coho coho : pa ien s
wi h s able co ona y a e y disease
The Be gen Co ona y Angiog aphy Coho (BECAC) includes 1580
adul s e e ed o elec i e co ona y angiog aphy because o suspec ed
s able angina pec o is ec ui ed a he Haukeland Uni e si y Hospi al in
Be gen, No way be ween 2000 and 2004. In o ma ion on ca dio ascu-
la dea hs was collec ed om he Cause o Dea h Regis y a he No -
wegian Ins i u e o Public Heal h, and e i ied agains hospi al medical
eco ds whene e a ailable. Du ing a median ollow-up o 4.6 yea s, a
o al o 81 pa ien s died om ca dio ascula disease. Baseline cha ac e -
is ics o he BECAC pa icipan s a e epo ed in Table 1and Supplemen-
a y ma e ial online, Table S1.
SPUM-ACS coho : pa ien s wi h acu e co ona y
synd omes
Special P og am Uni e si y Medicine—In lamma ion in Acu e Co ona y
Synd omes (SPUM-ACS) is a p ospec i e, mul i-cen e (Be n, Gene a,
Lausanne, and Zu¨ ich) coho s udy. Pa ien s wi h a p ima y diagnosis o
ACS and e e ed o in asi e managemen we e en olled a ou Swiss
uni e si y hospi als. Baseline cha ac e is ics o he SPUM-ACS pa ien s
a e summa ized in Table 1and Supplemen a y ma e ial online, Table S1.
A one-yea ollow-up, a o al o 51 pa ien s died om ca diac easons.
Clinical labo a o y analyses
S anda d lipids measu emen s we e de e mined using s anda d me hods
a ailable a each o he h ee s udy si es. In Co ogene subjec s, apolipo-
p o eins (AI, AII, and B), lipop o ein (a), lipop o ein-associa ed phospho-
lipase A2 ac i i y, and HDL and LDL pa icle numbe s and sizes we e
measu ed as desc ibed in Supplemen a y ma e ial online, Me hods.
Quan i ica ion o ce amides
The plasma le els o Ce (d18:1/16:0), Ce (d18:1/18:0), Ce (d18:1/24:0),
and Ce (d18:1/24:1) we e quan i ied on a 5500 QTRAP (SCIEX, F a-
mingham, MA) mass spec ome e equipped wi h an Eksigen 100-XL
UHPLC sys em as desc ibed ecen ly.
13
S a is ical analyses
Wilcoxon’s ank sum es was applied o g oup compa isons. Odds a-
ios (ORs) pe s anda d de ia ion we e es ima ed using logis ic eg es-
sion. Haza d a ios we e calcula ed using he Cox p opo ional haza d
model. The GRACE
14
isk sco e, consis ing o Killip class, sys olic blood
p essu e, hea a e, age, c ea inine, ca diac a es a admission,
ST-segmen de ia ion, and ele a ed ca diac enzyme le els ( oponin,
CK-MB), was used o calcula e he isk o long- e m mo ali y o
ACS pa ien s. The ollowing Ma schne sco e
15
a iables we e used in
R. Laaksonen e al.1968
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he modeling o s able CAD pa ien da a: o al choles e ol, HDL-C, age,
gende , smoking s a us, p e ious acu e MI, diabe es, hype ension, and
p io s oke.
Ne eclassi ica ion imp o emen was es ima ed as desc ibed by
Pencina e al.
16
Fo he 1-yea e en isk o he seconda y p e en ion
popula ion in he SPUM-ACS s udy we ca ego ized subjec s o low
isk (,1% e en p obabili y), in e media e (1–5%) isk o high- isk
(.5%) g oups. Fo he BECAC s udy he same ca ego iza ion was
used o 3-yea isk.
The ce amide isk sco e was calcula ed as ollows: Fo each indi idual, all
h eece amide a iosandeachconcen a ion (apa om Ce (d18:1/24:0))
we e compa ed wi h he whole s udy popula ion. I he a iable belonged
o he 3 d qua ile, he indi idual ecei ed +1 poin , and i o he 4 h qua -
ile, +2 poin s (Supplemen a y ma e ial online, Table S11). Thus, he sco e
anges om0 o12andbasedon hesco e, hesubjec swe espli in o
ou isk ca ego ies (0–2, 3–6, 7–9, and 10–12).
Mo e de ails on s a is ical me hods can be ound in Supplemen a y
ma e ial online.
Resul s
Ce amide concen a ions in high- and low-
isk pa ien s wi h co ona y a e y disease
In s able CAD pa ien s o he Co ogene s udy LDL-based ma ke s
such as LDL-C, LDL pa icle numbe (LDL-P), small dense LDL
(sdLDL), and apoB did no di e signi ican ly be ween cases who ex-
pe ienced co ona y dea h and con ols who emained ali e, and nei-
he did Lp(a) no Lp-PLA2. Howe e , he HDL- ela ed ma ke s
HDL-C, HDL pa icle numbe (HDL-P), small dense HDL (sdHDL),
and ApoA1 we e all signi ican ly (P,0.001) di e en be ween
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Table 1 Baseline cha ac e is ics o he subjec s in Co ogene, SPUM-ACS and BECAC s udies
Cha ac e is ic COROGENE SPUM-ACS BECAC
Cases Con ols Cases Con ols Cases Con ols
No o subjec s 80 80 51 1586 81 1506
Gende
Male, n(%) 60 (75%) 60 (75%) 42 (82%) 1223 (77%) 55 (68%) 889 (59%)
Age (yea s) 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 o dea h/ ollow-up ime (days) 528 (134–739) 1955 (1669–2173) 25 (7–216) 365 (359–365) 626 (196–1332) 1720 (1368–2111)
C ea inine (mmol/L) 98 (84–134) 79 (69–90) 100 (79–134) 75 (65–88) 98 (87–115) 87 (79–97)
Cu en smoke
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%)
Diabe es
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%)
Hype ension
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-lowe ing ea men
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%)
P e ious 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%)
P e ious s oke
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%)
Plasma ce amides p edic ca dio ascula dea h 1969
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he g oups wi h he medians being 217.1, 214.3, 219.0, and
212.9% lowe in cases, espec i ely. The di e ences be ween cases
and con ols in plasma ce amides and es ablished lipid ma ke s a e
p o ided in Table 2( he pe cen age o obse a ions o each ma ke
is p o ided in Supplemen a y ma e ial online, Table S2).
In he Co ogene s udy, he concen a ions o Ce (d18:1/16:0),
Ce (d18:1/18:0), and Ce (d18:1/24:1) we e signi ican ly di e en
(P,0.001 o all) be ween cases who had a a al MI du ing
he ollow-up pe iod and con ols (medians in cases +17.0%,
+10.3, and +11.2% highe han in con ols, espec i ely). In con-
as , he Ce (d18:1/24:0) beha ed di e en ly, wi h he median in
cases being 214.9% lowe han in con ols (P,0.001). Simila ly
o ou ea lie obse a ions,
4
highly signi ican di e ences, we e
obse ed o he h ee p ede ined ce amide a ios, wi h he med-
ians o cases anging be ween +25.7 and +34.8% (P,0.001)
ela i e o con ols. The di e ence be ween s able CAD pa ien s
and con ols is illus a ed in Supplemen a y ma e ial online,
Figu e S3.
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Table 2 Medians and in e qua ile anges o es ablished lipid ma ke s and ce amides in case and con ol g oups
a
BECAC SPUM-ACS
Cases (n581) Con ols (n51499) P- alue Cases (n551) Con ols (n51586) P- alue
Ce (d18:1/16:0)/Ce (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
Ce (d18:1/18:0)/Ce (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
Ce (d18:1/24:1)/Ce (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
Ce (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
Ce (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
Ce (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
Ce (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
Cases (n580) Con ols (n580) P- alue
Ce (d18:1/16:0)/Ce (d18:1/24:0) 0.132 (0.105–0.175) 0.105 (0.090–0.128) ,0.001
Ce (d18:1/18:0)/Ce (d18:1/24:0) 0.062 (0.047–0.077) 0.046 (0.037–0.062) ,0.001
Ce (d18:1/24:1)/Ce (d18:1/24:0) 0.703 (0.582–0.846) 0.556 (0.483–0.665) ,0.001
Ce (d18:1/16:0) (mmol/L) 0.275 (0.222–0.326) 0.235 (0.212–0.282) 0.007
Ce (d18:1/18:0) (mmol/L) 0.118 (0.094–0.152) 0.107 (0.092–0.137) 0.195
Ce (d18:1/24:0) (mmol/L) 1.923 (1.475–2.511) 2.235 (1.993–2.672) 0.008
Ce (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- eac i e p o ein (mg/L) 3.1 (1.6–8.7) 1.1 (0.7–2.8) ,0.001
a
Ce , ce amide; TC, o al choles e ol; TG, iacylglyce ols, LDL-C low-densi y lipop o ein choles e ol, HDL-C high-densi y lipop o ein choles e ol, sdLDL small dense low-densi y
lipop o ein choles e ol, LDL-P low-densi y lipop o ein pa icle numbe , sdHDL small dense high-densi y lipop o ein choles e ol, HDL-P high-densi y lipop o ein pa icle numbe ,
ApoB apolipop o ein B, ApoA1 apolipop o ein A1, Lp(a) lipop o ein (a), Lp-PLA2 lipop o ein-associa ed phospholipase A2. SI con e sion ac o s: To con e choles e ol o
mmol/L, mul iply alues by 0.0259; o con e iacylglyce ols o mmol/L, mul iply alues by 0.01129.
R. Laaksonen e al.1970
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Ce amide a ios in co ona y a e y
disease pa ien s
In he Co ogene s udy, he mos signi ican ORs o co ona y dea h
we e ound o HDL ma ke s and ce amide a ios. Ce amide ORs e-
mained p edic i e a e adjus men o con en ional lipid ma ke s
LDL-C, HDL-C, o al choles e ol, iacylglyce ols, and C- eac i e p o-
ein. Lp-PLA2 and Lp(a) had no signi ican associa ion wi h CV
mo ali y.
LDL-C and LDL pa icle numbe we e in e sely associa ed wi h
isk (LDL-C unadjus ed uppe qua ile OR 0.89 95% CI 0.37–2.14;
LDL-P uppe qua ile OR 0.67, 95%CI 0.29–1.59). Fo compa ison,
he unadjus ed uppe qua ile OR o he Ce (d18:1/16:0)/
Ce (d18:1/24:0) a io was 10.33 (95% CI 3.69–28.97). Figu e 1
shows non-adjus ed and adjus ed ORs o di e en lipid ma ke s
and ce amides in he Co ogene s udy.
Ce amides and isk in s able co ona y
a e y disease pa ien s
An independen assessmen o ce amides was pe o med in a co-
ho (BECAC) o s able pa ien s. We ound ha he p ede ined
ce amide a ios we e signi ican ly highe in 81 pa ien s who died ol-
lowing a CV e en wi hin 4.6-yea ollow-up compa ed wi h hose
who did no die du ing ollow-up (Table 2; Supplemen a y ma e ial
online, Table S2). Fo compa abili y wi h he Co ogene esul s, non-
adjus ed and adjus ed ORs o s anda d lipid ma ke s and ce amides
a e gi en in Supplemen a y ma e ial online, Table S3. The inc emen-
al imp o emen o disc imina ion o CV dea h was u he demon-
s a ed by calcula ing haza d a ios adjus ed o s anda d lipids and
he Ma schne sco e a iables (Table 3).
Adjus men o s a in ea men did no ha e a majo impac on he
esul s (Supplemen a y ma e ial online, Table S4). The odds a ios
we e calcula ed o ce amides and LDL-C also in pa ien s ha we e
on o no on s a in ea men bo h a baseline and a e 1-yea o
ollow-up (Supplemen a y ma e ial online, Table S5). Ce amides
we e p edic i e in bo h ins ances, al hough in pa ien s wi hou s a in
ea men he odds a ios we e be e . LDL-C did no show signi ican
p edic i e alue, con i ming ha he lack o a di ec associa ion
be ween LDL-C and CV dea h was no caused by in e e ence wi h
s a in ea men .
The inc emen al p ognos ic alue o ce amides was es ed by
compa ing he base model composed o he Ma schne sco e
Figu e 1 Ca dio ascula dea h odds a ios o pe s anda d de ia ion and 4 h qua ile o di e en lipid ma ke s and ce amides in Co ogene
s udy. Adjus men is made o o al choles e ol, iacylglyce ols, LDL-C, HDL-C, and C- eac i e p o ein.
Plasma ce amides p edic ca dio ascula dea h 1971
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a iables o a new model wi h he Ma schne sco e a iables
combined wi h he Ce (d18:1/16:0)/Ce (d18:1/24:0) a io. The
ce amides inc eased he c oss- alida ed c-s a is ics om 0.78
(0.75–0.80) o 0.80 (0.77–0.82). Fu he , he p edic ed p obabil-
i ies o a 1-yea e en isk by logis ic eg ession yielded an NRI o
0.15 (95% CI 0.06–0.25; 9.6% imp o emen o e en s and 5.8%
imp o emen o non-e en s).
Ce amides and isk p edic ion in acu e
co ona y synd omes pa ien s
Ano he independen assessmen o ce amides was pe o med in
he SPUM-ACS coho en olling ACS pa ien s. In 51 pa ien s who
died ollowing a ca diac e en wi hin one-yea - ollow-up he ce a-
mide a ios we e signi ican ly highe compa ed wi h hose who
su i ed du ing ollow-up (Table 2; Supplemen a y ma e ial online,
Table S2). Fo compa abili y wi h he Co ogene esul s, non-
adjus ed and adjus ed ORs o s anda d lipid ma ke s and ce amides
a e gi en in Supplemen a y ma e ial online, Table S3, and he e ec s
o s a in ea men a e accoun ed o he esul s in Supplemen a y
ma e ial online, Table S4. The inc emen al imp o emen o disc im-
ina ion o ca diac dea h was u he demons a ed by adjus ing o
s anda d lipids and he GRACE sco e (Table 4), and also by aking
in o accoun diabe es melli us and smoking s a us (Supplemen a y
ma e ial online, Table S6).
The inc emen al p ognos ic alue o ce amides was es ed by
compa ing he base model composed o he GRACE sco e o a
new model wi h he GRACE sco e and he Ce (d18:1/16:0)/
Ce (d18:1/24:0) a io on op. The ce amide a io inc eased
he c oss- alida ed c-s a is ics om 0.73 (0.70–0.77) o 0.82
.......................................... .......................................... ..........................................
...............................................................................................................................................................................
Table 3 Associa ion be ween ce amides and ca dio ascula dea h in BECAC
a
Uni a ia e model Mul i a iable
b
model 1
b
Mul i a iable
c
model 2
c
Haza d a io
d
(95% CI) P- alue Haza d a io
d
(95% CI) P- alue Haza d a io
d
(95% CI) P- alue
Ce (d18:1/16:0)/Ce (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
Ce (d18:1/18:0)/Ce (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
Ce (d18:1/24:1)/Ce (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
Ce (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
Ce (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
Ce (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
Ce (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
Ce deno es ce amide.
a
CV dea h deno es dea h om MI, s oke, and hea ailu e.
b
The model was adjus ed o TC, TG, HDL-C, and LDL-C.
c
The model was adjus ed as o model 1 wi h addi ional adjus men o he ollowing Ma schne sco e a iables: age, gende , smoking s a us, p e ious acu e MI, diabe es,
hype ension, and p io s oke.
d
Haza d a ios a e o 1 SD inc ease.
e
Na u al loga i hm o he ce amides and ce amide a io.
........................................... ........................................... ...........................................
...............................................................................................................................................................................
Table 4 Associa ion be ween ce amides and ca dio ascula dea h in SPUM-ACS
a
Uni a ia e model Mul i a iable
b
model 1
b
Mul i a iable
c
model 2
c
Haza d a io
d
(95% CI) P- alue Haza d a io
d
(95% CI) P- alue Haza d a io
d
(95% CI) P- alue
Ce (d18:1/16:0)/Ce (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
Ce (d18:1/18:0)/Ce (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
Ce (d18:1/24:1)/Ce (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
Ce (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
Ce (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
Ce (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
Ce (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
Ce deno es ce amide.
a
CV dea h deno es dea h om MI, s oke, and hea ailu e.
b
The model was adjus ed o TC, TG, HDL-C, and LDL-C.
c
The model was adjus ed as o model 1 wi h addi ional adjus men o he G ace sco e (Killip class, sys olic blood p essu e, hea a e, age, c ea inine, ca diac a es a admission,
ST-segmen de ia ion, and ele a ed ca diac enzyme le els).
d
Haza d a ios a e o one s anda d de ia ion inc ease.
e
Na u al loga i hm o he ce amides.
R. Laaksonen e al.1972
by gues on Sep embe 19, 2016h p://eu hea j.ox o djou nals.o g/Downloaded om
(0.79–0.85). Fu he mo e, he p edic ed p obabili ies o a 1-yea
e en isk ob ained by logis ic eg ession yielded an NRI o 0.17
(95% CI 0.07–0.27; 8.2% imp o emen o e en s and 9.1%
imp o emen o non-e en s).
The pe o mance o he ce amide a io Ce (d18:1/16:0)/
Ce (d18:1/24:0) in p edic ing non- a al MI was also in es iga ed by
calcula ing he haza d a ios bo h o Q-wa e and non-Q wa e
MIs. The a io showed a signi ican esul o Q-wa e MI, while
no signi ican esul s we e ob ained o non-Q wa e in a c ions
(Supplemen a y ma e ial online, Table S7).
Ce amides and C- eac i e p o ein
In he Co ogene and SPUM-ACS s udies, ce amides associa ed sig-
ni ican ly wi h LDL-C and C- eac i e p o ein. Pa icula ly, he CV
mo ali y- ela ed Ce (d18:1/16:0) and Ce (d18:1/18:0) we e posi-
i ely co ela ed wi h C- eac i e p o ein, while small nega i e co e-
la ions we e seen in bo h s udies be ween C- eac i e p o ein and
Ce (d18:1/24:0). Fu he mo e, he ‘p o ec i e’ Ce (d18:1/24:0)
had he s onges associa ions wi h LDL-C. Co ela ion coe icien s
o associa ions o ce amides and ce amide a ios wi h LDL-C and
C- eac i e p o ein a e p esen ed in Supplemen a y ma e ial online,
Tables S8 and S9.
Finally, he syne gy o C- eac i e p o ein and Ce (d18:1/16:0)/
Ce (d18:1/24:0) in isk s a i ica ion was in es iga ed by calcula ing
e en a es in di e en qua iles o bo h BECAC and SPUM-ACS.
Especially in he SPUM-ACS s udy he highes en ichmen o e en s
(11.4% 1-yea mo ali y) was obse ed i bo h he ce amide a io
and C- eac i e p o ein we e in he highes qua ile o he whole
popula ion (Supplemen a y ma e ial online, Table S10).
Ce amide sco e and isk o
ca dio ascula dea h
We ha e de eloped a en a i e isk sco e (Supplemen a y ma e ial
online, Table S11) based on ce amide concen a ions and hei a-
ios o model he clinical use o hese isk p edic o s. Based on
he sco e, he pa ien s we e placed in o ou isk ca ego ies
(low–mode a e–inc eased–high) and bo h in he BECAC and
SPUM-ACS s udies he isk inc eased along wi h he inc easing
sco e (Table 5). In he s able CAD and ACS pa ien s 4.2- and
6.0- old ela i e isk inc ease was obse ed when compa ing
he high- o low- isk ca ego y, espec i ely. When subjec s we e
so ed acco ding o hei LDL-C concen a ions and spli in o
ou ca ego iesin hesamep opo ionas o hece amide isk
sco e he en ichmen o high- isk pa ien s was no obse ed along
wi h inc easing LDL-C concen a ion.
Discussion
The p esen esul s p o ide e idence ha dis inc ce amide species
se e as signi ican p edic o s o ca dio ascula dea h beyond
cu en ly used lipid ma ke s in wo pa ien g oups—pa ien s wi h
s able CAD and highe isk ACS pa ien s . Impo an ly, he p edic-
ion also wo ks in pa ien s who a e al eady s a in ea ed and is
he e o e a po en ial indica o o esidual isk.
Ba es e al.
17
ecen ly pe o med a sys ema ic e iew o models
p edic ing ou come in pa ien s wi h s able CAD. The au ho s
concluded ha isk s a i ica ion should be imp o ed o p edic e-
cu en co ona y e en s and o op imize seconda y p e en ion
s a egies. Ou da a using ce amides add ess his unme need and
show obus pe o mance o p edic ing co ona y dea h bo h in
s able CAD and ACS pa ien s. The p esen esul s do no p o e
causali y. Howe e , i is emp ing o specula e ha ce amides a e
associa ed wi h plaque ulne abili y as hey a e known o uel
many cen al a he oscle osis p ocesses including lipop o ein agg e-
ga ion and up ake, in lamma ion, supe oxide anion p oduc ion, and
apop osis
8,18–21
(Supplemen a y ma e ial online, Figu e S1). Se e al
enzymes o he sphingolipid syn hesis ha e al eady been es ed as
po en ial d ug a ge s as inhibi ion o glycosphingolipid biosyn hesis
has been shown o dec ease a he oscle osis in mice.
10,22
E idence
is also accumula ing on ce amide chain-leng h-speci ic unc ions.
In a ecen s udy, he ela i e inc ease in long-chain species (C16)
bu no in e y-long-chain (C24-24:1) species was shown o
..................................................................................... ....................................................................................
...............................................................................................................................................................................
...............................................................................................................................................................................
Table 5 Ce amide sco e and isk o ca dio ascula dea h
BECAC (5-yea isk) SPUM-ACS (1-yea isk)
Sco e No dea h Dea h % Rela i e isk Sco e No dea h Dea h % Rela i e isk
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 dea h Dea h % Rela i e isk LDL-C (mg/dl) No dea h Dea h % Rela i e isk
≤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 Supplemen a y ma e ial online, Table S11 o in o ma ion on Ce amide Sco e calcula ion. To compa e Ce amide Sco e pe o mance wi h ha o LDL-C s udy, subjec s we e
so ed acco ding o hei LDL-C le els and spli in o ou ca ego ies in he same p opo ion as o he ce amide isk sco e.
Plasma ce amides p edic ca dio ascula dea h 1973
by gues on Sep embe 19, 2016h p://eu hea j.ox o djou nals.o g/Downloaded om
media e insulin esis ance in mice.
11,12
In Caeno habdi is elegans,
long-chain ce amides we e p o-apop o ic, and e y-long-chain ce -
amides we e an i-apop o ic.
9
Consis en ly in he p esen s udy,
long-chain species (d18:1/16:0 and d18:1/18:0) we e mo e ha m ul
han e y-long-chain (d18:1/24:0) species. Al e ed ce amide com-
posi ions may pa ially be explained by Ce S iso o ms, p o iding a
pu a i e biological explana ion o he use o ce amide a ios, and
possibili ies o medical in e en ion (Supplemen a y ma e ial on-
line, Figu e S2). In e es ingly, Ce (d18:1/24:1) beha ed di e en ly
compa ed wi h Ce (d18:1/24:0), emphasizing ha addi ional egula-
ion also akes place. While he cu en s udy e eals an associa ion
be ween ce amides and CV e en s, i will be a highly in e es ing
opic o u u e in es iga ions o es ablish i ce amide composi ion
can be in luenced and how i migh ansla e o ca dio ascula
bene i . The esponse o ce amides o lipid-lowe ing ea men s
such as s a ins has been documen ed in ou p e ious s udy.
4
We
ha e obse ed ha PCSK9 knock-ou mice ha e signi ican ly
educed plasma ce amide concen a ions and ha human PCSK9
loss-o - unc ion mu a ions a e associa ed wi h lowe plasma ce a-
mide concen a ions compa ed wi h indi iduals ca ying he majo
alleles.
4,23
S udy limi a ions in addi ion o he lack o causali y da a
include he limi ed numbe o e en s bo h in BECAC and
SPUM-ACS. Thus, he ce amide isk sco e de i ed om hese
s udies should be u he alida ed in sizeable coho s in o de o
ine- une he ela i e isk es ima es o di e en isk ca ego ies.
Finally, i is likely ha he ca e ul one- o-one case–con ol ma ching
in he Co ogene s udy is leading o somewha op imis ic bioma ke
esul s compa ed wi h a eal-li e pa ien ca e si ua ion whe e con-
olling o con ounding ac o s is mo e di icul .
The lack o a disce nible ela ionship be ween LDL- ela ed
pa ame e s and CV isk ac oss he s udies included he e, e en a e
s a in s a i ica ion, is hough -p o oking bu , in line wi h p e ious
epo s.
24 –26
Fo example, Sachde a e al.
24
analysed admission lipid
le els in a b oad popula ion o 136,995 pa ien s hospi alized o
CAD in 541 hospi als and obse ed ha nea ly hal o he admission
LDL-C concen a ions we e ,100 mg/dl al hough be o e admis-
sion only 21.1% pa ien s we e ecei ing lipid-lowe ing medica ions.
Fu he mo e, in he MIRACL ial, he plasma HDL-C, bu no
LDL-C, measu ed in he ini ial s age o ACS p edic ed he isk o
ecu en ca dio ascula e en s.
25
The lowe LDL-C in highe isk
pa ien s may no be a phenomenon o ACS solely as in he Sa u n
ial in es iga o s obse ed ha C- eac i e p o ein, bu no
LDL-C le els, we e associa ed wi h co ona y a he oma eg ession
and ca dio ascula e en s a e in ensi e s a in he apy.
26
Taken
oge he , i appea s ha he LDL-C concen a ions may be simila
o e en lowe in CAD pa ien s a high isk o u u e CV e en s
compa ed wi h pa ien s wi h mo e a o able p ognosis. This may
be a phenomenon ela ed o disease p og ession and culmina ion,
and should no de ac om he alue o applying LDL-C o gauge
he li e ime isk o de elop a he oscle o ic plaques. A po en ial ex-
plana ion o his is p o ided in Gie ens e al.
27
who demons a ed
ha in e leukin-6 (IL-6) ac i a es LDL- ecep o (LDL ) ansc ip-
ion and subsequen ly enhances LDL ac i i y in he li e leading
o an inc eased elimina ion o LDL-C om he ci cula ion. Thus,
ch onic, and in pa icula acu e bu s s o , in lamma ion in CAD
pa ien s may enhance LDL-C clea ance, esul ing in lowe ed blood
LDL-C concen a ions and impai ed isk p edic ion.
Ce amide measu emen in high- h oughpu quali y con olled
en i onmen s is s aigh o wa d and cos -e icien . Iso ope labelled
s anda ds enable p ecise quan i ica ion and analy ical s abili y. Mos
clinical labo a o ies a e equipped wi h obo ized sample handling
sys ems and also house mass spec ome y equipmen .
Thus, ce amide-based iden i ica ion o co ona y pa ien s a high
ca dio ascula isk will soon be possible. These high- isk pa ien s
should hen bene i om ea men s ha ex end beyond s anda d
ca e. The sugges ed ac ions could include mo e equen ollow-up
isi s and e icien li e-s yle counselling as well as he conside a ion
o highe s a in doses, eze imibe combina ions, o no el he apies
such as PCSK9 inhibi o s. In u u e, addi ional he apies may include
o he op ions, o example, ongoing andomized clinical ials a e
looking in o he e ec o me ho exa e and in e leukin-1binhibi ion
o ea ing ca dio ascula isk.
28
Indeed, ce amides a e closely
linked o in lamma o y p ocesses and ecen ly CERS6 has been
iden i ied as a a ge o me ho exa e.
29
I is hence plausible o
hink ha ce amide es ing could become inc easingly ele an ,
especially i he ials wi h an i-in lamma o y compounds u n ou
posi i e. A heal h economic dimension o ce amide es ing is i s abil-
i y o a ge mo e in ense, po en ially mo e expensi e ea men s
such as PCSK9 inhibi o s o hose a he highes isk. Ano he aspec
o ce amide es ing is i s po en ial o mo i a ing pa ien ’s adhe -
ence, whe he o medica ion o li e-s yle changes, due o i s a he
di ec linkage wi h CV mo ali y. I has been shown ha o e 40% o
he pa ien s p esc ibed s a ins a e non-adhe en , which may ans-
la e o many a oidable addi ional e en s and hospi aliza ions.
30
While he ce amide-based isk s a i ica ion ex ends beyond he
cu en lipid-based diagnos ics and add esses he unme need o
imp o ed iden i ica ion o high- isk CAD pa ien s he e a e u he
scien i ic and clinical issues ha need a en ion. The e a e wo majo
lines o u u e esea ch conce ning he p esen ce amide co el-
a ion and ca dio ascula isk. On one hand i is o in e es o pu sue
he biology o hese molecules and wo k ou hei molecula mech-
anism o ac ion in ca dio ascula disease. This will in ol e mul i-
disciplina y e o s o cell biologis s, biochemis s, gene icis s, and
clinicians de eloping app op ia e cell and animal models. E o s in
his ega d a e al eady being made o example in he scope o
he EU unded ‘EUFP7-A he o lux’ conso ium. The o he line o
ollow is o es ablish he u ili y o hese ma ke s in clinical p ac ice.
In he USA, he ce amide es ing is en e ing he clinic his yea and
only his eal-li e e alua ion will allow o a be e judgemen o he
ce amide u ili y and will es ablish hem as a new a mamen in he
clinical diagnos ic ool-ki .
Supplemen a y ma e ial
Supplemen a y ma e ial is a ailable a Eu opean Hea Jou nal online.
Au ho s’ con ibu ions
M. S.-A., M. H., M. S. pe o med s a is ical analysis; R. L., R. H., M. N.,
J. S., O. N., T. L., W. M. handled unding and supe ision; K. E., D. K.,
H. S., T. S., E. V., M.-L. L., R. K., C. M., T. H., P. J., N. R., L. R., S. W.,
B. G., E. R. P., G. S. T., F. M. acqui ed he da a; R. L., R. H., J. S., T. L.,
F. M., O. N. concei ed and designed he esea ch; R. L., T. V. d a ed
he manusc ip ; K. E., M. S.-A., M. H., R. H., D. K., W. M., H. S., T. S.,
R. Laaksonen e al.1974
by gues on Sep embe 19, 2016h p://eu hea j.ox o djou nals.o g/Downloaded om
E. V., M. L. L., M. N., R. K., C. M., T. H., P. J., N. R., L. R., S. W., E. P.,
G. T., B. G., F. M., J. S., O. N., T. L. made c i ical e ision o he manu-
sc ip o key in ellec ual con en .
Acknowledgemen s
We hank M s. Si pa Su ela-Tuominen and M s. Ri a Huuhilo o
expe echnical suppo and sample p epa a ion.
Funding
This wo k was suppo ed by he Eu opean Union’s Se en h F amewo k
P og amme FP7/2007-2013 RiskyCAD P ojec (305739
2
) and u he
by esea ch g an s o he Swiss Na ional Resea ch Founda ion (SPUM
33CM30-124112), he Swiss Hea Founda ion, bo h Be n Swi ze land,
he Founda ion o Ca dio ascula Resea ch—Zu¨ ich Hea House,
Zu¨ ich, Swi ze land as well as As aZeneca, Zug; Eli Lilly Indianapolis,
USA; and Ve nie , Med onic, Tolochenaz; Me ck Sha pe and Dohme,
Gla b ugg;Sano i,Ve nie ;andS .JudeMedical,Zu¨ ich (all Swi ze -
land). The Co ogene s udy was suppo ed by g an s om Aa no
Koskelo Founda ion, Helsinki Uni e si y Cen al Hospi al Special Go -
e nmen Funds (EVO #TYH7215, #TKK2012005, #TYH2012209, and
#TYH2014312), and Finnish Founda ion o Ca dio ascula esea ch.
The BECAC s udy was suppo ed by a g an om he Wes e n No way
Regional Heal h Au ho i y (911570). The unde s had no ole in he
design and conduc o he s udy; collec ion, managemen , analysis,
and in e p e a ion o he da a; p epa a ion, e iew, o app o al o he
manusc ip ; and decision o submi he manusc ip o publica ion.
Funding o pay he Open Access publica ion cha ges o his a icle
was p o ided by Zo a Biosciences.
Con lic o in e es : F.M. has ecei ed esea ch g an s o he ins i u-
ion om Amgen, As aZeneca, Bos on Scien i ic, Bio onik, Med onic,
MSD, Eli Lilly, and S . Jude Medical including speake o consul an ees.
S.W. has ecei ed esea ch g an s o he ins i u ion om Abbo , As a-
Zeneca, Bos on Scien i ic, Biosenso s, Bio onik, Co dis, Eli Lilly, Med-
onic, and S . Jude Medical. T.F.L. ecei ed esea ch g an s o he
ins i u ion om As aZeneca, Baye Heal h Ca e, Biosenso s, Bio onik,
Bos on Scien i ic, Med onic, Me ck, Sha pe and Dohme, Me ck, Inc.,
Roche, and Se ie , including lec u e ees. C.M.M. ecei ed esea ch
g an s o he ins i u ion om Eli Lilly, As aZeneca, Roche and MSD
and speake o consul an ees om Eli Lilly, Daiichi Sankyo, As aZene-
ca, Roche and MSD. W.M. is employed wi h Synlab Holding Ge many
GmbH, has owne ship in e es in Synlab Holding In e na ional GmbH
and has ecei ed esea ch g an s o he ins i u ion om Aege ion Pha -
maceu icals, Amgen, As azeneca, Danone Resea ch, Sano i/Genzyme,
Ho mann LaRoche, Numa es, Unile e , and BASF and speake and
consul ancy ees om Aege ion Pha maceu icals, Amgen, As azeneca,
Danone Resea ch, Sano i/Genzyme, Ho mann LaRoche, Me ck Sha p
andDohme,P ize ,Sano i,Synage a,Numa es,Unile e ,andBASF.
Zo a Biosciences holds pa en s o he diagnos ic use o ce amides
and R.H. and R.L. a e sha eholde s o Zo a Biosciences.
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