1-s2.0-S0735109712055222-main

Journal of the American College of Cardiology Vol. 61, No. 4, 2013© 2013 by the American College of Cardiology Foundation ISSN 0735-1097/$36.00

Cardiometabolic Risk

Remnant Cholesterol as a CausalRisk Factor for Ischemic Heart Disease

Anette Varbo, MD,*†‡ Marianne Benn, MD, PHD, DMSC,*†‡Anne Tybjærg-Hansen, MD, DMSC,†‡§� Anders B. Jørgensen, MD,†‡§Ruth Frikke-Schmidt, MD, PHD, DMSC,†‡§ Børge G. Nordestgaard, MD, DMSC*†‡§

Herlev and Copenhagen, Denmark

Objectives The aim of this study was to test the hypothesis that elevated nonfasting remnant cholesterol is a causal riskfactor for ischemic heart disease independent of reduced high-density lipoprotein (HDL) cholesterol.

Background Elevated remnant cholesterol is associated with elevated levels of triglyceride-rich lipoproteins and with reducedHDL cholesterol, and all are associated with ischemic heart disease.

Methods A total of 73,513 subjects from Copenhagen were genotyped, of whom 11,984 had ischemic heart disease diag-nosed between 1976 and 2010. Fifteen genetic variants were selected, affecting: 1) nonfasting remnant choles-terol alone; 2) nonfasting remnant cholesterol and HDL cholesterol combined; 3) HDL cholesterol alone; or4) low-density lipoprotein (LDL) cholesterol alone as a positive control. The variants were used in a Mendelianrandomization design.

Results The causal odds ratio for a 1 mmol/l (39 mg/dl) genetic increase of nonfasting remnant cholesterol was 2.8(95% confidence interval [CI]: 1.9 to 4.2), with a corresponding observational hazard ratio of 1.4 (95% CI: 1.3 to1.5). For the ratio of nonfasting remnant cholesterol to HDL cholesterol, corresponding values were 2.9 (95% CI:1.9 to 4.6) causal and 1.2 (95% CI 1.2 to 1.3) observational for a 1-U increase. However, for HDL cholesterol,corresponding values were 0.7 (95% CI: 0.4 to 1.4) causal and 1.6 (95% CI: 1.4 to 1.7) observational for a1 mmol/l (39 mg/dl) decrease. Finally, for LDL cholesterol, corresponding values were 1.5 (95% CI: 1.3 to 1.6)causal and 1.1 (95% CI: 1.1 to 1.2) observational for a 1 mmol/l (39 mg/dl) increase.

Conclusions A nonfasting remnant cholesterol increase of 1 mmol/l (39 mg/dl) is associated with a 2.8-fold causal risk forischemic heart disease, independent of reduced HDL cholesterol. This implies that elevated cholesterol contentof triglyceride-rich lipoprotein particles causes ischemic heart disease. However, because pleiotropic effects of thegenetic variants studied cannot be totally excluded, these findings need to be confirmed using additional genetic vari-ants and/or randomized intervention trials. (J Am Coll Cardiol 2013;61:427–36) © 2013 by the AmericanCollege of Cardiology Foundation

Published by Elsevier Inc. http://dx.doi.org/10.1016/j.jacc.2012.08.1026

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Remnant cholesterol is the cholesterol content of triglyceride-richlipoproteins, composed of very low-density lipoproteins andintermediate-density lipoproteins in the fasting state and of

From the *Department of Clinical Biochemistry, Herlev Hospital, CopenhagenUniversity Hospital, Herlev, Denmark; †The Copenhagen General Population Study,Herlev Hospital, Copenhagen University Hospital, Herlev, Denmark; ‡Faculty ofHealth and Medical Sciences, University of Copenhagen, Copenhagen, Denmark;§The Copenhagen City Heart Study, Bispebjerg Hospital, Copenhagen UniversityHospital, Copenhagen, Denmark; and the �Department of Clinical Biochemistry,Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark. This studywas supported by the Danish Medical Research Council, the Danish Heart Founda-tion, Herlev Hospital, Copenhagen University Hospital, Copenhagen County Foun-dation, and Chief Physician Johan Boserup and Lise Boserup’s Fund, Denmark. Dr.Nordestgaard has received lecture and/or consultancy honoraria from AstraZeneca,Merck, Pfizer, Karo Bio, Omthera, Abbott, Sanofi-Aventis, Regeneron, and ISISPharmaceuticals. All other authors have reported that they have no relationshipsrelevant to the contents of this paper to disclose.

HManuscript received July 12, 2012; accepted August 21, 2012.

these 2 lipoproteins together with chylomicron remnantsin the nonfasting state (1,2). Elevated nonfasting plasmatriglyceride is a marker of elevated nonfasting remnantcholesterol (1,3,4) and is associated with increased risk forcardiovascular disease (1–7). Because triglycerides per se areunlikely directly to cause cardiovascular disease (2,8), rem-

ant cholesterol is more likely to be the causal factor.

See page 437

Elevated remnant cholesterol is also associated with reducedhigh-density lipoprotein (HDL) cholesterol (2,9,10); how-ever, because HDL cholesterol increases have failed toreduce cardiovascular disease (11,12), elevated remnantholesterol may be a more likely causal factor than reduced
DL cholesterol.

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428 Varbo et al. JACC Vol. 61, No. 4, 2013Remnants and Ischemic Heart Disease January 29, 2013:427–36

We tested the hypothesis thatelevated nonfasting remnant cho-lesterol is a causal risk factor forischemic heart disease indepen-dent of reduced HDL cholesterol,using the Mendelian randomiza-tion approach (10,13,14): geneticvariants robustly associated with asingle lipoprotein, without effects on

other lipoproteins, were used to test the causal effect of lifelongexposure on risk for ischemic heart disease. Following a pre-specified plan and on the basis of current evidence from publishedresearch, we therefore carefully selected 15 genetic variants affect-ing levels of: 1) nonfasting remnant cholesterol alone; 2) nonfast-ing remnant cholesterol and HDL cholesterol combined; 3)HDL cholesterol alone; or 4) low-density lipoprotein (LDL)cholesterol alone as a positive control. Thus, several other geno-types were considered for inclusion but were a priori not included,exemplified by apolipoprotein E genotypes that affect levels of allthe lipoproteins studied here and therefore would be difficult touse for the present studies.

We genotyped 73,513 white subjects of Danish descentfrom Copenhagen, of whom 11,984 had ischemic heartdisease. Causal estimates for each lipoprotein on risk forischemic heart disease were calculated in the same subjectsand were thus directly comparable.

Methods

Studies were approved by institutional review boards andDanish ethical committees (H-KF-01-144/01, KF-100.2039/91, KF-01-144/01, KA-93125, and KA-99039)and conducted according to the Declaration of Helsinki.Informed consent was obtained from participants. All par-ticipants were white and of Danish descent. No subject wasincluded in more than 1 study.The CGPS (Copenhagen General Population Study).The CGPS is a prospective study of the general populationinitiated in 2003 with ongoing enrollment (1). Subjectswere selected on the basis of the national Danish CivilRegistration System to reflect the adult Danish populationage 20 to �80 years. Data were obtained from a question-naire, a physical examination, and blood samples includingdeoxyribonucleic acid extraction. Follow-up was 100% com-plete; that is, no subject was lost to follow-up. A total of57,719 participants were included at the time of analyses; ofthese, 10,368 were used as controls in the CIHDS (Copen-hagen Ischemic Heart Disease Study), leaving 47,351participants.The CCHS (Copenhagen City Heart Study). TheCCHS is a prospective study of the general populationinitiated from 1976 to 1978, with follow-up examinationsfrom 1981 to 1983, 1991 to 1994, and 2001 to 2003.Participants were recruited and examined exactly as in theCGPS. Blood samples for deoxyribonucleic acid extraction

Abbreviationsand Acronyms

CI � confidence interval

HDL � high-densitylipoprotein

LDL � low-densitylipoprotein

were drawn at the 1991 to 1994 and 2001 to 2003 r

examinations. A total of 10,609 participants were eligiblefor analyses.The CIHDS. This study comprises 5,185 patients withischemic heart disease from Copenhagen University Hos-pital during the period from 1991 to 2009 and 10,368controls without ischemic heart disease matched by age andsex from the CGPS. Besides a diagnosis of ischemic heartdisease, these cases also had stenosis or atherosclerosis oncoronary angiography and/or positive results on exerciseelectrocardiography.Ischemic heart disease. Information on diagnoses of isch-emic heart disease (World Health Organization Interna-tional Classification of Diseases-Eighth Revision, codes 410to 414; International Classification of Diseases-Tenth Re-vision, codes I20 to I25) were collected from 1976 through2010 by reviewing all hospital admissions and diagnosesentered in the national Danish Patient Registry and allcauses of death entered in the national Danish Causes ofDeath Registry, as previously described (1).Laboratory analyses. Nonfasting total cholesterol, triglyc-erides, and HDL cholesterol were measured using colori-metric assays (Boehringer Mannheim, Mannheim, Ger-many; Konelab, Thermo Fisher Scientific, Waltham,Massachusetts). LDL cholesterol was calculated using theFriedewald equation when plasma triglycerides were �4.0

mol/l and otherwise measured directly (Konelab). Non-asting remnant cholesterol was calculated as nonfastingotal cholesterol minus HDL cholesterol minus LDLholesterol.

enotypes. Genotyping was performed using TaqManApplied Biosystems, Carlsbad, California) or by restrictionnzyme assays. Genotypes were verified by genotyping ofandomly selected samples of each variant using 2 differentethods (TaqMan plus sequencing or restriction enzyme

ssay). Call rates for genotypes were �99.9% for all assays.ther covariates. Smokers were defined as current smok-

rs. Hypertension was defined as systolic blood pressure140 mm Hg (�135 mm Hg for patients with diabetes),

iastolic blood pressure �90 mm Hg (�85 mm Hg foratients with diabetes), and/or the use of antihypertensiveedications prescribed specifically for hypertension. Lipid-

owering therapy was self-reported.tatistical analysis. Data were analyzed using Stata/SEersion 11.2 (StataCorp LP, College Station, Texas). Two-ided p values �0.05 were considered statistically signifi-ant. Non-normally distributed variables were log trans-ormed to approach a normal distribution. Chi-square testsere used to evaluated Hardy-Weinberg equilibrium.In the prospective CGPS, CCHS, and CIHDS controls

ombined, Cox proportional hazards regression models withge as the time scale were used to estimate hazard ratios forschemic heart disease by lipoproteins in quintiles; patientsiagnosed with ischemic heart disease before study entryere excluded, and those dying or emigrating during

ollow-up were censored at their deaths or emigration dates,
espectively. Multivariate adjustment was performed for age,

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429JACC Vol. 61, No. 4, 2013 Varbo et al.January 29, 2013:427–36 Remnants and Ischemic Heart Disease

sex, smoking, hypertension, time since last meal, time of dayfor blood sampling, and lipid-lowering therapy. Analyseswere purposely not adjusted for variables that in themselvesthrough biological pathways affect lipoprotein levels, such asbody mass index, alcohol consumption, and diabetes. Con-ventional hazard ratios and confidence intervals (CIs) werecorrected for regression dilution bias using a nonparametricmethod (15), using lipoprotein values from 4,253 individ-als without lipid-lowering therapy participating in both the991 to 1994 and 2001 to 2003 examinations of the CCHS;his correction helps avoid underestimation of risk esti-

Figure 1 Remnant Cholesterol, Time Since Last Meal, and Tim

Remnant cholesterol as a function of time since last meal and time of day for blo

Characteristics of the Participants in the 3 StudiesTable 1 Characteristics of the Participants in the 3 Studies

Variable CGPS CCHS CIHDS

Number of participants 47,351 10,609 15,553

Women 28,806 (61%) 5,886 (55%) 4,418 (29%)

Age (yrs) 55 (46–65) 59 (46–69) 63 (56–71)

Triglycerides (mmol/l) 1.4 (1.0–2.1) 1.5 (1.1–2.2) 1.6 (1.1–2.3)

Remnant cholesterol(mmol/l)

0.6 (0.4–0.9) 0.7 (0.5–1.0) 0.7 (0.5–1.0)

HDL cholesterol (mmol/l) 1.6 (1.3–1.9) 1.5 (1.2–1.9) 1.4 (1.1–1.8)

LDL cholesterol (mmol/l) 3.2 (2.6–3.8) 3.7 (3.0–4.5) 3.2 (2.6–3.9)

Hypertension 32,081 (68%) 5,075 (48%) 7,741 (78%)*

Smoking, current 9,919 (21%) 4,589 (49%) 2,193 (23%)*

Statin use 4,620 (10%) 87 (1%) 1,008 (10%)*

Ischemic heart disease 4,582 (10%) 2,217 (21%) 5,185 (33%)

Data are from study enrolment from 2003 to 2009 for the CGPS, from the 1991 to 1994 or 2001to 2003 examinations of the CCHS when deoxyribonucleic acid was collected, and from studyenrollment from 1991 to 2009 in the CIHDS. Data are expressed as median (interquartile range)or as number of participants (percent). Number of participants varies slightly according toavailability of variables. To convert triglyceride values to milligrams per deciliter, multiply values inmillimoles per liter by 88. To convert cholesterol values to milligrams per deciliter, multiply valuesin millimoles per liter by 38.6. *Values available only for controls.

CCHS � Copenhagen City Heart Study; CGPS � Copenhagen General Population Study; CIHDS �

Copenhagen Ischemic Heart Disease Study; HDL � high-density lipoprotein; LDL � low-densityipoprotein.

mates, but it does not affect whether results are statisticallysignificant. Regression dilution ratios of 0.48, 0.63, 0.73, and0.60 were computed for nonfasting remnant cholesterol, theratio of nonfasting remnant cholesterol to HDL cholesterol,HDL cholesterol, and LDL cholesterol, respectively.

One-way analysis of variance was used to compare lipo-protein levels as a function of genotypes in the CGPS,CCHS, and CIHDS controls combined. Genotypes werecombined in 4 groups: 1) nonfasting remnant cholesterol–increasing alleles (TRIB1 rs2954029, GCKR rs1260326,nd APOA5 rs651821); 2) nonfasting remnant cholesterol–ncreasing and HDL cholesterol–decreasing alleles (LPLs1801177, LPL G188E, LPL rs268, and LPL rs328); 3) HDL

cholesterol–decreasing alleles (LIPC -480C�T, ABCA1N1800H, and ABCA1 R2144X), and, as a positive control;4) LDL cholesterol–increasing alleles (APOB rs5742904,LDLR W23X, LDLR W66G, LDLR W556S, and PCSK9rs11591147). The p values for trends were estimated usingCuzick’s extension of the Wilcoxon rank sum test.

Association of genotypes with observed risk for ischemicheart disease was by logistic regression estimating age-adjusted and sex-adjusted odds ratios in the CGPS, CCHS,and CIHDS combined. Theoretically predicted risk wasestimated from changes in lipoprotein values and knownconventional associations of lipoproteins with ischemicheart disease in the prospective study.

Potential causal relationships between genetically ele-vated or reduced levels of the different lipoproteins and riskfor ischemic heart disease were assessed by instrumentalvariable analyses using 2-stage least squares regression in anadditive model. Genotypes associated with specific lipopro-tein traits (i.e., nonfasting remnant cholesterol) were in-

ay for Blood Sampling

pling. Values are medians with interquartile ranges (error bars).

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cluded as individual instruments in both first-stage andsecond-stage regressions; thereby, each variant was weighted byfrequency and effect size. The strength of the separate andcombined instruments was evaluated by F statistics from thefirst-stage regression, where an F statistic �10 indicatessufficient strength to ensure the validity of the instrumentalvariable analysis (13). R2 values as percentages were usedas a measure of the contribution of genotypes to thevariation in the different lipoprotein levels. Lipoproteinvalues were not available for all cases in the CIHDS (7.6%missing) and were therefore imputed from age, sex, geno-types, and the known distribution of lipoproteins in theCGPS and the CCHS. The same was done for participantsfrom the CGPS (7.4%) and the CCHS (0.6%) usinglipid-lowering therapy; however, if subjects missing lipoproteinvalues or on lipid-lowering therapy were excluded instead,results were similar to those reported. Causal estimates frominstrumental variable analysis were compared with observedrisk for ischemic heart disease from conventional epidemiologyusing the Bland-Altman method.

Results

Table 1 shows baseline characteristics of participants in the3 studies. Participants in the CIHDS were older and moreoften men than in the CGPS and the CCHS. Genotypedistributions for all studies were in Hardy-Weinberg equi-librium (p � 0.10).Remnant cholesterol. Remnant cholesterol levels changedslightly as a function of time since last meal and time of dayfor blood sampling and had median levels of 0.55 mmol/lduring fasting and 0.67 mmol/l at 3 to 4 h after the last meal(Fig. 1); the highest and lowest levels were seen at 1 and 7PM, respectively. Increased levels of plasma triglycerideswere associated with increased levels of remnant cholesteroland with reduced levels of HDL cholesterol, while theassociation with LDL cholesterol was less pronounced (Fig. 2,top). Remnant cholesterol levels were highly correlated withnonfasting triglyceride levels (R2 � 0.96, p � 0.001),nversely correlated with HDL cholesterol levels (R2 �

0.45, p � 0.001), and less correlated with LDL choles-erol levels (R2 � 0.12, p � 0.001) (Fig. 2, bottom).

Risk for ischemic heart disease: observational estimates.Associations of lipoproteins in quintiles with risk for isch-emic heart disease in the CGPS, CCHS, and CIHDScontrols combined in a prospective design are shown inFigure 3. Observational hazard ratios for the fifth versus firstquintiles were 2.3 (95% CI: 1.7 to 3.1) for increasingnonfasting remnant cholesterol, 2.6 (95% CI: 2.1 to 3.2) forincreasing ratio of nonfasting remnant cholesterol toHDL cholesterol, 2.5 (95% CI: 2.1 to 3.0) for decreasingHDL cholesterol, and 1.8 (95% CI: 1.4 to 2.2) forincreasing LDL cholesterol.Genotypes and lipoprotein levels. Lipoprotein levels as afunction of increasing number of lipoprotein-increasing and
lipoprotein-decreasing alleles for the CGPS, CCHS, and
CIHDS controls combined are shown in Figure 4 forgroups of genotypes with similar effects. Corresponding effectsof the individual genotypes are shown in Online Figure 1.

For nonfasting remnant cholesterol alone, 3 to 6 versus 0or 1 alleles were associated with 15% (0.10 mmol/l [4mg/dl]) elevated nonfasting remnant cholesterol and 19%elevated ratio of nonfasting remnant cholesterol to HDLcholesterol. HDL and LDL cholesterol differed onlyslightly by genotype.

For nonfasting remnant cholesterol and HDL cholesterolcombined, 3 or 4 versus 0 or 1 alleles were associated with22% (0.15 mmol/l [6 mg/dl]) elevated nonfasting remnantcholesterol, 37% elevated ratio of nonfasting remnant cho-lesterol to HDL cholesterol, and 11% (0.18 mmol/l [7mg/dl]) reduced HDL cholesterol. LDL cholesterol differedonly slightly by genotype.

For HDL cholesterol alone, 2 or 3 versus 0 alleles wereassociated with 8% (0.13 mmol/l [5 mg/dl]) reduced HDLcholesterol; the ratio of nonfasting remnant cholesterol to

Figure 2 Associations and Correlations ofLipoprotein Cholesterol and Triglycerides

(Top) Lipoprotein cholesterol as a function of increasing levels of nonfastingtriglycerides. (Bottom) Correlation matrix of lipids and lipoproteins. Top panelreproduced with modifications, with permission, from Nordestgaard andTybjærg-Hansen (10) and from Chapman et al. (2). HDL � high-densitylipoprotein; LDL � low-density lipoprotein.

HDL cholesterol changed accordingly. Although nonfast-

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ing remnant cholesterol decreased slightly as a function ofgenotype, this was in the opposite direction of the usualinverse association between reduced HDL cholesterol andelevated nonfasting remnant cholesterol (2–4). LDL cho-esterol did not differ by genotype.

For LDL cholesterol alone, 3 versus 0 or 1 alleles weressociated with 90% (2.66 mmol/l [103 mg/dl]) elevatedDL cholesterol. Other lipoprotein classes did not differ byenotype.enotypes and risk for ischemic heart disease. Assuming

hat each of the lipoproteins is causally associated with riskor ischemic heart disease, we would theoretically expectenetically elevated or reduced levels to be associated withisk for ischemic heart disease in the same direction asbserved for conventional lipoprotein levels, as shown inigure 3. For nonfasting remnant cholesterol alone, foronfasting remnant cholesterol and HDL cholesterol com-ined, and for LDL cholesterol alone, the observed associ-tions of the genotypes with risk for ischemic heart diseaseere in the same direction and more pronounced than the

heoretically predicted risk for ischemic heart disease; however,

Figure 3 Risk for Ischemic Heart Disease: Observational Estim

Risk for ischemic heart disease as a function of lipoprotein levels in quintiles in thand Copenhagen Ischemic Heart Disease Study controls combined. The p values atein; LDL � low-density lipoprotein.

his was not the case for HDL cholesterol alone (Fig. 5). f

For nonfasting remnant cholesterol alone for 3 to 6 versusor 1 alleles, the observed risk was increased by 15% (95%I: 9% to 21%) and the theoretically predicted risk by 5%

95% CI: 4% to 6%) (Fig. 5). For nonfasting remnantholesterol and HDL cholesterol combined for 3 or 4 versusor 1 alleles, the corresponding risks were increased by 29%

95% CI: 18% to 42%) and 9% (95% CI: 7% to 11%).owever, for HDL cholesterol alone for 2 or 3 versus 0

lleles, we observed no association with risk for ischemiceart disease by genotype, despite a theoretically predicted

ncreased risk of 7% (95% CI: 5% to 8%). Finally, for LDLholesterol alone for 3 versus 0 or 1 alleles, correspondingisks were increased by 525% (95% CI: 314% to 845%) and5% (95% CI: 29% to 63%).Other risk factors for ischemic heart disease than lipoproteins

ere generally distributed equally among genotypes, confirminghat the genotypes are not confounded (Online Table 1).

isk for ischemic heart disease: causal estimates. Welso examined the potential causal association of the lipo-roteins with risk for ischemic heart disease in instrumentalariable analyses. For nonfasting remnant cholesterol alone,

pective Copenhagen General Population Study, Copenhagen City Heart Study,trends across quintiles. CI � confidence interval; HDL � high-density lipopro-

ates

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or the ratio of nonfasting remnant cholesterol to HDL

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cholesterol, and for LDL cholesterol alone, causal riskestimates for genetically elevated levels were in the samedirection and higher than corresponding risk estimates forcorresponding increases in conventional plasma levels of thesame lipoproteins; however, this was not the case for HDLcholesterol alone (Fig. 6).

The causal odds ratio for a 1 mmol/l (39 mg/dl) geneticincrease of nonfasting remnant cholesterol was 2.8 (95% CI:1.9 to 4.2), with a corresponding observational hazard ratioof 1.4 (95% CI: 1.3 to 1.5) (Fig. 6). For the ratio of

onfasting remnant cholesterol to HDL cholesterol, corre-ponding values were 2.9 (95% CI: 1.9 to 4.6) causal and 1.295% CI: 1.2 to 1.3) observational for a 1-U increase.

owever for HDL cholesterol, corresponding values were.7 (95% CI: 0.4 to 1.4) causal and 1.6 (95% CI: 1.5 to 1.7)bservational for a 1-mmol/l (39 mg/dl) decrease. Finallyor LDL cholesterol, corresponding values were 1.5 (95%I: 1.3 to 1.6) causal and 1.1 (95% CI: 1.1 to 1.2)bservational for a 1 mmol/l (39 mg/dl) increase.Results were similar for a 1 standard deviation increase or

Figure 4 Genotypes and Lipoprotein Levels

Lipoprotein levels as a function of genotype in allele counts in the Copenhagen GeDisease Study controls combined. Genetic variants included in each lipoprotein grbars, percent change in lipoprotein levels, and p values for trends across allele coprotein; LDL � low-density lipoprotein.

ecrease in genetic and plasma levels of the lipoproteins a

Online Fig. 2). Online Figure 3 illustrates that all geneticariants separately and combined for each lipoprotein stud-ed were valid instruments (F statistics �10). Figure 6hows causal odds ratios for the CGPS, CCHS, andIHDS combined; however, analyzing these studies sepa-

ately gave similar results (Online Fig. 4).

iscussion

his study suggests that a 1 mmol/l (39 mg/dl) increase inonfasting remnant cholesterol is associated with a 2.8-foldausal risk for ischemic heart disease, independent of re-uced HDL cholesterol. These findings are novel.That the causal risk for ischemic heart disease was higher

han the observational risk for elevated nonfasting remnantholesterol suggests that lifelong exposure through geneti-ally elevated levels may have a larger effect on risk thanuggested from observational data alone. This is in accor-ance with parallel results for LDL cholesterol in present

Population Study, Copenhagen City Heart Study, and Copenhagen Ischemic Hearte shown in brackets. Columns show mean lipoprotein levels with standard errorParticipants using lipid-lowering therapy were excluded. HDL � high-density lipo-

neraloup arunts.

nd former studies (16,17).

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Mechanistically, the explanation for a causal effect ofelevated nonfasting remnant cholesterol on ischemic heartdisease risk could be that remnants (i.e., triglyceride-richlipoproteins) enter and get trapped in the intima of thearterial wall (18,19). Like LDL trapping in the intima, thiswould lead to the accumulation of intimal cholesterol,atherosclerosis, and ultimately ischemic heart disease (2,9).

nlike LDL, remnants and triglyceride-rich lipoproteinsay not need to be oxidized to be taken up by macrophages

o cause foam cell formation and atherosclerosis (20).Previous case-control (21–31) and prospective (32–36)

tudies have also found observationally that elevated rem-ant cholesterol is associated with increased risk for cardio-ascular disease. However, these studies were relativelymall and, unlike the present study, were unable to investi-ate the causality of remnant cholesterol, because of con-ounding by various cardiovascular risk factors and becausef the inverse association with reduced HDL cholesterol2,10). To circumvent these problems, on one hand, we usedhe Mendelian randomization approach to avoid confound-ng from other cardiovascular risk factors: genetically deter-

Figure 5 Genotypes and Risk for Ischemic Heart Disease

Theoretically predicted risk and observed risk for ischemic heart disease as a funcHDL � high-density lipoprotein; LDL � low-density lipoprotein.

ined levels of lipoproteins are present from birth and are t

herefore generally not confounded by other risk factors13,14). On the other hand, by using carefully selectedenetic variants, we were able to deduct the causality ofipoproteins by using genetic variants as instruments. Vari-nts causing only lifelong elevated nonfasting remnantholesterol were associated with increased risk for ischemiceart disease. The same was seen for variants causing both

ifelong elevated nonfasting remnant cholesterol and re-uced HDL cholesterol; however, variants causing only

ifelong reduced HDL cholesterol were not associated withncreased risk for ischemic heart disease. Taken together,his implies that it must be the elevated nonfasting remnantholesterol and triglyceride-rich lipoproteins that are caus-lly related to increased risk for ischemic heart disease.

In our study, remnant cholesterol was determined in theonfasting state and thus included cholesterol in allriglyceride-rich lipoproteins, that is, very low-density lipo-roteins, intermediate-density lipoproteins, and chylomi-ron remnants combined. By using nonfasting remnantholesterol calculated as nonfasting total cholesterol minusDL cholesterol minus LDL cholesterol, remnant choles-

f genotypes in number of alleles. CI � confidence interval;
tion o
erol can be calculated directly from a standard lipid profile,

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provided it is taken in the nonfasting state, as has beenrecommended in Denmark since 2009. Thus, the presentlyused calculated nonfasting remnant cholesterol comes at noextra cost and is easily available.

The Mendelian randomization approach has potentiallimitations, with the most important being pleiotropy of thegenetic variants used; that is, the genetic variants may affectrisk for ischemic heart disease through mechanisms otherthan their effects on lipoprotein levels. For a proper Men-delian randomization study, a variant that exclusively affectsplasma remnant cholesterol is needed. Because remnantcholesterol is the result of multiple interrelated metabolicprocesses involving many genes, an ideal variant that affectsonly remnant cholesterol simply may not exist. Somepleiotropic effects are evident for the 3 genetic variantsassociated with remnant cholesterol and risk for ischemicheart disease. From previous studies, it is known thatTRIB1 and APOA5 are also associated with HDL choles-

Figure 6 Causal and Observational Risk Estimates for Ischemic

Observational risk estimates in hazard ratios for a 1 mmol/l or a 1 ratio unit increGeneral Population Study, Copenhagen City Heart Study, and Copenhagen Ischemsion, time since last meal, time of day for blood sampling, and lipid-lowering theragenetic lipoprotein levels in odds ratios are from the Copenhagen General PopulatStudy combined, estimated by instrumental variable analyses. The p values are fobetween observational and causal estimates. CI � confidence interval; HDL � hig

erol and LDL cholesterol and GCKR with HDL choles-

terol, fasting glucose levels, and risk for diabetes (37,38).Most important, remnant cholesterol is directly related toplasma triglycerides, because remnant cholesterol is thecholesterol content of triglyceride-rich lipoproteins. How-ever, because elevated triglycerides per se are unlikely to bethe direct cause of ischemic heart disease (2,8), the choles-terol content of the triglyceride-rich lipoproteins is morelikely to be the cause of the risk marked by high triglycer-ides. However, when we studied nonfasting plasma triglyc-erides instead of nonfasting remnant cholesterol, results andconclusions were largely similar (data not shown). Exceptfor triglycerides and other components of triglyceride-richlipoproteins, the presently used variants were not associatedto any large extent with other known risk factors, andchoosing more than 1 variant from different genes ondifferent chromosomes makes it unlikely that the variantshave the same pleiotropic effects (14). Nevertheless, ratherthan remnant cholesterol per se, it is possible that

rt Disease

decrease in plasma lipoprotein levels are from the prospective Copenhagenrt Disease Study controls combined, adjusted for age, sex, smoking, hyperten-usal risk estimates for a 1 mmol/l or a 1 ratio unit increase or decrease inudy, Copenhagen City Heart Study, and Copenhagen Ischemic Heart Diseaseficance of risk estimates, and p values for comparison are for differencesity lipoprotein; LDL � low-density lipoprotein.

Hea

ase oric Heapy. Caion Str signih-dens

triglyceride-rich lipoprotein particles per se or other unmea-


sured covariates of triglycerides could also be implicated asbeing causal for ischemic heart disease on the basis of ourfindings.

Also, it could be argued that a relatively larger populationvariation in remnant cholesterol compared with HDL andLDL cholesterol would make it easier to detect associationsof genetic variants with remnant cholesterol; however,because the variation over time was larger for remnantcholesterol than for HDL and LDL cholesterol, withregression dilution ratios of 0.48, 0.73, and 0.60, respec-tively, it is more likely that it is the opposite; that is, it ismore difficult to detect genetic variation associated withremnant cholesterol than with HDL and LDL cholesterol.Furthermore, analyses are limited to a number of geneticvariants and genes, for example, 4 variants in LPL forremnant cholesterol and HDL cholesterol, variants in 2genes for HDL cholesterol, and variants in 3 genes for LDLcholesterol. Because different biological pathways lead tovariability in these lipid fractions, the interpretation of theresults is limited to the processes regulated by the genesincluded in the analysis. For HDL cholesterol, Mendelianrandomization studies do not support a casual relationshipbetween common genetic variants near ABCA1, LCAT,LIPC, or LIPG (39–42), but it is possible that geneticvariation altering the expression or function of other genesin the HDL pathway, such as CETP and APOA1, mightalter risk for ischemic heart disease. Finally, because westudied white subjects only, our results may not necessarilyapply to other races.

Strengths of our study include sufficient statistical powerand no bias from admixture, because of the large sample sizeof all white subjects of Danish descent. Another strength ofthe present study is the control for the validity of the studydesign by the inclusion of a positive LDL cholesterolcontrol in all analyses; LDL cholesterol is well documentedas a causal risk factor for ischemic heart disease (10,16,43).Finally, although remnant cholesterol varies slightly morethan LDL cholesterol in the same subjects over 10 years(compare regression dilution ratios of 0.48 vs. 0.60), wecorrected for this regression dilution bias for all lipoproteins.

Conclusions

A 1 mmol/l (39 mg/dl) increase in nonfasting remnantcholesterol is associated with a 2.8-fold causal risk forischemic heart disease, independent of reduced HDL cho-lesterol. This implies that the elevated cholesterol content oftriglyceride-rich lipoprotein particles causes ischemic heartdisease. However, because we cannot totally exclude pleio-tropic effects of the genetic variants studied, our findingsneed to be confirmed using additional genetic variantsand/or randomized intervention trials. These findings mayhelp direct future efforts to reduce cardiovascular diseasebeyond the reduction achieved using statins. The focus offuture intervention studies should be not only on lowering
LDL cholesterol levels but also on lowering nonfasting
remnant cholesterol levels and triglyceride-rich lipoproteins,or alternatively on reduction in nonfasting apolipoprotein Bor non-HDL cholesterol, both of which include remnant ortriglyceride-rich lipoproteins.

Reprint requests and correspondence: Dr. Børge G. Nordest-gaard, Department of Clinical Biochemistry, Herlev Hospital,Copenhagen University Hospital, Herlev Ringvej 75, DK-2730Herlev, Denmark. E-mail: [email protected].

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Key Words: atherosclerosis y cardiovascular disease y lipoproteins ymyocardial infarction.

APPENDIX

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